Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a serious problem in the horse industry, and controlling EP is critical for international horse trading. EP is caused by two apicomplexan protozoan parasites, Theileria equi and Babesia caballi. Rapid and accurate methods that are suitable for detecting these parasites in the field are crucial to control the infection and spread of EP. In this study, we developed a card to detect antibodies against T. equi and B. caballi based on two colloidal gold immunochromatographic strips according to the principle of the double-antigen sandwich. The proteins equi merozoite antigen 1 (EMA1) and rhoptry protein BC48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. On the strip, the purified EMA1 or BC48 protein labeled with colloidal gold was used as the detector, and nitrocellulose membranes were coated with EMA1 or BC48 and the corresponding MAb as the test and control lines, respectively. The protocol takes 10 to 15 min and requires no specialized equipment or chemical reagents, and one test can detect two EP pathogens in one card. Specificity tests confirmed there was no cross-reactivity with sera positive for common equine pathogens. Using a commercial competitive enzyme-linked immunosorbent assay (cELISA) kit for comparison, 476 clinical samples were tested with the card. The coincidence rates were 96.43% and 97.90% for T. equi and B. caballi, respectively. The field trial feedback was uniformly positive, suggesting that this diagnostic tool may be useful for controlling the spread of T. equi and B. caballi. IMPORTANCE Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. EP is considered a reportable disease by the World Organization for Animal Health (OIE). The accurate diagnosis and differentiation of T. equi and B. caballi are very important for the prevention, control, and treatment of EP. Therefore, we developed a double-antigen sandwich colloidal gold immunochromatography assay (GICG) to detect T. equi and B. caballi. Two GICG strips were assembled side by side on one card for the detection of T. equi and B. caballi, and the two EP pathogens could be detected in one test. This method was simple, rapid, and specific for the detection of EP; therefore, compared to the previous methods, this method is more suitable for pathogen diagnosis in the field.

Immune response of adult horses, pregnant mares and foals to an experimental vaccine with recombinant EMA-2 protein of Theileria equi.

Equine theileriosis, caused by the Theileria equi protozoan, is a disease of worldwide importance. T. equi expresses surface proteins, of which the EMA-2 protein is a promising antigen for vaccine use. The aim of this study was to evaluate the immune response of adult horses, pregnant mares, and foals to an experimental EMA-2 protein of recombinant T. equi vaccine. A total of 46 horses were used in this study for vaccine trials and challenges. Twelve geldings, 14 pregnant mares, and 14 foals were divided into vaccinated and control groups. Total serum specific anti-rEMA-2 IgG, IgG subclasses, and transcription of cytokines related to the immune response were evaluated. For the vaccine challenge, six six-month-old foals were divided into vaccinated and control groups. For the challenge, blood from a horse with theileriosis was transfused to the foals. Geldings and pregnant mares maintained anti-rEMA-2 IgG levels at 130 and 140 days after vaccination, respectively. The most-detected IgG subclasses in vaccinated were IgG3/5, IgG4/7, and IgG1. IL2, IL10, IL12, IL17, IFN-γ, and TNF-α were the most-transcribed cytokines in PBMCs of vaccinated horses stimulated with rEMA-2. Challenge with T. equi demonstrated that vaccinated foals had an increase of 33% in total IgG four days after blood transfusion, while control foals had no significant response, suggesting that vaccine antibodies may have recognized EMA-2 protein of the native T. equi antigen. T. equi recombinant EMA-2 was shown to be a promising vaccine antigen by inducing humoral and cellular immunity similar to that observed in natural parasite infections.

Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton.

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and ß- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

Theileria secretes proteins to subvert its host leukocyte.

Theileria parasites are classified in the phylum Apicomplexa that includes several genera of medical and veterinary importance such as Plasmodium, Babesia, Toxoplasma and Cryptosporidium. These protozoans have evolved subtle ways to reshape their intracellular niche for their own benefit and Theileria is no exception. This tick transmitted microorganism is unique among all eukaryotes in that its intracellular schizont stage is able to transform its mammalian host leukocytes into an immortalised highly disseminating cell that phenocopies tumour cells. Here, we describe what is known about secreted Theileria-encoded host cell manipulators.

