Unveiling genotypic diversity of Theileria orientalis in lethal outbreaks among bovines in Karnataka, India.

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts.

Molecular occurrence and genetic identification of Babesia spp. and Theileria spp. in naturally infected cattle from Thailand.

Piroplasm including Babesia spp. and Theileria spp. in cattle can cause illness that affects livestock productivity, resulting in significant production losses, especially in tropical and subtropical regions such as Thailand. This study aimed to investigate the prevalence of bovine piroplasms and to identify these blood parasites based on the 18S ribosomal RNA gene in cattle in the northeastern part of Thailand. Piroplasmid infections among beef and dairy cattle were examined using nested PCR. Furthermore, amplicon DNA was sequenced and analyzed, and a phylogenetic tree was constructed to determine the genetic diversity and relationships of the parasite in each area. A total of 141 out of 215 (65.6%) cattle were positive for infection with Babesia or Theileria. DNA analysis revealed that infection by Babesia bigemina, Babesia bovis, Theileria orientalis, Theileria sinensis, and Theileria sp. were common piroplasms in cattle in this region, with a high sequence shared identity and similarity with each other and clustered with isolates from other countries. This study provides information on the molecular epidemiology and genetic identification of Babesia spp. and Theileria spp. in beef and dairy cattle to provide a better understanding of piroplasm infection in cattle in this region, which will help control these blood parasites. Moreover, this is the first report identifying T. sinensis circulating among Thai cattle.

Theileria orientalis Ikeda infection detected in red deer but not dogs or horses in New Zealand.

AIMS: To determine whether evidence for infection with Theileria orientalis (Ikeda) could be identified in samples of commercial red deer (Cervus elaphus), horses, and working farm dogs in New Zealand. METHODS: Blood samples were collected during October and November 2019 from a convenience sample of red deer (n = 57) at slaughter. Equine blood samples (n = 50) were convenience-sampled from those submitted to a veterinary pathology laboratory for routine testing in January 2020. Blood samples, collected for a previous study from a convenience sample of Huntaway dogs (n = 115) from rural regions throughout the North and South Islands of New Zealand between August 2018 and December 2020, were also tested. DNA was extracted and quantitative PCR was used to detect the T. orientalis Ikeda major piroplasm surface protein (MPSP) gene. A standard curve of five serial 10-fold dilutions of a plasmid carrying a fragment of the T. orientalis MPSP gene was used to quantify the number of T. orientalis organisms in the samples. MPSP amplicons obtained by end-point PCR on positive samples were isolated and subjected to DNA sequencing. The resulting sequences were compared to previously published T. orientalis sequences. RESULTS: There were 6/57 (10%) samples positive for T. orientalis Ikeda from the deer and no samples positive for T. orientalis Ikeda from the working dogs or horses. The mean infection intensity for the six PCR-positive deer was 5.1 (min 2.2, max 12.4) T. orientalis Ikeda organisms/µL. CONCLUSIONS AND CLINICAL RELEVANCE: Red deer can potentially sustain low infection intensities of T. orientalis Ikeda and could act as reservoirs of infected ticks. Further studies are needed to determine whether naïve ticks feeding on infected red deer can themselves become infected. ABBREVIATIONS: Cq: Quantification cycle; LOQ: Limits of quantification; MPSP: Major piroplasm surface protein; qPCR: Quantitative polymerase chain reaction.

Occurrence and genotyping of Theileria equi in dogs and associated ticks in Egypt.

