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 TH E BIOLOGY OF

MOSQUITOES
TRANSMISSION OF VIRUSES AND INTERACTIONS
WITH BACTERIA
 THE BIOLOGY OF

MOSQUITOES
This page intentionally left blank
 THE BIOLOGY OF

MOSQUITOES
VOLUME 3
TRANSMISSION OF VIRUSES AND INTERACTIONS
WITH BACTERIA

A.N. CLEMENTS
London School of Hygiene and Tropical Medicine

ab www.cabi.org
 CABI is a trading name of CAB International
CABI CABI
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Website: www.cabi.org

© A.N. Clements 2012. All rights reserved. No part of this publication may be reproduced
in any form or by any means, electronically, mechanically, by photocopying, recording or
otherwise, without the prior permission of the copyright owners.

A catalogue record for this book is available from the British Library, London, UK.

Library of Congress Cataloging-in-Publication Data

Clements, A. N. (Alan Neville)
The biology of mosquitoes. Volume 3, Viral and bacterial pathogens and bacterial
symbionts / A.N. Clements. p. cm.
Includes bibliographical references and index.
ISBN 978.1-84593-242-8 (alk. paper)
1. Mosquitoes. 2. Mosquitoes -- Pathogens. 3. Mosquitoes as carriers of disease. I. Title.
II. Title: Viral and bacterial pathogens and bacterial symbionts.

QL536.C557 2011
595.77'2-dc22
2010021495

ISBN-13: 978 1 84593 242 8

Commissioning editor: Rachel Cutts

Production editors: Tracy Head and Fiona Chippendale

Typeset by Columns Design XML in Goudy Oldstyle, 10.5/12.5 pt

Printed and bound in the UK by Cambridge University Press, Cambridge
 Contents

Acknowledgements vii
Preface viii
Introduction The traditional and revised classifications of aedine mosquitoes ix

41 Host/parasite interactions 1
41.1 Characteristics of infectious agents, their vertebrate hosts and mosquito vectors 1

41.2 Modes of transmission 4

41.3 Stable and non-stable infections in Drosophila 8
41.4 Simulation models of transmission cycles 11
41.5 Virulence 18

42 Immune responses of mosquitoes 22

42.1 Introductory matters 22
42.2 Immune response to viruses 24
42.3 Serine proteases 25
42.4 Recognition proteins 27
42.5 Regulation of the immune response in Anopheles gambiae 30
42.6 Antimicrobial peptides 32
42.7 Haemocyte types and their characteristics 34
42.8 Phagocytosis 36
42.9 Structural observations of encapsulation 37
42.10 Immune and other roles of melanin in mosquitoes 40
42.11 Modulation of host immune responses by salivary proteins 47

43 Viruses 52
43.1 Introduction 52
43.2 Characteristics, systematics and biology by family of viruses that infect culicids and are not
arboviruses 56
43.3 Mechanical transmission of pox viruses 78
43.4 Myxoma virus and myxomatosis 81

44 Arboviruses - characteristics and concepts 90

44.1 Characteristics and systematics by family 90
44.2 Arboviral transmission cycles 104
44.3 Serological and molecular-genetic assays for arboviruses 107
44.4 Modes of transmission to vertebrate hosts 116
44.5 Venereal transmission 119
44.6 Vertical transmission 125
44.7 Survival of arboviruses through climatically adverse seasons 136
44.8 Infection, replication and dissemination in mosquitoes 144
V
vi Contents

44.9 Pathology of infected mosquitoes 161

44.10 Pathology of infected vertebrates 162

45 Arboviruses - case studies of transmission 174

45.1 Transmission of Eastern equine encephalitis virus 175
45.2 Transmission of dengue viruses 196
45.3 Transmission of yellow fever virus 218
45.4 Transmission of Japanese encephalitis virus 251
45.5 Transmission of La Crosse virus 284
45.6 Transmission of Rift Valley fever virus 298
45.7 Transmission of West Nile virus 322

46 Pathogenic and symbiotic bacteria 362

46.1 Classification and species definition 362
46.2 Bacteria of the mosquito gut 365
46.3 Growth and sporulation 368
46.4 The bacterial genome 370
46.5 Bacillus thuringiensis 372
46.6 Lysini bacillus sphaericus 414
46.7 Clostridium bifermentans 428
46.8 Spiroplasma 430
46.9 Wolbachia 437

Appendix 1 Genera and higher taxa of the family Culicidae cited in this volume, with the
accepted abbreviations of generic names 466

Appendix 2 Correspondence between the names of Aedine species cited in this volume according
to the traditional and revised classifications 467

Appendix 3 Glossary of terms used in this volume relating to the epidemiology, pathology,
genetics or general biology of infectious agents and their hosts 470

Appendix 4 Abbreviations of terms and acronyms of names used in this volume 479

References 481

Species Index 551

Subject Index 563

 Acknowledgements

As with the earlier volumes, I sought the advice of a number of specialists, and this volume has benefited
greatly from their advice. I am very pleased to express my thanks to the individuals who contributed by
providing information or by scrutinizing and commenting on certain chapters. They are, I hope without
any omissions, Houssam Attoui, Barry Beaty, James Becnel, Max Bergoin, Shelley Cook, Neil Crickmore,
the late Christopher Curtis, Xavier Didelot, Richard Elliott, Paul Fine, Desmond Foley, Michael Gaunt,
Ernest Gould, Weidong Gu, Ralph Harbach, Stephen Higgs, Akira Igarashi, Ian Kitching, Laura Kramer,
Elena Levashina, Kenneth Linthicum, Ian Marshall, Bernard Mondet, William Reisen, Francois Rodhain,
Thomas Scott, Steven Sinkins, Dominique Soldati-Favre, Tom Solomon, Michael Turell and Scott Weaver.
The illustrations are the work of Mark de Pienne.
I express my appreciation to the following publishers who waived their copyrights and authorized
reproduction of certain illustrations - the American Mosquito Control Association, American Society for
Microbiology, American Society for Tropical Medicine and Hygiene, the Company of Biologists,
Cambridge University Press, Elsevier, the Society for General Microbiology and Wiley-Blackwell.

vii
 Preface

An intended single volume to have been titled The Biology of Mosquitoes expanded into two volumes,
published in 1992 and 1999, which broadly concerned mosquito physiology and mosquito behaviour,
respectively. A chapter on the pathogens and parasites of mosquitoes inserted into the projected third
volume expanded so greatly that it alone came to constitute this third volume, on the transmission of
viruses and interactions with bacteria, plus a fourth on the transmission of unicellular and multicellular
parasites, now partly written. The projected fifth volume is more distant.
An important development in this volume is adoption of the revised, phylogenetic classification of
mosquitoes of the tribe Aedini. Cladistic analysis of morphological data makes it possible to determine the
evolutionary relationships of organisms and to produce phylogenetic classifications. This scientific advance
has been adopted in recent decades by the taxonomists of most groups of living organisms, including
insects, but, curiously, not by most mosquito taxonomists. Over recent years a phylogenetic classification of
the culicid tribe Aedini was developed which regroups them into smaller genera with more distinct
geographical distributions and, in some cases, distinctive biology - developments that have been invaluable
in assembling and rationalizing a multitude of data for this review. This was most obvious in the division of
the traditional genus Aedes - which has well over 900 species - into its phylogenetically distinct lineages.
Because this advance in mosquito biology appears to be unknown to many, a description and justification
are provided in the Introduction which immediately follows this Preface. The changes to generic names
that resulted from the reclassification are detailed in Appendix 2.

Alan Clements

viii
 Introduction
The traditional and revised classifications of
aedine mosquitoes

A recent phylogenetic revision of the classification of the tribe Aedini necessarily led to changes in the
nomenclature of that group, introducing a number of restored or newly designated genera. Where they are
relevant, those genus names are used in this volume. Initial responses to the changes of classification and
nomenclature have generally not been welcoming, so it seems sensible, at the beginning of the volume, to
contrast the traditional and revised classifications, and to describe the scientific reasons for adoption of the
revised classification and the practical advantages that it provides.

THE TRADITIONAL CLASSIFICATION OF MOSQUITOES

By the end of the 19th century much was known of the biology of mosquitoes, and detailed descriptions
had been published of their life-cycle stages, adult feeding mechanisms, oviposition behaviour, internal
anatomy and histology. A classification had been developed, but based on only a small proportion of the
species that are now known. Discovery during the last two decades of the 19th century of the role of
mosquitoes in the transmission of microfilariae and malaria parasites triggered a drive to collect mosquitoes
wherever possible and to name and classify them. New species were described and many new genera
designated by mosquito taxonomists, notably Blanchard and Neveu-Lemaire in France, Theobald in
England, and Dyar and Knab in the United States. A Committee set up by The Royal Society in 1899 to
inquire into the causes and control of malaria appointed Theobald to prepare a monograph on the
mosquitoes of the world, and this was published between 1901 and 1910 in five volumes (Theobald, 1901-
1910). In North America, Howard, with Dyar and Knab, published reviews of the mosquitoes of North and
Central America in four volumes between 1912 and 1917 (Howard et al., 1912-1917).
It was a feature of these early taxonomic studies that genera and higher taxa were distinguished on the
basis of rather few characters. Theobald (1901a, 1901-1910) defined genera very largely on the shape and
arrangement of scales on the adults and, despite early criticism, continued to do so. In a survey of culicid
larvae, Dyar and Knab (1906) distinguished the subfamilies Anophelinae, Culicinae and Sabethinae, and
their genera and species, solely on larval characters. Dyar (1918) used the characteristics of male genitalia to
distinguish some culicid genera. Possibly due to such simplifications, certain taxonomists discarded many
previously designated genera, listing each as a junior synonym of a related genus that had historical
precedence. As early as 1906, Dyar and Knab subsumed 16 genera into Anopheles, 13 into Aedes and five
into Cutex; later, Dyar (1922) subsumed 37 genera into Anopheles, 49 into Aedes and 33 into Cutex.
F.W. Edwards succeeded Theobald at the British Museum and contributed further to mosquito
taxonomy; in 1932 he published a revised classification of the family Culicidae. The genera Anopheles, Cutex
and Aedes remained very species rich, while many of the early genera that had been put aside as junior

ix
x Introduction

synonyms were given the rank of subgenus in one or other of them. In the introduction to the 1932 work,
Edwards described 'the advantages of employing larger generic concepts', asserting that: (i) the wider
relationships of the species are more clearly indicated; (ii) limits can be more readily assigned to large genera
than to more numerous, smaller groups; (iii) avoidance of the duplication of specific names is ensured; (iv)
generic diagnoses should be applicable to both sexes; and (v) the use of subgeneric terms enables those who
wish to do so to make use of the smaller divisions. Most of these 'advantages' concerned expediency rather
than taxonomic correctness and, today, with our much greater knowledge of mosquitoes, some of the
supposed advantages are no longer pertinent.
Edwards (1932) retained the taxa Dixinae and Chaoborinae in the Culicidae as subfamilies, together
with the so-called true mosquitoes - the subfamily Culicinae, with its 30 genera, 89 subgenera and 1400
species. With some major alterations, i.e. the removal of the subfamilies Dixinae and Chaoborinae, and the
designation of the subfamilies Anophelinae, Culicinae and Toxorhynchitinae, Edwards' classification was
adopted in Stone et al.'s (1959) A Synoptic Catalog of the Mosquitoes of the World. The last major restructuring
of the traditional classification was in Knight and Stone's (1977) A Catalog of the Mosquitoes of the World, in
which the subfamily Culicinae was divided into ten tribes, one being the tribe Aedini. Each of the nine
genera within the Aedini consisted of one to three subgenera except for the genus Aedes, which had 38
subgenera. The growth in number of species since 1977 and the designation of further genera are
recognized now in the 'Systematic Catalog of Culicidae' (Gaffigan et al., 2011), which is compiled and
maintained online by the Walter Reed Biosystematics Unit (WRBU; http://wrbu.si.edu/) of the Walter
Reed Army Institute of Research (WRAIR) in Maryland.

CRITICISMS OF THE TRADITIONAL CLASSIFICATION OF MOSQUITOES

What are now perceived as defects of the traditional classification of mosquitoes arose partly because it was
produced before the development of modern taxonomic techniques. To some extent, phenetic methods
that rely on estimates of overall similarity were used; but, as Black (2004) pointed out, the morphological
characters that taxonomists used in dichotomous keys to identify species were also used to classify species
into higher taxonomic groups. Consequently, while certain genera are monophyletic, significant numbers
of genera are paraphyletic or polyphyletic.
Other problems arose because of the preference for large genera. Bates (1949) pointed out that, whereas
Theobald (1901-1910) had grouped 1050 species into 149 genera, Edwards (1932) had grouped 1400
species into only 30 genera. Belkin (1962) commented that 'Many of the subgenera of Aedes appear to be
heterogeneous complexes of superficially similar species, and it is very probable that they will have to be
subdivided into smaller natural groups', and argued that 'Lumping all small taxa into groups of a
convenient size but of indefinite affinities does nothing but obscure relationships'. He concluded that 'The
internal classification of the Aedini is in need of thorough revision'. Tanaka et al. (1979) described Aedes as
`A polymorphic genus; most characters extremely variable'.
The situation was given perspective when Zavortink (1990) contrasted the traditional classification of the
Culicidae with the classifications typical of most other groups of organisms. A number of authorities,
including Williams (1951), Dial and Marzluff (1989), Mayr and Ash lock (1991) and Scotland and Sanderson
(2004), had shown that a 'hollow curve' distribution is shown graphically when the number of known
species per genus is plotted against the number of genera with the corresponding number of species (Figure
I.1A). Generally, taxa of family rank consist of a number of monotypic (single species) genera, a smaller
number of ditypic genera, and progressively fewer genera at each further increment in number of species
per genus, culminating in the very few genera that are relatively species rich. Hyperbola-like or hollow-
curved distributions have been documented for higher taxa of plants, crustaceans, insects, fish, birds and
 Introduction xi

mammals. They were obtained whether cladistic analyses or other methods had been used to develop the
classifications, and whether the taxonomists had been `lumpers' or 'splitters' of species into genera.
By comparing the species richness of culicid genera with that of other groups of organisms, Zavortink
(1990) showed that the family Culicidae consisted of far fewer genera than did most families with a similar
number of species. Instead of a hollow curve, a plot of numbers of culicid genera against their species
richness yielded an almost flat line along the X axis (Figure L1B). By comparison with the classifications of
other organisms, Zavortink calculated that the 3146 mosquito species then recognized should have been
grouped into about 225 genera, not 37. The number of culicid genera was many fewer than would be
expected for a family that had achieved the beta level of taxonomic investigation (i.e. the level of
arrangement of species into hierarchical systems of higher categories or taxa). Zavortink concluded that 'we
have not even begun to develop a natural classification for the family'. This analysis had no influence on
mosquito taxonomists, and 20 years later the three largest culicid genera in the accepted classification were
still exceedingly large, with Anopheles comprising c. 475 species in seven subgenera, Cu lex c. 793 species in
27 subgenera and Aedes c. 927 species in 45 subgenera (data from the online Systematic Catalog of
Culicidae, Gaffigan et al., 2011).

Species per genus

55 B

45

35

25

15

6 genera

5 15 25 35 45 55 65 100 ...1000 species

Species per genus

Figure 1.1 (A) Hollow curve showing the decrease in the number of genera as the number of species per genus increases
for any group of organisms with a sound natural classification. (B) Number of mosquito genera (Culicidae) with the
number of species indicated. (Figures after Zavortink, 1990, with original captions.) The upper figure showing a hollow
curve was based on many published findings. In the lower figure from data for the family Culicidae in its traditional
classification, the plot takes the form of an almost straight line close to the X axis.
xii Introduction

PHYLOGENETIC CLASSIFICATIONS BASED ON CLADISTIC ANALYSES

Classifications of animal groups that have been developed from cladistic analyses of morphological data
reflect evolutionary relationships and are phylogenetic. Hennig (1950, 1966) redefined and clarified the
concepts of monophyly and of phylogenetic relationships, and introduced cladistic techniques that sought
patterns of similarity based only on shared evolutionarily novel features. Phylogenetic hypotheses were
developed from which the basis of a classification could be developed using monophyly, and the principle
of equivalent rank used as the criterion for recognizing and grouping taxa. A phylogenetic classification had
been achieved when all taxa were monophyletic and each subsidiary taxon had evolved from a single
ancestor. During the three decades that followed Hennig's contributions, the theory of phylogenetic
systematics was refined and cladistic methods were improved (Kitching et al., 1998).
Phylogenetic classifications are now almost universally accepted as the most useful general reference
system for biology. Andersen (2001) observed that the 'higher' Diptera now have a natural classification,
whereas the more primitive nematoceran families, including the Culicidae, are in need of cladistic,
phylogenetic analysis.

