The low-virulent African swine fever virus (ASFV/NH/P68) induces enhanced expression and production of relevant regulatory cytokines (IFNα, TNFα and IL12p40) on porcine macrophages in comparison to the highly virulent ASFV/L60

The impact of infection by the low-virulent ASFV/NH/P68 (NHV) and the highly virulent ASFV/L60 (L60) isolates on porcine macrophages was assessed through the quantification of IFNα, TNFα, IL12p40, TGFβ and ASFV genes by real-time PCR at 2, 4 and 6 h post-infection. Increased IFNα, TNFα and IL12p40 expression was found in infection with NHV, in which expression of TGFβ was lower than in infection with L60. Principal component analysis showed a positive interaction of cytokines involved in cellular immune mechanisms, namely IFNα and IL12p40 in the NHV infection. Quantification by ELISA confirmed higher production of IFNα, TNFα and IL12p40 in the NHV-infected macrophages. Overall, our studies reinforce and clarify the effect of the NHV infection by targeting cellular and cellular-based immune responses relevant for pig survival against ASFV infection

Design and descriptive epidemiology of the Infectious Diseases of East African Livestock (IDEAL) project, a longitudinal calf cohort study in western Kenya

BACKGROUND: There is a widely recognised lack of baseline epidemiological data on the dynamics and impacts of infectious cattle diseases in east Africa. The Infectious Diseases of East African Livestock (IDEAL) project is an epidemiological study of cattle health in western Kenya with the aim of providing baseline epidemiological data, investigating the impact of different infections on key responses such as growth, mortality and morbidity, the additive and/or multiplicative effects of co-infections, and the influence of management and genetic factors. A longitudinal cohort study of newborn calves was conducted in western Kenya between 2007-2009. Calves were randomly selected from all those reported in a 2 stage clustered sampling strategy. Calves were recruited between 3 and 7 days old. A team of veterinarians and animal health assistants carried out 5-weekly, clinical and postmortem visits. Blood and tissue samples were collected in association with all visits and screened using a range of laboratory based diagnostic methods for over 100 different pathogens or infectious exposures. RESULTS: The study followed the 548 calves over the first 51 weeks of life or until death and when they were reported clinically ill. The cohort experienced a high all cause mortality rate of 16% with at least 13% of these due to infectious diseases. Only 307 (6%) of routine visits were classified as clinical episodes, with a further 216 reported by farmers. 54% of calves reached one year without a reported clinical episode. Mortality was mainly to east coast fever, haemonchosis, and heartwater. Over 50 pathogens were detected in this population with exposure to a further 6 viruses and bacteria. CONCLUSION: The IDEAL study has demonstrated that it is possible to mount population based longitudinal animal studies. The results quantify for the first time in an animal population the high diversity of pathogens a population may have to deal with and the levels of co-infections with key pathogens such as Theileria parva. This study highlights the need to develop new systems based approaches to study pathogens in their natural settings to understand the impacts of co-infections on clinical outcomes and to develop new evidence based interventions that are relevant

Genetic Diversity and Population Structure of <i>Theileria annulata</i> in Oman

Background: Theileriosis, caused by a number of species within the genus Theileria, is a common disease of livestock in Oman. It is a major constraint to the development of the livestock industry due to a high rate of morbidity and mortality in both cattle and sheep. Since little is currently known about the genetic diversity of the parasites causing theileriosis in Oman, the present study was designed to address this issue with specific regard to T. annulata in cattle. Methods Blood samples were collected from cattle from four geographically distinct regions in Oman for genetic analysis of the Theileria annulata population. Ten genetic markers (micro- and mini-satellites) representing all four chromosomes of T. annulata were applied to these samples using a combination of PCR amplification and fragment analysis. The resultant genetic data was analysed to provide a first insight into the structure of the T. annulata population in Oman. Results: We applied ten micro- and mini-satellite markers to a total of 310 samples obtained from different regions (174 [56%] from Dhofar, 68 [22%] from Dhira, 44 [14.5%] from Batinah and 24 [8%] from Sharqia). A high degree of allelic diversity was observed among the four parasite populations. Expected heterozygosity for each site ranged from 0.816 to 0.854. A high multiplicity of infection was observed in individual hosts, with an average of 3.3 to 3.4 alleles per locus, in samples derived from Batinah, Dhofar and Sharqia regions. In samples from Dhira region, an average of 2.9 alleles per locus was observed. Mild but statistically significant linkage disequilibrium between pairs of markers was observed in populations from three of the four regions. In contrast, when the analysis was performed at farm level, no significant linkage disequilibrium was observed. Finally, no significant genetic differentiation was seen between the four populations, with most pair-wise FST values being less than 0.03. Slightly higher FST values (GST’ = 0.075, θ = 0.07) were detected when the data for T. annulata parasites in Oman was compared with that previously generated for Turkey and Tunisia. Conclusion: Genetic analyses of T. annulata samples representing four geographical regions in Oman revealed a high level of genetic diversity in the parasite population. There was little evidence of genetic differentiation between parasites from different regions, and a high level of genetic diversity was maintained within each sub-population. These findings are consistent with a high parasite transmission rate and frequent movement of animals between different regions in Oman