Theileriosis is a tick-borne protozoal disease caused by a piroplasm of the genus Theileria. Hard ticks are obligate hematophagous ectoparasites that serve as vectors of Theileria spp. Studies of the presence of theileriosis in Egyptian dogs and associated ticks are scarce. This study was conducted to detect and identify Theileria spp. in dogs and Rhipicephalus sanguineus ticks and to monitor the epidemiological data of this disease. The prevalence rates of Theileria equi infection were 12.02%, 0.73%, 2.93%, and 1.83% by microscopic examination of dog blood, tick hemolymph, tick midgut, and tick salivary smears, respectively. Conversely, the T. equi prevalence in dog blood and associated ticks assessed by PCR was 25.81% and 10.42%, respectively. Epidemiological data about Theileria infection revealed a significant difference in the infection between different seasons and different dog breeds (p value <0.05), whereas host, sex, and age of dogs had no significant effect on the infection. Sequencing of PCR products showed that all PCR positive samples were infected with T. equi. Transmission electron microscopy (TEM) described the different stages of Theileria in the midgut and salivary gland of infected ticks. The current study confirmed that T. equi is not specific to equine hosts, and confirmed that dogs are a susceptible host to T. equi.

Growth inhibitory effect of selected quinones from Indian medicinal plants against Theileria annulata.

Tropical Bovine Theileriosis, caused by the protozoan parasite Theileria annulata, poses a significant threat to cattle populations. Currently, Buparvaquone is the sole effective naphthoquinone drug commercially available for its treatment. In our research, we delved into the potential of naturally occurring quinones as alternative treatments. We isolated two quinones, emodin and chrysophanol, from Rheum emodi Wall, and two more, embelin and lawsone, from Embelia ribes Burm.f. and Lawsonia inermis L. respectively. We assessed the anti-Theileria efficacy of these quinones in vitro using MTT and flow cytometric assays on T. annulata-infected bovine lymphocytes. Additionally, we evaluated their safety on uninfected bovine Peripheral Blood Mononuclear Cells (PBMC) and Vero cells. Emodin emerged as a promising candidate, exhibiting an IC50 value of 4 µM, surpassing that of buparvaquone. Emodin also displayed relatively low LD50 values of 1.74 mM against uninfected PBMC and 0.87 mM against Vero cells, suggesting potential safety. Remarkably, emodin demonstrated a high cell absorption rate of 71.32%. While emodin's efficacy and bioavailability are encouraging, further research is imperative to validate its safety and effectiveness for treating Tropical Bovine Theileriosis.

Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle.

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

Comparative evaluation of cell-mediated immune response in calves immunized with live-attenuated and killed Theileria annulata vaccines.

Tropical theileriosis is a tick-borne disease caused by the protozoan Theileria annulata and transmitted by numerous species of Ixodid ticks of the genus Hyalomma. The main clinical signs are fever, lymphadenopathy, and anemia responsible for heavy economic losses, including mortality, morbidity, vaccination failure, and treatment cost. Development of poor cell-mediated immunity (CMI) has been observed in the case of many bovine pathogens (bacteria, viruses, and parasites). Quantification of CMI is a prerequisite for evaluating vaccine efficacy against theileriosis caused by T. annulata. The current study evaluated the CMI in calves administered with two types of T. annulata vaccine (live attenuated and killed). We prepared a live attenuated T. annulata vaccine by attenuation in a rabbit model and also prepared killed vaccine from non-attenuated T. annulata. For the evaluation of immune response in experimental groups including control, 20 calves were divided into four different groups (A, B, C, and D). They were either inoculated subcutaneously with live rabbit-propagated-Theileria-infected RBCs (5 × 106) (group A) or with killed T. annulata vaccine (2 × 109 schizonts) with Freund's adjuvant (group B), along with an infected group (group C) and a healthy control group (group D). The protection of vaccinated calves was estimated with challenge infection. Our results showed that with a single shot of live-attenuated and killed vaccine with a booster dose elicited cell-mediated immune responses in immunized calves. We observed a significant elevation in CD4 + and CD8 + T cells in immunized calves. A significant difference in the CD8 + T cell response between the post-challenge stage of killed and live vaccine (p < 0.0001) was observed, whereas no other difference was found at both pre- and post-immunization stages. A similar finding was recorded for the CD4 + T cells at a post-challenge stage, where a significant difference was seen between killed and live vaccine (p < 0.0001). Another significant difference was observed between the CD8 + T cells and CD4 + T cells at the post-challenge stage in the live vaccine group, where there was a significantly higher induction of CD4 + T cell response (p < 0.0001).