DEVELOPMENT OF A PHYLOGENETIC CLASSIFICATION OF AEDINE MOSQUITOES

In an early study, using characters of the female and male genitalia, and supplementary characters of larvae
and pupae from over 65% of known species of Aedes, Reinert (2000) divided the composite genus Aedes
into two genera: genus Aedes (with 22 subgenera) and genus Ochterotatus (with 21 subgenera). That first step
led on to cladistic analyses of the Aedini, the largest tribe in the family Culicidae, and involved examination
of a high proportion of known aedine species. The ranking of lineages as genera or subgenera involved the
criteria of monophyly and equivalent rank. As the investigation progressed through four phases, the
findings were published by Reinert et al. in 2004, 2006, 2008 and 2009.
The first phase assessed 119 exemplar species representing the 12 traditionally recognized aedine genera
and 56 subgenera, with examination of 172 characters from all life-cycle stages. Cladistic analyses yielded
eight most parsimonious cladograms from which a strict consensus tree was constructed that permitted two
possible phylogenetic classifications of the tribe Aedini. The principal problem was not in recognizing
monophyletic groups, but in deciding what taxonomic ranks should be assigned to them once their
phylogenetic relationships had been established. In one of the two possible classifications, the 11
traditionally accepted genera, i.e. Aedes (inclusive of Ochterotatus), Armigeres, Ayurakitia, Eretmapodites,
Haemagogus, Heizmannia, Opifex, Psorophora, Udaya, Verrallina and Zeugnomyia, would all be subsumed into
the genus Aedes, which would become the sole genus of the tribe Aedini. In the second and preferred
possible classification, all clades having phylogenetic equivalence with the 11 traditionally accepted genera
would have the rank of genus. With the restoration of many subgenera to their former rank and the
designation of new genera, the total number of genera would be raised to 46. A provisional, revised
classification of aedine mosquitoes was proposed on that basis (Reinert et al., 2004).
In the later phases, the investigation was extended to aedine groups that had not been examined in detail
earlier. The second phase concerned the genus Finlaya and associated taxa, and involved examination of
232 characters in 116 exemplar species (Reinert et al., 2006). The third phase concerned Ochlerotatus and
associated taxa, and involved examination of 297 characters from 159 exemplar species (Reinert et al.,
2008). In the final phase, the phylogeny and classification of the tribe Aedini were delineated based on a
cladistic analysis of 336 characters from eggs, 4th instar larvae, pupae, and adult females and males from 65
genera and 46 subgenera, habitat coded for 270 exemplar species (Reinert et al., 2009). The investigation
resulted in the recognition of 80 genera and 48 subgenera within the tribe Aedini, all of which, with the
exception of the genus Dendroskusea Edwards, were recovered as monophyletic taxa. As was usual with
 Introduction xiii

groups of Aedini at the generic level, all newly recognized genera and subgenera were polythetic taxa, i.e.
they were diagnosed by unique combinations of characters, none of which was diagnostic alone.
An updated, phylogenetic classification of the Aedini is available online in the Mosquito Taxonomic
Inventory (Harbach, 2011, http://mosquito-taxonomic-inventory.info/). Within the Inventory, a list
headed Resources includes two pertinent items. (i) The item `Aedini Classification' provides a pdf file (of
that name) in which all aedine genera and subgenera of the revised classification are listed, in parallel with
equivalent taxa of the traditional classification. (ii) The item 'Valid Species List' provides a pdf file (of that
name) in which the names of all valid mosquito species are recorded.

CRITICISMS MADE OF THE REVISED CLASSIFICATION OF AEDINE MOSQUITOES

The initial division of the composite genus Aedes into the genera Aedes and Ochlerotatus by Reinert (2000)
was opposed by Savage and Strickman (2004), who argued that 'Use of these names as genera complicates
mosquito identification and interferes with information retrieval among taxonomists, medical
entomologists, and vector control specialists'.
The Journal of Medical Entomology (JME, 2005; vol. 42, p. 511) carried a 'Letter from the Editor' entitled
`Journal Policy on Names of Aedine Mosquito Genera and Subgenera'. The 'Letter' started by citing, but
rephrasing, a paragraph from the Preamble to the International Code of Zoological Nomenclature. The
original paragraph reads: The objects of the Code are to promote stability and universality in the scientific
names of animals and to ensure that the name of each taxon is unique and distinct. All its provisions and
recommendations are subservient to those ends and none restricts the freedom of taxonomic thought or
actions'. Referring to that statement, the 'Letter' asserted that 'When reinterpreting the relationships
between species and groups of applied importance, systematists have responsibility to limit the impact on
nomenclature. Such caution was not exercised when the majority of known species and subgenera of Aedes
mosquitoes were transferred to the restored genus Ochlerotatus based on taxonomic characters that few
other workers have examined (Reinert, 2000)'. That opinion was expressly refuted by Polaszek (2006) in an
article published from the office of the International Commission on Zoological Nomenclature.
Publication of a first cladistic analysis and proposed phylogenetic classification of the tribe Aedini by
Reinert et al. (2004), with the restoration of some former genera and the designation of new genera,
prompted further criticism from Savage (2005), who considered that The entire approach to Aedini
systematics of these authors was flawed by an inordinate fear of paraphyletic taxa, or paraphylyphobia, and
their inability to distinguish between classification and cladistic analysis'. Referring to the cladistic analyses
of Reinert et al. (2004), the 'Letter' from the Editor in JME (2005) discouraged submission to that journal
of articles using the revised nomenclature, and reported support from the editors of several other journals
of medical entomology or tropical medicine. A virtually identical article was published in the American
Journal of Tropical Medicine and Hygiene (Weaver, 2005). That prohibitive attitude has persisted in certain
journals up to this time.
Considered and realistic criticism of Reinert et al. (2004) appeared online in a WRBU Forum which had
been established by a Mosquito Systematics Review Committee. Unfortunately, the Forum is no longer
accessible. The key criticisms, set out in the 'Summary of majority opinion' of the Committee, mostly
concerned technical aspects of the cladistic techniques that had been used and the interpretation of data,
matters to which I.J. Kitching published a detailed response in the Forum proceedings. Following its
deliberations, the Review Committee rejected Reinert et al.'s (2004) proposed classification of the Aedini
in the WRBU online Systematic Catalog of the Culicidae.
The critics could have found their comments and criticisms partly explained, or even agreed with, in the
following lines from the brief 'Final Comments' section in Reinert et al.'s (2004) 88-page article: The lack
of basal resolution and branch support in the cladograms makes it impossible to fully resolve the
xiv Introduction

relationships among the genera ... . Additional morphological data may provide a clearer view of the
relationships between closely related genera, but are unlikely to resolve deeper relationships within the
tribe. ... The next step will be to resolve the relationships and placement of taxa of uncertain taxonomic
position to achieve a more robust classification ...'. The first part of that last sentence was the only
indication that this article covered only the first phase of what was to become a very extensive investigation.
It might be taken as affirmation of the validity of the phylogenetic classification of the tribe Aedini that
no criticisms were published of the reports from the later phases of the investigation (i.e. from Reinert et
al., 2006, 2008, 2009). The taxon Ochlerotatus was accepted as having genus rank, possibly due to its
importance in North America. That only few entomologists in North America and Western Europe used
the remainder of the revised nomenclature in journal articles probably reflected not only the refusal of
journal editors to accept them but also a widespread ignorance of this development in taxonomy. Elsewhere,
however, the revised classification became adopted, as in important publications from China, Vietnam,
Thailand, Iran and Saudi Arabia.
No keys have been published that lead investigators to the newly named genera. However, in earlier
taxonomic articles that used the traditional nomenclature of aedine mosquitoes, keys to the traditional
subgenera of Aedes lead to taxa that now have genus rank, while keys to the species of the traditional
subgenera of Aedes that have been restored to or raised to genus rank will enable users to direct those
species to the new genera.
The hostility that met the change of the scientific name of the yellow fever mosquito, from Aedes
(Stegomyia) aegypti L. to Stegomyia aegypti L., is ironic. The mosquito to which Linnaeus assigned the name
Culex aegypti in 1792 was a species of Ochlerotatus, and definitely not the yellow fever mosquito (Gough,
1914; Patton, 1933). Earlier, mosquitoes used in the experimental transmission of yellow fever virus had
been given the valid name Aedes (Stegomyia) fasciata (Fabricius) (Howard, 1901). The name aegypti L. must
now be applied to the yellow fever mosquito instead of fasciata (Fab.) only because, at the request of medical
entomologists, the International Commission on Zoological Nomenclature (1964) used its plenary powers
to validate the specific name aegypti Linnaeus, to be interpreted by reference to a neotype, being a specimen
of the yellow fever mosquito from Malaya (now peninsular Malaysia).

PRACTICAL DIFFICULTIES THAT FOLLOW ADOPTION OF THE PHYLOGENETIC

CLASSIFICATION
From the first, critics correctly pointed out that the introduction of a phylogenetic classification, with many
new generic names, would cause problems with information transfer. To quote just one critic, it would
`inevitably create considerable confusion among teachers, students and researchers, with communication
difficulties and financial implications for republishing educational materials, keys, catalogues and
management of data bases'. In reality, the greatest medium- to long-term problems lie in the retrieval of past
information on those binomials in which the genus name has been changed, with an additional problem
where the change of genus name required a change of gender of the species name. The problem is less great
where the new genus names result from the restoration of subgenera to genus rank. It should not be
overlooked that, to a degree, this nomenclatural problem resulted from the practice of subsuming many
mosquito genera into a very few.
Interestingly, in his book The Natural History of Mosquitoes, Marston Bates (1949) had stated 'The (other)
cause of name changing results from the discovery of new relationships, of new and perhaps more
satisfactory methods of classifying a group of animals. Rearranging these animals under different generic
concepts may cause temporary inconvenience, but if the end result is an improved classification the
inconvenience is a small cost'.
 Introduction xv

In this volume, a high proportion of the species that are cited with genus names different from those in
the traditional nomenclature are species now assigned to Ochlerotatus, a genus that has been widely accepted
for some years. Another frequent change concerns the yellow fever mosquito, with a change of scientific
name from Aedes (Stegomyia) aegypti to Stegomyia aegypti. If these provide any measure of the effects of
adoption of the new nomenclature more generally, then the supposed difficulties have been
overemphasized.

BIODIVERSITY
Life is more abundant in the tropics than towards the poles. Over each hemisphere a 'latitudinal diversity
gradient' is apparent as a progressive increase in the numbers of species and of higher taxa from the polar
regions to the tropics. It has been postulated that, for the most part, taxa originate in the tropics and that,
while persisting there, extend their distribution over time into higher latitudes. If that is the case, then the
numbers of endemic genera should be greater at the lower latitudes (Jablonski et al., 2006).
Analysis of the global distribution of culicids revealed a latitudinal diversity gradient of species richness.
A frequency curve produced by plotting the mean number of mosquito species per km2 of the countries
intersected by bands of 10° of latitude, and extending across the northern and southern hemispheres,
revealed a progressive decline in species richness in each hemisphere as the bands of latitude moved away
from the equator and towards the poles (Figure 1.2). Excluding small island countries, the countries with
the largest numbers of total mosquito species, and of endemic mosquito species, were Panama, French
Guiana, Malaysia and Costa Rica, all tropical countries that are situated between 1° 2' N and 11° 12' N
(Foley et al., 2007).
The causes of the latitudinal diversity gradient remain a matter of conjecture, but it is possible to discern
changes of environmental structure with change of latitude which could affect the extent of biodiversity.
One is the greater spatial heterogeneity apparent in the tropics, which is associated with a greater variety of
microenvironments. Further, it is supposed that the greater seasonal stability of tropical environments has
led to the evolution of more specialized species with narrower ecological niches (Ca low, 1998). Culicids
occur in a variety of habitats, most distinctly during their aquatic stages, when their habitats range from
bodies of open water of a variety of forms to container habitats. In the subtropics and tropics, culicid larvae

10° bands of latitude

Figure 1.2 Latitudinal diversity gradients of culicid species over the northern and southern hemispheres. The vertical line
indicates the equator. The frequency curve shows the variation in species richness of culicids with latitude, as expressed by
the mean number of culicid species per km2 of countries intersected by bands of 10° of latitude. Northern hemisphere,
left; southern hemisphere, right. (The figure was generously provided by Dr Desmond Foley, who had calculated the values
of the data points from country records in the Systematic Catalog of the Culicidae, Gaffigan et al., 2011.)
xvi Introduction

make greater use of phytotelmata, including leaf axils, floral bracts, tank bromeliads, the pitchers of pitcher
plants and bamboo internodes, and also of natural cavities, including rock holes, swamp crypts, animal
footprints, crab holes and mollusc shells.
For any area or geographic region, interest in biodiversity extends beyond species richness to the
taxonomic identity of the species and the ecological niches that they occupy. The intensive studies that have
been undertaken on mosquito species and their ecology have made an exceptional contribution to the
knowledge of biodiversity in many countries. However, studies of biodiversity can be seriously disadvantaged
if the taxonomic classifications are not phylogenetic. Concealed within the genera and subgenera of the
traditional classification of mosquitoes are groups of species that cladistic analyses show to be distinct,
monophyletic lineages which have distinct distributions and biological characteristics. Consider Danielsia,
Hutecoetomyia and Phagomyia, genera designated by Theobald, but which in the traditional classification are
treated as junior synonyms of Aedes (Finlaya). Cladistic analysis showed them to be distinct lineages meriting
the rank of genus. It also revealed new monophyletic lineages within Aedes (Finlaya) that warranted genus
rank (Reinert et al., 2009).

END NOTE
This Introduction, in its original form as a longer review article, was rejected on principle by a leading
journal of medical entomology, but since then attitudes to the phylogenetic classification and
nomenclature of aedine mosquitoes have started to change. The same journal now welcomes articles that
use the revised nomenclature; articles of regional mosquito taxonomy that use the revised nomenclature
have been published in China, Korea, Thailand, Iran and Saudi Arabia; and a leading post-graduate
institution in the United Kingdom teaches the revised nomenclature solely.

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 41

Host/parasite interactions

41.1 Characteristics of infectious agents, their vertebrate hosts and

mosquito vectors 1

41.2 Modes of transmission 4

41.3 Stable and non-stable infections in Drosophila 8
41.4 Simulation models of transmission cycles 11

41.5 Virulence 18

41.1 CHARACTERISTICS OF INFECTIOUS Pathogens may be classed as obligate (facultative)

AGENTS, THEIR VERTEBRATE HOSTS or opportunistic. Obligate pathogens live in
AND MOSQUITO VECTORS intimate association with their hosts, and their
long-term survival depends on their ability to
41.1.1 Characteristics of infectious agents
replicate in the host. Opportunistic pathogens do
Terms associated with infection and parasitism may not require intimate association with their hosts
be defined and used rather differently by writers and can survive away from a host.
with different interests, e.g. by parasitologists and During the periods of their evolution, infectious
ecologists, so it is necessary to describe how they are agents have exploited some of the many mosquito/
used in this volume (see also Glossary, Appendix 3). vertebrate associations in which particular
The most basic term, infectious agent, embraces mosquito species feed on particular vertebrate
both viruses and cellular organisms. The term species. They undergo appropriate life-cycle phases
infectious organism applies to infectious micro- alternately in the mosquito and vertebrate and, in
organisms (bacteria and unicellular eukaryotes) and consequence, the mosquitoes function as
infectious multicellular eukaryotes. Infectious biological vectors. The infectious agents for which
agents may be classed as pathogens or parasites, two mosquitoes are biological vectors are: (i) viruses of
terms that are used interchangeably by some five families; (ii) some species of Hepatozoon (class
authorities but are distinguished as apart by others. Coccidea); (iii) some species of Plasmodium (class
Here, the term pathogen is applied to viruses and Hematozoea); and (iv) species of a number of
infectious bacteria that adversely affect their hosts, genera of Onchocercidae (superfamily Filarioidea).
while the term parasite is applied to infectious Parasites may be grouped in two categories -
eukaryotes, whether unicellular or multicellular, microparasites and macroparasites - which are
that adversely affect their hosts. The adjectival forms distinguished not just by size but by a number of
of those two terms are used less restrictively, even to biological characteristics (Anderson, 1979; Ander-
the extent of overlapping usage. For example, the son and May, 1991). Microparasites undergo
term pathogenic is applied to the deleterious effects asexual reproduction within a host, usually at very
of both pathogens and parasites, and the term high rates. They tend to be small and to have a
parasitic may be applied to the infectious lifestyle of short generation time. They are often intracellular.
both pathogens and parasites. Infections of invertebrates by microparasites
© A.N. Clements 2012. The Biology of Mosquitoes, Vol. 3: 1

Transmission of Viruses and Interactions with Bacteria (A.N. Clements)

