Molecular insights into the population structure and haplotype network of Theileria annulata based on the small-subunit ribosomal RNA (18S rRNA) gene.

The present study was conducted to elucidate the population genetic diversity and haplotype network of Theileria annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. In total, 52 sequences of the nuclear 18S rRNA gene used to assess the relationship of T. annulata with their country of origin identified 34 haplotypes. Haplotype 4 was widespread, occurring in India, China, Turkey and Iran, while the remaining haplotypes were singleton and unique to one country. Haplotype 4 displayed numerous single haplotypes around it and the stellate shape of the network suggested a rapid population expansion. India exhibited the largest number of haplotypes (h = 25) followed by Turkey (h = 6), China (h = 4), and Iran and Italy (h = 1). No geographical clustering of haplotypes was recorded. Nucleotide diversity was the highest in the Turkish followed by the Indian and Chinese populations. Similarly, haplotype diversity was the highest in China followed by Turkey, and the lowest in India. The overall dataset exhibited a low nucleotide diversity (0.00253 ± 0.00035), but high haplotype diversity (0.917 ± 0.034). It suggested the presence of only minor differences (01-11 nucleotide) between haplotypes which was also evident from the haplotype network. A high level of genetic diversity was documented within the Indian, Chinese and Turkish populations of T. annulata, whereas little genetic differentiation was noticed among these populations with a very high level of gene flow. Analysis of molecular variance (AMOVA) of T. annulata sequences revealed higher genetic variation within countries (83.58%) as compared to the variation among countries (16.42%). Neutrality indices, viz., Tajima's D, Fu and Li's F, Fu's Fs, and R2, along with the unimodal mismatch distributions demonstrated a recent population expansion of T. annulata in India and the overall dataset. However, the non-significant values of Tajima's D, Fu and Li's F, and Fu's Fs for the Chinese population along with a bimodal mismatch distribution signified a constant population size. For the Turkish population, the neutrality and mismatch distribution tests either indicated a constant or a slight increase in population size. The present study provides novel insights into the population genetics and haplotype network of T. annulata based on the 18S rRNA gene for the first time.

Genetic diversity in the Tams1 gene of Theileria annulata (Duschunkowsky and Luhs, 1904) infecting cattle.

The present study describes the genetic diversity in the Tams1 gene (733 bp) of Theileria annulata along with the sequence, phylogenetic and haplotype analyses of the Indian isolates. The phylogenetic analyses displayed distinct clustering of the Indian isolates into three groups suggesting the presence of three genotypes, hitherto designated as T. annulata genotypes 1-3 (G1-G3). Genotype 3 seems to be novel containing only two newly generated sequences. Indian isolates displayed 88.4-100% and 82.2-100% similarity with each other at nucleotide (nt) and amino acid (aa) levels, respectively. However, the newly generated sequences (n = 36) showed 90.5-100% and 84.3-100% identity between them at nt and aa levels, respectively. The most diverse and heterogeneous genotype, G1, exhibited the highest number of polymorphic sites (S = 148), haplotypes (h = 16) and nucleotide differences (k = 43.23) besides haplotype (Hd = 0.903 ± 0.031) and nucleotide (π = 0.059 ± 0.005) diversities. Neutrality indices suggested a respective decrease and increase in population sizes of G1 and G2 genotypes in India. The nucleotide sequence analyses indicated the presence of extensive sequence variations between nucleotide positions 1-124, 194-257 and 396-494. The N-terminus of Tams1 protein displayed a considerable sequence variability with extensive variations in two regions, between amino acid positions 1-39 and 127-172, as compared to the conserved carboxyl terminus.

Genetic characterization of Theileria species infecting bovines in India.

Genetic characterization of Theileria species infecting bovines in India was attempted targeting the 18S ribosomal RNA region of the parasite. Blood samples of bovines (n = 452), suspected for haemoprotozoan infections, from 9 different states of the country were microscopically examined for Theileria species infection. Four Theileria spp. positive blood samples from each state were randomly utilized for PCR amplification of the 18S rRNA gene (approx. 1529 bp) followed by cloning and sequencing. The sequence data analysis of all the 36 isolates revealed that 33 isolates had high sequence similarity with published sequences of T. annulata, whereas 3 isolates (MF287917, MF287924 and MF287928) showed close similarity with published sequences of T. orientalis. Sequence homology within the isolates ranged between 95.8 and 100% and variation in the length of targeted region was also noticed in different isolates (1527-1538 nt). Phylogenetic tree created for T. annulata sequences revealed that a total of 24 Indian isolates formed a major clade and grouped together with isolates originating from countries like China, Spain, Turkey and USA. Remaining 09 isolates clustered in a separate group and were closely related to the TA5 isolate of T. annulata (a new genotype) originating from India and also with the isolates from East Asian countries like Japan and Malaysia. All the three T. orientalis isolates had minimal intraspecific variation (99-100% homology) amongst themselves. Further, in the phylogenetic analysis T. orientalis Indian isolates were found to cluster away from other 14 isolates of T. buffeli/sergenti/orientalis originating from different countries (Australia, China, Indonesia and Spain). However, these 3 isolates clustered together with the T. buffeli Indian isolate (EF126184). Present study confirmed the circulation of different genotypes of T. annulata in India, along with T. orientalis isolates.

Immunoprophylactic evaluation of recombinant gametocyte 22 antigen of Eimeria tenella in broiler chickens.