Communication medium in theileriosis control: the factors that determine disease knowledge among smallholder farmers in Zimbabwe.

Theileriosis is one of the most important tick-borne diseases that has been affecting farmers and thousands of livestock in Zimbabwe. The main government strategy to combat theileriosis is use of plunge dips with anti-tick chemicals at specified times; however, an increase in number of farmers caused a strain on government services resulting in disease outbreak. One of the key issues that have been highlighted by department of veterinary is the strain in communication and knowledge of the disease with the farmers. Hence, it is important to evaluate the communication between farmers and veterinary services and identify possible areas of strain. A field survey was conducted with 320 farmers in Mhondoro Ngezi, a district badly affected by theileriosis. Face-to-face interviews with smallholders and communal farmers were conducted between September and October 2021, and the data were analyzed using Stata 17. Communal farmers relied mainly on oral communication and had limited knowledge of theileriosis; therefore, dead cattle % was high among them. Though veterinary extension officers were the prime source of information, oral communication medium affected knowledge transferred. The results of this study recommend adoption of communication mediums that encourage retention, such as brochures and posters by veterinary extension services. The government may also partner with private players to ease pressure of increased farming population due to land reform.

In vitro growth inhibitory effect of selected 18ß-glycyrrhetinic acid esters on Theileriaannulata.

Glycyrrhetinic acid (GA) is one of the important Pentacyclic Triterpenoids (PT) found in the roots of licorice. This study aimed to evaluate the in vitro growth inhibitory effect of 18ß-GA (18ß-Glycyrrhetinic acid) and C-30 esters against Theileria annulata, the causative agent of Tropical Bovine Theileriosis. C-30 esters of 18ß-GA were synthesized and their structures were elucidated using spectroscopy. The pharmacodynamic properties of 18ß-GA and its C-30 esters were predicted using DataWarrior and Swiss ADME tools. Cattle isolates of T. annulata schizont-infected bovine lymphoblastoid cells were cultured using standard conditions and the growth inhibitory effect of GA and its esters were evaluated using MTT assay. The isopropyl ester of 18ß-GA (GI50- 1.638 µM; R2- 0.818) showed improved anti-theileriosis efficacy than other 18ß-GA derivatives. The propyl (GI50 - 5.549 µM), ethyl (GI50 - 5.638 µM), and benzyl (GI50 - 7.431 µM) esters also showed considerable inhibitory effect. The GI50 value for 18ß-GA was recorded as 6.829 µM. This study throws light on the usefulness of 18ß-GA and its esters for the treatment of Tropical Bovine Theileriosis.

Theileriosis in naturally infected roan antelope (Hippotragus equinus).

Cases of Theileria-associated mortality are rarely reported in African wild artiodactyls. Descriptions of lesions are limited, particularly in endangered hippotraginids. Here, we analyzed retrospectively the gross and histologic findings in 55 roan antelope (Hippotragus equinus) with fatal natural theileriosis. The most frequently recorded gross findings in 40 cases were widespread petechiae and ecchymoses (72.5%), probable anemia (67.5%), icterus (60%), splenomegaly (60%), hepatomegaly (52.5%), and pulmonary edema (50%). Histologic lesions in 34 cases were characterized by multi-organ infiltrates of parasitized and nonparasitized mononuclear leukocytes (MLs), and fewer multinucleate giant cells (MNGCs). Liver, lung, kidney, adrenal gland, and heart were most consistently infiltrated, followed by spleen and lymph nodes. Leukocytes were phenotyped in lung, liver, kidney, and heart specimens from 16 cases, using immunohistochemistry to detect CD20, CD3, myeloid/histiocyte antigen (MAC387), IBA-1, and CD204 surface receptors. A roan polyclonal anti-Theileria sp. (sable) antibody was applied to the same tissues to identify intraleukocytic parasite antigens. Similar proportions of intravascular and extravascular IBA-1-, CD204-, and MAC387-reactive putative monocyte-macrophages and fewer CD3-positive putative T-lymphocytes were identified in all organs, especially the lungs in infected roan. CD20-positive putative B-lymphocytes were significantly scarcer than in uninfected controls. Intraleukocytic Theileria parasites labeled consistently in affected tissues. Some parasitized and nonparasitized MLs and the MNGCs failed to label with selected leukocyte markers. Fatal theileriosis in roans may largely be the result of multi-organ monocyte-macrophage activation with associated tissue injury and overwhelming systemic inflammation. The identity of the parasitized leukocytes and characteristics of the lymphohistiocytic response require further clarification in roans.

