A locus conferring tolerance to Theileria infection in African cattle.

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa's most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

Systematic Determination of TCR-Antigen and Peptide-MHC Binding Kinetics among Field Variants of a Theileria parva Polymorphic CTL Epitope.

CTLs are known to contribute to immunity toward Theileria parva, the causative agent of East Coast fever. The Tp967-75 CTL epitope from the Muguga strain of T. parva is polymorphic in other parasite strains. Identifying the amino acids important for MHC class I binding, as well as TCR recognition of epitopes, can allow the strategic selection of Ags to induce cellular immunity toward T. parva In this study, we characterized the amino acids important for MHC class I binding and TCR recognition in the Tp967-75 epitope using alanine scanning and a series of variant peptide sequences to probe these interactions. In a peptide-MHC class I binding assay, we found that the amino acids at positions 1, 2, and 3 were critical for binding to its restricting MHC class I molecule BoLA-1*023:01. With IFN-γ ELISPOT and peptide-MHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 5-8 in the epitope were required for TCR recognition. Only two of eight naturally occurring polymorphic Tp9 epitopes were recognized by both CTLs. Finally, using a TCR avidity assay, we found that a higher TCR avidity was associated with a stronger functional response toward one of two variants recognized by the CTL. These data add to the growing knowledge on the cross-reactivity of epitope-specific CTLs and specificities that may be required in the selection of Ags in the design of a wide-spectrum vaccine for East Coast fever.

Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt.

BACKGROUND: Tick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene. RESULTS: We collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries. CONCLUSION: This study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi.

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64-90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation.

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.

Expression profile of microRNAs in bovine lymphocytes infected with Theileria annulata and treated with buparvaquone.

Several miRNA-based studies on Theileria-transformed bovine cells have been conducted; however, the mechanism by which transformed cells exhibit uncontrolled proliferation is not yet fully understood. Therefore, it is necessary to screen more microRNAs that may play a role in the transformation process of host cells infected with Theileria annulata to better understand the transformation mechanisms of Theileria-infected cells. RNA sequencing was used to analyze miRNAs expression in the host bovine lymphocytes infected with T. annulata at different time points after buparvaquone (BW720) treatment and DMSO treatment (control groups). Differential miRNAs related to cell proliferation and apoptosis were identified through comparison with gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and a regulatory network of miRNA-mRNA was constructed. In total, 272 differentially expressed miRNAs were found at 36, 60 and 72 h. The miRNAs change of bta-miR-2285t, novel-miR-622, bta-miR-2478, and novel-miR-584 were significant. Analysis of 27 of these co-differential expressed miRNAs revealed that 15 miRNAs were down-regulated and 12 miRNAs were up-regulated. A further analysis of the changes in the expression of each of these 27 miRNAs in the three datasets suggested that bta-miR-2285t, bta-miR-345-5p, bta-miR-34a, bta-miR-150, and the novel-miR-1372 had significantly changed. Predicted target genes for these 27 miRNAs were analyzed by KEGG and the results demonstrated that EZR, RASSF, SOCS1 were mainly enriched in the signaling pathway microRNAs in cancer. MAPKAPK2, RELB, FLT3LG, and GADD45B were mainly enriched in the MAPK signaling pathway, and some genes were enriched in Axon guidance. This study has provided valuable information to further the understanding of the regulatory function of miRNAs in the host microenvironment and host-parasite interaction mechanisms.

The first study of the prevalence and genetic diversity of Theileria equi and Babesia caballi in horses in Russia.

