A comprehensive molecular survey of vector-borne blood parasites in cattle in Kyrgyzstan with a note of the first molecular detection of Anaplasma bovis and Candidatus Anaplasma Camelii.

Vector-borne pathogens continue to increase their impact on the livestock industry worldwide. To protect animals against these pathogens, it is very important to identify the species that cause the disease and understand their prevalence. This study aimed to investigate the presence and prevalence of vector-borne pathogens in apparently healthy cattle in different parts of Kyrgyzstan using molecular diagnostic techniques. For this purpose, 531 blood samples were collected from the Osh, Jalal-Abad, and Batken oblasts of Kyrgyzstan. The blood samples were investigated for vector-borne pathogens using PCR, RLB, and RFLP. Moreover, DNA sequence analyses were used to confirm the results of molecular techniques and phylogenetic analyses of these pathogens. 359 (67.61%) out of 531 samples were found to be infected with at least one pathogen, whereas 172 (32.39%) were detected to be negative. Thirteen vector-borne pathogens were detected in cattle blood samples, and the prevalence of these pathogens was as follows: Theileria orientalis (47.83%), T. annulata (25.61%), Babesia major (0.19%), B. occultans (0.38%), Anaplasma phagocytophilum-like 1 (3.20%), A. capra (3.01%), A. centrale (2.82%), A. bovis (1.13%), (A) ovis (0.19%), Candidatus Anaplasma camelii (0.94%), Trypanosoma theileri (19.21%), Mycoplasma wenyonii (6.03%), and Ca. Mycoplasma haemobos (2.64%). Among the positive samples, one pathogen was identified in 189 cattle (35.59%), and co-infections (two or more pathogens) were determined in 170 (32.01%) animals. Theileria parva, T. mutans, (B) bigemina, B. bovis, B. divergens, and A. marginale could not be detected in the study. Anaplasma bovis and Ca. Anaplasma camelii were detected for the first time in the country. This molecular survey provides important epidemiological and genetic data for the vector-borne pathogens in cattle. The results of the study showed that vector-borne pathogens have a significant spread and distribution in cattle in Kyrgyzstan.

Evaluation of Oxidative Stress, Thyroid Hormones, Trace Elements and Some Biochemical Markers in Goats Naturally Infected with Theileria ovis.

BACKGROUND: Theileriosis is a tick-borne disease caused by protozoon species in the Theileria genus of the Theileriidae family. The biochemical changes induced by infection are considered to be an important understanding of the pathophysiology of caprine theileriosis. In this study, it was aimed to determine oxidative stress, thyroid hormones, trace elements, and biochemical parameters in theileriosis infection. MATERIALS AND METHODS: A sample of 14 goat was used for this purpose, of which 7 were healthy and 7 were infected with Theileria ovis. Theileria infection was diagnosed from the polymerase chain reaction (PCR). Sera from blood samples was tested for total antioxidant capacity (TAC), total oxidant capacity (TOC), oxidative stress index (OSI), alanine aminotransferase (ALT), aspartate transaminase (AST), gamma glutamyl transferase (GGT), total protein, albumin, triglyceride, cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), urea, blood urea nitrogen (BUN), creatinine, triiodothyronine (T3), thyroxine (T4), copper (Cu), zinc (Zn), cobalt (Co), manganese (Mn), selenium (Se), iron (Fe). RESULT: TOC, OSI, AST, ALT and GGT values were higher in the patient group than in the healthy group (P < 0.05). On the other hand, there were decreases in TAC, T3, T4, total protein, albumin, creatinine, Cu, Zn, Se, and Co values (P < 0.05). However, there was not found to be a statistical difference between the healthy and patient groups in terms of triglyceride, cholesterol, HDL, LDL, urea, BUN, Mn, and Fe values (P > 0.05). CONCLUSIONS: It can be stated that oxidative stress is a complication of caprine theileriosis and it may be accompanied with hypothyroidism and deficits in trace minerals.

Molecular prevalence, associated risk factors and phylogenetic evaluation of Theileria lestoquardi in the blood samples of small ruminants.