2 Host/parasite interactions

frequently prove fatal, but not when that host also for part of its life cycle and in which it attains
serves as a vector. Vertebrate hosts that recover sexual maturity. An intermediate host is one in
from infection by microparasites have usually which the infectious organism lives for part of its
developed immunity against reinfection, often for life cycle, and in which either it does not become
life. Usually, the duration of infection is short sexually mature or only the asexual stages occur.
relative to the average lifespan of the vertebrate Species that are susceptible to infection by a
host, so for the survivors most microparasitic particular pathogen fall into one or other of two
infections are transient. For that reason, the categories - competent and incompetent. When
population growth of microparasites that infect infected, competent hosts develop a viraemia or
vertebrates requires recruitment of susceptible parasitaemia that is of sufficient titre and duration
individuals to the host population. Most obligate to infect blood-feeding vectors in the natural
bacterial pathogens have the characteristics of habitat. When infected, incompetent hosts do not
microparasites. Many viruses and protozoan and develop such a viraemia or parasitaemia, and in
fungal pathogens share these characteristics, but the natural habitat cannot infect blood-feeding
less completely. vectors. In practice, the term 'competent host' is
Macroparasites undergo sexual but not asexual now often replaced by amplifying host, and the
reproduction within the definitive host. Typically, term 'incompetent host' is replaced by dead-end
they are larger than microparasites and are usually host (Section 44.2.2).
extracellular. They grow within the host, and their The causative agents of some diseases of wild
generation times are relatively long, frequently animals can be transmitted to humans, and such
lasting for an appreciable fraction of the host's life- diseases are termed zoonoses. The species from
span. Their transmission stages (eggs or larvae) are which infectious agents can be transmitted must
often produced in very large numbers, and either be competent hosts that generate a parasitaemia or
pass into the external environment or are ingested viraemia of sufficient magnitude and duration to
by vectors. The immune responses of vertebrates permit infection of vectors. The best single
to macroparasites tend to be of a relatively short indicator of the involvement of a vertebrate species
duration. Macroparasite infections typically are in a transmission cycle is frequent isolation of the
persistent, with hosts being continually reinfected. infectious agent from free-ranging individuals.
The growth of infrapopulations within individual From serological evidence, it is known that Rift
hosts results from the gradual accumulation of Valley fever virus (RVFV) infects mammals of a
new infections. Relatively few macroparasites number of orders (Section 45.6.2.a). However,
sequentially infect invertebrate and vertebrate seropositivity for RVFV indicates no more than
hosts; among these, digeneans and some cestodes that an individual has been infected, and for only
undergo asexual reproduction in the intermediate very few species is there virological evidence that
host. Acanthocephalans, parasitic platyhelminths, indicates whether they could be amplifying hosts
nematodes and arthropods have the characteristics of RVFV. For effective transmission from wild
of macroparasites. hosts to humans, the vector species must be
abundant and must feed frequently on both the
wild host and on humans.
41.1.2 Characteristics of different types of
Infections of host populations vary in their
vertebrate host
epidemiological characteristics, but usually may be
Some pathogens or parasites sequentially infect classed as enzootics, epizootics or epidemics. An
two different hosts during a life cycle, and those enzootic is a disease that is constantly present in
hosts are functionally distinguishable by the uses (wild) animal populations within a specified area,
made of them by the infectious agent. A definitive and usually at a low rate of prevalence. Virulence
host is one in which an infectious organism lives to the host species is generally but not necessarily
 Characteristics of infectious agents, their vertebrate hosts and mosquito vectors 3

low, and the host and pathogen populations coexist A number of terms have been coined to
in a balanced state; consequently the disease is distinguish between species whose capabilities as
always present. An enzootic system can become vectors differ quantitatively or in other ways. The
epizootic through a change in one or more of the terms 'primary vector' and 'secondary vector' are
key components - host, pathogen or environment. flawed (Smith, 1967), and the following terms,
An epizootic is an outbreak of an infectious proposed by White (1982), are used here. A main
disease in a (wild) animal population that is vector is a widespread species capable of maintain-
characterized by an exceptionally large number of ing transmission of a particular infectious agent.
cases relative to the number occurring earlier. In some locations there can be more than one
Epizootics are sporadic and limited in duration, main vector, but with differences in seasonality,
show sudden changes in incidence and then ecology or behaviour. Thus, in parts of Africa
prevalence, and are usually caused by pathogens where Anopheles gambiae s.l. and Anopheles funestus
that have a short generation time. The change in are sympatric, both can be main vectors of
prevalence depends first on massive reproduction Plasmodium falciparum, but An. funestus becomes
of the causal organism, reinforced by environ- the more important when the larval habitats of
mental and host factors. At the beginning of an An. gambiae are flushed out by prolonged heavy
epizootic the host population must largely be rains. A subsidiary vector will be one of three
susceptible to infection by the causal agent. When types - incidental, local or bridge. (i) An 'inci-
the proportion of susceptible hosts has fallen dental vector' is a species that is incapable of
sufficiently, the high multiplication rate of the maintaining endemicity on its own but that
causal agent declines (Fuxa and Tanada, 1987). regularly supplements transmission where it is
The term epornithic may be used for an outbreak sympatric with a main vector. (ii) A 'local vector' is
of an infectious disease in a wild bird population. a species of limited distribution that has sufficient
An epidemic is the equivalent of an epizootic but vectorial capacity to maintain localized endemicity
occurs in a human population or, as used by some without the main vector. In coastal regions of
authors, among domesticated livestock. Africa, Anopheles melas and Anopheles merus, which
develop in saltwater habitats, can be local vectors
of P. falciparum where they have sufficient contact
with humans. (iii) A 'bridge vector' is a species that
41.1.3 Characteristics of mosquito vectors
can transmit an arbovirus from an amplifying host
An individual infectious agent might be trans- to a dead-end host. It is important, where possible,
mitted by one, a few or many species of mosquito. to assess putative vectors quantitatively by measur-
Within a zoogeographical region, a number of ing their vectorial capacity, i.e. the average number
mosquito species might show vector competence of potentially infective bites that will ultimately be
for an infectious agent in the laboratory, i.e. delivered by all the vectors that feed upon a single
susceptibility to infection and permissiveness for infective host in one day (Glossary, Appendix 3).
replication of that agent and its transmission in Mosquito species that are considered main
saliva. Not all species that show vector competence vectors of infectious agents should satisfy the
in the laboratory are vectors in the field. Other following criteria. (i) The mosquito and vertebrate
factors are important, including sympatry with the hosts of the infectious agent occur sympatrically.
vertebrate host species, co-occurrence with the They dwell in the same habitat at the same season,
host species in time, and an appropriate host and in some cases occupy the same microhabitat.
preference of the adult females. Species with vector (ii) The mosquitoes feed preferentially on one or
competence that display these other characteristics more species of amplifying host. (iii) The infectious
are said to have vectorial capacity. (See also agent has been isolated from wild mosquitoes. (iv)
Glossary, Appendix 3) The mosquito population attains a sufficient size
4 Host/parasite interactions

relative to that of one or more amplifying host 41.2.1 Mechanical and biological transmission
species. (v) In the laboratory, the mosquitoes are
When infectious agents are transferred from one
readily susceptible to infection by the infectious
vertebrate host to another by haematophagous
agent, do not block its full development and trans-
arthropods, the mode may be mechanical or bio-
mit it efficiently. (vi) The lifespan of a sufficiently
logical. In mechanical transmission, infectious
high proportion of wild females exceeds the
agents are transferred from infected to uninfected
extrinsic incubation period of the infectious agent.
vertebrate hosts on the mouthparts of haemato-
For a mosquito species to be confirmed as a
phagous arthropods. A blood meal on an infected
main vector of an infectious agent in a given area
host must be followed immediately or within a few
it should, ideally, show the six characteristics
hours by a second blood meal on an uninfected
listed above, but it is difficult to measure all of
host. In such cases, the infectious agents do not
them. In practice, a putative vector is expected to
invade or reproduce within the arthropods, which
show characteristics (iii) and (v) at least. The
cannot be described as hosts but are mechanical
ability of a mosquito species to transmit an
vectors. The best-known examples of mosquitoes
infectious agent, under experimental conditions,
serving as mechanical vectors are in the trans-
from infective to uninfected hosts does not, alone,
mission of certain pox viruses, notably myxoma
establish that it is a natural vector. For example,
virus (Section 43.3). Mechanical transmission is a
some mosquitoes that can transmit yellow fever
form of direct transmission, because the infectious
virus or certain Plasmodium species from host to
organism does not need to replicate in the
host in the laboratory (Bates, 1949; Huff, 1965)
arthropod vector (in this example, in the
have no association with those infectious agents
mosquito).
in nature.
Infectious agents that of necessity have two
hosts in the life cycle, one a haematophagous
arthropod and the other a vertebrate, pass between
41.2 MODES OF TRANSMISSION
these hosts by biological transmission. The
`Transmission' is a generic term used for the haematophagous arthropods are both hosts and
processes by which infectious agents, whether vectors. Infectious agents that are ingested when
viruses or cellular organisms, symbionts or para- the arthropod feeds on an infective vertebrate
sites, pass from one host to another. Transmission invade and reproduce within the arthropod host;
occurs in a variety of ways, which may be denoted when the arthropod feeds again later, the infec-
by contrasting pairs of terms: direct or indirect tious agents pass to and invade the vertebrate.
transmission; mechanical or biological trans- Infectious agents that have invaded a competent
mission; horizontal or vertical transmission. arthropod host must be able to survive in it,
When transmission is direct, the transmission traverse a variety of barriers and reproduce. Most
stages of an infectious agent pass from one host often they invade the salivary glands of the
individual to another of the same species (without arthropod, and are carried into a new vertebrate
the need for replication in another species). In the host in saliva during a blood meal. For a vector
case of infectious agents that necessarily infect population, the mean time between ingestion of
more than one species of host during the life cycle, an infected blood meal and attainment of
indirect transmission occurs when the trans- competence to transmit the parasite by bite is the
mission stages of the infectious agent pass from `extrinsic incubation period'. Mosquitoes are the
one host species to a different host species (repli- definitive hosts (Section 41.1.2) and vectors of
cation occurring in one of the species). Different many arboviruses, certain apicomplexans and
modes of indirect transmission are described in certain filarioid worms. For a very few arboviruses
the following sections. (defined in the Glossary, Appendix 3), mechanical
 Modes of transmission 5

transmission by mosquitoes supplements bio- different forms (Fine, 1974, 1975). Most often,
logical transmission (Section 44.4.2). vertical transmission involves the passage of an
infectious agent from an adult female host to her
progeny, but a few infectious organisms can also be
41.2.2 Horizontal transmission
transferred in gametes from adult male hosts to
The term horizontal transmission concerns trans- their progeny, e.g. sigma virus in Drosophila
ference of an infectious agent between any two (Section 41.3.2.b). Infectious agents that are
individuals that are not related as parent and perpetuated by vertical transmission alone satisfy
offspring. It embraces all modes of biological one or more of a number of conditions: (i) they
transmission other than vertical transmission are transmitted to the progeny of both their male
(direct transference from a parent to its progeny). and female hosts; and (ii) if transmitted by female
Horizontal transmission can occur between hosts hosts alone, either the filial infection rate is
of the same generation or from hosts of one consistently 100% or infected hosts have a selective
generation to those of the next. It can involve advantage over uninfected hosts. The last situation
transmission between hosts of the same species is found in Wolbachia, in which infected y x
and between hosts of different species. aposymbiotic cS crosses are fertile, whereas infected
Horizontal transmission of mosquito parasites 5' x aposymbiotic y crosses are sterile (Section
and pathogens between their hosts may occur in 46.9.4.b, Figure 46.21).
any of five different ways. (i) Ingestion by mosquito Theoretical analyses of virulence suggest that,
larvae of viral particles or microsporidial spores when transmission of a parasite is vertical, selective
released on the death of infected hosts. (ii) forces will act to reduce virulence. A parasite that
Invasion by the motile stages of a parasite that are is transmitted largely or exclusively vertically
released on the death of infected hosts, e.g. should not harm its host, because the number of
theronts of Lambornella. (iii) Uptake of arbovirus new infections depends upon the fecundity and
or Plasmodium by mosquitoes feeding on infective fertility of the host (Sabelis and Metz, 2002). This
vertebrate hosts. (iv) Transference of arbovirus or is consistent with many empirical observations.
Plasmodium from infective mosquitoes to vertebrate
hosts during blood feeding. (v) Transference of
arbovirus in semen from infected male mosquitoes
(b) Definitions of terms
to uninfected females during copulation, so-called
venereal transmission (Section 44.5). Terms relevant to different aspects of vertical
transmission were defined by Gross (1949),
Burgdorfer and Varma (1967), Fine (1975) and
41.2.3 Vertical transmission
Turell (1988). They are scattered through the
(a) Biology Glossary (Appendix 3), but are brought together
here. Definitions are always open to refinement,
Vertical transmission is the direct transference of
and some changes have been made for this
an infectious agent from one generation of its host
volume.
to the next, i.e. from a parent organism to his or
her progeny. The term 'vertical transmission' was Vertical transmission - transference of an infectious
coined to describe the mode of transference of a agent from a parent organism to his or her
virus (Mouse mammary tumor virus, Betaretrovirus, progeny.
family Retroviridae) from infected female mice to Transovarian transmission - a means of vertical
their sucklings in milk (Gross, 1949). Later, transmission in which infectious agents that have
vertical transmission was shown to occur widely in invaded oocytes when within the ovary are
the animal and plant kingdoms, and in many transmitted to a female's progeny.
6 Host/parasite interactions

Transovum transmission - a postulated means of COMMENTS

vertical transmission in which, during ovulation,
(i) The adjective 'ovarian' is standard in medical
infectious agents invade chorionated ova (oocytes)
dictionaries; `ovarial' is a much older form (Shorter
and are transmitted to a female's progeny.
Oxford English Dictionary, 6th edn). Thus, 'trans-
Trans-stadial transmission - passage of an infectious
ovarian transmission' replaces `transovarial trans-
organism from one developmental stage of a host
mission'.
individual to a later stage.
(ii) The term 'maternal infection rate' replaces the
Maternal infection rate - the proportion of a cohort
terms `transovarial infection rate' and `transovarial
or population of adult females that is infected at
transmission rate', being more appropriate.
emergence, the females having infected germ cells.
(iii) The term 'filial infection rate' concerns the
Filial infection rate - the proportion of the progeny
progeny of an individual female. It has sometimes
of an infected female that is infected through been used incorrectly, e.g. when the mean value
vertical transmission. (The developmental stage of
for a number of filial infection rates was cited. The
the progeny is not specified.)
mean of a number of filial infection rates approxi-
Vertical-transmission rate - the proportion of the
mates the vertical-transmission rate.
progeny from a cohort or population of infected
(iv) In the definitions of 'filial infection rate' and
females that is infected through vertical trans- `vertical-transmission rate' the developmental stage
mission.
of the progeny is not specified.
Effective vertical-transmission rate - the proportion of
(v) For mosquitoes, the term 'vertical-transmission
the progeny from a cohort or population of rate' is limited to transference of an infectious
females, both infected and uninfected, that is agent through the female line. It does not include
infected through vertical transmission. (Proposed
possible venereal transmission after transference
new term.)
through the male line.
Paternal vertical-transmission rate - of a cohort or
(vi) The terms 'paternal vertical-transmission rate'
population of infected males that had mated with
and 'maternal vertical-transmission rate' are not in
uninfected females, the proportion of their
common use; they are included here because of
progeny that is infected through vertical trans- their use in certain simulation models (Section
mission.
41.4).
Maternal vertical-transmission rate - of a cohort or
(vii) The quantitative terms defined above are not
population of infected females that had mated rates but proportions (see Glossary, Appendix 3,
with uninfected males, the proportion of their for both these words).
progeny that is infected through vertical trans- (viii) An oocyte (or ovum) is a female gamete, in
mission.
which meiosis occurs. Very soon after an oocyte
Stable infection - an infection in which virtually all
has been laid, female meiosis is complete, at which
primordial germ cells of the host are infected so
stage the oocyte becomes an egg.
that all or virtually all gametes derived from them
are infected. A female with a stable infection
transmits the infectious agent to essentially all of
(c) Occurrence in mosquitoes
her progeny.
Non-stable infection - an infection in which no Among the viruses and microorganisms that infect
primordial germ cells of the host are infected and mosquitoes, two variants of vertical transmission
in which relatively few or no germ cells become have been described. (i) Transmission cycles are
infected late in or after the completion of maintained by vertical transmission only. Infections
gametogenesis. A female with a non-stable of the rickettsial mutualist Wolbachia in mosquitoes
infection transmits the infectious agent to only a are maintained in this way (Section 46.9). (ii)
proportion or to none of her progeny. Transmission cycles involve both vertical and
 Modes of transmission 7

horizontal transmission, notably La Crosse virus concept of 'stable infections', in which the
(Section 45.5.6), and for some microspordia. The infectious agent infects primordial germ cells and
situation in the few insect-only flaviviruses known hence is present in all gametes (Section 41.3.2.b).
to infect mosquitoes is variable (Section 43.2.2). (ii) Through simulation models in which the
Three means of vertical transmission through variables are the relative rates of fertility, viability
the female line have been postulated. (i) Trans- and vertical transmission (Section 41.4.1).
ovarian transmission: infectious agents invade female Wolbachia infections in mosquito populations are
gametes, either by entering primordial germ cells maintained by vertical transmission alone because
or by entering oocytes before they have become virulence is low and also because, in situations in
enclosed by chorions; they survive through the egg which infected and uninfected host populations
stage and undergo trans-stadial transmission to the occur sympatrically, they provide a selective advan-
F1 adult host. (ii) Transovum transmission: infectious tage for infected over uninfected host populations
agents invade chorionated oocytes during ovu- (crosses between aposymbiotic females and
lation and while they are within the female genital infected males are infertile (Section 46.9)).
ducts (possibly entering through the micropyle); Transmission cycles that include phases of both
they survive through the egg stage and undergo horizontal and vertical transmission have been
trans-stadial transmission to the F1 adult host investigated in detail in two groups of infectious
(Section 44.6.1.b). (iii) Contamination of the oocyte organisms: microsporidia and viruses. Among the
surface: during ovulation and while the oocytes are microsporidian parasites of mosquitoes, the alter-
within the female genital ducts, infectious agents nation of phases of vertical and horizontal trans-
adhere to the chorion and remain attached mission has two variants. (i) Transmission cycles
throughout oviposition. Later, they are ingested by comprise two phases of proliferation which are
and infect newly hatched larvae. No evidence in passed in different individuals of the same
support of this hypothesis has been produced, and mosquito species. One phase starts with the
it is not considered further. infection of larvae by vertical transmission; growth
When vertical transmission occurs, the and multiplication of the parasite within the host
infectious agents are equally distributed between larvae lead to death of the larvae and the release of
male and female progeny. In Drosophila melanogaster spores. The second phase, of horizontal trans-
infected with sigma virus (which is not an mission, starts with the ingestion of such spores by
arbovirus), vertical transmission to the following other larvae of the same species and results in non-
generation is possible through both female and fatal infection. This is known to occur for Cuti-
male lines (Section 41.3.2.b). With mosquito- cospora magna infecting Cutex restuans (Volume 4,
borne arboviruses, only vertical transmission Chapter 53). (ii) Transmission cycles comprise two
through the female line is known. Infection of proliferative phases, one of which is passed in a
spermatozoa and direct transference from adult mosquito host and the other in a different organ-
male mosquitoes to their progeny has never been ism. Species of Amblyospora and Parathelohania
described, but it has been looked for in only one usually infect mosquitoes as the definitive host
or two species, so a blanket dismissal of the and copepods as the intermediate host. Most of
possibility would be rash. Some arboviruses can be the parasite species are transmitted vertically from
transmitted horizontally by venereal transmission, adult female mosquitoes to their progeny via binu-
i.e. from male to female mosquitoes during copu- cleate spores, then horizontally from the infected
lation, and this can lead to indirect transference of mosquito larvae to copepods via meiospores, and
arbovirus from adult male hosts to their progeny. horizontally from copepods to mosquito larvae via
Infectious agents can be perpetuated in the long uninucleate spores.
term by vertical transmission alone, but only if Coupling of vertical and horizontal trans-
certain conditions are satisfied. The phenomenon mission has been reported for viruses (non-arbo-
has been approached in two ways. (i) Through the viruses) of the families Baculoviridae and Iridoviridae
8 Host/parasite interactions

