The chemotactic response of murine peritoneal exudate cells to Trypanosoma brucei

A modified Boyden technique was used to assess the chemotactic activity of unstimulated mouse peritoneal exudate cells in the presence of Trypanosoma brucei. Live parasites, the soluble fraction obtained from disrupted trypanosomes, and specific mouse hyperimmune T. brucei sera were unable to generate chemotactic activity. Complexes of whole parasites and hyperimmune sera gave similar control values, whereas significant activity was obtained using a mixture of live trypanosomes and untreated mouse plasma. Immune complexes prepared by pre-incubation of soluble trypanosome fractions with either untreated or heat inactivated (56°C for 30 min) hyperimmune sera, or with mouse plasma, also resulted in enhanced chemotactic activity.     

Tsetse flies as vectors of trypanosomes

Glossina spp. can be naturally infected with trypanosomes belonging to the subgenera Duttonella, Nannomonas and Trypanozoon; rates of infection vary but are, in general, highest with those trypanosomes which have the simplest cycle of development in the insect and lowest in those with the most complicated cycle of development. Differences in rates of infection have mainly been accounted for in terms of such factors as the maintenance temperature of puparia and adults, the age of the fly at the time of the infective feed and, perhaps most important, the type of host animals on which the flies feed. Differences in infectibility occur between species of Glossina and may occur between different individuals of a single species. The nature of the mechanism involved is unknown; there is no evidence that trypanosomes have any pathogenic effect on Glossina. Cyclical transmission of a strain of Trypanosoma brucei through Glossina seems to have little effect on the antigenic characters of the strain, the tsetse fly acting only as a carrier of the strain which either remains unaltered throughout the period required for cyclical development and for the rest of the life of the fly or reverts to a so-called basic antigenic type.     

The isolation ofTrypanosoma brucei fromHippopotamus amphibius in the luangwa valley,Zambia

Summary Trypanosoma brucei was isolated from four out of75 hippopotami by means of intraperitoneal mouse inoculations. This appears to be the first isolation of trypanosomes fromHippopotamus amphibius and a new host record forT. brucei. The four strains of polymorphic trypanosomes isolated were identified asT. brucei by the blood incubation infectivity test. In500 wet and390 dry blood films (total890) of370 hippopotami examined no trypanosome was found. The value of blood film examination for the diagnosis of trypanosome infections in hippopotami is shown to be limited.

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Sumario ElTrypanosoma brucei fue aislado en cuatro de 75 hipopotamos por medio de la inoculación intraperitoneal en ratones. Este parece ser el primer aislamiento de trypanosomas deHippopotamus amphibius y un nuevo hospedero para el record delT. brucei. Las cuatro cepas de trypanosomas polimórficos aisladas fuereon identificadas comoT. brucei por la prueba de infectividad de incubación de sangre (PIIS). En 500 frotices mojados de sangre y en 390 frotices secos (890 en total) de 370 hipopotamos examinados no se encontraron trypanosomas. El valor del examen del frotiz de sangre para el diagnóstico de las infecciones por trypanosomas en hipopotamos es demostrado ser limitado.

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Résumé Trypanosoma brucei a été isolé de quatre hippopotames sur 75, par inoculation à la souris par voie intrapéritonéale. Il semble que ce soit la première fois que des trypanosomes ont été isolés d’Hippopotamus amphibius et qu’ils s’agisse d’un nouvel h?te pourT. brucei. Les quatre souches de trypanosomes polymorphes isolés ont été identifiées commeT. brucei par le test d’infectiosité du sang incubé (BIIT). Aucun trypanosome n’a été trouvé dans 890 préparations de sang (500 de sang frais et 390 frottis) de 370 hippopotames. Il a été démontré que la valeur de l’examen de frottis de sang pour le diagnostic d’infections à trypanosomes chez l’hippopotame est limitée.

     

Trypanosomes

Most immunological studies on trypanosomes have been made on species in the subgenus Trypanozoon which includes those causing sleeping sickness in man, but members of the subgenera Duttonella and Nannomonas are more important as the cause of disease in cattle. Wherever possible, studies on these subgenera in cattle are discussed in this paper. The antigenic structure of trypanosomes is complex and common antigens between species, strains and populations occur in homogenates. Antigens variation is responsible for trypanosome surface alter during infection and this antigenic variation is responsible for many of the immunological problems of trypanosmiasis. The characteristics humoral response to infection is a rapid and sustained rise in IgM followed by a later increase in IgG but little is known about cell-mediated immunity in trypanosomiasis. The altered immunoligical state of the infected animal may be of significance in the pathogenesis of the disease in which anemia is marked and kinin production occurs. Possible methods of stimulating protective immunity are discussed and the suppression of immune response to other antigens is analysed.     