Equine piroplasmosis (EP) is a global worldwide infection, which can lead to the death of animals. Despite the causative agents of EP being well studied, there are no data on the distribution and genetic characteristics of EP agents in any region of Russia. In this study, blood samples from 750 horses from Novosibirsk province, Irkutsk province, and Altai region of Russian Siberia were examined for the presence of EP agents. Theileria equi and Babesia caballi were detected in all examined regions, with mean prevalence rates of 60.4% and 7.2%, respectively. The identified pathogens were genetically characterized by the 18S rRNA gene. The determined T. equi sequences were highly conserved and belonged to genotypes A and E, with genotype E being found in 88.6% of genotyped samples. In contrast to T. equi, B. caballi sequences were genetically diverse. Seven sequence variants of B. caballi were identified, and only two of them matched known sequences from the GenBank database. The determined B. caballi sequences belonged to four distinct branches within genotype A. Mixed infections with several variants of B. caballi or with T. equi and B. caballi were common. The conducted phylogenetic analysis based on all available B. caballi sequences of the 18S rRNA gene (> 900 bp) from GenBank and from this study first demonstrated the presence of five monophyletic clusters within genotype A and three clusters within genotype B. Thus, the genetic study of B. caballi from Siberia has significantly expanded the data on the genetic diversity of this pathogen.

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.

Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat, India.

BACKGROUND OBJECTIVES: Vector-borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at the identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region in India using microscopy and Polymerase Chain Reaction (PCR). METHODS: A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. RESULTS: The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45 % sensitive and 100 % specific). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. INTERPRETATION CONCLUSION: Despite holding much significance to the animal sector, little work has been undertaken in regional parts of India regarding camel parasites. The present study offers first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

Molecular and immunological studies on Theileria equi and its vector in Egypt.

Equine piroplasmosis is not fully understood regarding pathogenicity, prophylaxis, host immune response expression, and specific vectors. Accurately identifying the parasite vector is crucial for developing an effective control plan for a particular infection. This study focused on morphologically identifying two Hyalomma species (H. anatolicum and H. marginatum) and one Rhipicephalus annulatus (R. annulatus) at the species level. The identification process was followed by phylogenetic analysis using the neighbor-joining method based on the cytochrome oxidase subunit 1 (COXI) gene as a specific vector for Theileria equi (T. equi) in horses. T. equi was diagnosed morphologically and molecularly from infected blood samples and crushed tick species using conventional PCR. Subsequently, phylogenetic analysis based on the amplification of the 18 S rRNA gene was conducted. The obtained sequence data were evaluated and registered in GenBank under accession numbers OR064161, OR067911, OR187727, and OR068139, representing the three tick species and the isolated T. equi, respectively. The study demonstrated that T. equi infection leads to immune system suppression by significantly increasing the levels of oxidative stress markers (CAT, GPx, MDA, and SOD) (P ≤ 0.0001), with this elevation being directly proportional to parasitemia levels in infected blood cells. Furthermore, a correlation was observed between parasitemia levels and the expression of immune response infection genes (IFN-gamma, TGF-ß1, and IL-1ß cytokines) in infected horses compared to non-infected equine. Common macroscopic symptoms indicating T. equi infection in horses include intermittent fever, enlarged lymph nodes (LN), and tick infestation.

Molecular identification, risk factor assessment, and phylogenetic analysis of tick-borne pathogens in symptomatic and asymptomatic cattle from South-Eastern Iran.