that infect culicid hosts: vertically infected larvae virus (SIGMAV) was classified with the family
die, releasing virions that may be ingested by Rhabdoviridae but was not assigned to any genus
healthy larvae (Section 43.2.3.d). Horizontal trans- (Fauquet, 2005). Later, closely related viruses were
mission between mosquito and vertebrate hosts isolated from D. obscura and D. affinis. Genomic
and viral amplification in the vertebrate hosts are sequencing and phylogenetic analysis showed the
important elements in the transmission cycles of three species forming a deep-branching Glade in the
mosquito-borne arboviruses. Vertical transmission Rhabdoviridae tree that merited recognition as a new
in the mosquito host has been reported from the genus. All three species are transmitted vertically, in
field for a small number of bunyaviruses, and may males through sperm and in females through
be important for their perpetuation (Section oocytes (Longdon et al., 2010, 2011). Host/virus
44.6.2). The evidence for stable infections of interactions have been investigated in colonized and
arboviruses in mosquitoes is slender (Section wild populations of D. melanogaster and sigma virus
44.6.4), and it is thought that most such infections in France, and the concept of stable and non-stable
are non-stable - in which case the vertical infections arose from those studies.
transmission rate is <1.0 and the prevalence rate of Whether or not infections with yellow fever
infection declines from one host generation to the virus in certain of its mosquito hosts are stable or
next. Therefore, perpetuation of the virus in the unstable could be important for explanations of
medium term depends upon its amplification the ability of the virus to survive through long, dry
during phases of horizontal transmission. seasons. Evidence for stable infections of arbo-
viruses in mosquitoes is discussed in Section
44.6.4. Infections of mosquitoes by Wolbachia have
41.3 STABLE AND NON-STABLE
the characteristics of stable infections (Section
INFECTIONS IN DROSOPHILA
46.9).
41.3.1 Introduction
Stable infections are those in which virtually all 41.3.2 Laboratory investigations of sigma virus
primordial germ cells of the host are infected, so in Drosophila
that all or virtually all gametes derived from them
(a) Hypersensitivity to carbon dioxide
are infected. A female with a stable infection
transmits the infectious agent to virtually all her Flies of certain strains of Drosophila melanogaster
progeny. In non-stable infections, no primordial when placed in an atmosphere rich in CO2
germ cells of the host are infected and germ cells become inert within a few seconds but recover
become infected late in gametogenesis or after its entirely on return to a normal atmosphere, even
completion. A female with a non-stable infection after 5 h exposure. The flies of some other strains
transmits the infectious agent to a proportion of fail to recover after even a short exposure,
her progeny. These two types of infection have been especially at lower temperatures. This phenom-
characterized most fully with infections of sigma enon was first observed by L'Heritier and Teissier
virus in Drosophila, and it is from comparisons with (1937), who reported that flies of a strain carrying
those characteristics that claims for stable infections the gene ebony were 'sensible' (sensitive) to CO2,
in mosquitoes should be assessed. Note that the dying upon exposure, whereas other strains were
`stability of a virus', measured by the loss of unaffected, or insensitive. The sensitivity trait was
infectivity when outside a host, is a different found to be inherited, but independently of the
concept and is not considered here. chromosomes; it was ascribed to the presence in
Our knowledge of stable infections derives the cytoplasm of a factor named 6 (L'Heritier and
principally from studies of infections of Drosophila Teissier, 1938a,b). The susceptibility to CO2 that
melanogaster with sigma virus, a subject that is leads to death was never transferred from sensitive
reviewed in detail in the following sections. Sigma to insensitive strains by contact, but could be
 Stable and non-stable infections in Drosophila 9

transferred by the injection of haemolymph, or by depends on the efficiency of its transmission from
the transplantation of ovaries or cerebral ganglia adults to offspring. Low efficiency transmission
from sensitive into insensitive flies of either sex. was regarded as equivalent to the classical con-
Some progeny of the recipient females, but not of dition of virus multiplication within host cells and
recipient males, were sensitive. L'Heritier and de classed as a 'non-stabilized' state. High efficiency
Scoeux (1947) postulated that sensitivity was due transmission was taken to be a veritable inte-
to a 'virus like cytoplasmic hereditary unit'. Later, gration of the virus with the host cells, and was
this agent was shown to be a virus that multiplies classed as 'stabilized' (Brun and Sigot, 1955; Brun
in host cytoplasm; this was named sigma virus. and Plus, 1980). Those terms are still widely used,
The action of CO2 on the flies is chemically as in 'stabilized females', 'stabilized males' and
specific; no other gas produces comparable effects. `stabilized line'. They will be replaced in this
In a test developed by Plus (1954), flies were volume by the terms 'stable infection' and 'non-
exposed to pure CO2 at 13 °C for 15 min, and any stable infection', which are thought to be more
fly which could not right itself and walk after 15 appropriate.
min in an atmosphere of pure air was classed as In flies with stable infections, virtually all
sensitive. primordial germ cells are infected with sigma virus,
Later, the infectious agent was shown to be a but by chance a proportion of 0.01 to 0.001
virus that multiplies in host cytoplasm, and it was remain free from virus particles. Consequently,
named sigma virus. The action of CO2 is specific within the ovaries of adult females, virtually all of
to that compound; no other gas produces com- the primordial germ cells and oogonia are infected,
parable effects. The effects of CO2 on virus- and all oocytes produced by those ovaries are
infected flies are the result of its actions within the infected. Therefore, a female with a stable
thoracic ganglia. Sensitivity allows easy separation infection transmits the virus to virtually all her
of uninfected flies, which quickly recover from progeny. Early embryos are rich in virions, and
brief exposure (L'Heritier, 1970; Brun and Plus, their pole cells (the germ-line cells) are infected
1980). from the outset. If such an embryo develops to an
In North America, sampling revealed moderate adult female, all its oocytes will have been infected
rates of CO2 sensitivity in wild-caught flies of very early and the stable infection will be
Drosophila affinis (18-39% in small samples from 17 perpetuated. In D. melanogaster, once initiated, the
populations) and Drosophila athabasca (3-20% in stable infection with sigma virus will be
six populations). A large sample of D. melanogaster maintained for generations through the maternal
from Nebraska showed 1.6% sensitivity. Lines that lineage (Figure 41.1). In contrast, in D. affinis and
were 100% sensitive were developed from single D. athabasca, CO2 sensitivity is not only maternally
sensitive females of D. affinis and D. athabasca inherited but is inherited by males, which have
(Williamson, 1961). The causal agent, which was inherited their sensitivity only from their fathers
heritable and transmissible by inoculation, was (Williamson, 1961).
taken to be sigma virus (Brun and Plus, 1980). If the embryo develops to an adult male, the
infection will be stable, but only some of its
spermatozoa will transmit the virus. When a male
(b) Stable and non-stable infections
with a stable infection inseminates a non-infected
Sigma virus is perpetuated solely by vertical 0/0 female (see below), none of the progeny will
transmission; infection by horizontal transmission have stable infections, but some will have non-
has never been reported and no possible mech- stable infections. The term 'valence' (English,
anism for the natural horizontal transmission of valency), when used with reference to the parental
the virus is known. In populations of D. melano- males in such crosses, is quantified as the
gaster in which the virus is endemic, perpetuation frequency of infected flies among their progeny.
10 Host/parasite interactions

(iii) (iv) oogenesis and during fertilization by virus present

in infected sperm. Embryos that develop from
,-d 9-d 9-d 9-d such oocytes are not rich in viral genomes, and in
only a proportion of them do the germ-line cells
become infected. Three types of adult female
A\ progeny can be distinguished: (i) females that are
I I I

uninfected; (ii) females whose oocytes become

infected late in oogenesis and which acquire non-
Ii Stable infection stable infections; (iii) females whose oocytes
become infected early in oogenesis and which
9d 1 I Non-stable infection
acquire stable infections (a process termed neo-
9d 1 i Not infected stabilization) (Figure 41.1). When embryos that are
poor in viral genomes give rise to adult males, the
Figure 41.1 Diagram illustrating the transmission of
sigma virus to the progeny of Drosophila melanogaster spermatocytes do not become infected during
when infected females have mated with uninfected spermatogenesis, and the males do not transmit
males, and when infected males have mated with the virus to their offspring (Bregliano, 1970; Brun
uninfected 0/0 (permissive allele) females. The and Plus, 1980; Fleuriet, 1982a, 1988).
outcomes are further affected by whether the infections
are stable or non-stable. (After Fleuriet, 1988.)
Left. The parents of one or other sex have stable (c) Genetics in Drosophila
infections.
(i) The female parents have stable infections, and the In D. melanogaster, the gene ref(2)P is polymorphic
maternal vertical-transmission rate is 1. Outcome: all and its several alleles can be classified as permissive
progeny have stable infections. or restrictive. Two alleles, ref(2)P° and ref(2)PP
(ii) The male parents have stable infections, and the
paternal vertical-transmission rate (valency) is variable.
(sometimes designated 0 and P), have been
Outcome: any infections are non-stable. extensively studied. The presence of the P allele
Right. The parents of one or other sex have non-stable causes interference with the multiplication of
infections. sigma virus in the fly. The permissive and restric-
(iii) The female parents have non-stable infections. tive alleles are codominant, and their products
Outcome: the maternal vertical-transmission rate is
compete with one another to permit or restrict
variable; some of the progeny develop stable infections
(neostabilization), others develop unstable infections multiplication of sigma virus. In heterozygotes, the
and the rest are uninfected. presence of a permissive allele counteracts the
(iv) The male parents have non-stable infections. activity of a restrictive one. All natural populations
Outcome: the paternal vertical-transmission rate is 0. of D. melanogaster examined were polymorphic for
these alleles, P being in the minority (Fleuriet,
When infected males from French populations 1982b, 1988; Wyers et al., 1995).
were mated with uninfected females of the same Genes of both the virus and its host contribute
origin, the F1 valencies ranged from 0 to 1. to the maintenance of sigma virus in Drosophila.
In flies with non-stable infections, virions are Two types of sigma virus coexist in nature. Type I
disseminated throughout the adult body, and the virus survives in flies carrying permissive (0)
amount of virus is higher than in flies with stable alleles, but is very sensitive to the restrictive (P)
infections. However, the embryo contains few viral alleles. Type II virus is more resistant to the P
genomes, and the primordial germ cells are not alleles, but a wide range of sensitivities to P is
infected from the outset. In females, a few oocytes found among Type II clones. In a fly population
may become infected in one or other of two ways: perpetuating both P° and PP alleles, Type I virus
either during late oogenesis by some of the virus cannot persist whereas Type II can be maintained
that is disseminated throughout the body, or after and can spread (Fleuriet, 1982b).
 Simulation models of transmission cycles 11

41.3.3 Field studies of sigma virus in Drosophila viability of the progeny of infected females was
significantly lower than that of the progeny of
The prevalence rate of sigma virus in populations
uninfected females. This viability was largely
of D. melanogaster can vary widely, both geo-
unaffected when infected females had mated with
graphically and in time, reflecting interactions
0/P males (Fleuriet, 1994). In contrast to these
between virus and host. In northern and central
results with initially wild-caught flies, laboratory
France during the 1970s, the prevalence rate was
clones of sigma virus often were harmful to their
about 0.15-0.20. It was lower in some other parts
hosts (Fleuriet, 1988).
of the world. In contrast, when experimental
Transmission through male as well as female
populations were reared in cages, the virus was
hosts can make possible the survival of an
able to infect almost all of the flies (Fleuriet,
infectious agent from generation to generation by
1982b, 1988). Analyses of 38 natural populations
vertical infection alone (Section 41.4.1). For that
of D. melanogaster in the Languedoc region of
reason, neostabilization is essential for the
southern France between 1983 and 1991 revealed
perpetuation of sigma virus infections in wild
a year-by-year increase in the prevalence of infected
populations of D. melanogaster. Virtually all the
flies, followed by a progressive decrease. Between
progeny of females with stable infections of sigma
1983 and 1988 the mean prevalence rate of
virus will carry stable infections, but the progeny
infection rose from 0.15 to 0.65; between 1989
of crosses between uninfected females and males
and 1991 it fell from 0.65 to 0.30. In all French
with stable infections either have non-stable
populations the efficiency of transmission by
infections or are uninfected. However, neo-
males decreased. These changes in prevalence were
stabilization of infections occurs among the
associated with changes in both virus and host. (i)
progeny of the F1 daughters that had acquired
Adaptation of the virus - between 1983 and 1991,
non-stable infections (Figure 41.1). For 12 French
the prevalence rate of Type I virus, which is
populations, neostabilization values of 0.92-0.97
sensitive to the P allele, declined from 0.47 to
were found; the mean value in France was about
0.03, while that of Type II virus increased from
0.75 (Fleuriet, 1982a, 1988).
0.53 to 0.97. The effect of this change was
enhanced by a decrease in the sensitivity of the
Type II virus to the flies' restrictive allele (P). (ii)
Adaptation of the flies - the occurrence of the 41.4 SIMULATION MODELS OF
restrictive allele (P) increased slightly but pro- TRANSMISSION CYCLES
gressively in fly populations from 1984 to 1991
41.4.1 Modelling cycles that are maintained by
(Fleuriet, 1990; Fleuriet et al., 1990; Fleuriet and
vertical transmission alone
Periquet, 1993).
Flies that had been collected from wild French Fine (1975) developed a general model for trans-
populations, and that were infected or not infected mission cycles of infectious organisms maintained
with sigma virus, were compared for physiological through successive generations of their host
traits that included viability of the developmental populations by vertical transmission alone. The
stages, male and female fertility, female longevity model assumes that an infection is present in a
and sexual selection. Infection modified the flies' proportion of the adult members of a sexually
fitness only slightly; the only significant difference reproducing population. It assumes also that (i)
that was found was a lower viability, in some infection persists throughout the lifetime of
replicates, of the developmental stages of the vertically infected individuals, (ii) infection has
progeny of infected females (Fleuriet, 1981). similar effects on males and females of the host
Further experiments showed that, when females species, and (iii) mating occurs at random in the
had mated with 0/0 males, the egg-to-adult host population. Development of the model led to
12 Host/parasite interactions

a 'fundamental vertical transmission equation' right-hand side of the equation, one can iterate the
(Eqn 41.1, shown in the caption to Figure 41.2), calculation and so determine the prevalence rates of
which indicates the quantitative contribution of infection that would be found in successive
vertical transmission to the prevalence rates (see generations of hosts. The results of two such
Glossary) of infection in subsequent generations. iterations are illustrated in Figure 41.2; both have
As noted in Figure 41.2, given any set of values the initial prevalence rate set at 0.5. Under one set
for the five variables listed in the caption, the of variables (solid circles) the prevalence rate
solution of the fundamental vertical transmission increases and would stabilize at approximately 0.83,
equation can readily be obtained. For a vector compatible with infection being perpetuated by
population B, by repeatedly substituting the solu- vertical transmission alone. Under the second set of
tion Ba' (the prevalence rate of inherited infection variables (hollow circles) the prevalence rate declines
among adult members of the progeny generation) steadily, ultimately to zero, so infection cannot be
for Ba (the prevalence rate - or proportion - of maintained by vertical transmission alone. This
infection among adult ovipositing vectors) in the latter condition is typical of transmission cycles in