Identification of a 40S ribosomal protein (S17) that is differentially expressed between the macroschizont and piroplasm stages of Theileria annulata

The nucleotide and protein sequence of the 40S ribosomal protein S17 (RibS17) of the protozoan parasite Theileriaannulata has been determined. Southern blot analysis showed the gene was single copy and comparative sequence analysis revealed that the predicted polypeptide had high sequence homology with the RibS17 from other organisms. Northern blot analysis showed that there was a 3-fold increase in the level of RibS17 RNA between the macroschizont and the piroplasm stage of the lifecycle, whereas, there was no difference in expression between the sporozoite and the macroschizont stages. Antisera to the purified fusion protein, corresponding to the terminal 50 amino acids of the protein sequence, were raised in rabbits. Western analysis detected a polypeptide of the predicted size that was more abundant in the piroplasm stage compared with the macroschizont stage. Immunofluorescence analysis with the same antisera revealed a strong signal in the macroschizont and piroplasm stages, but the antiserum did not cross-react with the bovine host cells. The antisera did, however, cross-react with Toxoplasmagondii tachyzoites and Plasmodiumfalciparum merozoites. The possible functional significance of the stage related increase in abundance of a ribosomal protein is discussed

Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda

Recent advances in genomic technology have focused many veterinary researchers on the possibility of producing one multivalent recombinant vaccine against all the haemoparasites that infect cattle in the tropics. Before such a vaccine is developed it is essential to define target cattle populations as well as the range of anti-pathogen vaccines required in order to control disease. To further this objective, we have evaluated a reverse line blot (RLB) assay, which simultaneously detects the principal tick transmitted protozoan and rickettsial cattle pathogens, in different epidemiological scenarios in Uganda. A critical question is the sensitivity, particularly in relation to detecting carrier animals. As Theileria parva is considered to be the most important pathogen in the region, we assessed the sensitivity of the RLB assay for T. parva and showed that 1–2×103 parasites per ml of blood could be detected—a level comparable with previously developed PCR methods and well below conventional microscopic detection. We applied the RLB assay to evaluate the differences in pathogen profiles between crossbred and indigenous cattle and show that there were different profiles, with a low prevalence of T. parva and Theileria taurotragi in the indigenous cattle compared to a high prevalence in the crossbred cattle. In contrast, we show higher prevalences of Theileria mutans and Theileria velifera in the indigenous compared to the crossbred cattle. Interestingly Anaplasma marginale, Babesia bovis and Babesia bigemina were of low prevalence but a high prevalence of Ehrlichia bovis was seen, raising the question of whether this rickettsial species could be pathogenic in cattle. Analysis of animals with clinical symptoms of East Coast Fever showed that, while T. parva is a major cause of these symptoms, T. mutans and possibly T. taurotragi and T. velifera, may also cause clinical disease. Overall, the results presented here highlight the complexity of tick-borne pathogen infections in cattle in Uganda

Population genetic analysis and sub-structuring of Theileria parva in Uganda

In recent years the population structures of many apicomplexan parasites including Plasmodium spp., Toxoplasma gondii and Cryptospordium parvum have been elucidated. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of another apicomplexan species (Theileria parva) to investigate the levels of diversity of this parasite and the role of genetic exchange in three geographically separate populations. The principal hindrance to carrying out such a study on field isolates was the high proportion of blood samples that contain multiple genotypes, making it impossible to determine the genotypes of the parasites directly. This problem was overcome by sampling only young indigenous calves between 3 and 9 months of age in which approximately 60% of the T. parva infected calves contained a single/predominant allele at each locus, making it possible to undertake population genetic analyses. Blood samples were collected from calves in three geographically distinct regions of Uganda and were analysed using 12 polymorphic mini and microsatellite markers that were evenly dispersed across the four chromosomes. We have identified 84 multilocus genotypes (MLG) from these samples, indicating high levels of diversity in the parasite. Analysis of linkage disequilibrium between pairs of loci provides evidence that the population in Lira district had an epidemic structure. The population in Mbarara was substructured containing two genetically distinct sub-groups and the larger sub-group also had an epidemic population structure. The population from Kayunga was in linkage disequilibrium. Genetic distances and Wrights fixation index (F&lt;sub&gt;ST&lt;/sub&gt;) indicate that there is evidence for geographical sub-structuring between the Lira and the Kayunga populations