Nested PCR assay for detection of Theileria annulata in Hyalomma anatolicum infesting cattle from coastal Odisha, India.

Ticks are economically important obligatory blood feeding arthropods that have a pivotal role in transmission of infection. The present study was conducted in ixodid ticks collected from four districts of coastal Odisha, India to investigate the prevalence of Theileria annulata. Adult semi engorged Hyalomma anatolicum ticks (n = 178) were dissected, the salivary gland was isolated and DNA was extracted. A nested PCR targeting the Tams1 gene of T. annulata, utilizing two sets of primers (N516F, N517R, and Ta14136iF, Ta249R) was utilized for detection of the parasite. The PCR products were then sequenced and subjected to BLAST analysis, alignment, and phylogenetic study. Two sequences deposited in GenBank were assigned Accession No MH477290.1 and Accession No MH477291.1. The molecular investigation of T. annulata revealed an overall prevalence of 14.6% in tick vectors, and nested PCR was found to have significant (p < 0.05) higher results than primary PCR. A significant higher presence (p < 0.05) was recorded in female ticks compared with male ticks. This is the first report of detection of the parasite in tick vectors in the state of Odisha.

Establishment and application of a qPCR diagnostic method for Theileria annulata.

Bovine theileriosis caused by several Theileria species including Theileria annulata, Theileria parva, Theileria orientalis, Theileria mutans, and Theileria sinensis is a significant hemoprotozoan tick-borne disease. Among these, Theileria species, T. annulata, which causes tropical theileriosis (TT), is regarded as one of the most pathogenic and is responsible for high mortality. At present, most conventional diagnostic methods for tropical theileriosis are time-consuming and laborious and cannot distinguish newfound T. sinensis in China. Therefore, a high sensitivity and specificity real-time quantitative PCR method based on the TA19140 target molecule was developed, and the method was found to be specific for T. annulata. No cross-reaction was observed with T. sinensis, T. orientalis, Babesia bovis, Babesia bigemina, or Hyalomma anatolicum which is negative for T. annulata. A total of 809 field samples from different regions of China were analyzed by using the developed qPCR and conventional PCR. The positive samples for T. annulata detected by real-time qPCR and conventional PCR were 66/809 (8.16%) and 20/809 (2.47%), respectively, and all positive amplicons by qPCR were confirmed by Sanger sequencing. The results showed that the developed qPCR for the T. annulata 19,140 gene was more sensitive than conventional PCR. In addition, we first discovered that TA19140 was mainly expressed at the schizont and merozoite stages of T. annulata by relative quantification. The protein encoded by the TA19140 gene may be used as a potential diagnostic antigen for tropical theileriosis. In conclusion, a real-time quantitative PCR diagnostic method targeting the TA19140 gene was successfully established and could be used for both the quantitative and qualitative analysis of T. annulata infection from cattle and vector ticks, which will greatly help to control and diagnosis of tropical theileriosis.

Molecular identification of Theileria species in naturally infected sheep using nested PCR-RFLP.