Raising small ruminants is the main source of income for farmers in Pakistan especially in rural areas of Dera Ghazi Khan in Punjab. Despite having large sheep population, the prevalence of intra-erythrocytic protozoa, Theileria (T.) lestoquardi, has never been reported from this area. This study was conducted to fill this knowledge gap and 333 blood samples of apparently healthy small ruminants (168 sheep and 165 goats) along with their epidemiological data were collected from Dera Ghazi Khan district during August till November 2022. The polymerase chain reaction (PCR) analysis amplified a 785 base pair amplicon specific for the Merozoite surface antigen (ms 1-2) gene of T. lestoquardi in 2 out of the 168 (3.3%) sheep blood samples, while no goat blood sample out of 165 was found to be infected with T. lestoquardi. DNA sequencing confirmed the presence of Theileria lestoquardi in both samples and phylogenetic analysis revealed that these amplicon resembled the partial ms 1-2 gene sequences detected in small ruminants from Pakistan, India Iran and Egypt. All the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, composition of herd, size of herd and Tick burden on sheep) were not found associated with the prevalence of T. lestoquardi. In conclusion, this study reports a low prevalence of T. lestoquardi infection in the Dera Ghazi Khan District of Punjab, Pakistan. The data generated from this work will help pave the way for the prophylactic detection and control of ovine and caprine theileriosis in the region.

Molecular detection of tick-borne piroplasmids in camel blood samples collected from Cairo and Giza governorates, Egypt.

Piroplasmosis, a tick-borne disease affecting livestock, including camels, is caused by intracellular apicomplexan parasites belonging to the order Piroplasmida. Despite its importance, there's limited research on piroplasmosis among Egyptian camels. This study aimed to fill this gap by investigating tick-borne piroplasmids in camels from Cairo and Giza Governorates. Out of 181 blood samples collected between October 2021 and March 2022 from apparently healthy one-humped camels (Camelus dromedarius), PCR assays revealed a 41.4 % infection rate with various piroplasmids. Detected species included B. bovis (17.7 %), B. bigemina (12.2 %), B. caballi (8.3 %), B. naoakii (11.6 %), B. microti (1.7 %), T. equi (4.4 %), and Theileria spp. (28.7 %). Phylogenetic analysis revealed the first detection of T. equi genotype E in Egypt and identified a novel B. caballi genotype. Additionally, B. microti isolates were identified as the US-type. These findings shed lights on piroplasmosis among Egyptian camels, and provide valuable information for devising effective control strategies, especially B. microti, a pathogen with potential human health risks.

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.

Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle.

BACKGROUND: Serious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle. METHODS: Molecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia. RESULTS: A total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83-81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98-100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle. CONCLUSION: We present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

Clinical and hematologic features of experimental theileriosis in roan calves (Hippotragus equinus).

Theileria sp. (sable) infection commonly causes significant calf mortality in endangered roan antelopes (Hippotragus equinus). Schizont-induced leukocyte transformation and systemic immune dysregulation with associated cytopenias characterizes theileriosis in livestock. Data on related hematologic alterations are scarce in roan antelopes. The objective of this study was to investigate temporal changes in selected clinical parameters and hematologic measurands in experimentally infected (EI) roan calves. Six of eight calves developed theileriosis after inoculation with a Theileria sp. (sable)-infected tick stabilate. Consecutive measures of rectal temperature, lymph node size, white blood cell count (WBC), packed cell volume (PCV), hemoglobin concentration, differential leukocyte counts, leukocyte and erythrocyte morphology and percentage parasitemia were recorded. Data were compared with 15 age-matched PCR-negative control calves and nine older immune animals that had recovered from natural infection. Two non-pyrexic EI calves recovered uneventfully. Six pyrexic calves were treated, four of which died. Time to pyrexia and/or observation of schizonts and piroplasms was approximately two weeks. Total WBCs were unchanged post-infection (PI); neutrophils and typical monocytes decreased whereas typical lymphocytes (Ls) and atypical mononuclear leukocytes (AMLs), which were grouped together, increased. Parasitized and non-parasitized lymphocyte and AML (L/AML) size increased significantly PI. Piroplasms occurred intermittently at low frequencies («1 % of erythrocytes) after infection. Fatally infected calves were dehydrated, anemic, and icteric with hemorrhages and multi-organ infiltration by AMLs. The PCV and hemoglobin concentration increased PI, and platelet clumps were consistently observed. Experimental acute theileriosis in roan antelopes follows a similar pattern of disease progression to that in domestic livestock. Parasitized and non-parasitized AMLs are pivotal to the pathogenesis and require phenotypic characterization if we are to further our understanding of disease progression and severity in roan antelopes.