P F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12

Host generation

Figure 41.2 Calculated changes in the prevalence rates of an infection when maintained through 12 generations of
its hosts by vertical transmission alone. The prevalence rate in the parental generation was set at 0.5, and two sets of
variables were applied. The curves were obtained by iteration of the 'fundamental vertical transmission equation'
(Eqn 41.1; see below). (Modified from Fine, 1975.) Each point on the curves is a prevalence rate (Ba) for the
generation specified on the horizontal axis.
The 'fundamental vertical transmission equation' (41.1) provides an initial measure of the quantitative contribution
of vertical transmission to the prevalence rates of an infection through successive generations of the host:
P[Baad(1-Ba +Baa)+Baae(1- Ba + Baa - Baad)]
Ba - 41.1
ABaad(1-Ba + Baa) + Baae(1-Ba + Baa - Baad)] + (1- Ba + Baa -Baad)(1-Ba + Baa - Baav)
where
13, ' is the prevalence rate of inherited infection among adults of the progeny generation;
Ba is the proportion (or prevalence rate) of the adult ovipositing members of the vector population (B) that are infected;
d is the maternal vertical-transmission rate, i.e. the prevalence rate of infection among the progeny of infected
females when mated with uninfected males (symbol r in Fine, 1975);
v is the paternal vertical-transmission rate, i.e. the prevalence rate of infection among the progeny of infected males
when mated with uninfected females;
a is the relative fertility (number of progeny) of infected adults compared with their uninfected peers;
/6 is the survival rate (to reproductive age) of vertically infected individuals compared with uninfected individuals.
Given any set of values for Ba, d, v, a and fl, the solution of the fundamental equation is straightforward. By
repeatedly substituting the solution Barfor Ba in the right-hand side of the equation, one can iterate the calculation,
determining the prevalence rates of infection that would be found in successive generations of hosts, as long as the
assumptions hold. The results of two such iterations are illustrated in this figure.
 Simulation models of transmission cycles 13

which infectious agents infect both vertebrate hosts (2a) If d = 1, then infected females transmit the
and arthropod vectors, and that include phases of agent to all their progeny. Many microorganisms
both horizontal and vertical transmission. are thought to be maintained in this manner,
There is a simpler method of predicting whether notably bacterial mutualist symbionts of arthro-
an infectious agent can be maintained in succes- pods, which are so intimately associated with their
sive generations of its host by vertical transmission hosts that they never need to escape the confines
alone. Where of the host organism. Unless the infectious agent
confers some advantage on its host, it is likely that,
afi(d + v) >1 (41.2) because of drift, the agent will eventually be
eliminated. Where Drosophila simulans contains the
the conditions are sufficient for survival from gener-
symbiont Wolbachia, infected host populations
ation to generation solely by vertical transmission. In
this expression, a is the relative fertility of infected gain advantage over aposymbiotic populations
compared with uninfected adults, fi is the relative because crosses between aposymbiotic females and
viability of infected compared with uninfected indi- infected males are infertile (Section 46.9.6.c).
viduals, d is the maternal vertical-transmission rate and (2b) If d < 1, then the prevalence rate of infection
v is the paternal vertical- transmission rate (see Table steadily decreases towards zero.
41.2 for fuller definitions). In cases where the
Whether venereal transmission, which has been
expression
demonstrated experimentally in mosquitoes
ap(d + v) <1 (41.3) (Section 44.5), can enhance significantly the
maintenance of an infectious agent by vertical
applies, the prevalence rate would decline steadily, transmission can be tested by use of a simulation
and ultimately to zero, in the absence of amplifi- model (Eqns 44.7, 44.8). Assuming an efficiency of
cation during phases of horizontal transmission venereal transmission of 0.75, and a daily survival
(Fine, 1975). Fleuriet (1988) pointed out that there rate of 0.90, the model indicates that, when vertical-
are several possible situations, all assuming that transmission rates at emergence range from 0.002
infection does not greatly affect host fitness, i.e. afl to 0.08, venereal transmission increases the
1. summed contribution of vertically infected females
(1) Vertical transmission occurs through both to egg deposition in the first and second gono-
female and male gametes. trophic cycles by 0.24-0.26 (24-26%) (Table 44.5).
At much higher vertical-transmission rates, the
(la) If d + v > 1, then the infectious agent persists extent of this contribution by venereally infected
in the population. It is transmitted through both females is greatly reduced. Thus, when the vertical-
female and male gametes, and if the combined transmission rate at emergence is 0.95, which
maternal and paternal vertical-transmission rates approaches the rate of 1.0 needed for perpetuation
are sufficient the agent may be maintained in a solely by vertical transmission, venereal transmission
population by vertical transmission alone, even if would increase vertical transmission to the next
infection reduces host fitness (a < 1, fi < 1), pro- generation by only 0.013 (1.3%).
vided that a/3(d + v) > 1. That was the case with
sigma virus in Drosophila melanogaster in France,
where infections persisted at different prevalence 41.4.2 Modelling cycles that involve both vertical
rates in different host populations (Section 41.3.3). and horizontal transmission
(lb) If d + v < 1, then the infectious agent is Fine and LeDuc (1978) developed quantitative
eliminated. models of the natural transmission cycle of
(2) Vertical transmission is through female gametes Keystone virus, a cycle that involves Ochlerotatus
only. atlanticus as mosquito host and vector, and certain
14 Host/parasite interactions

small mammals as vertebrate hosts. The filial infec- _c

tion rate among the progeny of infected female Oc. .E 10
atlanticus was taken to be <1.0 (characteristic of 8
non-stable infections), therefore the vertical- 6
transmission rate was <1.0 and amplification of
5 2
virus during phases of horizontal transmission was 1
1

1 1

essential for its perpetuation. More recently, some

0
authors have described certain infections of mos- Jun Jul Aug Sep Oct

quitoes by an arbovirus as stable. If their claim is Figure 41.3 Relative numbers of adult female Ochtero-
confirmed, additional simulation models will be tatus atlanticus at Pocomoke Cypress Swamp, Maryland,
needed. during 1971, based on light-trap samples. (After LeDuc
et al., 1975c.) The arrows show the dates of isolation of
Keystone virus from those mosquitoes, while the
(a) Ecology of Keystone virus and its hosts associated numerals show the numbers of isolates. As in
the original figure, the datum points are spaced evenly
Keystone virus (KEYV), a serotype of the along the x-axis although the intervals between sampling
California antigenic group (Orthobunyavirus, family varied; consequently, the apparent durations of the
monthly periods are irregular.
Bunyaviridae), is maintained over a wide area of the
south-eastern USA in a sylvatic cycle that involves
vertebrates as amplifying hosts and Ochlerotatus larvae of Oc. atlanticus, and from adult males and
atlanticus as vector. The system has been investi- females reared from wild-caught larvae, confirmed
gated most thoroughly at the Pocomoke Cypress that vertical transmission occurs in nature (LeDuc
Swamp in Maryland (Figure 45.3), where the virus et al., 1975c).
is endemic but near the northern limit of its Ochlerotatus atlanticus is known to be an
range. Here, Oc. atlanticus usually has just one opportunistic feeder which will attack mammals,
generation per year. This mosquito overwinters in reptiles and birds, and this was confirmed at the
the egg stage, the eggs having been laid in heavily Pocomoke Cypress Swamp (LeDuc et al., 1972).
shaded, shallow depressions in the ground. Among the range of its wild mammalian hosts,
Hatching is triggered by the first major summer rabbits and grey squirrels became viraemic after
rain, which may occur at any time between June experimental infection (Table 41.1). Studies using
and September. In this area, a minimum rainfall rabbits as sentinels suggested that a high propor-
of 3 inches (7.6 cm) in 24-48 h is needed to tion of the susceptible mammalian hosts became
produce suitable temporary pools. Such rains may infected with KEYV when vertically infected
occur only once a summer, and in most years only females first fed, and developed a viraemia that
a single brood emerges. The aquatic stages develop lasted 2-4 days. Surviving females were said to
rapidly and the adults emerge almost simul- blood feed repeatedly. Some mosquitoes taking
taneously (Figure 41.3) (LeDuc et al., 1975a,c). second or later blood meals became infected by
Characteristically for a floodwater mosquito feeding on viraemic hosts, and in that way
species, the flight period for Oc. atlanticus is short, horizontal transmission of the virus led to an
lasting about a month during the summer, increase in the prevalence rate of infection in the
although small numbers of adults can be found vector population. During the summer months,
earlier and later. The first isolation of KEYV between 11 June and 8 September 1971, the overall
occurs on virtually the first appearance of adults, MIR (minimum infection rate)/1000 of females
when the females are unlikely to have taken a captured in light traps was 4.0 (n = 7964) (LeDuc
blood meal (Figure 41.3), so the vector population et al., 1975a,b; Fine and LeDuc, 1978). In each
must be vertically infected at the time of emer- gonotrophic cycle, females laid eggs determined
gence. The isolation of KEYV from wild-caught for winter dormancy, and some of the eggs
 Simulation models of transmission cycles 15

Table 41.1 Interactions between Ochlerotatus atlanticus, Keystone virus and

mammalian hosts in Pocomoke Cypress Swamp, Maryland. (From LeDuc et al.,
1975c.)

Host Engorged Seropositive for KEYV Viraemia after

C. atlanticus* experimental
(%) (%) n infection

Whitetail deer 34 9.9 121 -t

Rabbit (domesticated) 12 33.3 3
Grey squirrel 6 29.6 27 +*
Racoon 4 17.9 39 + (low)
Opossum 2 0 18
Turtles 15 0 227
Others 27

", Total of 436 blood meals.

t, A few wild whitetail deer had detectable neutralizing antibody to KEYV.
t, Up to 105 PFU m1-1.
- indicates test not done.

deposited by infected females contained virus. are potentially infective to vertebrates at their first
Because not all eggs laid by infected females blood meal, i.e. before any horizontal transmission
contained virus, the filial infection rate among (Figure 41.4). Because d < 1, then Bad < Ba. The
dormant eggs was lower than the maternal predicted difference in prevalence rate of infection
infection rate (Fine and LeDuc, 1978). between parental mosquitoes and their progeny,
(Ba - Bad), must be compensated for by amplifi-
cation during horizontal transmission in each
(b) A simplistic model
mosquito generation.
In developing a simulation model of the trans-
mission cycle of Keystone virus, Fine and LeDuc
(c) An advanced model
(1978) first formulated a 'purposefully over
simplistic' model that involved five variables (a, fi, A more detailed and advanced simulation model
v, d, Ba), which are defined in Table 41.2. It was of Keystone virus transmission was also formulated
assumed that infection with KEYV has no selective by Fine and LeDuc (1978). Definitions of the
effect on its vector, Oc. atlanticus, and that all symbols used are given in Table 41.2. The model
infections are non-stable. We note that Ba is the comprised two phases.
prevalence rate of infection among ovipositing Phase (i). Transmission to amplifying hosts
females, regardless of age, and that d the is during the vectors' first gonotrophic cycle (see
maternal vertical-transmission rate among the pro- Glossary, Appendix 3). The expression Bad
geny of uninfected y x infected d'5'. It follows described the prevalence rate of infection among
that the prevalence rate of vertically transmitted newly emerged mosquitoes; it specifies the
infections among the progeny of each generation proportion of mosquitoes which are potentially
is equivalent to Ba x d, and that at the end of any infective at their first blood meal and before any
season Bad will be the prevalence rate among both horizontal transmission. (The fundamental
the overwintering eggs and the newly emerged vertical-transmission equation (Eqn 41.1, in
adults that have developed from them. Thus, the Figure 41.2) could also be used.) After females of
model specifies the proportion of mosquitoes that Oc. atlanticus had taken their first blood meal,
16 Host/parasite interactions

Table 41.2 Symbols of variables used in models of transmission cycles of Keystone virus maintained by both horizontal
and vertical transmission. (From Fine and LeDuc, 1978.)
a = relative fertility rate (infected relative to uninfected adults)
fl = relative survival rate to reproductive age (of infected progeny relative to uninfected)
B a = prevalence rate of viral infection among adult ovipositing females of a vector population B; equivalent to the
proportion of all egg batches that are deposited by infected females if fertility is impaired)
Bad = prevalence rate of vertical infection among newly emerged mosquitoes (i.e. the initial prevalence rate)
Badi = probability that a mosquito taking its first blood meal is infectious
1 - Badi = probability that a mosquito taking its first blood meal is not infectious
(1 - Bad i ) n = probability that none out of n mosquitoes, taking their first blood meals, is infectious
1 - (1 - B ad i ) n = probability that at least one out of n mosquitoes, taking their first blood meals, is infectious

d = maternal vertical-transmission rate (prevalence rate of infection among the progeny of a cohort of infected
females when mated with uninfected males)
f = proportion of mosquitoes that become infected by a single blood meal on a viraemic host
h, = effective incidence rate of virus infections in the vertebrate population after the first mosquito blood meals
hm2 = incidence rate of virus infections in the mosquito population at the time of the second blood meal
i = proportion of infected mosquitoes that are infective to vertebrates
M = number of vectors that oviposit at least once
n = number of vectors per susceptible host at time of initial blood feeding
p = probability that a female mosquito survives through a gonotrophic cycle
S = proportion of all vector blood meals that are taken from susceptible host species
s = proportion of individuals of susceptible host species that lack immunity to virus at onset of transmission season
Ss = proportion of blood meals taken on susceptible and potentially viraemic hosts
73, 72, 73, etc. = proportion of females of vector species that are infected when taking their first, second, third, etc.
blood meals
v = paternal vertical-transmission rate (prevalence rate of infection among the progeny of a cohort of infected males
when mated with uninfected females

the effective incidence rate (ho) of KEYV gonotrophic cycle was taken to be the prevalence
infections in the vertebrate population was rate of vertical infection (Bad).
expressed as Phase (ii). Transmission from the amplifying
hosts to infected and uninfected mosquitoes during
hy = Ss {1 - (1 -B,di)'} (41.4) the second gonotrophic cycle. The incidence rate
where S was the proportion of blood meals taken of infection among mosquitoes that succeeded in
on susceptible host species; s was the proportion of taking a second blood meal (hm2) can be equated
individuals of susceptible host species that lacked with the incidence rate of viraemia in the vertebrate
acquired immunity at that time; the product Ss population, and the probability (f) that they will
was the proportion of all blood meals taken on become infected; therefore
hosts able to both become infected and become h ., = f Ss {1- (1 - Ba di)" } (41.5)
sufficiently viraemic to transfer virus back to mos- The same incidence rate can be expressed
quitoes; i was the proportion of infected mos- differently as
quitoes that were infective; and n was the number
of vectors per susceptible vertebrate host at the B (1- d)
time of feeding. The symbol h., was derived from hm2 = a
(41.6)
p(1 - Bad)
the phrase 'horizontal transmission to vertebrates'.
The prevalence rate of infection among mos- where p is the probability of a female mosquito
quitoes surviving to oviposit at the end of the first surviving through a gonotrophic cycle. Combin-
 Simulation models of transmission cycles 17

Parental generation Filial generation

Second
a)
blood meals
Ec
(1) Vertical
(1) transmission
To c rniSSion
o
Bad
ooav A
tt
Hatching Emergence Oviposition

Figure 41.4 Diagram illustrating the relationship between the prevalence rates of Keystone virus infection in its
mosquito host, Ochlerotatus atlanticus, and the developmental stages of two generations of that host. (After Fine and
LeDuc, 1978.) Each box is constructed as an age x prevalence graph for a generation of vectors. The prevalence rate of
infection (Bad) is low throughout the aquatic stages, but increases when adult mosquitoes feed on viraemic vertebrates.
The effective prevalence rate of infection among ovipositing females is then raised to Ba. Of the eggs laid by an infected
female, only a proportion (d) carry virus, so the prevalence rate falls to Bad at the beginning of the next generation. The
prevalence rate must be increased by the amount (Ba - Bad) during each generation. No direct estimates of d had been
made for KEYV in Oc. atlanticus, so the shape of the curves is speculative.

ation of a number of epidemiological elements led Two assumptions are inherent in the 'basic
to the 'basic transmission model', which predicts transmission model' (Eqn 41.7): (i) all vector-to-
the average number (n) of first blood meals that vertebrate transmission occurs at the first blood
must be taken by the mosquito population on meal; and (ii) all vertebrate-to-vector transmission
each susceptible vertebrate host to maintain the occurs at the second blood meal. Because, prob-
transmission cycle of Keystone virus in the ably, these assumptions are not strictly true, an
Pocomoke Swamp. Thus equation was developed describing the prevalence
rate of infection (Ba) among ovipositing females
pf Ss(1 -B d)- B (1- d) in all gonotrophic cycles. Thus
log
pfSs(1 -B d) (41.7)
n= B=
A471-14- N41)71-2 4- N4p2773 4- N4p371-4 +."
(41.8)
log(1- B di)
M / (1 -1))

The implications of this basic transmission where M is the number of vectors that oviposit at
model (Eqn 41.7) are illustrated in Figure 41.5, least once, and 77-1, 7r2, etc. are the proportion of
which shows the relationship between the number females of vector species that are infected when
of first blood meals that need to be taken on taking their first, second, third, etc. blood meals.
each susceptible host (n) and the product pfSs' This simplifies to
at two different values of the maternal vertical- Ba = (1- p)(7r, +pn-2 +p27,3 +p37,4 +...) (41.9)
transmission rate (d). The extent to which the
maternal vertical-transmission rate affects the Fine and LeDuc (1978) commented that math-
number of first blood meals that must be taken on ematical models can be useful in highlighting the
each vertebrate host for the transmission cycle to types of field data needed for evaluating virus
be maintained is evident. The product term `pfSs' transmission, and in assessing the validity of such
is a measure of the proportion of the initial female data, and that, further, they provide a rigorous
mosquito population that both survives to take a means of testing qualitative hypotheses about how
second blood meal and becomes infected, and complex systems function in nature. Their 'sim-
which, therefore, produces infected diapause eggs. plistic model' of KEYV transmission was designed
18 Host/parasite interactions