Tick-borne pathogens (TBPs) represent a substantial threat to cattle globally, exerting adverse impacts on production, health, and economic viability. This study delves into the prevalence and implications of TTBPs in cattle sourced from resource-limited smallholder livestock farms situated in southeastern Iran, proximate to Afghanistan and Pakistan. Blood and tick specimens were systematically collected from a cohort of 230 cattle, comprising 150 asymptomatic and 80 symptomatic individuals. Genomic DNA isolated from blood samples underwent rigorous examination for the presence of key TBPs, including Anaplasma marginale, A. phagocytophilum, A. bovis, A. centrale, Babesia bigemina, and Theileria annulata, utilizing multiple genetic markers. Nucleotide sequence analysis facilitated the reconstruction of phylogenetic relationships. The study also evaluated various potential risk factors, such as clinical status, gender, age, breed, tick infestation, and management practices, to elucidate their associations with TTBPs. Among the cattle cohort, a staggering 87.8% (202/230) tested positive for at least one pathogen. Prevalence statistics encompassed A. marginale (72.2%), T. annulata (68.3%), A. phagocytophilum/A. platys-like complex (66.1%), A. centrale (16.7%), B. bigemina (10.0%), and A. bovis (6.1%). Remarkably, mixed infections involving two, three, and four pathogens were detected in 23%, 52.1%, and 2.2% of animals, respectively. Notably, all asymptomatic cattle were positive for at least one TBP. Tick infestation was observed in 62.2% (143/230) of cattle, predominantly caused by Hyalomma anatolicum (82.5%), Rhipicephalus (Boophilus) annulatus (13.1%), and R. sanguineus sensu lato (4.4%). Risk factors linked to TBPs encompassed tick infestation, older age, and crossbred animals. Clinical presentations among symptomatic cattle encompassed fever, anemia, weight loss, anorexia, jaundice, and enlarged superficial lymph nodes. This study underscores the pivotal role of asymptomatic carriers in the propagation of TTBPs within endemic regions. Furthermore, it emphasizes the potential for the implementation of molecular diagnostics to unmask subclinical infections, thereby affording the opportunity for targeted interventions aimed at ameliorating the burden of TTBPs in resource-constrained smallholder dairy farms.

The interplay of cytokines in bovine tropical theileriosis: a mini review.

Studying cytokine profiling in Theleria annulata infection enhances our understanding of how the immune response unfolds, the intricate interactions between the host and the parasite, the strategies employed by the parasite to evade the immune system, and potential avenues for developing treatments. The generation of pro-inflammatory cytokines plays a pivotal role in the immune response against T. annulata infection. Elevated concentrations of these cytokines potentially contribute to the manifestation of clinical symptoms associated with the disease, such as fever, anemia, exophthalmia, and weight loss. The production of anti-inflammatory cytokines potentially serves as a regulatory mechanism for the immune response, preventing the development of severe disease. Nevertheless, in animals afflicted by T. annulata infection, there is often a notable decrease in the levels of these cytokines, suggesting that they may not be as effective in mitigating the disease as they are in uninfected animals. This knowledge can be harnessed to develop improved diagnostic methods, treatments, and vaccines for tropical theileriosis. The objective of this current mini review is to achieve the same goal by consolidating the available knowledge of cytokine interactions in Bovine Tropical Theileriosis (BTT).

Identification of novel bovine leukocyte antigen alleles and association of BoLA-DRB3.2*020:02:01 with resistance to Theileria orientalis infection in crossbred Kedah-Kelantan cattle: a pilot study.

The bovine leukocyte antigen (BoLA) gene is a significant genetic part of the immune system and has been used as a disease marker in cattle. In this study, we detected Theileria orientalis, T. sinensis, Anaplasma marginale, Anaplasma platys, Candidatus Mycoplasma haemobos and Trypanosoma evansi by PCR amplification and sequencing of the amplicons. The allelic association of the BoLA-DRB3.2 gene with blood pathogen disease resistance and susceptibility in 87 Kedah-Kelantan x Brahman (KKB) and 38 Bali cattle was determined by Fisher's exact test and Cochran Mantel Haenszel (CMH) correction test. Sequence-based typing of the BoLA-DRB3.2 gene identified 43 alleles (27 previously reported alleles and 16 novel alleles) across the two cattle breeds. Alignment analysis of the 16 novel alleles revealed 90.7-95.8% and 85-92% nucleotide and amino acid identities, with the reference allele, BoLA-DRB3*016:01 cDNA clone NR-1. BoLA-DRB3*009:02 (25.6%) and BoLA-DRB3*036:01 (36%) were the most frequent alleles in KKB and Bali cattle, respectively. In KKB cattle, BoLA-DRB3*020:02:01 was significantly associated with resistance to T. orientalis whereas *007:01 and *009:02 were significantly associated with resistance to C. Mycoplasma haemobos. Also, DRB3*017:01 was associated with susceptibility to T. orientalis in KKB cattle. In the Bali cattle, BoLA-DRB3*015:01 was found to be a genetic marker of susceptibility to C. Mycoplasma haemobos infection. Therefore, this study identified BoLA-DRB3.2 alleles associated with resistance and susceptibility to T. orientalis infection in KKB cattle and susceptibility to C. Mycoplasma haemobos infection in Bali cattle for the first time. Therefore, this study suggests that these BoLA-DRB3 resistance alleles could be used as candidate markers for selection, whereas susceptibility alleles could be used as candidate markers for culling in the beef industry.