An alternative cold chain for storing and transporting East Coast fever vaccine.

East Coast fever (ECF) is an often fatal, economically important cattle disease that predominantly affects eastern, central, and southern Africa. ECF is controlled through vaccination by means of simultaneous injection of oxytetracycline and cryogenically preserved stabilate containing live, disease-causing parasites. Storage and transportation of the stabilate requires liquid nitrogen, a commodity that is commonly unreliable in low-resource settings. Here we show that storage of conventionally prepared stabilate at -80 °C for up to 30 days does not significantly affect its ability to infect cultured peripheral blood mononucleated cells or live cattle, suggesting an alternative cold chain that maintains these temperatures could be used to effectively manage ECF.

Seroprevalence and risk factors associated with Equine piroplasmosis in North Egypt.

Equine piroplasmosis (EP) is caused by Theileria equi and/or Babesia caballi and has economic importance particularly in equines reared in poor management systems. This study is based on cELISA test to study the seroprevalence of EP among 370 horses and 150 donkeys in four Governorates north Egypt. Additionally, its risk factors were studied for the first time. The seroprevalence rates 36.5 %, 20 %, and 5.6 % for T. equi, B. caballi, and mixed infections, respectively. The highest antibody levels against T. equi were detected in Kafr ElSheikh (40 %) and Giza (40.1 %) Governorates, whereas those of B. caballi were detected in Qalyubia (25 %) and Kafr ElSheikh (24.1 %) Governorates. Concerning T. equi, animals >10 years (OR = 2.06) were more likely to be infected with EP than those <5 years old. In addition, the seropositivity increased among grazing (OR = 5.7, 95 % CI: 1.73-19.27) males (OR = 1.8, 95 % CI: 1.23-2.61) infested with ticks (OR = 2.3, 95 % CI: 1.60-3.48) during summer (OR = 4.3, 95 %CI: 2.53-7.46); whereas the seropositivity of animals for B. caballi increased among grazing equines (OR = 7.8, 95 % CI: 1.05-58.25) over 10 years old (OR = 2.08, 95 % CI: 1.10-3.94) and infested with ticks (OR = 2.4, 95 % CI: 1.54-3.76) during summer (OR = 7.12, 95 % CI: 3.15-16.06). Therefore, EP is an important prevalent disease in Egypt and deserves further attention regarding the management system, treatment, and vector control.

Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.

Factors associated with the prevalence of antibodies against Theileria equi in equids of Western Pará, Brazil.

The State of Pará has one of the largest herds of equids (horse, donkey and mule) in Brazil, most of these animals are found on cattle farms. Equine theileriosis is a tick-borne disease caused by the parasite Theileria equi and is characterized by fever, anaemia, icterus, intravascular haemolysis, haemoglobinuria, spleen and hepatomegaly, and even death. The present study aimed to determine the prevalence of antibodies against T. equi in equids in the western region of the State of Pará, Brazil, and to identify potential risk factors associated with parasite infection. A cross-sectional study was conducted with cluster sampling of farm horses from 18 municipalities. In the cities visited, samples from sport and carthorses were also included. Serum was obtained to detect T. equi-specific antibodies using an indirect enzyme-linked immunosorbent assay (iELISA) based on a crude parasite antigen. In order to identify possible risk factors of the infection which are associated with the prevalence of antibodies, a chi-squared test was carried out. Of 1,117 equids, 373 tested positive for T. equi antibodies with an overall prevalence of 33.4% (31.3%-37.0% for the 95% confidence interval). Sex, animal species and breed were found not to be associated with the presence of T. equi antibodies, whereas age, the presence of dogs or ticks were associated with seropositivity (p < 0.05). Horses with ticks were 2.4 more likely seropositive than horses without ticks. The presence of dogs in the equid habitat and the presence of ticks resulted in a higher T. equi seropositive rate probably because dogs are hosts for vector ticks of T. equi. Our study represents the first report of T. equi antibodies in equids of western Pará revealing a widespread distribution of seropositive animals.

Antigenicity assessment of the Theileria equi merozoite antigen (EMA-2) expressed in Pichia pastoris in mice and horses.