Ovine theileriosis is an important tick-borne haemoprotozoan disease of sheep in tropical and subtropical regions, causing severe productivity and economic loss. There is a paucity of information related to molecular studies of ovine theileriosis from India. The present study identified different Theileria spp. in naturally infected sheep using nested PCR-restriction fragment length polymorphism (nPCR-RFLP). Blood samples and ticks were collected from 204 sheep in different agro-climatic zones of Haryana state, India, during the tick active season. Microscopic examination of thin blood smears revealed 33.3% (68/204) infections with Theileria spp., while 44.6% (91/204) of blood samples were positive by nPCR assay. Different Theileria spp. were identified based upon RFLP patterns using four restriction enzymes: Hpa II, Bsh 1285I, Hae II and Rsa I. Out of 91 positive samples, 50.5% (46/91), 23.08% (21/91), 11% (10/91) and 2.2% (2/91) were positive for T. ovis, T. lestoquardi, T. luwenshuni (Theileria sp. China 1/Theileria sp. China) and T. annulata, respectively. Mixed infection was detected in 13.2% (12/91) of cases. Based upon HpaII enzymatic digestion pattern, two samples with T. lestoquardi and T. annulata, nine samples with T. lestoquardi and T. ovis and one sample with T. ovis and T. annulata were detected. The presence of these Theileria spp. was further confirmed by sequence analysis. The majority of ticks collected from sheep were identified as Rhipicephalus spp. followed by Hyalomma anatolicum and Hemaphysalis spp. The present investigation depicts the first comprehensive molecular report of naturally infected sheep with T. ovis, T. lestoquardi, T. annulata and T. luwenshuni from northern India.

Comparative expression profiling of cytokine genes in Theileria annulata-infected and healthy cattle.

Theileriosis is one of the top ten economically important diseases in cattle in India. Cytokines are considered important mediators and regulators of the immune response to an infection. In the present study, the gene expression profiles of fourteen cytokines (IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12A, IL12B, IL16, TGFB1, TNFA, IFNA and IFNB) were compared in Theileria annulata-infected and healthy crossbred cattle. Blood samples were obtained from the District Disease Diagnostic Laboratory, Karnal. The presence/absence of T. annulata infection in the animals was determined on the basis of blood smear examination and molecular detection through PCR using the genus-specific primers. Total RNA was extracted from peripheral blood mononuclear cells, which was further reverse transcribed to cDNA. Primer3 software was employed to design the primers for Real-Time qPCR. The results were examined using 2-∆∆Ct method with RPS15 and GAPDH as the reference genes. The expression of IL1B, IL6, IL8, IL10, IL12A, IL12B, TNFA, IFNA and IFNB was significantly higher, whereas the expression of IL2 was lower in the infected animals. The transcript levels of IL1A and TGFB1 were also higher in the diseased animals, but the results were non-significant. This study profiles the expression kinetics of various pro-inflammatory and anti-inflammatory cytokine genes in response to bovine theileriosis.

In vitro inhibition studies of coumarin derivatives on Bos taurus enolase and elucidating their interaction by molecular docking, molecular dynamics simulations and MMGB(PB)SA binding energy calculation.

