p67 gene alleles sequence analysis reveals Theileria parva parasites associated with East Coast fever and Corridor disease in buffalo from Zambia.

Theileriosis caused by Theileria parva infections is responsible for high cattle mortalities in Zambia. Although infected buffalo are a risk to cattle, the characterization of T. parva parasites occurring in this host in Zambia has not been reported. Furthermore, considering the advances in the development of a p67 subunit vaccine, the knowledge of p67 genetic and antigenic diversity in both cattle and buffalo associated T. parva is crucial. Therefore, blood samples from buffalo (n=43) from Central, Eastern and Southern provinces, and cattle (n=834) from Central, Copperbelt, Eastern, Lusaka, and Southern provinces, were tested for T. parva infection and the parasites characterized by sequencing the gene encoding the p67 antigen. About 76.7 % of buffalo and 19.3 % of cattle samples were PCR positive for T. parva. Three of the four known p67 allele types (1, 2 and 3) were identified in parasites from buffalo, of which two (allele types 2 and 3) are associated with T. parva parasites responsible for Corridor disease. Only allele type 1, associated with East Coast fever, was identified from cattle samples, consistent with previous reports from Zambia. Phylogenetic analysis revealed segregation between allele type 1 sequences from cattle and buffalo samples as they grouped separately within the same sub-clade. The high occurrence of T. parva infection in buffalo samples investigated demonstrates the risk of Corridor disease infection, or even outbreaks, should naïve cattle co-graze with infected buffalo in the presence of the tick vector. In view of a subunit vaccine, the antigenic diversity in buffalo associated T. parva should be considered to ensure broad protection. The current disease control measures in Zambia may require re-evaluation to ensure that cattle are protected against buffalo-derived T. parva infections. Parasite stocks used in 'infection and treatment' immunization in Zambia, have not been evaluated for protection against buffalo-derived T. parva parasites currently circulating in the buffalo population.

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.

TurboID mapping reveals the exportome of secreted intrinsically disordered proteins in the transforming parasite Theileria annulata.

Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.

Characterisation of field tropical Theileriosis and associated risk factors in two bioclimatic areas of Algeria.

Tropical theileriosis (TT) is a tick-borne disease caused by Theileria annulata and commonly infects cattle in tropical and subtropical regions, including Algeria. It is a significant obstacle to cattle breeding programs established to improve production in Algeria. The present investigation aimed to estimate the current molecular prevalence, risk factors, and genetic characterisation of T. annulata in two bioclimatic areas of Algeria. In a cross-sectional study, 679 blood samples (629 from healthy cattle selected on farms and 50 from diseased cattle identified by veterinarians) were collected from the humid (n = 307+50) and semi-arid (n = 322) areas and screened by blood smear examination followed by polymerase chain reaction targeting cytochrome oxidase subunit 3 (cox III) mitochondrial and the 18S ribosomal RNA (18S rRNA) genes for Theileria spp. Seventy-six positive samples (56 clinically healthy and 20 with clinical signs) for Theileria spp. were confirmed to be T. annulata by the merozoïtes surface antigen-1 (Tams1) gene showing a rate of 8.9 % in clinically healthy and 40.0 % in suspected cattle. Among the 307 bloods samples collected from healthy cattle in the humid area, 25 cattle (8.1 %) were positive for T. annulata. Of the 322 healthy cattle from the semi-arid site, 31 (9.6 %) were carriers of T. annulata DNA. In subclinical population, demographic and environmental parameters analysis indicated that T. annulata infection was higher in adult crossbred cattle raised in the intensive and semi-intensive system (P<0.001). The multiple logistic regression analysis showed that age, breed, farming system, and bioclimatic area are potential risk factors for T. annulata infection in cattle (P<0.05). Multiple alignments of cox III sequences of T. annulata showed high heterogeneity with 25 polymorphic sites (nucleotide diversity π = 0.02402), resulting in two haplotypes with a low genetic diversity index (Hd) of 0.533. The 18S rRNA sequence alignment revealed only one T. annulata genotype with 100 % identity to the strains isolated from cattle and ticks in Mediterranean and Asian countries. Our preliminary results will serve as a basis for further studies on the genetic diversity and molecular epidemiology of T. annulata.

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.

Exploring the Transcriptome Dynamics of In Vivo Theileria annulata Infection in Crossbred Cattle.