Whole genome sequencing of Theileria parva using target capture.

Protozoan parasite isolation and purification are laborious and time-consuming processes required for high quality genomic DNA used in whole genome sequencing. The objective of this study was to capture whole Theileria parva genomes directly from cell cultures and blood samples using RNA baits. Cell culture material was bait captured or sequenced directly, while blood samples were all captured. Baits had variable success in capturing T. parva genomes from blood samples but were successful in cell cultures. Genome mapping uncovered extensive host contamination in blood samples compared to cell cultures. Captured cell cultures had over 81 fold coverage for the reference genome compared to 0-33 fold for blood samples. Results indicate that baits are specific to T. parva, are a good alternative to conventional methods and thus ideal for genomic studies. This study also reports the first whole genome sequencing of South African T. parva.

Molecular prevalence of Theileria infections in cattle in Yanbian, north-eastern China.

Bovine Theileria are tick-borne protozoan parasites that invade bovine erythrocytes and lymphocytes. Three main bovine Theileria species have been identified in China: T. orientalis, T. sinensis, and T. annulata. To examine the prevalence of bovine theileriosis in Yanbian, a total of 584 bovine blood samples were collected from five localities from 2017 to 2019 and analyzed by PCR. Six pairs of oligonucleotide primers directed against the 18S rRNA gene of Theileria spp., Tams-1 gene of T. annulata, MPSP gene of T. orientalis, and T. sinensis, were used to detect these parasites. A sequence analysis of the amplified genes confirmed that the Theileria species were T. orientalis and T. sinensis, without T. annulata. The overall prevalence of Theileria in cattle was 42.81% (250/584). Out of the 584 samples, 159 (27.23%) and 157 (26.88%) were positive for T. sinensis and T. orientalis, respectively, and the mixed infection rate was 11.30% (66/584). The total prevalence of bovine Theileria species in Helong, Hunchun, Longjing, Yanji, and Dunhua was 66.28%, 49.68%, 23.81%, 28.15%, and 0%, respectively. These results provide epidemiological data for the prevention and control of bovine Theileria species in Yanbian, China.

TITLE: Prévalence moléculaire des infections à Theileria chez les bovins à Yanbian, dans le nord-est de la Chine. ABSTRACT: Les Theileria bovins sont des parasites protozoaires transmis par les tiques qui envahissent les érythrocytes et les lymphocytes des bovins. Trois espèces principales de Theileria de bovins ont été identifiées en Chine, T. orientalis, T. sinensis et T. annulata. Pour examiner la prévalence de la theilériose bovine à Yanbian, un total de 584 échantillons de sang bovin ont été collectés dans cinq localités de 2017 à 2019 et analysés par PCR. Six paires d'amorces oligonucléotidiques dirigées contre le gène d'ARNr 18S de Theileria spp., le gène Tams-1 de T. annulata et le gène MPSP de T. orientalis et T. sinensis, ont été utilisées pour détecter ces parasites. Une analyse de séquence des gènes amplifiés a confirmé que les espèces de Theileria étaient T. orientalis et T. sinensis, sans T. annulata. La prévalence globale des Theileria chez les bovins était de 42,81 % (250/584). Sur les 584 échantillons, 159 (27,23 %) et 157 (26,88 %) étaient positifs pour T. sinensis et T. orientalis, respectivement, et le taux d'infection mixte était de 11,30 % (66/584). La prévalence totale des espèces bovines de Theileria à Helong, Hunchun, Longjing, Yanji et Dunhua était respectivement de 66,28 %, 49,68 %, 23,81 %, 28,15 % et 0 %. Ces résultats fournissent des données épidémiologiques pour la prévention et le contrôle des espèces de Theileria de bovins à Yanbian, en Chine.