Applications of PCR-based tools for detection and identification of animal trypanosomes: a review and perspectives

This paper aims to review the applications of the polymerase chain reaction (PCR) for the detection and identification of trypanosomes in animals. The diagnosis of trypanosomes, initially based on microscopic observations and the host range of the parasites, has been improved, since the 1980s, by DNA-based identification. These diagnostic techniques evolved successively through DNA probing, PCR associated to DNA probing, and currently to PCR alone. Several DNA sequences have been investigated as possible targets for diagnosis, especially multi-copy genes such as mini-exon, kinetoplastid mini-circles, etc., but the most favoured target is the nuclear satellite DNA of mini-chromosomes, which presents the advantages, and the drawbacks, of highly repetitive short sequences (120-600 bp). Several levels of specificity have been achieved from sub-genus to species, sub-species and even types. Random priming of trypanosome DNA has even allowed "isolate specific" identification. Other work based on microsatellite sequences has provided markers for population genetic studies. For regular diagnosis, the sensitivity of PCR has increased with the advancement of technologies for sample preparation, to reach a level of 1 trypanosome/ml of blood, which has brought to field samples a sensitivity two to three times higher than microscopic observation of the buffy coat. Similarly, PCR has allowed an increase in the specificity and sensitivity of diagnosis in vectors such as tsetse flies. However, because of the diversity of Trypanosoma species potentially present in a single host, PCR diagnosis carried out on host material requires several PCR reactions; for example, in cattle, up to five reactions per sample may be required. Research is now focusing on a diagnosis based on the amplification of the internal transcribed spacer-1 (ITS-1) of ribosomal DNA which presents the advantages of being a multi-copy locus (100-200), having a small size (300-800 bp), which varies from one taxon to another but is conserved in size in a given taxon. This may lead to the development of a multi-species-specific diagnostic protocol using a single PCR. By reducing the cost of the PCR diagnosis, this technique would allow a greater number of field samples to be tested in epidemiological studies and/or would increase the variety of Trypanosoma species that could be detected. Further investigations are required to develop and optimise multi-species-specific diagnostic tools for trypanosomes, which could also serve as a model for such tools in other pathogens.     

Validation of the usefulness of 26S rDNA D1/D2, internal transcribed spacer, and intergenic spacer 1 for molecular epidemiological analysis of Macrorhabdus ornithogaster

Macrorhabdus ornithogaster (MO) is an infectious fungus that causes gastric damage in birds. In this study, we established nested and seminested polymerase chain reaction (PCR) methods that specifically amplify the domain D1/D2 region (D1/D2) of 26S ribosomal DNA (rDNA), internal transcribed spacer (ITS) of rDNA, and intergenic spacer (IGS) 1 region from avian feces. Phylogenetic analysis of MO collected from Japanese pet birds showed little genetic variation; analysis based on these regions did not distinguish between host species order, differences in MO shape, or host gastrointestinal symptoms. These regions were found to be unsuitable for molecular epidemiological studies of MO and further investigation into other genetic regions is required.     

Aggregate diversity: New approach combining within- and between-breed genetic diversity

Between-breed genetic diversity is classically considered as a major criterion to be taken into account when setting priorities for conservation of domestic animal breeds. However, it has been argued that methods based on the between-breed component of genetic diversity may not be optimal because they ignore the within-breed component of variation. The paper considers the most common methods used to evaluate those two components when genetic diversity is evaluated on the basis of genetic markers, and proposes to define an aggregate diversity combining linearly the two components. This implies defining for each breed (or population) its contributions to the between-breed and to the within-breed diversity. When defining an aggregate diversity, one can weight these contributions by F_ST and 1−F_ST, respectively, since the fixation index F_ST of Wright represents the proportion of the total genetic variation which is due to differences in allelic frequencies between populations. Such an approach is valid when the objective is genetic improvement by selection within a so-called “meta-population”. However, in a more general context of animal breeding, when heterosis and complementarities between breeds have to be considered, as well as adaptation to specific environments, more weight should be given to the between-breed variation. The proper weight to apply may require solutions adapted to each particular situation. In a long-term conservation perspective, priorities should also take into account the degree of endangerment of each breed. By combining diversity contributions and probability of extinction, a cryopreservation potential (or priority) may be estimated for each breed. The problem is illustrated on a sample of 11 European pig breeds typed for 18 microsatellite loci.     