The molecular changes occurring in the host in response to in vivo Theileria annulata parasitic infection are not well understood. Therefore, the present study investigated the differential expression profiles of peripheral blood mononuclear cells (PBMCs) across Theileria annulata-infected and non-infected crossbred cows. The differential expression profiles from PBMCs of infected and non-infected crossbred cows were generated by RNA sequencing. A marked difference in the expression of genes associated with innate immunity (FTH1, ACTB, ISG15) was observed between the two groups. The over-represented pathways in Theileria annulata-infected cows were associated with the immune system and regulation of the mitotic cycle. Enriched genes and pathways in non-infected animals were associated with the maintenance of chromatin integrity and cell structure. The highly connected genes identified in this study form potential candidates for further investigation into host-parasite interactions in cattle. An improved understanding of the transcriptomic dynamics during theileriosis would lead to underpinning molecular level differences related to the health status of cattle.

Differential diagnosis of theileriosis through blood smear examination and polymerase chain reaction in small ruminants from Pakistan.

Background: Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity. Aim: To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan. Methods: The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp. Results: Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively. Conclusion: Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan.

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).

First Molecular Confirmed Outbreak of Malignant Ovine Theileriosis in Sheep from North India.

CONTEXT: Malignant ovine theileriosis is a tick-borne disease of sheep and goats, caused by protozoan Theileria lestoquardi. The disease has serious economic implications for small ruminant production around the world. METHODS: An outbreak of malignant ovine theileriosis in a sheep flock was investigated from Hisar district of Haryana, India, in March 2022. The etiological agent was identified using polymerase chain reaction assay with genus specific primers targeting 18S rRNA gene and subsequently confirmed by sequencing. RESULTS: The morbidity, mortality and case fatality rate reported in the outbreak were 22.2, 18.8 and 85%, respectively. The phylogenetic analysis clustered the present study T. lestoquardi isolate in the same clade with T. lestoquardi from Iraq, Iran and Pakistan with maximum nucleotide identity of 99.37% with strains from Iraq. The tick vector Hyalomma anatolicum recovered from dead animals was implicated in the disease's transmission. CONCLUSIONS: Malignant ovine theileriosis resulted in high case fatality rate. This study presents the first molecularly confirmed outbreak of malignant ovine theileriosis outbreak in the North Indian region, with characteristic post-mortem findings.

Prevalence and Risk Factors Associated with Theileria annulata Infection in Two Bovine Portuguese Autochthonous Breeds.

Tropical Bovine Theileriosis is an important tick-borne disease. This study aims to assess the occurrence of Theileria annulata infection in two indigenous Portuguese cattle breeds. A total of 843 blood samples collected from animals of Alentejana (n = 420) and Mertolenga (n = 423) breeds were analyzed. The detection of Theileria annulata was determined by amplification of a fragment of the merozoite-pyroplasm surface antigen gene with 319 base pairs (bp). The prevalence found (10.8%) is lower than that reported in previous studies (21.3%). A statistically significant difference was found for positivity between breeds (p < 0.05). There is also a higher probability of older animals being positive compared to younger ones (p < 0.05). The region where Mertolenga animals are located is shown to have a significant impact on positivity (p < 0.05). Thus, the development of sustainable T. annulata control strategies and their implementation, adapted to the epidemiological conditions of higher risk, will be extremely important.

Molecular detection of Theileria annulata, Theileria mutans and Theileria velifera but no evidence of Theileria parva infected or vaccinated cattle in Nigeria despite extensive transboundary migrations.

The extensive livestock management system predominant in Nigeria necessitates active disease surveillance for the early detection and prompt control of transboundary animal diseases. Theileriae are obligate intracellular protozoa which infect both wild and domestic bovidae throughout much of the world causing East Coast Fever (Theileria parva), Tropical or Mediterranean theileriosis (Theileria annulata) or benign theileriosis (Theileria mutans; Theileria velifera). This study aimed to detect and characterize Theileria spp. infecting cattle in Nigeria using conventional PCR and sequencing approach. Five hundred and twenty-two DNA samples obtained from different cattle blood samples were subjected to PCR targeting the 18S rRNA gene of piroplasmida and specifically, the p104 kDa and Tp1 genes for the evidence of infection or vaccination respectively, with T. parva. A total of 269 out of 522 (51.5%) of the cattle tested PCR- positive for DNA of piroplasmida. Nucleotide sequence and phylogenetic analyses showed that the cattle were infected with T. annulata, T. mutans and T. velifera. Piroplasmida DNA was associated with sex (ꭓ2 = 7.2; p = 0.007), breed (ꭓ2 = 115; p = 0.000002) of animals and the state where the samples were collected (ꭓ2 = 78.8; p = 0.000002). None of the samples tested positive for T. parva DNA or showed evidence of vaccination (Tp1 gene). This is the first report on the molecular detection and characterization of T. annulata in the blood of cattle from Nigeria. Continuous surveillance of Nigerian cattle for East Coast Fever (ECF) is encouraged considering the recent report of the disease in cattle in the neighboring country, Cameroon, where unregulated transboundary cattle movement into Nigeria has been observed.