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.

Stress conditions do not affect Theileria equi parasitemia levels in sub-clinically infected horses.

Stress has been suggested as a risk factor for Theileria equi peracute disease and may lead to relapse in clinical signs in chronically infected horses. The aim of this study was to assess the effect of stress on T. equi parasitemia in sub-clinically infected horses in two settings: horses hospitalized at a veterinary teaching hospital and horses from an endurance farm. Blood samples were collected from the hospitalized horses (n = 32) upon admission (T0) and at discharge (T1) from the hospital, and results were compared between horses that underwent surgery (stress) and other hospitalized horses (control). Blood samples were collected from an endurance farm (n = 20) six weeks before (T0) and two days after (T1) participation in an 80-km endurance event, and results were compared between horses that participated (stress) or did not participate (control) in the event. Theileria equi parasite load was determined using qPCR, and T1/T0 ratio was calculated for each horse. Mean parasite load at both time points did not differ statistically between the stress group and the controls in both settings. Theileria equi genotype was determined based on the 18S rRNA gene, when possible. Parasite genotypes were similar to strains previously characterized in the region and classified as genotypes A and D. The results of this study contradict the common assumption that stress may lead to increased parasite load in horses with a subclinical T. equi infection.

Serological and molecular detection of selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan.

In Sudan, donkeys are important animals, providing transportation and income possibilities. However, the prevalence of parasites in donkeys in Sudan has not been thoroughly characterized. Accordingly, in this study, we aimed to detect selected hemoprotozoan parasites in donkeys in West Omdurman, Khartoum State, Sudan, wherein people depend mainly on donkeys for their daily life. In total, 198 blood samples collected from donkeys in a local market in West Omdurman, were screened using serological and molecular diagnostic techniques. Serologically, 52 (26.3%), 56 (28.3%), and 19 (9.6%) samples were positive for trypanosomosis using Card Agglutination Test for Trypanosoma evansi, Trypanosoma evansi crude antigen -based enzyme-linked immuno sorbent assay (ELISA) and recombinant Trypanosoma evansi GM6-4r-based ELISA, respectively. ELISA for equine piroplasmosis revealed 156 (78.8%) and 10 (5.1%)Theileria equi- and Babesia caballi-positive samples, respectively. PCR detected Trypanosoma congolense, subgenus Trypanozoon, Theileria equi, and Babesia caballi in 18 (9.1%), 77 (38.9%), 18 (9.1%), and 8 (4%) samples, respectively. Of the 77 Trypanozoon-positive samples, 35 (45.5%) were confirmed as Trypanosoma evansi type A. To our knowledge, this is the first report of detection of Trypanosoma congolense in donkeys outside of tsetse-infested areas in Sudan.

First Report Regarding the Simultaneous Molecular Detection of Anaplasma marginale and Theileria annulata in Equine Blood Samples Collected from Southern Punjab in Pakistan.

AIM: The present study was designed to check the molecular detection of Anaplasma marginale and Theileria annulata in blood samples of horses and donkeys collected from Dera Ghazi Khan District in Punjab and to document their phylogenetic origin and their association with studied epidemiological factors (sex and age) and complete blood count parameters, if any. METHODS AND RESULTS: A total of 195 blood samples were collected from apparently healthy horses (N = 141) and donkeys (N = 54). A. marginale DNA was detected by PCR in 4.9% (7/141) horse and in 9.2% (5/54) of donkey blood samples. Prevalence of T. annulata was 5.6% (8/141) and 11.1% (6/54) in horse and donkey samples, respectively. While 1.4% (N = 2) horses and 3.7% (N = 2) donkeys were found co-infected with both parasites. Representative amplicon for both parasites was confirmed by DNA sequenced and partial DNA sequence of the major surface protein-1b encoding gene of A. marginale and cytochrome b gene from T. annulata were submitted to the GenBank database under the accession number MK792344-MK792348. Epidemiological data analysis revealed that female horses were more prone to A. marginale (P = 0.02) while female donkeys were more susceptible to A. marginale (P < 0.001) and T. annulata (P < 0.001) infection. It was observed that horse and donkey infected either with Anaplasma marginale or Theileria annulata had significantly disturbed red and white blood cell counts and their associated parameters. CONCLUSION: This is a first ever study regarding molecular detection of A. marginale and T. annulata in equine blood samples from Pakistan. We recommend that this multiplex PCR protocol should be used for the detection of Anaplasma marginale and Theileria annulata in livestock for their proper diagnosis and treatment.