Equine theileriosis is a severe equine disease caused by the protozoan Theileria equi, which is prevalent in tropical and subtropical areas. In this study, a recombinant equi merozoite antigen-2 (rEMA-2) of T. equi was used as an immunogen. Two groups of 10 mice each were divided into control and vaccinated groups. Sixty mares seronegative for theileriosis were divided in two groups, one vaccinated and another group as a control animal. Mice and mares of the vaccinated groups were inoculated with 150 µL of the vaccine containing 50 µg of rEMA-2 and 2 mL of the vaccine containing 200 µg of rEMA-2, respectively, at days 0 and 21. The immunogenicity of rEMA-2 was evaluated by ELISA and fluorescent antibody test (IFAT) using serum from vaccinated mice, mares and antigenicity in naturally infected horse. At every point throughout the ELISA study, there were significant differences between the vaccinated and control groups (p < 0.05). The vaccine induced 3- and 4-fold IgG increases in mice at the 14th and 28th day, respectively, compared to the control group. The horses' IgG dynamics showed a significant (p < 0.05) increase in the total IgG titer as early as day 7, which increased until day 28 at which time a more significant (p < 0.001) IgG titer was observed. In evaluating the isotypes, we observed a trend similar to that of total IgG, where IgG(T) (IgG3-5) were significantly (p < 0.05) more elevated than the other isotypes analyzed, followed by IgGb (IgG4-7) and IgGa (IgG1). Positive fluorescence was detected by IFAT, suggesting that the protein is immunogenic and conserves some epitopes identical to the native T. equi antigens present in the equine blood smear. Thus, our results suggest that rEMA-2 can be a promising vaccinal antigen.

A Seroepidemiological Survey of Theileria equi and Babesia caballi in Horses in Mongolia.

Equine piroplasmosis caused by Theileria equi and Babesia caballi is an economically important disease with a worldwide distribution. The objective of the present study was to investigate the seroepidemiology of T. equi and B. caballi in horses reared in various Mongolian provinces. Serum samples prepared from blood collected from horses in 19 Mongolian provinces were screened for antibodies specific to T. equi and B. caballi using enzyme-linked immunosorbent assays based on recombinant forms of T. equi merozoite antigen-2 and the B. caballi 48-kDa merozoite rhoptry protein, respectively. Of 1,282 horses analyzed, 423 (33%) and 182 (14.2%) were sero-positive for T. equi and B. caballi, respectively. Additionally, 518 (40.4%) were positive for at least 1 parasite species, of which 87 (16.8%) were co-infected with both parasites. Both T. equi and B. caballi were detected in all surveyed provinces, and on a per province basis the positive rates ranged from 19.0 to 74.2% and 4.5 to 39.8%, respectively. Theileria equi- and B. caballi-positive rates were comparable between male horses (31.9 and 14.1%, respectively) and female horses (34.5 and 14.3%, respectively). However, the positive rates were higher in the >3-yr-old age group (37.7 and 15.6%, respectively) compared with the 1-3-yr-old age group (19.4 and 10.0%, respectively). These findings confirmed that T. equi and B. caballi infections are widespread among horses all over Mongolia, and that horse age is a risk factor for infection in this country. Our results will be useful for designing appropriate control measures to minimize T. equi and B. caballi infections among Mongolian horses.

Evolution and diversity of the EMA families of the divergent equid parasites, Theileria equi and T. haneyi.

The equine parasite Theilera equi continues to curtail global equine commerce due primarily to its ability to persist indefinitely in the immunocompetent horse. Details regarding the parasite life cycle, pathogenesis and mechanism of persistence remain unclear. The recently discovered T. haneyi is also capable of persistence in the horse, creating a potential reservoir for additional infections. These two divergent parasites share a unique gene family that expresses surface merozoite antigens, or equi merozoite antigens (EMAs). The EMA family was maintained in number and size in both parasites despite a species divergence of over 30 million years ago. This family is unique amongst Theilerias in number, structure and biochemical properties. In silico analysis revealed no evidence of selection for diversity within this family, indicating a role in host adaptation and persistence rather than antigenic variation and immune escape. Biochemical analysis revealed the presence of a conserved domain, homologous to the hemolysin toxin found in cobra venom. This finding combined with data from protein interaction prediction models may indicate interaction with the structural components of the host erythrocyte and a role in merozoite entry or escape. Additional predicted protein interactions focus on disruption of the enzymatic functions of the host cell, potentially resulting in enhanced parasite survival.