In vitro efficacy of plumbagin and thymol against Theileria annulata.

Phytochemical compounds, plumbagin and thymol were evaluated for their efficacy against Theileria annulata using MTT cell viability assay. Plumbagin and thymol were found to be effective in preventing the proliferation of Theileria annulata infected bovine lymphocytes. The IC50 values of plumbagin and thymol were 0.019 µM and 0.009 µM, respectively. Plumbagin and thymol were found to be non-cytotoxic to the bovine peripheral blood mononuclear cells. However, both the compounds were found to have inhibitory effect on vero cell proliferation. Plumbagin had primarily anti-theilerial activity but thymol had primarily anti-mitotic activity. The in vitro efficacy and cell toxicity studies indicate the potential application of plumbagin, purified from Plumbago indica as a lead therapeutic molecule against T. annulata infection in cattle. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-022-01550-x.

Bovine cerebral theileriosis: first molecular report in cross bred cattle calf in India.

Bovine tropical theileriosis caused by Theileria annulata, is a serious constraint to Indian dairy industry with more fatal infections in exotic cattle and substantial losses to cross-bred and indigenous zebu cattle. The present communication is to place on record the first report of molecular based confirmed case of cerebral theileriosis caused by T. annulata coupled with its morphological detection, clinical manifestations, haematological alterations and therapeutic management in a cross bred cattle calf from India. After preparation of peripheral thin blood smear from cross bred cattle calf at the site of collection and fixation with methanol, blood sample brought to Department of Veterinary Parasitology, College of Veterinary Science and A.H, Jabalpur and stained by standard protocol for Giemsa staining. Genomic DNA was isolated from the collected blood sample using QIAamp® DNA blood mini kit following the manufacturer's recommendations and PCR was performed. The cross bred cow calf revealed high rise in temperature (105.5°F), increased heart rate, labored breathing with seromucous nasal discharge, enlargement of prescapular lymph node and animal exhibited tonic clonic convulsions in response to any sudden noise. Giemsa stained thin blood smear revealed intraerythrocytic piroplasm and Koch'sblue bodies of T. annulata within the cytoplasm of lymphocytes. The species of Theileria was confirmed by molecular amplification of genomic DNA as T. annulata.

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 Degradome Analysis of the Bovine Piroplasmid Pathogens Babesia bovis and Theileria annulata.

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.

Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis.

Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

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.

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.

Theileria terrestris nov. sp.: A Novel Theileria in Lowland Tapirs (Tapirus terrestris) from Two Different Biomes in Brazil.

The low-land tapir (Tapirus terrestris) is the largest wild terrestrial mammal found in Brazil. Although T. terrestris has been already reported as a host of hemoparasites, the occurrence and genetic identity of Piroplasmida agents in this species is still cloudy. Although it is reported that Theileria equi, an endemic equid-infective agent in Brazil, is occurring in lowland tapirs, these reports are probably misconceived diagnoses since they are solely based on small fragments of 18S rRNA that may not achieve accurate topologies on phylogenetic analyses. The present study aimed to detect and investigate the identity of Theileria spp. in tapirs from Pantanal and Cerrado biomes. Blood-DNA samples from tapirs were screened for a partial (~800 bp) 18S rRNA gene fragment from Piroplasmida and 64 (64/122; 52.46% CI: 43.66-61.11%) presented bands of expected size. Samples were submitted to different protocols for molecular characterization, including near-full length 18S rRNA gene (~1500 bp), and the ema-1 gene from T. equi. Eight sequences were obtained for extended fragments (1182-1473 bp) from the 18S rRNA gene. Moreover, three sequences from partial cox-1 and five from partial hsp70 gene were obtained. None of the samples presented amplifications for the ema-1 gene. Phylogenetic and distance analyses from the 18S rRNA sequences obtained demonstrated a clear separation from tapirs' Theileria spp. and T. equi. Phylogenetic analyses of cox-1 and hsp70 sequences obtained herein also showed a unique clade formed by tapir's Theileria spp. Theileria terrestris sp. nov. is positioned apart from all other Theileria species in 18S rRNA, cox-1, and hps70 phylogenetic analyses. This novel proposed species represents a new Piroplasmida clade, yet to be characterized regarding biological features, vectors involved in the transmission cycles, additional vertebrate hosts, and pathogenicity.

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.