FTA-Sodium hydroxide-based polymerase chain reaction (PCR): An efficient and cheaper option for Theileria parva detection in dairy cattle in Mbarara, Uganda.

East Coast fever is caused by Theileria parva, and poses serious concerns for dairy farmers owing to massive economic losses. In the current study, we compared three methods (DNA extraction kits, FTA-NaOH and FTA-TENT) of DNA extraction to identify the most economical and reliable method. A survey for T. parva prevalence was conducted in dairy cattle in Mbarara, Uganda. Cytochrome C oxidase subunit I (COI) and T. parva-p104 genes were amplified to compare the methods. FTA-NaOH-based polymerase chain reaction (PCR) yielded the best detection rate for both COI gene and p104 gene. Prevalence of T. parva was 45.0% and 83.3% at animal and farm-level, respectively. FTA-NaOH based-PCR is simple, highly sensitive and cost-effective tool for T. parva diagnosis in resource constrained settings.

Molecular Detection of Theileria ovis and Theleiria equi in Livestock from Palestine.

Theileria and Babesia are intracellular protozoan parasites infecting a wide range of animals. In Palestine, there is limited information on the prevalence of Theileria and Babesia spp. in livestock. We used PCR of the 18S ribosomal RNA gene followed by DNA sequencing to detect and identify parasite DNA in blood samples from sheep (n = 49), goats (n = 48), horses (n = 40), camels (n = 34), donkeys (n = 28) and mules (n = 2) from four districts of Palestine. DNA of T. ovis and T. equi was detected in 19 and 2 ovine blood samples, respectively. None of the camels, donkeys, and goats were positive for T. ovis. Sheep had a significantly higher rate of infection than other animals (P < 0.05). Theileria ovis is highly prevalent in sheep, while T. equi DNA was detected in a small proportion of the equids in Palestine.

Assessment of the prevalence of Theileria lestoquardi in sheep from the Sudan using serological and molecular methods.

Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered to be among the most important tick borne diseases in the Sudan. Information on the prevalence of the disease in different parts of the Sudan is limited. The purpose of this study was to estimate the prevalence of the disease in five states of the Sudan using molecular and serological assays. A total of 393 blood and serum samples from clinically asymptomatic sheep were analysed using nested reverse line blot (nRLB) and loop mediated isothermal amplification (LAMP), as well as an enzyme-linked immunosorbent assay (ELISA). The results indicated a sero-prevalence of 33.8% while RLB and LAMP assays revealed molecular prevalences of 29.5 and 22.6% respectively. The prevalence of Theileria lestoquardi varied significantly according to the geographical origin of the infected animals, whereas age and gender did not have a significant effect. RLB data indicated that T. lestoquardi usually occurred as a co-infection with the non-pathogenic Theileria ovis. Using RLB as a gold standard, a sensitivity of 68.1% and a specificity of 96.4% were recorded for LAMP and a sensitivity of 75.9% and a specificity of 83.8% for ELISA. The Kappa coefficient between nRLB and LAMP indicated a significant level of agreement (0.692), but only moderate concordance (0.572) between nRLB and ELISA. The results of the present study confirm and extend earlier findings regarding the widespread of T. lestoquardi infections in sheep in the Sudan. The data provide evidence that should enable the veterinary authorities to deploy appropriate control measures.

First Report of Theileria Infection of Bactrian Camels (Camelus bactrianus) in Xinjiang, China.