300 population interacts with a population of its

natural host, the extent of harm caused to infected
7,1
0 individuals is an important factor in the dynamics
_c
of both populations. Two descriptive terms
-8 200 pertinent to such situations are: pathogenicity -
the ability to cause pathological changes or disease;
EI'3-

co and virulence - the degree of pathogenicity of a

E
parasite as indicated by the cruelty of the disease
produced. Parasitologists are likely to treat
8 100
d = 0.1 pathogenicity and virulence as aspects of host/
(7)
parasite interaction that result in costs to the
0
d fitness of both parasite and host. Virulence is the
z
more useful of these terms - all parasites, as
0
0 0.1 0.5 1.0
distinct from other symbionts, are necessarily
pfSs pathogenic. In epidemiological studies, virulence
Figure 41.5 Plots of the average numbers (n) of first
may be one variable, but often any quantitative
blood meals that need to be taken on each susceptible measurements of virulence are crude, e.g. the rates
host in a vertebrate population to maintain the of morbidity or death in host populations.
Keystone virus transmission cycle; shown at two What is usually perceived as a pathological state
different vertical transmission rates (d). Solid curve, d = or a disease of the host is the consequence of
0.1; broken curve, d = 0.5. The graph represents
solutions to Eqn 41.7, with the assumption that the
modifications of the host induced in it by the
prevalence rate of viral infection (Ba) among adult parasite to its own advantage, to enable it to
mosquitoes lies within the range 0.001 < Ba < 0.005, as survive, develop and multiply within the host. Not
found in field isolations. In nature, the independent all modifications of the host are pathological;
variable `p fS s ' (see text) is probably <0.1. (From Fine some may reduce the reproductive capability of the
and LeDuc, 1978.) host. These actions of the parasite are genetically
to test the hypothesis that the prevalence rate of regulated. The perceived virulence of a parasite
infection among ovipositing females is higher than population is an aggregate of the mechanisms that
that among females of the following generation make up its disease-producing capability. Infection
(because only a proportion of the eggs laid by an of a host triggers a response in the form of
infected female carry virus), and that, con- activation of innate immune mechanisms, which
sequently, amplification within vertebrate hosts is also are genetically regulated (Chapter 42). The
required for perpetuation of the virus (Figure refractoriness of a host population is an aggregate
41.4). Their more advanced models concerned of the defensive mechanisms employed against the
particular phases of the transmission cycle. In the parasites infecting it. Where in nature populations
absence of much essential quantitative field data, of a host and a parasite occur together, the
these models of KEYV transmission have not been interactions between them generate selective forces
tested, so the concepts of transmission that they that affect the virulence of the parasites and the
explore remain hypothetical. refractoriness of the hosts, with the possibility of
modification of either; in other words, there is a
trade-off between those two variables (Section
41.5 VIRULENCE 42.1.2). This is usually perceived as a trade-off
between the aggressiveness of the parasites and the
41.5.1 Characteristics
diseased state of the hosts.
In this section, the term `parasite' is used for any Such interactions have been extensively
form of infective agent. Where in nature a parasite modelled, but we examine just the equation for
 Virulence 19

the basic reproductive rate, which was devised for resulting in intermediate levels of virulence. Nor
measurement of the transmission of malaria do host-parasite relationships evolve to zero
parasites (Macdonald, 1955) and developed for virulence with no disease, because the functional
analysis of the population dynamics of micro- causes of virulence are beneficial to the parasite,
parasites (Anderson and May, 1981). Estimates of increasing its transmission success during its
the basic reproductive rate of myxoma virus were lifetime. However, virulence can carry a cost in the
used by Anderson and May (1982) in their study form of premature host death, which shortens the
of interactions between myxoma virus and lifetime of the infection. Consequently there is a
populations of the European rabbit in Australia, trade-off between how fast and for how long the
where mosquitoes served as vectors through parasite transmits. This is thought to result in
mechanical transmission (Section 43.4.3.b; Eqn maximum 'lifetime transmission' occurring in
43.2). Here, that equation is elaborated and the parasites that cause an intermediate degree of
notation modified as suggested by Frank (1996). virulence. Selective forces may drive virulence
upwards, but only to the point where reductions
/3(v)N
Ro = (41.10) in transmission caused by host death begin to
+ v + c(v) outweigh the benefits of transmission (Frank,
where Ro is the basic reproductive rate of the 1996).
parasite, 8 is the host's disease-free death rate, v is A very large epidemiological survey, the Garki
the disease-induced mortality rate (virulence), c is Project, was carried out in a malaria-endemic area
the rate at which hosts recover by clearing the in Nigeria where P. falciparum predominated
infection, fi is the rate of transmission of the disease (Molineaux and Gramiccia, 1980). Later analysis
between infected and susceptible hosts, and N is the of the field data revealed strong positive correl-
total population size of the host. ations between asexual multiplication, trans-
The terms in the equation have simple intuitive mission rate and infection length, and host
meanings. Transmission, fi(v)N, is the number of morbidity and mortality. The expected total
new infections per unit time produced by one lifetime transmission of the parasite was maximal
infected individual introduced into a population in young children, in whom the fitness cost to the
of N uninfected hosts. The denominator terms, 1/ parasite of host mortality balanced the fitness
[(5 + v + c(v)1, describe how long an infection is benefits of higher transmission rates and slower
expected to persist within a host: the host may die clearance rates, a clear example of trade-off
as a result of parasite virulence, v; or the infection (Mackinnon and Read, 2004).
may be cleared by the host, c(v); or the host may In cases of direct transmission, i.e. of a pathogen
die of other causes, 6. The product of transmission from one host individual to another of the same
and residence time in the host determines the species, it is necessary that the infective hosts
total number of new infections emanating from an maintain their motility; and for that reason, it is
infected host. Equation 41.10 indicates that a thought, selection acts against any exploitation of
higher rate of transmission increases parasite fit- a host that causes severe pathogenicity. In cases of
ness, whereas higher virulence decreases parasite indirect transmission in which the two hosts are a
fitness because it damages the parasites' food vertebrate and a haematophagous arthropod, the
supply (the host). Parasite fitness is governed by latter being the vector, both hosts are liable to
the balance between the benefits of high trans- suffer pathogenic effects, but selective forces act
mission and the costs of increased virulence differently on the two. It is not essential for
(Frank, 1996). transmission that infective vertebrate hosts retain
As noted in the following section, selection does their motility, therefore intensive exploitation of
not produce a balance between increasing success the vertebrate host should carry lower costs to the
of the parasite and increasing costs to the host, pathogen than in the case of directly transmitted
20 Host/parasite interactions

pathogens. In contrast, the continued motility of the parasite, increasing its transmission success
infected vectors and protection of their lifespans during its lifetime. For example, by extracting
are essential for transmission. Within populations more resources from the host, the parasite is able
of humans at risk of infection with some 60 to produce more of its transmissible forms per
infective agents (viruses, bacteria or protozoans), unit time. Also, where a mechanism that reduces
mortality rates were markedly and significantly clearance of a parasite by its host becomes
higher with the vector-borne infective agents than enhanced, the duration of infection is longer so
with those that were directly transmitted (Ewald, increasing lifetime transmission. The idea that a
1983; Ewald and De Leo, 2002). well-adapted parasite should necessarily cause only
slight or moderate virulence is now regarded as
erroneous. Theory and empirical evidence support
41.5.2 Genetic variability and evolution
the idea that natural selection can drive a host-
Microparasites have short generation times, and parasite relationship to any position along the
usually show considerable genetic variability; there- range between commensalism and lethality
fore, natural selection can cause rapid changes in (Ewald, 1994, 1995; Ewald and De Leo, 2002).
the genetic make-up and phenotypic characteristics Empirical observation shows virulence to
of their populations. Vertebrate hosts have much continue in many host-parasite relationships of
longer generation times, and so can respond only very long standing, but the phenomenon that
relatively slowly to parasite-imposed selection hosts are not severely affected by infection (Section
pressure by developing resistance. Both parasites 41.1.2) suggests that virulence can decline to a
and hosts exploit their genetic heterogeneity to maintained, relatively harm-free level. Two examples
adapt, and eventually evolve, in response to one that involve mosquitoes as vectors indicate that,
another and to their environment. on a time scale longer than historical time, some
For many years it was thought that parasites that vertebrate-pathogen associations have evolved to a
harm their hosts have reduced chances of survival relatively harm-free state. (i) The effects of
and that, in consequence, parasitic species that infection with yellow fever virus on African
cause serious disease are disadvantaged over time. monkeys are mild, and few infected monkeys die.
Individual traits may have positive, neutral or In contrast, among New World monkeys, first
negative effects on the fitness of a parasite or a exposed to yellow fever virus in the 16th century,
host. It was predicted that selection acting on such species of Atouatta, Saguinus and Aotus still suffer
traits would produce a balance between increasing high fatality rates (Sections 45.3.2.b; 45.3.6.c). (ii)
success of the parasite and increasing costs to the After the arrival of Cutex quinquefasciatus in the
host - leading to its death, and resulting in inter- Hawaiian Islands in 1826, epizootics, first of 'bird
mediate levels of virulence. That was shown not to pox' and later of avian malaria due to Plasmodium
be the case, notably with myxoma virus infecting relictum capistranoae, led to the decline of many
rabbits (Section 43.4.3), and the view long held by native bird species and the local extinction of
parasitologists that all parasites evolve to become others. Introduced species of birds were relatively
mild for their hosts has been rejected (Bull, 1994; tolerant of infection (Section 51.9.2).
Sabelis and Metz, 2002). Observational evidence Attempts to elucidate the evolutionary history of
and modelling have shown that host/parasite host-parasite relationships are fraught, because the
relationships do not develop to benignity on short evidence must be based on a variety of approaches.
time scales; nor do they evolve towards it on Two examples concerning P. falciparum, both cited
evolutionary time scales. from Rich and Ayala (2000), illustrate that point.
Equally, host-parasite relationships do not Divergence between Plasmodium reichenowi, the
evolve to zero virulence with no disease, because chimpanzee parasite, and P. falciparum, the human
the functional causes of virulence are beneficial to parasite, is estimated to have occurred 8-12 million
 Virulence 21

years ago, which is roughly consistent with the time age of genes because they reflect the mutation rate
of divergence between the two host species, and the time elapsed since their divergence from a
chimpanzees and humans. This suggests that P. common ancestor. That approach revealed a scarcity
falciparum is an ancient human parasite, associated of synonymous polymorphisms in ten P. falciparum
with our ancestors since the divergence of humans genes. Of five possible explanations, the most
from the great apes. Another approach to eluci- satisfactory pointed to a recent population bottle-
dating the evolutionary relationships of P. falci- neck, indicating that extant world populations had
parum involved silent (i.e. synonymous) nucleotide been recently derived from a single ancestral strain
polymorphisms, which can be used to estimate the (Rich and Ayala, 2000).
 42
Immune responses of mosquitoes

42.1 Introductory matters 22

42.2 Immune response to viruses 24
42.3 Serine proteases 25
42.4 Recognition proteins 27
42.5 Regulation of the immune response in Anopheles gambiae 30
42.6 Antimicrobial peptides 32
42.7 Haemocyte types and their characteristics 34
42.8 Phagocytosis 36
42.9 Structural observations of encapsulation 37
42.10 Immune and other roles of melanin in mosquitoes 40
42.11 Modulation of host immune responses by salivary proteins 47

This chapter contains descriptions of the types of pathogens, e.g. the lipopolysaccharides of Gram-
immune response to infection found in mos- negative bacteria and lipotechoic acids of Gram-
quitoes. The descriptions are cross referenced to positive bacteria. For insects, which have a
the sections of other chapters that include more relatively short lifespan, this is the more effective
detailed accounts of the responses to particular form of defence. Specific (or adaptive) immune
pathogens or parasites. mechanisms, found in vertebrates, involve the
highly specific recognition of foreign antigens by
means of immunoglobulins, major histo-
42.1 INTRODUCTORY MATTERS compatibility complex molecules and T-cell
receptors. A cellular memory provides the ability
42.1.1 Types of immunity
to raise an enhanced response, as with antibodies,
Two types of immune mechanism are recognized - when a pathogen is re-encountered. It is possible
innate and specific. Innate immune mechanisms, that, within their limitations, insect immune
which function in insects as in other animals, systems have greater capabilities than is generally
involve a variety of defences, including release of accepted. Putative cases of cellular memory in
antimicrobial peptides and phagocytosis by blood insects, in which the hosts raised an enhanced
cells. They are activated within seconds of immune response following an initial encounter
encountering an elicitor. The responses to invasive with an elicitor, are discussed by Pham and
organisms recognized as `non -self are relatively Schneider (2008).
non-specific and no cellular memory is required. The innate immune mechanisms of insects
`Pattern recognition receptors', which trigger share some similarities with those of mammals,
responses, do not recognize unique antigens but but they will be mentioned only very briefly. Our
are specific for a few, highly conserved structures knowledge of the insect immune system largely
that are present in large groups of potential derives from genetic studies on Drosophila
22 © A.N. Clements 2012. The Biology of Mosquitoes, Vol. 3:
Transmission of Viruses and Interactions with Bacteria (A.N. Clements)
 Introductory matters 23

melanogaster and data obtained from a few populations were possible. First, a stable equi-
lepidopteran species such as Manduca sexta, the librium with intermediate levels of investment by
size of which make biochemical studies feasible. host and parasite, both partners investing less as
Comparison of the innate immune mechanisms the intensity of transmission increases. Second,
found in Anopheles gambiae with those known from where the cost of investing into resistance is
D. melanogaster have proved informative. sufficiently high and transmission is sufficiently
The immune responses of mosquitoes can be intense, the host invests no resources in its immune
very effective in countering invasion by prokaryotic response and, accordingly, the parasite invests
and eukaryotic parasites. They involve humoral and nothing in a counter-response. Third, where the
cellular components and combinations of both. cost of immunity is high and transmission is
The humoral component includes inducible anti- intermediate, no stable equilibrium is achieved but
microbial peptides and a phenol oxidase cascade the levels of investment by host and parasite cycle
system that both yields reactive oxygen and nitrogen around intermediate levels.
intermediates and is involved in encapsulation. The Most investigations into mosquito immune
cellular component involves haemocytes that are systems are undertaken in laboratories distant
involved in phagocytosis or in encapsulation. Most from natural mosquito populations. Often model
investigations into the immune responses of mos- systems are used, i.e. host/parasite associations that
quitoes have concerned parasites that infect both do not occur in nature but that can be produced in
humans and mosquitoes - namely malaria parasites the laboratory. Even if host/parasite associations
and filarial nematodes of which mosquitoes are that occur in nature are used in the laboratory,
hosts and vectors. Antimicrobial peptides have been there has been no continuous association between
studied mostly for their antibacterial activity. the colonized hosts and the parasites that permitted
trade-offs between them. Comparisons between
findings from the laboratory and field are
42.1.2 Comparison of immune responses
enlightening. When laboratory-reared Anopheles
observed in laboratories and in nature
gambiae were infected with Plasmodium falciparum,
Where, in nature, populations of a host and a typically many oocysts formed in each female and
parasite occur together, the interactions between most became encapsulated (Collins et al., 1986). In
them generate selective forces that affect the contrast, in regions of Africa where malaria was
virulence of the parasite to the host and the holoendemic, in wild-caught females of An. gambiae
refractoriness of the host to the parasite. There is a infected with P. falciparum very few parasites
trade-off between those two - the virulence and the survived the hosts' defences to develop to the
refractoriness. A host mosquito invests resources oocyst stage, and of those oocysts none or only very
into its immune mechanisms, while the parasite few were encapsulated (Hogg and Hurd, 1997;
invests resources into suppressing or evading those Taylor, 1999; Schwartz and Koella, 2002).
immune responses. Both investments have a cost, Experimental infection of An. gambiae with the
the cost to the host being measurable in the rodent parasite Plasmodium berghei (not a natural
reduction in reproductive success. combination) led to the formation of many
Koella and Boete (2003) developed a mathe- oocysts, most of which matured and produced
matical model of the coevolution of immunity and sporozoites. The roles of three recognition proteins
immune evasion based on perceived interactions (CTL4, CLTMA2 and LRRM1) were examined by
between populations of malaria parasites and their gene silencing. Knockout of the genes for CTL4 or
mosquito hosts. The model was considered appro- CTLMA2 resulted in the encapsulation of high
priate also for other parasites that are indirectly percentages of the parasites, indicating that those
transmitted by intermediate hosts. Three outcomes proteins normally protect malaria parasites against
of the interactions between parasite and host encapsulation. In contrast, knockout of the gene
24 Immune responses of mosquitoes

for LRRM1 led to a 3.6-fold increase in oocyst informatic analysis and 'manual curation' of the
numbers with no encapsulations, suggesting that immune repertoire identified 91 trios and 57
LRRM1 is normally involved in the killing of a pairs, plus a combined total of 589 paralogous
substantial proportion of invading ookinetes, genes in the three species. Probably, orthologues
possibly by lysis, before oocyst formation (Osta et serve corresponding functions whereas paralogues
al., 2004). Hemingway and Craig (2004) asserted may have acquired different functions. Plotting
that those findings were 'the clearest example yet phylogenetic distances between orthologous
of coevolution between the parasite and its insect immune genes in the Drosophila-St. aegypti pair
vector'. The investigation was continued in and in the Drosophila-An. gambiae pair revealed, on
Cameroon, where it involved colonized females of average, significantly greater divergence between
the local Yaounde strain infected by feeding on P. the orthologous immune genes of that trio of
falciparum gametocyte carriers in Mfou, 30 km species than between the totality of trios of
from Yaounde city. Knockout of the genes for orthologous genes in their collective genomes.
CTL4, CTLMA2 or LRRM1 had no significant Inter-species divergence was illustrated with two
effect on development of the parasites. Whether types of recognition receptor that belong to
the differences in outcomes between the labora- distinct structural classes - thioester-containing
tory and field investigations were due to the proteins (TEPs) and leucine-rich repeat proteins
difference in species of malaria parasite or a result (LRRs). Members of both classes are associated
of coadaptation of the genotypes of the parasite/ with the killing and disposal of parasites by lysis or
vector populations was uncertain (Cohuet et al., encapsulation (Section 42.4). AgTEP1, for
2006). example, binds to the surface of both Plasmodium
Findings from laboratory investigations into the and bacteria. Bioinformatic analysis showed that
immune responses of mosquitoes must be TEPs form one orthologous trio and two groups:
interpreted with caution. They can identify the one group includes Drosophila and mosquito
different immune processes that are available to peptides, while the other comprises only mosquito
the mosquito, and they can elucidate the mech- species-specific clades. The three LRR proteins
anisms by which those processes function, but they that recognize malaria parasites are produced by
do not reveal the relative importance of the An. gambiae but not by St. aegypti (which is not a
different immune processes in wild mosquitoes or natural host of malaria parasites) or Drosophila.
permit the possibility of trade-off between mos- From this and other evidence, Waterhouse et al.
quito and parasite populations. (2007) concluded that the acquisition of new
functions for recognition proteins, such as
recognition of malaria parasites, is through the
42.1.3 Evolutionary genetics of innate immune
modification of genes bearing powerful and
systems
ancient recognition domains.
To explore the evolutionary dynamics of innate
immunity in D. melanogaster, An. gambiae and
42.2 IMMUNE RESPONSES TO VIRUSES
Stegomyia aegypti, of which the complete genomes
had been analysed, Waterhouse et al. (2007) Infection by a virus involves penetration of cells,
undertook a meta-analysis of data concerning 31 replication and dissemination to other tissues. The
gene families and functional groups implicated in first step in cell penetration is the binding of
innate immunity or defensive functions. Bio- `attachment protein' molecules on the surface of
informatic analysis identified 4951 orthologous the virus to specific 'receptor' molecules on the
trios of genes in the three species, and 886 cell's surface. Once bound, viruses may be
`mosquito-specific' orthologous pairs of genes that internalized by a variety of mechanisms (Section
were absent from Drosophila. Combined bio- 43.1.1). The concept of non-structural barriers to
 Immune responses to viruses 25

