Control of contagious bovine pleuropneumonia: knowledge, attitudes, perceptions and practices in Narok district of Kenya.

CBPP is an important transboundary disease in sub-Saharan Africa whose control is urgent. Participatory data collection involving 52 focus group discussions in 37 village clusters and key informant interviews, a cross-sectional study involving 232 households and a post-vaccination follow up involving 203 households was carried out in 2006-2007 in Narok South district of Kenya. This was to investigate knowledge, attitudes, perceptions and practices (KAPP) associated with control of CBPP as well as the adverse post-vaccination reactions in animals in order to advice the control policy. The community perceived trans-boundary CBPP threat to their cattle. They had traditional disease coping mechanisms and were conversant with CBPP prevention and control with 49.8% (95%CI: 42.8-56.7%) giving priority to CBPP control. However, 12.9% (95%CI: 9.0-18.1%) of pastoralists had no knowledge of any prevention method and 10.0% (95%CI: 6.5-14.7%) would not know what to do or would do nothing in the event of an outbreak. Although 43.5% (95%CI: 37.1-50.2%) of pastoralists were treating CBPP cases with antimicrobials, 62.5% (95%CI: 52.1-71.7%) of them doubted the effectiveness of the treatments. Pastoralists perceived vaccination to be the solution to CBPP but vaccination was irregular due to unavailability of the vaccine. Vaccination was mainly to control outbreaks rather than preventive and exhibited adverse post-vaccination reactions among 70.4% (95%CI: 63.6-76.5%) of herds and 3.8% (95%CI: 3.5-4.2%) of animals. Consequently, nearly 25.2% (95%CI: 18.5-33.2%) of pastoralists may resist subsequent vaccinations against CBPP. Pastoralists preferred CBPP vaccination at certain times of the year and that it is combined with other vaccinations. In conclusion, pastoralists were not fully aware of the preventive measures and interventions and post-vaccination reactions may discourage subsequent CBPP vaccinations. Consequently there is need for monitoring and management of post vaccination reactions and awareness creation on CBPP prevention and interventions and their merits and demerits. CBPP vaccine was largely unavailable to the pastoralists and the preference of the pastoralists was for vaccination at specified times and vaccine combinations which makes it necessary to avail the vaccine in conformity with the pastoralists preferences. In addition, planning vaccinations should involve pastoralists and neighbouring countries. As the results cannot be generalized, further studies on CBPP control methods and their effectiveness are recommended.

Genotypic diversity, a survival strategy for the apicomplexan parasite Theileria parva.

The tick-borne protozoan parasite Theileria parva causes East Coast fever (ECF), a severe lymphoproliferative disease of cattle that is a major constraint to the improvement of livestock in eastern, central and southern Africa. Studies in cattle experimentally infected with T. parva have shown that the protective cytotoxic T lymphocyte (CTL) response is tightly focused, with individual animals recognizing only one or two dominant antigens, the identity of which varies with MHC class I phenotype. It is well known that cross-protection between T. parva stocks is limited, but precise evaluation of genetic diversity in field populations of the parasite has been hampered by a lack of molecular markers spanning the genome. A recently described panel of satellite markers has provided evidence for substantial genotypic diversity and recombination but does not provide cover for large segments of the genome. To address this deficiency, we undertook to identify additional polymorphic markers covering these regions and we report herein 42 newly identified PCR-RFLP markers distributed across the 4 T. parva chromosomes, as well as 19 new satellite markers for chromosomes 1 and 2. This brings the total number of available polymorphic markers to 141 for the 8.5 Mb genome. We have used these markers to characterise existing parasite stabilates and have also shown that passage of the parasite through naïve cattle and ticks can lead to substantial changes of parasite populations in resulting stabilates. These markers have also been used to show that passage of mixed parasites through an immunised calf results in the removal of the immunising genotype from the parasite population produced by ticks fed on this animal.

Infection of cattle with Theileria parva induces an early CD8 T cell response lacking appropriate effector function.