Molecular characterization of breeds and its use in conservation

The conservation of farm animal resources is important for coping with future breeding needs and for facilitating the sustainable use of marginal areas. The increasing availability of molecular markers for most farm animal species and the development of techniques to analyse molecular variation is widening our capacity to characterise the genetic variation of breeds. In this paper we review the most popular molecular markers used in conservation and animal breeding studies, the different measures of genetic diversity that they provide, and their application for managing within-breed genetic diversity and for setting between-breed conservation priorities. We also address the relationship between genomic and marker heterozygosity, the relationship between molecular and quantitative measures of genetic diversity, and the characterization of breeds based on non-neutral markers.     

Recent breeding history of dog breeds in Sweden: modest rates of inbreeding,extensive loss of genetic diversity and lack of correlation between inbreeding and health

One problem in modern dogs is a high occurrence of physical diseases, defects and disorders. Many breeds exhibit physical problems that affect individual dogs throughout life. A potential cause of these problems is inbreeding that is known to reduce the viability of individuals. We investigated the possible correlation between recent inbreeding and health problems in dogs and used studbook data from 26 breeds provided by the Swedish Kennel Club for this purpose. The pedigrees date back to the mid‐20th century and comprise 5–10 generations and 1 000–50 000 individuals per pedigree over our study period of 1980–2010. We compared levels of inbreeding and loss of genetic variation measured in relation to the number of founding animals during this period in the investigated dog breeds that we classified as ‘healthy’ (11 breeds) or ‘unhealthy’ (15) based on statistics on the extent of veterinary care obtained from Sweden's four largest insurance companies for pets. We found extensive loss of genetic variation and moderate levels of recent inbreeding in all breeds examined, but no strong indication of a difference in these parameters between healthy versus unhealthy breeds over this period. Thus, recent breeding history with respect to rate of inbreeding does not appear to be a main cause of poor health in the investigated dog breeds in Sweden. We identified both strengths and weaknesses of the dog pedigree data important to consider in future work of monitoring and conserving genetic diversity of dog breeds.     

Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife

Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.     

Signatures of functional constraint at Fgfr1a Genes in schizothoracine fishes (Pisces: Cypriniformes): The dermal skeleton variation adapted to high‐altitude environments

Fishes harbor a huge resource of phenotypic diversity and are useful for understanding the genetic basis of morphological variation. However, it is difficult to transfer classical genetic mapping to most non‐model species. In this study, we performed a comparative sequence analysis of Fgfr1a to first interpret the evolution of this candidate scale‐loss gene in 15 schizothoracine fishes with various scale phenotypes. While considerable amino acid (AA) substitutions were observed, molecular evolution analysis indicates that the overall coding regions were subject to functional constraint. We also identified extra copies of Fgfr1a in 4 scale‐loss fishes and detected accelerated evolution in one AA substitution specific to these duplicates. We speculate that Fgfr1a had accumulated mutations in the ancestral lineage of scale‐loss schizothoracine fishes before experiencing duplication events, which was further followed by the diversification of species. In silico mutation analysis predicted deleterious effects of the mutations while no disruptive molecular mechanism was detected. Collectively, our results highlight the important role of Fgfr1a gene in the adaptive evolution of schizothoracine fishes during their radiation in the Qinghai‐Tibetan Plateau.     

The infection ofGlossina morsitans weid byTrypanosoma brucei in relation to the parasitaemia in the mouse host

Summary G. morsitans were fed upon white mice infected withT. brucei TREU667, within 24 hours of emergence from the puparium. Infection rates in the flies averaged 5.8 per cent mature infections with a maximum of 15.4 per cent in one experiment. Irradiation of mice with 400r X-rays 24 hours before infection was found to slightly enhance the infection rate in flies. Flies are more readily infected when the blood-meal contains 50–20,000 trypanosomes per mm³ and 10–20,000 short stumpy forms per mm³. With the strain ofT. brucei used, suitable conditions in the mouse occur subsequent to 12 days after infection.

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Sumario MoscasG. morsitans fueron alimentadas sobre ratones blancos infectados conT. brucei TREU 667, dentro de las 24 horas de su emergencia del puparium. Las tasas de infección en las moscas promediaron 5.8 por ciento de infecciones maduras con un maximo de 15.4 por ciento en un experimento. La irradiación de ratones con 400r de rayos X por 24 horas antes de la infección acrecentó ligeramente la tasa de infección en las moscas. Las moscas son mas facilmente infectables cuando la sangre de los ratones contiene de 50–20,000 tripanosomas por mm³ y 10–20,000 formas cortas de mu?ón por mm³. Con la cepa deT. brucei usada, las condiciones adecuadas en el ratón occurren subsecuente a los 12 dias despues de la infección.