Serological and molecular detection of Theileria equi and Babesia caballi in Philippine horses.

Theileria equi and Babesia caballi are tick-borne protozoan parasites that can cause anemia in horses. In the Philippines, serological detection of these parasites has only been reported in the Northern area (Luzon). In this study, 105 horses from Cebu and Bohol, Philippines were tested using peripheral blood smear examination (PBSE), immunochromatographic test (ICT) strips, and PCR. Clinical history, presenting clinical signs and complete blood count were obtained. Results revealed that although all horses were negative using PBSE, 23 (21.9%) were positive (12 for T. equi, and 11 for B. caballi) using ICT. PCR revealed 26 and 2 horses positive for T. equi and B. caballi, respectively. All positive horses showed no clinical signs. Partial DNA sequences of representative amplicons were found 100% identical to GenBank registered T. equi and B. caballi sequences. Statistical analyses revealed that location was found associated with T. equi PCR positivity and B. caballi seropositivity. This study documents the first serological detection of T. equi and B. caballi in horses in the southern area of the Philippines, and their first molecular detection and characterization in the country.

Immune parameters to p67C antigen adjuvanted with ISA206VG correlate with protection against East Coast fever.

East Coast fever (ECF) is a lymphoproliferative disease caused by the tick-transmitted protozoan parasite Theileria parva. ECF is one of the most serious cattle tick-borne diseases in Sub-Saharan Africa. We have previously demonstrated that three doses of the C-terminal part of the sporozoite protein p67 (p67C) adjuvanted with ISA206VG confers partial protection against ECF at a herd level. We have tested the efficacy of two doses of this experimental vaccine, as reducing the vaccination regimen would facilitate its deployment in the field. We reconfirm that three antigen doses gave a significant level of protection to severe disease (46%, ECF score < 6) when compared with the control group, while two doses did not (23%). Animals receiving three doses of p67C developed higher antibody titers and CD4+ T-cell proliferation indices, than those which received two doses. A new panel of immune parameters were tested in order to identify factors correlating with protection: CD4+ proliferation index, total IgG, IgG1, IgG2 and IgM half maximal titers and neutralization capacity of the sera with and without complement. We show that some of the cellular and humoral immune responses provide preliminary correlates of protection.

Adding injury to infection: The relationship between injury status and genetic diversity of Theileria infecting plains zebra, Equus quagga.

Asymptomatic tick-borne infections are a common feature in wild herbivores. In human-dominated habitats, snare injuries to wild herbivores are common and are likely to co-occur with enzootic infections. The influence of injury on pattern, course and outcome of enzootic infection in wild herbivores is unknown. We identified Theileria species infecting zebra and assessed the relationship between host injury-status and parasitaemia, parasite diversity and selection regimes. We also determined host leucocyte differential as this can reveal mechanisms by which injuries influence infections. Theileria infecting zebra was identified using PCR and sequencing of the V4 region of the 18 s rRNA gene and confirmed with phylogenetic analyses. The influence of injury status on parasite infection patterns, genetic diversity and selection were assessed using population genetic tools. Parasitaemia estimated from prevalence and leucocyte differential were determined from microscopic examination of Giemsa stained thin blood smears. Phylogenetic and sequence analyses revealed that the zebra population studied was infected with three Theileria equi haplotypes. Parasitaemia was lower among injured compared to non-injured animals and lower during dry than wet season. Mean (±SD) genetic diversity was 0.386 (±0.128) in injured and 0.513 (±0.144) in non-injured zebra (P = .549). Neutrality tests indicated that T. equi is under strong purifying selection in injured females (Li & Fu's D* = -2.037) and demographic expansion in all zebra during the wet season (Tajima D = -1.904). Injured zebras had a higher median per cent of neutrophils (64% vs 37%) a lower median per cent of basophils (0% vs 1%) and eosinophils (2% vs 4.5%) than non-injured animals, suggesting a heightened immune response and a shift from a Th2 to Th1 T-Cell response favoring the elimination of intracellular parasites in injured animals. This study demonstrates the utility of population genetics in revealing factors influencing parasite diversity and infection patterns.