Tropical theileriosis is among the most common vector-borne diseases and caused by Theileria parasites. Theileria annulata is an obligate intracellular protozoan parasite and transmitted to especially Bos taurus and Bos indicus by Hyalomma tick vectors. C8 ([4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one); C9 (4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one); C21 (4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one) were identified as potent Theileria annulata enolase (TaEno) inhibitors in our previous studies. An ideal drug compound must inhibit the target parasite enzyme without inhibiting its homolog in the host. In this study, the inhibitory effect of the compounds previously evaluated on TaEno were tested on the host Bos taurus enolase (BtEno3) by in vitro studies. The interactions of enzyme-coumarin and enzyme-coumarin-substrate by in silico studies were also performed. All of the coumarin derivatives tested showed very low inhibitory effects on B. taurus enolase; 36,87% inhibition at 100 µM concentration for C8, 8,13% inhibition at 100 µM concentration for C9 and 77,69 µM of IC50 value for C21. In addition, these three coumarin derivatives and substrate 2PG were docked into the BtEno3 using molecular docking methods. Molecular interactions between enolase-coumarin and enolase-coumarin-substrate complexes were analyzed using molecular dynamics simulation methods for 100 ns. Estimated free energy of bindings of the substrate 2PG and coumarin derivatives to the BtEno3 were calculated by MM-GB(PB)SA methods. In comparison to the inhibition studies performed on TaEno, C8 and C9 coumarin derivatives remain the possible inhibitor candidates as they inhibit the host enolase at very high concentrations. These two promising compounds will be further analyzed by in vitro and in vivo studies towards developing an alternative drug against tropical theileriosis.

Identification of benzamide inhibitors of histone deacetylase 1 from Babesia and Theileria species via high-throughput virtual screening and molecular dynamics simulations.

Theileria and Babesia species are eukaryotic protozoan parasites classified under the order Piroplasmida of the phylum Apicomplexa. Tick vectors transmit these microorganisms in tropical and subtropical regions to a wide range of animals, including ruminants, causing fatal and life-threatening diseases such as bovine babesiosis and theileriosis. Resistance to commercially available drugs requires the search for new drug candidates. Histone deacetylase (HDAC) has a potential to be utilized as a drug target; therefore, it may be considered as an effective alternative. Previous studies revealed that HDAC inhibitors, identified for human use, show promising anti-parasitic effects. We have herein focused on the class I HDAC enzyme, HDAC1, of the Babesia and Theileria species to discover potential benzamide inhibitors by following a streamlined workflow of computer-aided drug design methodology. Molecular docking and molecular dynamics simulations revealed that benzamide derivatives stably interacted with the HDAC1 active site in both parasites as hypothesized. Furthermore, specific residue insertions at the entry point of the active site cleft of parasitic HDAC1 could enable ways to design parasite-specific drugs without adversely affecting host enzymes.

Mitochondrial genome of Theileria uilenbergi endemic in sheep and goats in China.

Mitochondrial genomes provide new insights that help elucidating biological features, genetic evolution, and classification of protozoans. Theileria uilenbergi (T. uilenbergi), transmitted by Haemaphysalis qinghaiensis and H. longicornis, is considered as highly pathogenic to sheep and goats in China. This study reports and outlines features of its mitochondrial genome. The T. uilenbergi mitochondrial genome is a linear monomeric molecule of 6.0 kb length, which encodes three protein-coding genes named cytochrome c oxidase I (cox1), cytochrome b (cob), and cytochrome c oxidase III (cox3), as well as six large subunit (LSU) rRNA gene fragments, and ends in terminal inverted repeats (TIRs). The array structure and organization of the mitochondrial genome of T. uilenbergi is identical to that of T. parva. Phylogenetic analysis based on the amino acid sequences of cox1, cob, and cox3 genes suggests that T. uilenbergi is distantly related to the group of transforming Theileria species such as T. parva. This study contributes to a comprehensive understanding of the phylogeny and evolution of the mitochondrial genome of piroplasms and provides useful information of diagnostic marker for T. uilenbergi.

Migratory Gaddi sheep and goats as potential carriers of Theileria infection: a molecular survey.