Gametocyte proteins are being explored as potential vaccine candidates against Eimeria sp. in chicken since they are the components of the resilient oocyst wall. The aim of this study was to investigate the immunoprophylactic efficacy of recombinant Eimeria tenella gametocyte antigen 22 (EtGam22) in chickens against homologous oocyst challenge. Broiler chicks were subcutaneously immunized individually with 100 µg of recombinant EtGam22 adjuvanted with Montanide ISA 71 VG at 7 days of age and boosted 2 weeks later. The immunized chickens were challenged individually with 1 × 104 sporulated oocysts of E. tenella 1 week post-booster immunization. The anti-EtGam22 IgY and serum cytokine response was measured post-immunization. The results showed that the anti-EtGam22 IgY antibody, serum IFN-γ, IL-2, TGF-ß, and IL-4 levels in chickens vaccinated with recombinant protein were significantly increased post-immunization as compared to unimmunized challenged controls (P < 0.05). The peripheral blood lymphocyte proliferation activity was also found significantly higher in EtGam22-immunized group on day 28, i.e., pre-challenge (P < 0.05). Upon homologous oocyst challenge, chickens immunized with rEtGam22 exhibited a significant drop in the total oocyst output per bird (246.78 ± 36.9 × 106, 45.23% reduction) and a significantly higher weight gain (497.7 ± 19.2 g) as compared to unimmunized challenged controls. Taken together, these data indicate that EtGam22 is a potent immunogen for use as a subunit vaccine against cecal coccidiosis in chickens as it induces a diverse and robust immune response involving multiple cytokines and strong antibody titers.

Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines.

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

A clinically rare occurrence of rectal mucosal prolapse associated with tenesmus in a calf caused by Eimeria sp.

Eimeria sp. is a host-specific intracellular parasite that mostly affects young animals. This parasite causes great economic losses in livestock sector. A 6 weeks old calf was brought to Referral Veterinary Polyclinic, ICAR-Indian Veterinary Research Institute, Izatnagar with the history of inappetance and passage of foul smelling diarrhoeic feces rich in occult blood and mucous for the last 3 days. On clinical examination, calf was found to be present in lateral recumbency and showed severe tenesmus, prolapsed rectal mucosa along with small quantity of blood mixed feces sticking to the perineum and tail regions. Diagnosis was done based on clinical observations and fecal examination which showed oocysts of Eimeria sp. The prolapsed rectal mucosa was corrected aseptically by manual procedure. The calf was treated with a combination of sulfadimidine and amprolium along with supportive therapy. The calf recovered clinically after 5 days of therapy and further fecal examination showed no evidence of oocysts of Eimeria sp.

Detection of theileriosis in cattle and buffaloes by polymerase chain reaction.

Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease of great economic importance in tropical and subtropical regions of the world. The present study was undertaken to detect theilerosis in cattle and buffaloes by polymerase chain reaction (PCR). The diagnosis of theileriosis is usually carried out by blood smear staining technique, which is not sufficiently sensitive to detect the piroplasms in the carrier animals. In this study, a total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Screening of blood smears by Giemsa staining detected 15 samples (12.93 %) positive for Theileria piroplasms out of 116 samples. However, the PCR based screening using the specific primers from the major merozoite-piroplasm surface antigen sequence of T. annulata (Tams1) gene detected 74 samples (63.79 %) positive for T. annulata which included 59 samples found negative by Giemsa staining. Our study suggests that the PCR based screening is more sensitive and accurate method for diagnosis of tropical theileriosis in cattle and buffaloes.

Qualitative and quantitative assessment of Theileria annulata in cattle and buffaloes Polymerase Chain Reaction.

Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease associated with high morbidity and mortality in the livestock. The conventional method of diagnosis is by the demonstration of the parasite stages by microscopic examination. This method suffers from low sensitivity, making it even more difficult to detect piroplasms in the carriers. PCR based assays are known to be more sensitive. The present study was undertaken to detect and quantify T. annulata in the blood of clinically infected and carrier animals using a quantitative PCR protocol targeting the gene encoding the major merozoite piroplasm surface antigen Tams 1. A total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Of these, 74 samples (63.79%) were positive for T. annulata by real-time PCR, including the 15 samples that were positive by Giemsa staining. The parasite load ranged from 1.39 x 10(6) to 3.35 x 10(9) and 0.35 x 10(6) to 2.83 x 10(7) ml(-1) of blood in cattle and buffalo samples, respectively by qPCR. Our study suggests that real-time PCR assay can be used to detect and quantify the load of T. annulata in the blood of cattle and buffaloes. It also serves as a support to clinical diagnosis and assessment of carrier status in apparently healthy animals.

Qualitative and quantitative assessment of Theileria annulata in cattle and buffaloes Polymerase Chain cattle and buffaloes Polymerase Chaincattle and buffaloes Polymerase Chain Reaction

Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease associated with high morbidity and mortality in the livestock. The conventional method of diagnosis is by the demonstration of the parasite stages by microscopic examination. This method suffers from low sensitivity, making it even more difficult to detect piroplasms in the carriers. PCR based assays are known to be more sensitive. The present study was undertaken to detect and quantify T. annulata in the blood of clinically infected and carrier animals using a quantitative PCR protocol targeting the gene encoding the major merozoite piroplasm surface antigen Tams 1. A total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Of these, 74 samples (63.79%) were positive for T. annulata by real-time PCR, including the 15 samples that were positive by Giemsa staining. The parasite load ranged from 1.39 x 106 to 3.35 x 109 and 0.35 x 106 to 2.83 x 107 ml-1 of blood in cattle and buffalo samples, respectively by qPCR. Our study suggests that real-time PCR assay can be used to detect and quantify the load of T. annulata in the blood of cattle and buffaloes. It also serves as a support to clinical diagnosis and assessment of carrier status in apparently healthy animals.