Theileria parva causes an acute lympho-proliferative disease in cattle, which can result in death of susceptible animals within 2-3 weeks of infection. Analyses of the cellular response in the lymph node draining the site of infection demonstrated an early T cell response, with the appearance of large numbers of uninfected lymphoblasts between 6 and 9 days p.i., coinciding with initial detection of parasitised cells. There was a marked increase in the representation of CD8(+) T cells and the emergence of a sizable sub-population of CD2(-) CD8(+) alpha/beta T cells during this period. Analysis of T cell receptor beta chain variable (TCR BV) gene expression did not reveal any evidence for the involvement of a superantigen in stimulating the response. Responding lymph node cells were found to produce increased quantities of IFNgamma and IL-10, and both the CD2(+) CD8(+) and CD2(-) CD8(+) populations expressed IFNgamma transcripts. Purified CD2(+) CD8(+) cells proliferated when stimulated in vitro with autologous parasitised cells or non-specific mitogens, whereas CD2(-) CD8(+) cells were refractory to these stimuli. In contrast to the parasite-specific cytotoxic activity associated with T cell responses in immune cattle, the responses to primary infection exhibited variable levels of non-specific cytotoxic activity. Stimulation of purified CD2(+) CD8(+) T cells in vitro with autologous parasitised cells also failed to reveal evidence of specific cytotoxic activity. These findings indicate that primary infection with T. parva induces an aberrant T cell response that lacks appropriate effector activity.

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells.

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.

Cloned Theileria parva produces lesser infections in ticks compared to uncloned T. parva despite similar infections in cattle.

Experimental transmissions of cloned Theileria parva in cattle with Rhipicephalus appendiculatus ticks were compared to transmissions with uncloned T. parva during studies on the potential for genetic recombination during syngamy of Theileria to produce antigenic diversity for evasion of bovine immunity. Prevalence and abundance of T. parva infection in adult ticks, which resulted from the feeding of nymphs on the calves, were significantly higher in the uncloned compared to the cloned T. parva. Development of sporoblasts of T. parva in the ticks to produce infective sporozoites was similar. There was no statistically significant difference in the clinical course of infection in cattle between cloned and uncloned T. parva. It was concluded that cloned T. parva has characteristics that reduce its viability during the tick stages of its life cycle.

Theileria parva and the bovine CTL response: down but not out?

Theileria parva is a tick-borne intracellular protozoan of cattle, with obligate sequential differentiation stages in lymphocytes and erythrocytes. Immunity is mediated by cytotoxic T lymphocytes (CTL) that target and clear parasitized lymphocytes but allow persistence of infected erythrocytes, which are required for transmission to the tick. The life cycle of T. parva is haploid with the exception of a brief diploid stage in the tick vector during which sexual recombination occurs. There is evidence for antigenic diversity in field parasite populations, although broad immunity can be acquired following exposure to a limited number of strains. The CTL response in individual animals is tightly focused and its specificity is strongly influenced by major histocompatibility complex (MHC) phenotype. This review discusses the issue of how CTL immunity is likely to impact on parasite population structure in the light of available information on diversity of the parasite and its ability to recombine.

Current status of vaccine development against Theileria parasites.

The tick-borne protozoan parasites Theileria parva and Theileria annulata cause economically important diseases of cattle in tropical and sub-tropical regions. Because of shortcomings in disease control measures based on therapy and tick control, there is a demand for effective vaccines against these diseases. Vaccines using live parasites have been available for over two decades, but despite their undoubted efficacy they have not been used on a large scale. Lack of infrastructure for vaccine production and distribution, as well as concerns about the introduction of vaccine parasite strains into local tick populations have curtailed the use of these vaccines. More recently, research has focused on the development of subunit vaccines. Studies of immune responses to different stages of the parasites have yielded immunological probes that have been used to identify candidate vaccine antigens. Immunisation of cattle with antigens expressed in the sporozoite, schizont or merozoite stages has resulted in varying degrees of protection against challenge. Although the levels of protection achieved have not been sufficient to allow exploitation for vaccination, there are clearly further lines of investigation, relating to both the choice of antigens and the antigen delivery systems employed, that need to be pursued to fully explore the potential of the candidate vaccines. Improved knowledge of the molecular biology and immunology of the parasites gained during the course of these studies has also opened up opportunities to refine and improve the quality of live vaccines.

Conservation of promoter, coding and intronic regions of the non-classical MHC class II DYA gene suggests evolution under functional constraints.

The major histocompatibility complex (MHC) in ruminants contains a unique pair of class II genes (DYA and DYB) of unknown function. As functional genes show higher levels of nucleotide conservation than pseudogenes we compared the DYA genes from sheep and cattle, two species which diverged from a common ancestor approximately 20 million years ago. Comparative analysis identified levels of nucleotide conservation in immediate promoter (97%), coding (94%) and intronic regions (91%) comparable with functional MHC genes. The Ovar-DYA transcript revealed an open reading frame encoding a 288 amino acid protein compared with a 253 amino acid protein associated with the BoLA-DYA transcript. A dinucleotide deletion in exon 4 of the Ovar-DYA transcript combined with alternative exon 5 splice sites introduces unusual diversity to the cytoplasmic domain of the Ovar-DYalpha polypeptide. The degree of conservation between these class II MHC genes is consistent with evolution under purifying selection suggesting that these genes retain a unique function in ruminants.