Risk factor analysis associated with Theileria equi infected equines in semi-arid and sub-humid ecological enzootic zones of India.

Equine piroplasmosis is a haemoprtozoan disease of equines and enzootic in tropical and subtropical countries. A cross-sectional study on sero-prevalence of Theileria equi, the causative agent of EP, was performed in semi-arid and sub-humid ecological endemic zones of India including Rajasthan, Haryana and Gujarat states, in order to evaluate the enzootic status/level of exposure to equine population due to this infection and addressed associated risk factors. Serum samples were collected from a total of 1021 equids that comprised of 792 horses, 168 donkeys and 61 mules and evaluated for T. equi specific antibodies in equine merozoite antigen-2 (EMA-2) based on indirect ELISA. The state with high sero-prevalence rate was Rajasthan (71.40%), followed by Haryana (60.39%) and Gujarat (48.92%). Overall T. equi sero-positivity in equines was 64.44%. Species-wise T. equi sero-prevalence was 66.29%, 51.19% and 91.80% in horses, donkeys and mules respectively. The association and risk factor among age, gender and species in relation with T. equi infection was statistically analyzed at 95% level of significance (p<0.05). A very high T. equi sero-positivity was recorded in 0-1year age group of equines (60%), indicating that this naïve age group contacts with T. equi infected ticks and remains infected throughout its lifetime. The sero-prevalence rate was significantly associated with the species of animal (p<0.05). The risk factor analysis kept mules at higher risk (Odd's ratio; 5.696; 95% confidence interval: 2.25-14.38) of getting infection as compared to horses and donkeys. This study has demonstrated high enzootic nature of T. equi infection in semi-arid and sub-humid ecological zones of India. Mules, in comparison to horses and donkeys were found at higher risk of getting T. equi infection, indicating that disease prevalence is associated with species of the infected host.

Sero-prevalence and risk factor analysis of Theileria equi infection in equids from different agro-climatic zones of Punjab (India) by Indirect Immunofluorescence Antibody test.

In the Indian sub-continent, equine piroplasmosis (EP) is an imperative tick-borne disease of equids predominately caused by obligatory intra-erythrocytic protozoa Theileria equi. Present exploration is the first epidemiological survey report based on Indirect Immunofluorescence Antibody test (IFAT). A total of 120 equines (98 horses and 22 donkeys/mules) from five districts of two main agro-climatic zones of Punjab were screened. The overall prevalence was 58.33%, with western plane zone at higher infection risk for T. equi (74.50%, OR = 7.45, 95% CI = 62.24-85.76); followed by western zone (46.38%, OR = 4.63, 95% CI = 34-57.7). District wise highest and lowest prevalence was recorded in district Fazilka (80.76%) and Bathinda (35.29%) respectively. IFAT revealed elevated risk of infection of T. equi in the farms with inappropriate management structure. The risk factor connected with the type of host species had an odds ratio of 1.67 for donkeys/mules versus horses for T. equi infection. Followed by presence/absence of tick on animal. This study reveals the endemicity of EP in Punjab with prominence of various odds of infection, associated with latent infection of T. equi detectable by serological techniques as it was noticed that western plane zone and western zone are more prone to EP infection.

Prevalence and effect of Theileria orientalis infection in homebred calves in the Gloucester region of New South Wales, Australia.

OBJECTIVES: To examine the prevalence of Theileria orientalis in the Gloucester area of New South Wales and its effect on individual animals. METHODS: Blood samples (EDTA and clotted blood) were collected from a total of 55 calves and their dams from 6 properties over a 16-week period. A total of 202 and 190 blood samples were collected from the calves and dams, respectively, and were examined via blood film for the presence of intraerythrocytic T. orientalis piroplasms. Packed cell volume (PCV) was measured to determine infection resulting in anaemia. The presence of antibodies against the T. orientalis major piroplasm surface protein (MPSP) was tested using ELISA. RESULTS: The overall prevalence of T. orientalis infection in both dams and calves from all herds examined was 95%. Mean peak parasitaemia was observed in calves between 6 and 9 weeks of age (P = 0.051), coinciding with a decline in mean PCV. Only 3 (6%) of the blood samples collected from the dams were positive for Theileria-associated antibodies and no significant relationship (P > 0.05) was found between the presence of antibodies in the dams and PCV levels in the calves. There was no evidence that passive transfer of antibodies from dams to calves protected the calves against a decline in PCV. CONCLUSION: This study confirmed a high prevalence of low-level Theileria infection, but low MPSP seroconversion rates, in dams and calves in an area where the disease has been endemic for a number of years.