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

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

Significance of Anaplasma phagocytophilum, Babesia caballi, and Theileria equi as etiologic agents in horses with clinical manifestations from the metropolitan area of Rio De Janeiro, Brazil.

Equine Piroplasmosis (EP) and Equine Granulocytic Anaplasmosis (EGA) are diseases that affect horses, transmitted by ixodid ticks, causing a nonspecific febrile syndrome. Equine Piroplasmosis is endemic in Brazil, and most horses are in enzootic stability. Serological and molecular studies carried out on horses in Brazil have shown the presence of Anaplasma phagocytophilum, however, the clinical relevance of this infection has not yet been established. The present study aims to evaluate the importance of Babesia caballi, Theileria equi, and A. phagocytophilum as etiological agents in horses with clinical manifestations suggestive of these diseases in the metropolitan mesoregion of Rio de Janeiro. A total of 45 animals with clinical signs were submitted to DNA extraction followed by qPCR test. Anaplasma phagocytophilum, Neorickettsia risticii and Theileria haneyi were not found in any of the horses with clinical signs, however 62.2% were infected with at least one agent of EP. Theileria equi was the most frequent etiologic agent (35.5%), followed by coinfection (15.5%) and B. caballi (11.2%). These results suggest that A. phagocytophilum has minor clinical importance in the region, while EP is frequently found in symptomatic horses, representing an important differential diagnosis in suspected cases.

Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

Babesia bigemina and Theileria annulata infections in cattle: molecular detection, phylogenetic analysis, and assessment of risk factors.

Babesia bigemina and Theileria annulata are tick-borne protozoans that cause piroplasmosis in cattle, resulting in huge damages to the livestock industry. The prevalence of these infections depends on various intrinsic and extrinsic risk factors. In Pakistan, there is no information regarding the molecular characterization of Babesia bigemina and the risk factors associated with piroplasmosis. This study aimed to molecularly characterize Babesia spp. and Theileria spp. infecting various cattle breeds in Khyber Pakhtunkhwa, Pakistan, and to shed light on risk factors associated with these infections. Altogether, 219 blood samples were collected from various symptomatic cattle breeds, including Holstein Friesian (65.3%; 143/219), Jersey (21.5%; 47/219) and Sahiwal (13.2%; 29/219). Isolated genomic DNA from these blood samples was used in PCR for the amplification of the 18S rRNA fragment of apicomplexan parasites. Obtained 18S rDNA sequences from cattle hosts showed 99.5% identity with B. bigemina, or 100% with T. annulata. Having an overall infection rate of 61.6% (135/219), the highest infection rate was recorded for T. annulata (43.8%; 95/219), followed by B. bigemina (18.3%; 40/219). Phylogenetic analysis of 18S rDNA sequences revealed that B. bigemina clustered with corresponding species reported from Bolivia, and South Africa, while T. annulata grouped with same species from Italy, India, and Turkey. Among the different risk factors, the breed, season, and tick infestation were found to have a significant (P < 0.05) association with the piroplasmid infections. The information obtained in this study can be employed for effective surveillance and control of babesiosis and theileriosis in Pakistan. In addition to confirming our previous molecular detection of T. annulata in cattle, this study provides the first molecular surveillance and phylogenetic position of B. bigemina and associated risk factors in the study region.

The antigen recognition portion of African buffalo class I MHC is highly polymorphic, consistent with a complex pathogen challenge environment, and the 3' region suggests distinct haplotype configurations.