Immunopathology of Chlamydophila abortus infection in sheep and mice.

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.

Cellular immunity against Theileria parva and its influence on parasite diversity.

Theileria parva, a tick-borne parasite of African cattle, causes a fatal disease known as East Coast fever. Cattle that recover from the disease develop strong parasite-specific MHC-class I-restricted cytotoxic T-lymphocyte responses. Protection can be transferred between immune and naïve calves in the CD8+ T cell fraction emanating from a responding lymph node. In vitro studies suggest that this response requires input from activated CD4+ T cells. The T parva life cycle involves developmental stages in mammalian and tick hosts and can lead to a number of different endemic scenarios for the disease. These range from a stable situation with high prevalence of herd infection, but low fatality rates, to a low prevalence/high fatality scenario. The impact on endemic stability is an important consideration for the design of vaccine implementation strategies. For subunit vaccines targeted at T parva schizonts, the principal issue in this regard is whether development of the piroplasm stage is blocked by immunity.

Transcription of the unique ruminant class II major histocompatibility complex-DYA and DIB genes in dendritic cells.

Dendritic cells (DC) constitute the most effective immune cell population for priming and recalling T cell responses to foreign antigens. DC patrol the peripheral tissues collecting foreign antigen for subsequent presentation by classical class II MHC molecules to T cells in the draining lymph nodes. Since the description of the DYA and DIB class II MHC genes, which are unique to ruminants, no transcript or protein have been reported. Here we provide evidence that these genes are transcribed in cattle and that paired transcription is restricted in afferent lymph to a functionally distinct population of DC. Analysis of lymph node, lung and thymus suggests that tissue DC also transcribe both genes. Cytokine-induced differentiation of cultured monocytes to a DC phenotype is linked with induction of both DYA and DIB transcription. This is consistent with an association of their products with the potent antigen presenting capacity of these cells in cattle.

The CD45 locus in cattle: allelic polymorphism and evidence for exceptional positive natural selection.

Cattle in Africa are a genetically diverse population that has resulted from successive introduction of Asian Bos indicus and European B. taurus cattle. However, analysis of mitochondrial genetic diversity in African cattle identified three lineages, one associated with Asian B. indicus, one with European B. taurus, and a third ascribed to an indigenous African sub-species of cattle. Due to their extended coevolution, indigenous African herbivores are generally tolerant to endemic African pathogens. We are interested in identifying alleles derived from the indigenous African cattle that may be associated with tolerance to African pathogens. An analysis of the locus which encodes the abundant plasma membrane-associated tyrosine phosphatase, CD45, identified three highly divergent allelic families in Kenya Boran cattle. Analysis of allelic distribution in a diverse range of cattle populations suggests a European B. taurus, an Asian B. indicus, and an African origin. This demonstrates not only significant allelic polymorphism at the CD45 locus in cattle but also convincing autosomal evidence for a distinct African sub-species of cattle. Furthermore, maximum-likelihood analysis of selection pressures revealed that the CD45 locus is subject to exceptionally strong natural selection which we suggest may be pathogen driven.

Rapid changes occur in the percentage of circulating bovine WC1(+)gamma delta Th1 cells.

gamma delta T cells found in the peripheral blood of cattle include a major subpopulation distinguished by expression of WC1. These cells are distinct from the WC1(-)gamma delta T cell population based on T cell receptor gene usage. We documented that a group of 6-month-old calves allowed free-range grazing and access to their mothers had a significantly greater proportion of total gamma delta T cells in their blood, attributable to the WC1(+)gamma delta T cell subpopulation, compared to age and breed-matched calves held in conventional housing. When the animals with the greater proportion of gamma delta T cells were transferred to conventional housing there was a decrease in the WC1(+)population so that by 3 weeks after transfer there was no longer a significant difference between the two groups. To investigate the biological activities of WC1(+)gamma delta T cells, the cells were purified by flow cytometric sorting. In vitro, they responded to stimulation by irradiated monocytes in autologous mixed leukocyte reaction (AMLR) cultures but not to direct stimulation through the T cell receptor (T c R) by anti-delta monoclonal antibody. After stimulation in the AMLR, WC1(+)gamma delta T cells had a Th1 cytokine profile characterised by production of IFN -gamma and lack of IL -4. Thus we propose that higher levels of the WC1(+)gamma delta T cells may provide calves with a mechanism to produce Th1 cytokines and that the level of these cells may be modulated according to environment or stress since both groups of calves were apparently disease-free.

