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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Co-infection with tick-borne disease agents in cattle in Russia.

Tick-borne diseases cause significant livestock losses worldwide. In Russia, information concerning single or mixed infections with different Anaplasma, Theileria and Babesia species in cattle is very limited. This study was conducted to determine the level of co-infection with protozoan pathogens (Theileria spp. and Babesia spp.) and rickettsial pathogens (A. marginale and A. phagocytophilum) in cattle in central Russia. Blood samples were examined with real time polymerase chain reaction (RT-PCR) for A. marginale and A. phagocytophilum, and by amplifying the V4 hypervariable region of the 18S rRNA gene, followed by cloning, DNA sequencing, and phylogenetic analyses, for Babesia and Theileria species. In total 67% of examined blood samples were positive for Theileria spp. or A. marginale, and 19% of the animals were co-infected with Theileria spp. and A. marginale. Seasonal variation in prevalence was found for Theileria spp. Phylogenetic analysis based on 18S rRNA gene sequences revealed the presence of five Theileria species: T. annulata, T. orientalis, T. buffeli, T. sergenti, and T. sinensis. No samples were positive for Babesia spp. or A. phagocytophilum. The data obtained for prevalence of bovine theileriosis and anaplasmosis in the central part of Russia underscore the need for improved surveillance and control programs to reduce tick-borne diseases in cattle.

Increased Trypanosoma spp. richness and prevalence of haemoparasite co-infection following translocation.

BACKGROUND: Understanding how fauna translocation and antiparasitic drug treatment impact parasite community structure within a host is vital for optimising translocation outcomes. Trypanosoma spp. and piroplasms (Babesia and Theileria spp.) are known to infect Australian marsupials, including the woylie (Bettongia penicillata). However relatively little is known about these haemoparasites, or how they respond to management practices such as translocation. We monitored haemoparasites infecting woylies for up to 12 months during two fauna translocations to supplement existing woylie populations in three different sites (Dryandra, Walcott and Warrup East) within south-western Australia between 2014 and 2016, with the aim of investigating (i) how haemoparasite prevalence, Trypanosoma spp. richness and Trypanosoma spp. community composition varied over time and between different sites following translocation; and (ii) whether ivermectin treatment indirectly impacts haemoparasite prevalence. Using molecular methods, 1211 blood samples were screened for the presence of trypanosomes, and a subset of these samples (n = 264) were also tested for piroplasms. RESULTS: Trypanosomes and piroplasms were identified in 55% and 94% of blood samples, respectively. We identified five Trypanosoma species, two Theileria species, a single species of Babesia and a novel Bodo species. Trypanosoma spp. richness and the prevalence of haemoparasite co-infection increased after translocation. Prior to translocation, Trypanosoma spp. community composition differed significantly between translocated and resident woylies within Walcott and Warrup East, but not Dryandra. Six months later, there was a significant difference between translocated and resident woylies within Dryandra, but not Walcott or Warrup East. The response of haemoparasites to translocation was highly site-specific, with predominant changes to the haemoparasite community in translocated woylies occurring within the first few months following translocation. Ivermectin treatment had no significant effect on haemoparasite prevalence. CONCLUSIONS: This study contributes to our understanding of haemoparasite dynamics in woylies following translocation. The highly site-specific and rapid response of haemoparasites to translocation highlights the need to better understand what drives these effects. Given that haemoparasite prevalence and composition of translocated and resident animals changed significantly following translocation, we propose that parasite monitoring should form an essential component of translocation protocols, and such protocols should endeavour to monitor translocated hosts and cohabiting species.

The haematological, proinflammatory cytokines and IgG changes during an ovine experimental theileriosis.

Malignant ovine theileriosis is caused by Theileria lestoquardi, which is highly pathogenic in sheep. Theileriosis involves different organs in ruminants. Little is known about the role of proinflammatory cytokines in the pathogenesis of T. lestoquardi infection. The aim of this study was to measure concentration changes of proinflammatory cytokines and immunoglobulin G (IgG) during an ovine experimental theileriosis and correlate it with clinical and haematological parameters. During an experimental study, seven healthy Baluchi sheep (four females and three males) about 6-8 months old were infected with T. lestoquardi by feeding of infected unfed ticks on the sheep's ears. The infected sheep were clinically examined during the study and blood samples were collected on days 0, 2, 5, 7, 10, 12, 14, 17 and 21. The haematological parameters were analysed by an automatic veterinary haematology cell counter and the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IgG were measured by enzyme-linked immunosorbent assay. All infected sheep had temperatures above 40 °C on days 3-4 post infection (PI). The maximum temperature was noted on day 7, and it remained high until day 21. The parasitaemia of T. lestoquardi infection increased from 0.01% (day 7 PI) to 3.3% (day 21 PI). The mean white blood cell (WBC), red blood cell (RBC), lymphocyte, neutrophil and platelet values slightly increased on day 2 PI and decreased by day 17 and day 21 PI. The percentage parasitaemia and fever had a negative correlation with the numbers of WBCs, RBCs, lymphocytes, neutrophils and platelets. The serum concentration of IL-6, TNF-α and IFN-γ cytokines increased and peaked on day 12 and thereafter decreased to levels lower than 0. Out of all tested cytokines, the concentration of IL-6 was significantly higher, as early as day 2 PI. No significant changes were observed for the IgG levels during the course of disease. A significant and strong correlation was observed between IL-6, TNF-α and IFN-γ values and a moderate correlation between IL-6 and the numbers of lymphocytes in the present study. A strong correlation was determined between the percentage parasitaemia and haematological parameters in T. lestoquardi-infected sheep. In addition, preliminary results indicate that the measurement of the serum concentrations of IL-6 in combination with haematological parameters could be considered a good marker to estimate the pathogenicity of T. lestoquardi strain.

Genetic Diversity of Theileria equi From Horses In Different Regions of Brazil Based On the 18S rRNA Gene.

Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (∼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.