invasion or dissemination of viruses has some They can bind specifically to some other RNAs and
support in the descriptively named `midgut either increase or decrease their activity. The RNAi
infection barrier' and `midgut escape barrier' pathway is initiated by short, double-stranded RNA
(Section 44.8.3.a), but their mechanisms are not (dsRNA) molecules in a cell's cytoplasm, which
known. interact with argonaute, a component of the 'RNA-
When the natural mammalian hosts of arbo- induced silencing complex' (RISC).
viruses become infected they either die or recover Most mosquito-borne arboviruses are ssRNA
and remain immune for life. In contrast, the (single-stranded RNA) viruses (Table 43.1).
mosquito hosts of the same viruses remain infected However, replication of the ssRNA O'nyong-nyong
for the rest of their lives. Examples in which this virus (Togaviridae, Alphavirus) in infected BHK cells
has been established include St. aegypti infected led to replicative intermediates, i.e. dsRNA forms.
with dengue virus, Cu lex univittatus infected with Following inoculation of An. gambiae with
West Nile virus (WNV) and Haemagogus O'nyong-nyong virus (ONNV) that had been
janthinomys infected with yellow fever virus (Section engineered to carry a GFP (green fluorescent
44.8.1.g). If such lifelong persistence is character- protein) marker, the virus spread to other tissues
istic of all infections of mosquitoes with an over a 9-day incubation period. When the ONNV-
arbovirus, it follows that the innate immune eGFP was co-inoculated with dsONNV, the
system of mosquitoes is incapable of eliminating ONNV-eGFP titres were significantly lower after
arboviruses. However, there is evidence that 3 and 6 days. But when ONNV-eGFP was
mosquitoes can reduce the amount of virus in co-inoculated with dsRNA from AgAgo2, a gene
their bodies. that silences RNAi, the virus titres were 16-fold
Oral infection of St. aegypti with Sindbis virus higher than the controls at 3 and 6 days. Keene et
induced the Toll pathway-related Rell tran- al. (2004) concluded that RNAi is an antagonist of
scription factor (Section 42.4.2) in midgut tissue ONNV replication in An. gambiae.
(Sanders et al., 2005). Infection of St. aegypti with Dengue virus (DENV) (Flaviviridae, Flavivirus) is
DENV-2 induced a set of genes corresponding to another ssRNA virus. Although oral infection of
the Toll pathway. Activation of Toll and Imf St. aegypti with DENV2 generated dsRNA and
pathways in St. aegypti through RNAi-mediated production of DENV2-specific siRNAs, virus repli-
silencing of Cactus and Caspar caused a reduction cation and release of infectious virus persisted,
in the extent of infection with dengue virus that suggesting viral circumvention of RNAi. However,
appeared to be controlled primarily by the Toll silencing certain genes in the RNAi pathway (dcr2,
pathway. Repression of the Toll pathway through r2d2, agog) increased virus replication in the host
MyD-gene silencing resulted in higher dengue and decreased the extrinsic incubation period
virus infection levels. Activation of the Toll required for virus transmission. Sanchez-Vargas et
pathway was supported by the up-regulation of al. (2009) concluded that RNAi is a major
Spaetzle (Spz), Toll and Re 11A, and the down- determinant of DENV transmission by St. aegypti.
regulation of the negative regulator Cactus. The RNAi functions not only in regulating gene
results suggested that the infection of mosquitoes expression but also as an innate defence
with DENV-2 induces the Toll pathway, which mechanism, protecting cells against viruses and
then exerts an anti-dengue effect (Xi et al., 2008). transposons; experimental evidence has shown
RNA interference (RNAi), once termed 'post- that the RNAi pathway is present and active in
transcriptional gene silencing', is a pathway that anopheline and culicine mosquitoes. When
enables cells to control which genes are active and initiated, it leads to the destruction of any mRNA
how active they are. Two types of small RNA that has sequence identity with the dsRNA trigger.
molecules are central to RNAi, namely microRNA After inoculation of An. gambiae with o'nyong-
(miRNA) and small interfering RNA (siRNA). nyong virus (ONNV) that had been engineered to
26 Immune responses of mosquitoes