Theileriosis caused by parasites of the genus Theileria, is a vector-borne haemoprotozoan parasitic disease of critical concern in small ruminants. This study aimed to explore the infection status of migratory Gaddi sheep and goats with parasites from the Theileria genus in concurrence with ectoparasite infestations using molecular methods. Seventy three apparently healthy animals were randomly sampled from different flocks of migratory Gaddi sheep and goats and were systematically screened for ectoparasitic infestations. Molecular investigation for theileriosis was conducted using the genus wide polymerase chain reaction (PCR) technique. Out of 56 (76.71%) animals positive for the genus Theileria, 2 randomly selected amplicons were sequenced and subjected to BLAST analysis and were showing 99.71% identity with Theileria luwenshuni, a pathogenic Theileria species of small ruminants. To confirm the presence of T. luwenshuni, species-specific PCR was attempted to identify that 38 (52.05%) animals were infected by T. luwenshuni. On analysing the molecular prevalence data of Theileria to the ectoparasitism, it was evident that the infection existed in the animals irrespective of the type of ectoparasitic infestation and even T. luwenshuni was found in non-infested animals also. This is the first report of subclinical infections of T. luwenshuni in sheep and goats of Northern India and its potential carrier status. The asymptomatic carrier status of these nomadic animals is a matter possessing serious implications on the disease transmission rates and the production economics of small ruminant production in this region.

The phenotypic and haemato-biochemical appraisal of tropical theileriosis in newborn calves.

Tropical theileriosis is one of the major causes of newborn calves mortality. Observation of clinical manifestations is important while making the presumptive/tentative diagnosis of tropical theileriosis in newborn calves. The phenotypic and haemato-biochemical appraisals of tropical theileriosis could be of great help to make a holistic therapeutic plan for diseased newborn calves. Therefore, the present study aimed to evaluate the haemato-biochemical and phenotypic diagnostic markers of tropical theileriosis in newborn calves. A total of 43 newborn calves naturally infected with Theileria annulata and 16 age-matched healthy calves were enrolled. The percentage distribution of clinical markers was generalized lymph nodes enlargement (100%), pyrexia (97.67%), respiratory distress (95.34%), tick infestation (90.69%), anorexia (88.37%), pica (81.39%), pallor mucous membrane (67.44%), hyperlacrimation (58.13%) and exophthalmia (30.22%). Haemograms including TEC, Hb and HCT were found to be significantly (P ≤ 0.001) lowered in diseased calves. Remarkable alterations in the leukogram panels were not observed. Serum glucose, total protein, albumin and globulin concentrations of calves with theileriosis were significantly (P ≤ 0.001) lower than healthy ones, whereas triglycerides and total cholesterol levels of diseased calves were significantly (P ≤ 0.001) higher. Significantly (P ≤ 0.001) elevated activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) enzymes were observed in diseased calves. An evaluation of clinical phenotypes could be helpful to initiate quick treatment of diseased calves in field conditions and save the lives of sick calves of economically poor farmers. Altered haemato-biochemical panels to be appraised by veterinary clinicians while making a therapeutic plan of tropical theileriosis.

Knowledge, attitude and perception of bovine piroplasmosis by cattle owners in Constantine, North-East of Algeria, using participatory epidemiology.

Cattle piroplasmoses are tick-borne diseases, spread worldwide that cause significant economic losses. A participatory epidemiological study was conducted individually or in focus groups with 73 cattle owners in Beni Hamidene locality (district of Constantine, Algeria). The aim of this study was to study cattle owners' knowledge, attitude and perception on cattle piroplasmosis. Proportional piling technique was used to determinate most common cattle diseases, and to evaluate economic impact of diseases according to the interwieved farmers. Theileriosis (49/73; 67.1%) and babesiosis (44/73; 60.3%) were considered the most important bovine diseases. No zoonotic disease was cited by the interviewed cattle owners. According to the majority of cattle owners, theileriosis and babesiosis are deadly diseases (87.3 and 78.1%, respectively). All cattle owners (73/73) cited fever as the most common symptom of tropical theileriosis. Some of them (14/73; 19.2%) do not make distinction between theileriosis and babesiosis. According to cattle owners (65/73; 89.0%), the use of acaricide is the most appropriate tick control method. This study provides information about knowledge on bovine piroplasmoses in Algeria. These information could be considered when performing control programmes by both animal decision-makers and field veterinarians.