Seroprevalence of Theileria equi and Babesia caballi in horses in Spain.

Equine piroplasmoses are enzootic parasitic diseases distributed worldwide with high incidence in tropical and subtropical regions. In Spain, there is insufficient epidemiological data about equine piroplasmoses. The main aim of the present study was therefore to estimate the prevalence of Theileria equi and Babesia caballi in five regions and obtain information about the risk factors. This study was conducted in the central and south-western regions of Spain, using indirect fluorescence antibody testing (IFAT) in 3,100 sera samples from apparently healthy horses of different ages, breeds, coat colours, genders and geographical locations. The overall seroprevalence was 52%, consisting of 44% seropositive for T. equi and 21% for B. caballi. There was a significant association between age (p < 0.0001), breed (p < 0.004), geographical location (p < 0.0001) and the seroprevalence, but neither the coat colour nor the gender was significantly associated with prevalence. In addition, it was proved that most of the geographic areas showed a moderate to high prevalence. The statistical κ value was used to compare the results obtained by the IFAT and the competitive enzyme-linked immunosorbent assay (cELISA) utilised to test some samples (n = 108) and showed a higher concordance for T. equi (κ = 0.68) than for B. caballi (κ = 0.22). Consequently, this revealed the importance of developing an appropriate technique to detect each haemoparasite.

Genotyping of Theileria lestoquardi from sheep and goats in Sudan to support control of Malignant Ovine Theileriosis.

Theileriosis, caused by parasitic protozoa of the genus Theileria parasites, are among the major tick-borne diseases of ruminant livestock. The largest economic losses are attributed in particular to those caused by the leukoproliferative species of Theileria: T. parva, T. annulata and T. lestoquardi. Theileria lestoquardi is transmitted by Hyalomma ticks and causes malignant ovine theileriosis (MOT), a disease that is particularly prevalent in Sudan. The disease is considered of a high economic importance in Sudan, where export of sheep is a major component of the national economy. A live vaccine based on a Sudanese isolate of T. lestoquardi (Atbara strain) was previously developed for the control of MOT in Sudan, but not yet deployed in the field. The present study aims to genetically characterize and compare samples of T. lestoquardi circulating in Sudan as well as the live vaccine isolate in order to understand vaccine breakthroughs and failure that may occur. Sheep and goats blood samples were collected from six regions in Sudan that are known to be endemic for T. lestoquardi infection or have experienced outbreaks of MOT. Blood samples infected with T. lestoquardi were identified by PCR or RLB. Genotyping was carried out by (1) sequencing the homologues of two T. parva CD8+ T cell antigen genes, Tp1 and Tp2, and (2) using a panel of seven micro- and mini-satellite markers. A total of 100 T. lestoquardi positive field samples and the T. lestoquardi (Atbara) vaccine were genotyped. The results showed that all samples had mixed genotypes, with several alleles identified at one or more loci. The gene diversity ranged from 0.7840 (TS8) to 0.2133 (TS12) with mean values of 0.5470. PCA revealed three clusters of the parasite in Sudan; interestingly one independent cluster was clearly seen, corresponding to the vaccine isolate. The T. lestoquardi Tp1 homologue showed higher homology with T. annulata than with T. parva sequences included the defined single CD8+ T cell target epitope region. The result indicates that multiple genotypes are a common feature of T. lestoquardi infection in Sudan. Both genotyping and the sequencing results clearly showed that the vaccine isolate is highly distinct from the field samples. This finding raised the question whether vaccination with the prepared lived vaccine will effectively protect animals against challenges by the field isolates of T. lestoquardi. The results of this work will inform on the best approach for controlling MOT in Sudan.