African buffalo (Syncerus caffer) have been distinct from the Auroch lineage leading to domestic cattle for 5 million years, and are reservoirs of multiple pathogens, that affect introduced domestic cattle. To date, there has been no analysis of the class I MHC locus in African buffalo. We present the first data on African buffalo class I MHC, which demonstrates that gene and predicted protein coding sequences are approximately 86-87% similar to that of African domestic cattle in the peptide binding region. The study also shows concordance in the distribution of codons with elevated posterior probabilities of positive selection in the buffalo class I MHC and known antigen binding sites in cattle. Overall, the diversity in buffalo class I sequences appears greater than that in cattle, perhaps related to a more complex pathogen challenge environment in Africa. However, application of NetMHCpan suggested broad clustering of peptide binding specificities between buffalo and cattle. Furthermore, in the case of at least 20 alleles, critical peptide-binding residues appear to be conserved with those of cattle, including at secondary anchor residues. Alleles with six different length transmembrane regions were detected. This preliminary analysis suggests that like cattle, but unlike most other mammals, African buffalo appears to exhibit configuration (haplotype) variation in which the loci are expressed in distinct combinations.

Investigation of haematological, inflammatory and immunological response in naturally infected cattle with Theileria annulata.

In this study, we aimed to investigate haematological, pro-inflammatory, inflammatory, anti-inflammatory and immunological responses in naturally Theileria annulata-infected cattle. The study material consisted of 25 Simmental cattle, 2-4 years of age, one of which was a control group consisting of healthy animals (Control group, n = 10), and the other was a Theileria group that include animals positive for Theileria annulata (Theileria group, n = 15). Haematological analysis (red blood cell [RBC], haemoglobin [HGB], haematocrit [HCT]), pro-inflammatory (tumour necrosis factor-α [TNF-α], nuclear factor kappa B [NF-ĸB] and interleukin-1 beta, [IL-1ß]), inflammatory (neutrophil-lymphocyte ratio [NLR]), anti-inflammatory (interleukin-10 [IL-10]) and antimicrobial peptide (CAMP) analyses were performed by using ELISA kit from blood samples. It was found that the rectal temperature of the Theileria group was found to be significantly higher (p < .001) than that of the control group. Haematological and biochemical analysis revealed that the RBC and HGB count and HCT percentage decreased (p < .001), while NF-ĸB (p < .001), TNF-α (p = .002), IL-1ß (p < .001), IL-10 (p = .012), NLR (p < .001) and CAMP (p = .037) levels increased in Theileria group compared to the control group. There was a strong correlation between NF-ĸB and TNF-α, NF-ĸB and IL-10, NLR and IL-1ß, NF-ĸB and CAMP, TNF-α and CAMP and IL-10 and CAMP. As a result of this study, it was revealed that a pro-inflammatory and immunological response also occurs along with the anti-inflammatory response in the inflammatory process.

Circulatory cytokines during the piroplasm stage of natural Theileria annulata infection in cattle.

The objective of the present study was to investigate the inflammatory and anti-inflammatory cytokine response from a broad perspective in cattle with natural Theileria annulata infection. Ten cattle naturally infected with T. annulata and eight healthy cattle were included in this study. A total of 11 cytokines, including tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and IL-17 were evaluated in serum samples using commercially available enzyme-linked immunosorbent assay (ELISA) kits. There was no statistical significance for serum TNF-α, IL-1ß, IL-5, IL-6, and IL-17 levels between the T. annulata infected and healthy cattle. In contrast, the median serum levels of IFN-γ (p = .023), IL-2 (p = .066), IL4 (p = .0016), IL-10 (p = .00087), IL-12 (p = .00018), and IL-13 (p = .023) were significantly higher in T. annulata-infected cattle than in healthy cattle. The results of the present study revealed that in the intraerythrocytic stage of tropical theileriosis, a very pronounced anti-inflammatory response occurs as well as an ongoing inflammatory process.