INTRODUCTION: Protozoan Theileria is transmitted by tick vectors to some animals worldwide and causes considerable economic damage. The detection of pathogens in camels is not only crucial for the preservation of species but also provides important information on the epidemiologic chain of diseases. MATERIALS AND METHODS: We conducted a molecular detection in Xinjiang, China to assess the impact of Theileria infection in Bactrian camels and ticks in 2016. The 18S rRNA and MPSP gene sequences of T. sinensis obtained from Bactrian camels and ticks in Xinjiang, China have been deposited in the GenBank database. RESULTS: PCR revealed that 6.56% of the Bactrian camel blood samples, 2.75% of Rhipicephalus sp., 3.81% of Hy. asiaticum, 4.32% of Hy. dromedarii, and 0% of D. niveus tick samples were positive for T. sinensis, respectively; no other Theileria and Babesia were found in these samples and D. niveus samples. The results showed that one Theileria parasite, Theileria sinensis, was found in Bactrian camels (Camelus bactrianus), Rhipicephalus sp., Hy. asiaticum, and Hy. dromedarii in Xinjiang, respectively. CONCLUSION: This is the first detection of T. sinensis DNA in Bactrian camels from China. Our results provide important data to increase the understanding of theileriosis of Bactrian camel, and will aid in taking effective measures to control theileriosis transmission to camels, cattle and other ruminants in Xinjiang, China.

First molecular survey of piroplasm species in cattle from Kyrgyzstan.

Bovine piroplasmosis is a tick-borne disease caused by apicomplexan hemoparasites of the genera Theileria and Babesia. This study was carried out to assess the presence and frequency of piroplasm parasites in apparently healthy cattle in Kyrgyzstan. A total of 454 blood samples were collected from animals of various ages in eight villages located in the Chu valley and around the Lake Issyk Kul. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers specific targeting members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic and species-specific oligonucleotide probes were covalently linked. The results revealed the presence of three piroplasm species (Theileria orientalis, Babesia major, Theileria annulata). Theileria orientalis was the most prevalent species (32.8%; CI 28.5-37.3). Babesia major was the only species of Babesia found in any of the samples (1.3%; CI 0.5-2.8). The co-existence of Theileria annulata and T. orientalis was detected in nine animals (1.9%; CI 0.9-3.7). BLAST search revealed that the Theileria sequences shared 100% identity with the recently reported sequences for T. buffeli and T. annulata. The sequence of B. major was also 100% identical to an existing B. major sequence. This molecular survey provides important epidemiological data for control of bovine piroplasmosis caused by T. orientalis, B. major, and T. annulata in Kyrgyzstan.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 µM concentration as baseline revealed nine effective compounds against four tested parasites. Two "hit" compounds, namely MMV021057 and MMV675968, that showed IC50 < 0.3 µM and a selectivity index (SI)> 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs.

Establishment and Expression of Cytokines in a Theileria annulata-Infected Bovine B Cell Line.

This study aimed to establish a pure single-cell Theileria annulata-infected B cell line for the assessment of cytokine production in transformed and lipopolysaccharide (LPS)-stimulated cells. Several studies have aimed to identify cell surface markers in T. annulata-transformed cells; however, no information on cytokine production in these cells is available. To investigate the potential of the transformed cells to produce cytokines and their potential responses to antigen-stimulation, we purified mature B cells (CD21) from the whole blood of cattle experimentally infected with the T. annulata Kashi strain by magnetic separation. The purity and specificity of the established cell line was assessed by the identification of specific cell surface markers (CD21, IgM, and WC4) by flow cytometry analysis. The transcript levels of the cytokines IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL16, LTA, TGFB1, TNFA, IFNA, and IFNB in transformed, buparvaquone (BW720c)-treated cells, and antigen-stimulated cells were analyzed by quantitative polymerase chain reaction (qPCR) using cDNA from these cells. A T. annulata-infected bovine B cell line was successfully established with a purity of ~98.8% (CD21). IL4 and IL12A were significantly (p < 0.01) upregulated in the transformed cells. In BW720c-treated transformed cells, IL12B, TGFB1, and IFNB were significantly (p < 0.01) upregulated. Notably, no significant (p > 0.05) upregulation of cytokines was observed in LPS-stimulated transformed cells. Moreover, IL1A, IL1B, IL8, and IL16 were significantly (p < 0.01) upregulated in LPS-stimulated B cells. Our data signify the potential use of this cell line for cytokine production, observance of immunoglobulins, and production of an attenuated vaccine against tropical theileriosis.