A statistically derived index for classifying East Coast fever reactions in cattle challenged with Theileria parva under experimental conditions.

A statistically derived disease reaction index based on parasitological, clinical and haematological measurements observed in 309 5 to 8-month-old Boran cattle following laboratory challenge with Theileria parva is described. Principal component analysis was applied to 13 measures including first appearance of schizonts, first appearance of piroplasms and first occurrence of pyrexia, together with the duration and severity of these symptoms, and white blood cell count. The first principal component, which was based on approximately equal contributions of the 13 variables, provided the definition for the disease reaction index, defined on a scale of 0-10. As well as providing a more objective measure of the severity of the reaction, the continuous nature of the index score enables more powerful statistical analysis of the data compared with that which has been previously possible through clinically derived categories of non-, mild, moderate and severe reactions.

Bovine CD4+ T-cells lines reactive with soluble and membrane antigens of Cowdria ruminantium.

Cowdria-specific CD4+ T-cell lines generated from immunised cattle respond to both soluble and membrane proteins of the agent. Furthermore, the lines produced the Cowdria-inhibitory cytokine IFN-gamma in response to soluble antigens fractionated by gel filtration and FPLC. Activity eluted as a single peak around fraction 15 for all T-cell lines tested. This fraction induced the highest production of IFN-gamma by the lines and was shown by SDS-polyacrylamide gel electrophoresis and silver staining analysis to contain less than 10 different bands ranging from 22 to 32 kDa. Given their high sensitivity and specificity, these short-term CD4+ T-cell lines will be valuable tools for the identification of Cowdria antigens for incorporation in a subunit vaccine.

Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies.

Theileria parva is an intracellular sporozoan parasite that infects and transforms bovine lymphocytes, causing a severe lymphoproliferative disease known as East Coast fever in eastern, central and southern Africa. In this article, Declan McKeever and colleagues summarize the current understanding of immune mechanisms provoked by the parasite with regard to their role in both pathogenesis and protection. In particular, the influence of genomic polymorphism in parasite and host on the development of immunity is discussed, along with the evolution of current vaccine development strategies as a result of immunological research on the disease.

Immune responses to Cowdria ruminantium infections.

Understanding the basis of protective immunity to Cowdria ruminantium will facilitate the development of an effective subunit vaccine against heartwater in ruminants and contribute to a better definition of protective immune mechanisms to obligate intracellular pathogens in general. Until recently, immunological studies of heartwater in ruminants concentrated solely on antibody responses. Since 1995, the mechanisms underlying cell-mediated immunity of heartwater have been analysed. Progress achieved in these areas is discussed here by Philippe Totté and colleagues, with special emphasis on ruminants, the natural hosts of C. ruminantium.

Bovine gammadelta T-cell responses to the intracellular protozoan parasite Theileria parva.

T cells bearing the gammadelta antigen receptor (gammadelta T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that gammadelta T cells are involved in immune responses against Theileria parva. gammadelta T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of gammadelta T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the gammadelta T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the gammadelta T-cell lines and clones. These gammadelta T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of gammadelta T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine gammadelta T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.

Variation in the number of expressed MHC genes in different cattle class I haplotypes.

Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes.

Immunohistochemical detection of parasite antigens in Theileria parva-infected bovine lymphocytes.

Theileria parva is the causal agent of East Coast fever (ECF), a fatal disease of cattle characterized by pyrexia, transient lymphadenopathy and panleukopenia. We have evaluated monoclonal antibodies (mAb) against three distinct antigens (p67, PIM and p32) of the parasite as immunohistological reagents for monitoring the kinetics of infection in cattle. Bovine lymphocytes were stained with the mAb at various intervals after infection in vitro and in vivo. The p67 sporozoite surface antigen was detected in only a small percentage of both, in vitro and in vivo infected cells. In contrast, expression of the polymorphic immunodominant molecule (PIM) of the parasite proved a useful indicator of infection and staining was correlated with the results of Giemsa analysis. PIM was detected from day 3 in in vitro-infected cells, but was not detected until day 5 in vivo after challenge with a 70% lethal dose of stabilized sporozoite. The p32 antigen was expressed only late in infection in vivo and its expression was associated with the development of merozoites. Less than 20% of in vitro-infected cells expressed p32. The immunohistochemical staining with anti-PIM mAb was found to be a useful tool for analysis of T. parva infection kinetics in cattle.