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Résumé DesG. morsitans écloses depuis moins de 24 heures ont été nourries sur souris blanche infectée avecTrypanosoma brucei. Les taux d'infection chez les mouches ont atteint une moyenne de 5,8 p. 100 de formes infectantes avec un maximum de 15,4 p. 100 dans une expérience. L'irradiation des souris aux rayons X à la dose de 400 r, 24 heures avant l'infection, a augmenté légèrement le taux d'infection chez les mouches. Les mouches sont plus aisément infectées quand le repas de sang contient de 50 à 20,000 trypanosomes par mm³ et 10 à 20,000 formes courtes et trapues par mm³. Avec la souche deT. brucei utilisée, ces taux d'infection favorables apparaissent chez la souris 12 jours après l'infection.

     

群内遗传多样性在畜禽保种中的应用

畜禽遗传资源的保存对应对未来的育种需要和畜牧业的可持续利用是非常重要的。分子标记广泛应用于大多数畜禽品种及分子变异分析技术的发展,促进了分析品种遗传变异的能力。作者对用于度量畜禽遗传资源的群内遗传多样性评价方法及其在确定种内遗传多样性,以及保种方案中的应用进行了综述。     

Analysis of host genetic factors influencing African trypanosome species infection in a cohort of Tanzanian Bos indicus cattle

Trypanosomosis caused by infection with protozoan parasites of the genus Trypanosoma is a major health constraint to cattle production in many African countries. One hundred and seventy one Bos indicus cattle from traditional pastoral Maasai (87) and more intensively managed Boran (84) animals in Tanzania were screened by PCR for the presence of African animal trypanosomes (Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei), using blood samples archived on FTA cards. All cattle screened for trypanosomes were also genotyped at the highly polymorphic major histocompatibility complex (MHC) class II DRB3 locus to investigate possible associations between host MHC and trypanosome infection. Overall, 23.4% of the 171 cattle tested positive for at least one of the three trypanosome species. The prevalence of individual trypanosome species was 8.8% (T. congolense), 4.7% (T. vivax) and 15.8% (T. brucei). The high prevalence of T. brucei compared with T. congolense and T. vivax was unexpected as this species has previously been considered to be of lesser importance in terms of African bovine trypanosomosis. Significantly higher numbers of Maasai cattle were infected with T. brucei (23.0%, p=0.009) and T. congolense (13.8%, p=0.019) compared with Boran cattle (8.3% and 3.6%, respectively). Analysis of BoLA-DRB3 diversity in this cohort identified extensive allelic diversity. Thirty-three BoLA-DRB3 PCR-RFLP defined alleles were identified. One allele (DRB3*15) was significantly associated with an increased risk (odds ratio, OR=2.71, p=0.034) of T. brucei infection and three alleles (DRB3*35, *16 and *23) were associated with increased risk of T. congolense infection. While further work is required to dissect the role of these alleles in susceptibility to T. brucei and T. congolense infections, this study demonstrates the utility of FTA archived blood samples in combined molecular analyses of both host and pathogen.     

Identification of a novel filovirus in a common lancehead (Bothrops atrox (Linnaeus, 1758))

I performed metaviromic analysis of publicly available RNA-seq data from reptiles to understand the diversity of filoviruses (family Filoviridae). I identified a coding-complete sequence of a filovirus from the common lancehead (Bothrops atrox (Linnaeus, 1758)), tentatively named Tapajós virus (TAPV). Although the genome organization of TAPV is similar to mammalian filoviruses, our phylogenetic analysis showed that TAPV forms a cluster with a fish filovirus. However, TAPV is still distantly related to all the known filoviruses, suggesting that TAPV can be assigned as a species of a novel genus in Filoviridae. To our knowledge, this is the first report identifying a filovirus in reptiles, and thus contributes to a deeper understanding of the diversity and evolution of filoviruses.     

Mining the genetic diversity of Ehrlichia ruminantium using map genes family

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host.In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster.We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands).These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host–vector–pathogen interactions that could be crucial for developing broad-spectrum vaccines.     