carry a green fluorescent protein (GFP) marker, contrast, the serine proteases of St. aegypti include
the virus was seen to spread to other tissues. When many fewer CLIPAs and many more CLIPBs. In
the ONNV-eGFP was co-inoculated with dsRNA An. gambiae, activation of phenol oxidases by
from AgAgo2, a gene that silences RNAi, the virus limited proteolysis of their pro-phenol oxidase
titres were 16-fold higher than the controls at 3 zymogens is induced by a protease cascade, mostly
and 6 days. Keene et al. (2004) concluded that of CLIPBs, which is regulated, positively and
RNAi is an antagonist of ONNV replication in negatively, by a network of inactive protease
An. gambiae. Silencing certain genes in the RNAi homologues - CLIPAs, CTLs (C-type lectins) and
pathway (dcr2, r2d2, agog) increased DENV-2 SRPNs (serine proteinase inhibitors). Reverse
replication in St. aegypti, leading Sanchez-Vargas et genetic analyses identified a set of regulators of the
al. (2009) to conclude that RNAi is a major melanin synthesis associated with encapsulation of
determinant of DENV transmission by St. aegypti. P. berghei or of Sephadex beads (viz. one SPRN,
two CTLs, three CLIPAs and eight CLIPBs)
(Waterhouse et al., 2007, review).
42.3 SERINE PROTEASES
Five serine proteases were identified as haemo-
References are made in this and some other lymph proteins of An. gambiae. Four were CLIP
chapters to serine proteases that are involved in proteases, of which three (Spl4A, Spl4Dl,
immune responses or developmental processes. Spl4D2) showed changes in transcript abundance
Some proteins, including enzymes, require acti- following immune challenge, and one (Spl8D)
vation before they can function - activation involv- showed no such response. The fifth serine protease,
ing limited proteolysis of a precursor molecule by a Sp22D, was expressed constitutively in adult
serine protease. In many cases, the activity of haemocytes, fat body cells and midgut epithelial
serine proteases is regulated by serpins, a class of cells. It had a complex multi-domain structure
protease inhibitor. including a trypsin-like serine protease domain,
two putative chitin-binding domains, a mucin-like
42.3.1 Characteristics domain, and two cysteine-rich domains (Gorman et
al., 2000a,b; Gorman and Paskewitz, 2001).
Serine proteases are members of a large family of The actions of proteases are restricted to specific
extracellular endopeptidases in which the active functions by protease inhibitors, notably serpins.
centre contains a catalytically active serine residue. Serpins constitute a protein superfamily of
When these enzymes cleave peptide bonds, an proteins, many of which are serine proteinase
ester is briefly formed between the hydroxyl group inhibitors (SRPNs). Serpins bind tightly to
of the active serine and the carboxyl group of the activated proteases, blocking their activity.
cleaved peptide bond.
Some serine protease molecules contain a
42.3.2 Functions
domain of approximately 30-60 amino acids that
includes three disulphide bonds which give it a Serine proteases are known to function in the
`paperclip'-like configuration. They are termed immune responses of mosquitoes by activation of
`CLIP-domain serine proteases' (clip-SPs) and recognition proteins, activation of antimicrobial
function in developmental and immune processes. peptide synthesis and activation of certain phenol
The CLIP domain is thought to regulate the oxidases involved in the biosynthesis of melanin.
activity of the catalytic-protease domain. From An. In their various life-cycle stages malaria parasites
gambiae two classes of CLIP have been defined: also make use of serine proteases, for example in
class CLIPA, which consists of ten catalytically effecting secondary severance of surface proteins
inactive putative serine protease homologues (Volume 4, Chapter 51).
(CLIPA1-10); and class CLIPB, which consists of Shortly after septic injury of An. gambiae, the
17 catalytically active enzymes (CLIPB1-17). In recognition protein TEP1 is proteolytically cleaved
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 PLEURISY.
Cause.—Exposure to sudden changes of temperature, confinement in ill-
ventilated, damp stables, wounds penetrating the chest, fractured ribs, heart
diseases. It also occurs in conjunction with Bronchitis, Influenza, etc.
Symptoms.—Generally only one side of the lungs is affected and that being
the right, although it may affect both sides at the same time. First you will
notice the animal distressed, uneasy, shivering, the affected side is painful to
pressure of the hand. The breathing is short and quick, and the flanks heave
—which shows that the animal tries to breathe as much as possible, by the
action of the muscles of the abdomen and not by the movement of the ribs.
The nostrils are dilated. There is usually a short, dry, painful cough present,
which is repressed by the animal as much as possible, so as not to shake the
inflamed parts. Often when expelling air from the lungs the horse gives a
painful grunt especially when made to move. The pulse is generally hard and
faster than usual. The temperature in early stages may rise from 104° to 106°
F. If the ear is applied to the affected side a dry crackling or friction sound can
be heard; a groove along the lower portion of the ribs will extend back to the
flank. Within two or three days the pulse will be softer and weaker,
temperature will fall to 101° or 102° F. and there will be fluids form and the
painful short breathing will disappear. The liquids may now undergo
absorption if properly treated, and the case terminate favorably in a week or
ten days.
Frequently large quantities of fluid accumulate in the chest cavity that
cannot be absorbed, the breathing becomes more difficult, short and quick,
pulse becomes weak and rapid and the animal dies from exhaustion.
Treatment.—Place the animal in a comfortable, roomy stall; blanket if the
weather is chilly, permit fresh air, but no drafts, as this is very important.
Apply a paste made from Mustard and cold water over the chest cavity.
Internally, administer Ammonium Iodide, Chlorate of Potash, Nitrate of
Potash, each four ounces. Make into thirty-two powders and give one powder
every two or three hours in gelatin capsule and administer with capsule gun.
The diet is a proper means of keeping up the animal and is very important.
Coax the animal to eat grass or vegetables, hot bran mashes or steam rolled
oats. If there is a cough present, give the same treatment as recommended
for Acute and Chronic Coughs.
 PIN WORM, THREAD OR MAW WORM.
(Oxyuris Curvilis)
This worm when full grown is about one and three-quarter inches in length;
its tail is thin and whip-like and head thick and terminating in a curve
somewhat resembling the crook of a stick. The presence of these parasites
may be detected by a light-yellow substance (the eggs of the worms) which
adheres to the skin below the anus. Pin Worms like Round Worms frequently
come away with the feces.
Treatment.—Dissolve four tablespoonfuls Common Salt in one gallon of warm
water and inject it into the rectum. When this has been expelled, follow with
an injection per rectum of Turpentine, four ounces, to one-half gallon Linseed
Oil. Elevate the horse’s hind quarters so as to retain the injection longer. This
will expel the worms and their eggs that cling to the walls of the rectum. The
worms sometimes make their way so far forward that it is impossible to reach
them with an injection. In this case treat same as for Round worms.
Where there is irritation produced about the tail the horse continually rubs
and it is well to apply Mercurial Ointment to both tail and the anus.
 POLL EVIL.
Poll Evil is so-called because it occurs in the region of the poll. It is not a
constitutional disease, but comes, no doubt, from well marked causes, as
from inflammation set up and involving the bones and muscles in the region
of the poll, and perhaps of the larger ligament. Owing to the low vitality of the
parts and the action of the head in taking food, etc., the pus is apt to burrow
deep into the muscle.
Cause.—Direct or indirect injury. A common cause is striking the head
against a low doorway or an ill-fitting halter or bridle.
Symptoms.—Swelling just back of the ears on one or both sides of the head.
The animal stands with the nose out; slight heat in the parts, pain on
pressure. In the first stages, it is merely inflammatory action. The second
stage is suppuration, or there may be great swelling in some cases when
there is but little pus formed.
In other cases there is profuse suppuration and the pus makes its way out
and discharges to the surface and sinuses are formed, which extend in various
directions. Any abscess in this region is called Poll Evil.
Treatment.—When the enlargement is first noticed in the region of the poll, I
would advise the following: Red Iodide of Mercury, four drams; Lard, four
ounces, rub in well over the enlargement and perhaps this will prevent
sinuses from forming, but when the cases are long standing and so-called
pipes are formed, I would advise removing all diseased material and treat as
an ordinary wound.
 PETECHIAL FEVER.
(Purpura Haemorrhagica)
Cause.—Constitutional weakness following some debilitating disease such as
Distemper, Pink Eye, Catarrh and even following operations, when an animal
becomes weak from want of exercise, in which case it generally appears
during his recovery. It is not infectious and cannot be transmitted by
inoculations.
Symptoms.—There is a slight swelling of the limbs, most likely to be about the
hocks. The swelling may disappear by exercising, but will soon return. The
swellings present a very abrupt appearance, nearly the same as if a string
were tied around the limbs and swell very quickly, and symptomatic of
Purpura. Exudations take place in which, if on white limbs, you will see little
red spots, from which a liquid is oozing. The swelling is very painful and the
entire limb may be swollen. Small vesicles appear on the limbs and also in the
mucous membranes, and it is well to look at the mucous membranes before
giving your opinion, as you will, no doubt, detect these spots, which may
extend into the lungs. These spots increase and may run into each other. The
mucous membranes of the nose may become a mass of corrupt matter. The
upper lip may hang pendulous, which is due to the want of nervous stimulus.
If the nostrils are swollen very badly, there is difficulty in breathing and if the
animal is not able to take food, the symptoms are considered very bad. The
pulse varies much in some cases; although the swelling is very great, the
pulse may not be more than forty or fifty per minute. The temperature is
elevated one to three degrees above normal, there may be a cough and a
brownish colored discharge from the nostrils. The mouth and eyes become
affected and, together with the discharge from the nose, the horse is a
loathsome looking object. In milder cases the appetite is retained, or the
animal may take food one day and the next refuse it. The bowels are
constipated as a general rule in the first stages of the disease and the urine
may be of a dark color, may even contain blood. There may be a peculiar
dropsical swelling of these petechial spots or it may show itself in connection
with the eyes and there may be blood extravasation without outer symptoms.
This disease may effect the bowels, liver, lungs, etc. The animal usually
stands, perhaps from the difficulty in moving the limbs. It is necessary to
watch the case closely, for flies will attack him and he will be filled with
maggots. Sloughing may take place; the entire sheath or patches upon the
body may slough off and there may be paralysis of the penis.
Treatment.—Place the animal in a clean, light, comfortable stall. If the
weather is cold, blanket. The following medicine is recommended because of
its particular effect on the blood in this disease: Chlorate of Potash, eight
ounces; Iodide of Potash, eight ounces; Quinine Sulphate, eight ounces. Make
into thirty-two capsules and give one capsule every six hours. Also administer
one ounce capsules filled with Spirits of Turpentine three or four times a day.
Moisten the capsules with Sweet Oil and give with capsule gun. Feed hot bran
mashes containing two or three ounces of pure Flaxseed meal. Also, feed
vegetables, green grass, if possible.
 QUITTOR.
(Fistula of the Foot)
Cause.—Injuries. Horses working on rough stony roads are subject to
punctures, pricks, bruises, corns, treads, etc., which end in pus formation
which does not get a pendant opening and destroys the tissues with which it
comes in contact. Finally it bursts, forms sinuses and pipes, as commonly
called, at the top of the hoof.
Symptoms.—Extreme lameness, heat, pain and swelling will show themselves
about the top of the hoof. As a rule a Quittor develops slowly and is more or
less painful during the first stages. After the sinus is formed and the pus
discharges, the inflammation generally subsides. Its healing process is often
delayed due to the diseased portion of the cartilages inside the horny hoof.
Treatment.—Apply Flaxseed or hot Bran poultices to relieve the inflammation
and hasten the formation of sinuses or pipes. Then with an ordinary syringe
inject the following: Silver Nitrate, ten grains; Water, one ounce. Inject fifteen
to twenty drops twice daily. Keep the food clean and the animal as quiet as
possible. It is very disagreeable, as stated before, and the healing is very
slow, but this must be naturally expected, as we are unable to provide the
sinuses with good drainage.
 RED WORM.
(Strongylus Tetracanthus)
The Red Worm varies in length from one-third to three and one-quarter
inches, and is sometimes white, though it usually appears to be red because
of the blood it contains. This parasite is found in all parts of the world. Its
favorite haunt is marshy land.
Symptoms.—Paleness of all visible membranes, eyes watery and inflamed,
swelling of the sheath, legs, and lower surface of the belly; fetid diarrhoea,
dullness, debility, emaciation, rough coat, and the presence of worms in the
feces. The worms when first passed are bright red in color but after being
exposed to the air they turn dark and may easily escape the notice of the
casual observer.
Treatment.—Withhold all food for twenty-four hours, then place the following
drugs in a gelatin capsule: Calomel, two drams, Barbadoes Aloes, three
drams; Ferri Sulphate, two drams. Give with capsule gun. Also place the
following tonic in their feed: Pulv. Quassia, one ounce; Ferri Sulphate, two
ounces; Pulv. Anise Seed, two ounces. Mix and make sixteen powders. Give
one powder two or three times a day in the feed.
 RHEUMATISM.
Cause.—Exposure to cold rains, drafts, lying on damp ground when the
blood is in poor condition. Also due to over-stimulating food.
Symptoms.—Lameness, swelling or soreness which may shift from one place
to another, then finally locate in or near one of the joints of the limbs.
Treatment.—Take away all grains and feed laxative foods such as potatoes,
carrots, apples, kale and good hay. If the weather is warm turn out to
pasture, but confine in warm stable at night. It is advisable to give a physic,
as Aloin, two drams; Gentian, one dram; Ginger, one dram. Place in gelatin
capsule and give at one dose with capsule gun, as its action on the blood has
a very good effect. When the swellings are painful, apply Camphorated
Liniment once or twice daily. Also, administer the following tonic: Potassi
Iodide, one ounce; Nitrate of Potash, two ounces; Chlorate of Potash, two
ounces; Pulv. Gentian Root, one ounce; Ferri Sulphate, one ounce; Pulv. Anise
Seed, four ounces. Mix well and make into twenty powders. Give one powder
three times a day in bran or place in capsule and give with capsule gun.
 RING BONE.
Cause.—Faulty conformation—a narrow or straight pastern joint is
considered faulty. Be very careful in selecting a sire when breeding, as faulty
conformation is hereditary. Ringbone may also result when young animals are
put to work on hard roads, or running in stony pastures sometimes produces
Ringbone before the bones have become properly hardened. Other causes are
injury to tendons or ligaments, bruised joints, blows, calking, or picking up a
nail.
Symptoms.—Lameness will manifest itself when the horse first starts out in
the morning; this may become less noticeable or even disappear temporarily
as the animal works. They gradually grow lamer and examination will disclose
an enlargement at or around the top of the hoof. This may appear in one or
more feet, but the front feet are more often affected.
Treatment.—If the Ringbone is very much inflamed, reduce the heat by
applying cold water or ice packs to the part. Clip off all hair from around the
top of the hoof and rub in well for twenty minutes the following: Red Iodide of
Mercury, two drams; Pulv. Cantharides, two drams; Turpentine, one dram;
Pine Tar, two drams, and mix in two ounces of Lard. Apply the ointment every
two days for a week and repeat same treatment in two weeks. Keeping the
animal as quiet as possible will hasten its recovery. If the animal is
comparatively young, recovery is certain, although the enlargement may
never disappear.
 ROUND WORM.
(Ascaris Megalcephala)
Resembles the Earth Worm somewhat in shape, yellowish-white in color,
stiff and elastic. When full grown, it varies in length from six to sixteen inches.
These worms are usually found in the small intestines, although they
sometimes invade the stomach, and when numerous seriously disturb the
animal’s health.
Symptoms.—The animal’s general health is affected as is evident from the
morbid state of his appetite, rough coat, pot-belly, liability to colic and slight
diarrhoea. Some of these worms are often expelled with the feces. As they
increase in number, they block up the small intestines, giving rise to colic, and
may in time kill the horse. They sometimes cause perforation of the bowels.
Treatment.—Withhold all food from eighteen to twenty-four hours, then
administer the following: Ferri Sulphate, two drams; Antimony Tartrate, two
drams; Pulv. Quassia, two drams. Place in gelatin capsule and give with
capsule gun. Follow this from six to eight hours with Aloin, two drams; Ginger,
two drams, and give as above directed. It is a good plan to repeat the above
treatment in ten days to insure the removal of any worms which may have
survived the first treatment.
 SCROTAL RUPTURE.
(Inguinal Hernia)
Cause.—Abnormal size of the upper ring through which a part of the
intestines or its connecting membrane descends into and through the canal
leading from the abdomen to the scrotal cavity. There is little danger of
strangulation from this form of rupture which may occur at birth and
disappear with age. A careful examination should therefore be made of the
scrotum before castration.
Symptoms.—In most cases, this condition is easily detected. The scrotum will
be somewhat enlarged. Sometimes the intestines will become strangulated
and colicky symptoms appear. When a young male colt shows signs of colic,
examine him for Scrotal Rupture.
Treatment.—The trouble usually disappears with age although in some cases
it is well to operate. Where colicky symptoms are present, roll the colt on its
back, manipulating the scrotum. Diet carefully.
 SHOE BOIL.
Cause.—Injuries, bruises or pressure when lying on a rough floor. Sharp
heeled shoes and kicks also have a tendency to produce it.
Symptoms.—A hot painful swelling of the Elbow joint when first noticed.
When neglected, it takes on a white fibrous or callous growth.
Treatment.—First remove the cause. Do not lance the enlargement; let it
come to a head of its own accord, by applying Red Iodide of Mercury, two
drams; Pulv. Cantharides, three drams; Lard, two ounces. Mix well together
and apply twice a week. When the swelling is hot and painful it is well to
apply cold water or ice packs before applying the above mentioned
prescription.
 SPLINTS.
Cause.—This disease is chiefly produced by trotting or running on hard
ground, etc. It is evident that horses with high knee action and heavy bodies
are more liable to this disease. Jumping is also a common cause of splints,
but the more accustomed a horse is to jumping the less liable he is to throw
splints, because practice teaches the animal to regulate his movements so as
to more or less diminish the disagreeable if not actually painful effect of
concussion.
Symptoms.—A splint is detected by grasping the horse’s leg with the fingers
upon one side and the thumb upon the other, and tracing the inner and outer
splint bones from their heads downward to their tapering extremities. Any
actual enlargement will at once arrest the hand; any rising or irregularity will
create suspicion and lead to close examination. Horses, especially young ones
which have lately been put to work, not infrequently develop splints before
any swelling appears. For this reason, in examining a case of obscure
lameness, particularly if the animal is young, do not fail to look for the sign of
splint lameness, namely; that the lameness is abnormally greater at a trot
than at a walk and that the animal usually fails to bend the knees freely and
grows worse with exercise. The last mentioned condition is also present with
corns, but an examination of the foot will determine the question of their
existence. In young horses splints are sometimes mistaken for coffin-joint
lameness or navicular disease. To avoid this error, it should be remembered
that, when brought on by navicular disease, the action of the limb improves
with exercise; also that horses of five years of age or less very rarely suffer
from coffin-joint disease. Some horses, owing to unusual development of the
inner splint bones of the fore legs may appear to have splints, although
careful examination may prove both limbs to be free from any bony deposit.
When deciding such a point, note if the two inner splint bones are of the
same size. Any swelling perceptible in a limb recently affected with splint-
lameness is usually attended by heat and pain.
Treatment.—If there is heat present, foment with hot or cold water; when
heat has subsided, apply the following ointment: Red Iodide of Mercury, two
drams; Turpentine, twenty drops; Lard two ounces, and mix. Apply every
forty-eight hours until three applications have been applied. Rub in for twenty
minutes each time.
 During this treatment use the horse for slow work on soft roads, etc. As a
rule the splints will not disappear at once, but gradually. I may add that
common splints are not considered an unsoundness.
 SPASMODIC COLIC.
Cause.—Horses seem to be predisposed to this form of colic on account of
the great length of their intestines which are apt to be telescoped, twisted or
their circular muscular fibers spasmodically contracted. Perhaps the principal
cause is a change of food, sudden change of temperature, constipation,
drinking cold or too large a quantity of water, especially if the animal is warm;
overloading the stomach with frozen or mouldy food. Worms frequently
produce colic.
Symptoms.—If the animal is tied it will become uneasy, paw, point its nose to
the flank, twitch the tail, lie down and get up frequently. If the animal is loose
it will walk around, paw, kick at its belly with the hind feet, make attempts to
lie down, roll on its back and remain in that position for a while. The pulse
increases with the pain, temperature rises from one-half to one degree,
breathing labored and fast, the animal sweats in spots, there may be
diarrhoea present, but this does not frequently occur.
Unfavorable symptoms of spasmodic colic are cold legs to the feet, points of
the ears cold, trembling of the muscles, cold sweats, mucous membranes of
the nose, mouth and eyes have a dark color due to the congestion.
Treatment.—In all cases of spasmodic colic, except where there is diarrhoea
present or mares heavily in foal, give Aloin, two drams; Ginger, two drams.
Place in gelatine capsule and give with capsule gun. It is advisable to give
rectal injection of Warm Water and Glycerine. They are soothing and cooling
to the intestinal canal. Also give the following prescription: Pulv. Nux Vomica,
four ounces; Carbonate of Ammonia, four ounces; Asafoetida, four ounces.
Make into six powders; place one powder in gelatin capsule and give with
capsule gun every two hours until relieved. The former prescription removes
the cause as it is a physic. The latter contains medicines blended so as to
counteract the spasmodic contractions of the bowels. It is also a heart
stimulant, just what is needed in colic to keep up the animal’s vitality. Beware
of colic remedies that are given in drop doses. They contain drugs to relieve
the pain only and do not remove the cause. When their effects are worn off,
the disease has progressed; the animal’s heart action has been weakened and
chances are that the animal will die. If drenching is resorted to, it must be
done with great precaution. Remember a horse cannot breathe through its
mouth.
 SIDE BONES.
Cause.—The chief causes of Side Bones are: Deprivation of frog pressure,
injuries, high heeled shoes, the use of which is almost entirely confined to
draft horses. A high heeled shoe prevents the frog from resting on the ground
which is its natural support.
Symptoms.—Enlargement just above the hoof, usually affecting the front feet,
or may affect only one side of one of the feet. The pain which produces the
lameness is due to pressure on the soft tissues between the newly formed
side bone and the hoof. Sometimes the enlargement has a tendency to spread
the hoof. In such a case the lameness is not so severe.
Treatment.—Clip the hair from over the Side Bone and rasp the foot below
the enlargement, so that the hoof will be flexible on pressure from the fingers.
Then apply the following to both the enlargement and the rasped surface of
the hoof: Red Iodide of Mercury, two drams; Pulv. Cantharides, four drams.
Mix well in two ounces of Lard and apply every forty-eight hours until three
applications have been applied.
If you must work the animal, put it to some easy work where it has soft
ground to walk upon.
 STAGGERS.
(Forage Poisoning—Inflammation of the Brain)
(Cerebral Meningitis)
Cause.—Certain plants or stagnant water are most commonly instrumental in
producing staggers; frequently seen in the early autumn months when the
grass in the pastures becomes dry and certain forage remains green which
contains toxic principles. These plants are ravenously eaten by horses on
account of being green and tender. This is one of the common causes of the
disease, although moldy, indigestible or highly nitrogenous foods are
frequently productive of staggers. This form of staggers is not contagious,
although what produces staggers in one horse will also produce it in another.
In this way several horses may become affected with staggers at the same
time. Inflammation of the brain may occur as a complication of some
infectious or digestive disease. Other causes are blows on the head, tumors in
or on the brain, which naturally cause the animal to stagger, as the brain
controls the horse’s power of locomotion.
Symptoms.—These vary to a certain extent, but a careful observer will detect
some trouble connected with the nervous system, as the animal walking
unsteadily, stepping high and keeping the legs spread apart, bracing itself to
keep from falling. There is also great depression, dullness and sleepiness with
little or no inclination to move about. The head may be placed against a wall
or fence and the legs kept moving as if the horse were trying to walk. As the
disease progresses and no attempts are made to relieve it, they will become
fractious, nervous, easily excited, pawing and eventually fall, keeping the feet
moving as if walking, throwing their heads about in a delirious manner and
eventually death follows. The horse as a rule eats and drinks ravenously when
the first signs of staggers are noticed, but in its latter stages the tongue and
gullet become paralyzed and although the animal attempts to eat and drink
he cannot swallow. The pulse varies. It is strong, but subnormal when the
first symptoms of staggers are noticed, that is to say, it is as slow as twenty
to twenty-five beats per minute. As the disease progresses, however, it
becomes weaker and faster. Constipation frequently accompanies this disease,
also paleness tinged with yellow about the mucous membranes of the mouth
and eyes. In many instances I believe that the poisonous forage eaten by
horses depresses the heart action to such an extent that it results in the brain
not receiving the proper blood supply, causing dizziness or staggers.
Treatment.—Place the animal in a clean, dark stall, keeping the surroundings
as quiet as possible. In its first stages it is easily treated, but as the horse
becomes easily excited and his swallowing becomes difficult, treatment
becomes more difficult. When the first signs are noticed, administer a physic
as: Aloin, two or three drams; Ginger, two or three drams, according to the
size of the animal. Place in gelatin capsule and give with capsule gun. This
physic removes the irritant from the intestines and prevents its absorption into
the blood. Also administer the following: Bromide of Potassium, twelve
ounces; Nitrate of Potash, four ounces; Iodide of Potash, three ounces. Make
into twenty-four capsules and give one capsule every four hours.
My method of administering medicine to animals places me in a position to
treat them and compel them to take the medicine even though paralysis may
exist. When animals will eat, feed food that is easily digested, as hot wheat
bran mashes, steamed rolled oats and vegetables and give small quantities,
but not often, of clean fresh water. It is necessary to give stimulants and
tonics as soon as they are on the road to recovery, as Pulv. Nux Vomica, four
ounces; Pulv. Gentian Root, four ounces; Sulphate of Iron, two ounces. Make
into sixteen capsules and give one capsule three times daily.
 STIFLE JOINT LAMENESS.
(Dislocation of the Patella)
Although dislocations are infrequent, this is the most common form which
occurs in the horse.
Cause.—Young loose jointed horses are predisposed to dislocation of the
stifle on account of the comparative want of strength of their ligaments. They
are much more liable to this accident than older horses, especially if they are
in poor health or in rough hilly pastures; the nature of which would naturally
make them susceptible to this injury, which, however, may take place as a
result of accident at any age. Young horses that suffer, off and on from
dislocation, often lose their liability with increasing strength and age. This
dislocation may be partial or complete. In the former instance and the most
common is where the patella, or the little stifle bone that glides in the groove
composed of the lower hip and upper thigh bones, has become partially
dislocated or removed from its natural position.
Symptoms.—When the dislocation is complete the affected limb is drawn
forward, while the foot from the pastern down is drawn backward, and the
animal may throw weight on it when made to move, which is accomplished
with great difficulty. When the dislocation is partial, the symptoms are about
the same as mentioned, only the limb is less rigid. If the horse is moved, the
stifle makes a clicking sound. In this form both limbs may be affected.
Treatment.—In partial dislocation, the stifle bone may be replaced by
drawing the leg forward, and with the hand pressing in on the stifle. In
complete dislocation, tie a rope around the pastern of the affected leg, then
draw the rope through a collar placed around the horse’s neck and draw
forward as far as possible and tie. Then press with both hands inward. After
the stifle is placed back into position use the following liniment: Aqua
Ammonia Fort., four ounces; Oil of Turpentine, four ounces; Raw Linseed Oil,
four ounces. Mix and apply well over the stifle joint once or twice a day for
two or three days. Feed nourishing food and put the animal to slow, easy
work or turn out to good pasture.
In chronic cases of Stifle Joint Lameness, treatment is of no value,
therefore, care for the animal as soon as the catch in the walk or lameness
appears.
 STRING-HALT.
Cause.—Several theories have been put forth as to the cause of String-Halt
which is generally supposed to be a nervous disease; a condition opposite to
paralysis. The exact cause of this disease is hard to determine, but it is likely
to occur in highly nervous horses. It sometimes follows an injury which may
have irritated the nerves in some way. I believe that castration causes it in
many instances, due to the severe struggle when being thrown, or pulling
down severely on the spermatic cord when removing the testicle.
Symptoms.—Spasmodic contraction of one or both limbs. This sign varies, as
sometimes it is very violent, while in others it may be so slight that it is hard
to detect when stepping the horse forward, but on backing or turning the
horse around the signs are easily noticed. All symptoms are better marked in
the winter than in the summer, as some show it in the winter that do not
show it in the summer at all.
Treatment.—The feeding of laxative foods that are easily digested relieves
String-Halt in many instances.
 SORE THROAT.
(Pharyngitis—Laryngitis)
Cause.—Exposure to cold weather or rain when the animal is not
accustomed to it; drenching with irritating medicines or inhaling irritating
smoke or gases.
Symptoms.—At first the animal generally chills, the legs and ears are cold, but
eventually they become very warm as the temperature increases, coughing,
grinding of the teeth, saliva oozing from the mouth; the animal will hold its
head in a stiff straight position, moving it as little as possible. There will be
great difficulty in masticating and swallowing, as the food will come from the
mouth in the form of wads, and as this soreness of the throat progresses food
will also come from the nostrils. This is a bad sign, as extensive inflammation
is no doubt present. Water, also, runs through the nostrils freely when the
animal attempts to drink, due to the swollen condition of the throat. The
animal forces the water back into the mouth, but is unable to swallow and
hence the water gushes out through the nostrils. The animal evinces great
pain when pressure is applied from the outside and he breathes with great
difficulty. Although the pulse is not much affected at this stage, the
temperature is elevated from one to two degrees above normal. The urine
becomes scanty and highly colored, the eyes congested and discharging.
Eventually the throat becomes greatly swollen and abscesses may form and
discharge. As a rule constipation is associated with this disease.
Treatment.—Mild attacks of sore throat are easily treated, but when serious
cases develop, treatment is unsuccessful. Place the animal in a clean,
comfortable stall; permit as much fresh air as possible, but avoid all drafts. If
the weather is chilly, blanket the animal, hand rub the legs and bandage with
woolen cloths or bandages. Administer a mixture made from Chlorate of
Potash, three ounces; Nitrate of Potash, three ounces; Tannic Acid, one-half
ounce; Molasses, one-half pint; Pine Tar, one-half pint. Mix well and place
about one tablespoonful on the tongue every two hours in severe cases; in
mild attacks, give less frequently. When they will eat, feed food that is easily
digested, as hot wheat bran mashes and steamed rolled oats containing two
or three ounces of pure ground flaxseed. It is always necessary to apply
strong liniments to the throat, as they relieve inflammation and stimulate the
formation of an abscess. The following liniment will be found very beneficial:
Aqua Ammonia Fort., four ounces; Oil of Turpentine, four ounces; Sweet Oil,
six ounces; shake well and apply two or three times daily. If the swelling is
extreme between the jaws, so as to interfere with the animal’s breathing, it is
well to lance the abscess if a soft spot can be found. Just cut through the skin
with a knife; then use a clean blunt instrument to locate the pus cavity.
Otherwise, severe hemorrhage may be produced.
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