Test for personality characteristics in dogs used in research

The great variation in morphological phenotypes displayed by dogs offers not only excellent opportunities for genetic analyses but also a challenge regarding between-breed and even within-breed variation. Also, behavioral responses may vary between individuals, and are to be taken into account in experimental situations. To our knowledge, no standardized test for scoring personality characteristics (TFPC) in dogs maintained for research under controlled conditions has yet been developed. The present article describes a protocol consisting of 9 test situations that are likely to arise in experimental contexts. The intent was to establish an easy-to-use standardized test protocol. Sixteen beagles were used, all housed in constant and controlled conditions. The results revealed considerable individual differences in response to certain stimuli. The largest within-group variation was found when being caged; the responses varied from passivity to escape attempts (score range: 2-5 in a 5-step scale). Substantial variation was also seen in locomotion and food consumption after exposure to stress (score range: 1-5 in a 5-step scale). In a new environment, the females showed more frequent changes in attention (focusing) compared with males (P < 0.01). There was an age-related reaction to sudden sounds (Spearman r_sp = ?0.52, P < 0.05). We also describe application of the TFPC to a study of food intake in response to pancreatic polypeptide performed with 6 of the male dogs. A within-group rank-order procedure was used, and interesting correlations between personality characteristics and food intake behavior were identified. We discuss how the TFPC may contribute to improvement of experimental studies in dogs.     

Control of tsetse flies and trypanosomes using molecular genetics

TSETSE FLIES (DIPTERA: Glossinidae) are important agricultural and medical vectors transmitting the African trypanosomes, the agents of sleeping sickness disease in humans and various diseases in animals (nagana). While the prevalence of disease has increased to epidemic proportions, lack of a mammalian vaccine and affordable and effective drugs have hindered disease control. Trypanosomiasis management relies heavily on the control of its single insect vector, the tsetse fly. Despite the effectiveness of some of these tools, their impact on disease control has not been sustainable due to their local nature and extensive dependence on community participation. Recent advances in molecular technologies and their application to insects have revolutionized the field of vector biology, and there is hope that such new approaches may form the basis for future tsetse interventions. The success of the genetic approaches aiming to disrupt the transmission cycle of the parasite in their invertebrate host depends on full understanding of the interaction between tsetse and trypanosomes. This article reviews the biology of trypanosome development in the fly and the multiple bacterial symbionts that inhabit the same gut environment. The availability of a genetic transformation system for the midgut symbiont allows for gene products to be expressed in vivo in the tsetse gut where they can produce a hostile environment for pathogen transmission. The characterization of gene product(s) with anti-pathogenic properties and their expression in vivo is discussed. A strategy is outlined where the replacement of susceptible insect phenotypes with their engineered refractory counterparts can result in decreased disease transmission.     

Estimating the probability of effective transmission of trypanosomes using the poisson distribution

The trypanosome infection rate in cattle is determined by the probability of effective transmission (PET) of trypanosome from Glossina spp. to susceptible cattle. The PET in turn is a function of the rate of contact between cattle and Glossina spp. and was calculated using a Poisson probability distribution. Seven parameters determined the final form of the PET. These were: probability of at least one contact with Glossina spp. (PTC), probability that Glossina spp. will feed on the host (PFF), proportion of Glossina spp. which are non-tenerals (PNT), proportion of infected Glossina spp. (PTI), proportion of infected Glossina spp. which are infectious (PMTI), proportion of successful transmissions of the infective agent in inoculum of infectious Glossina spp. (PII), and finally, the probability for the presence of a susceptible host animal (PC). The product of these proportions provided an estimate of effective transmission of trypanosomes from infectious Glossina to susceptible cattle. The probability of this event varied between (0.0007 and 0.0009) or 7–9 infections per 10 000 susceptible cattle per year at a steady state.     

Assessment of the dynamics of microparasite infections in genetically homogeneous and heterogeneous populations using a stochastic epidemic model

The aim of this paper was to explore the effect of genetic heterogeneity in host resistance to infection on the population-level risks and outcomes of epidemics. This was done using a stochastic epidemiological model in which the model parameters were assumed to be genetically controlled traits of the host. A finite locus model was explored, with a gene controlling the transmission coefficient (i.e., host susceptibility to infection) and a gene controlling the recovery period. Both genes were simulated to have 2 alleles with underlying additive or dominance inheritance and an independent assortment of alleles. The model was parameterized for a viral pig disease (transmissible gastroenteritis), and complete homogeneous mixing among genotypes was assumed. Mean population genotype dramatically affected epidemic outcomes, and subtle effects of heterogeneity on epidemic properties were also observed. Genetic variation in the transmission coefficient led to probabilities of epidemics occurring that were slightly greater than expected, but genetic variation in the recovery rate had no such effect. Epidemics were generally less severe in genetically heterogeneous populations than expected from the constituent subpopulations. Furthermore, the genotype of the initial infected animal had a marked effect on epidemic probabilities, particularly when genetic variation was for recovery rate. The results of this model provide useful information to determine the optimum population structures and to exploit genetic variation in resistance to infection. Applications of the proposed model in genetically heterogeneous populations for identifying practical disease management strategies are also discussed.