Identification, molecular characterization and risk factors of Theileria infection among sheep: a first comprehensive report from North India.

The present study was aimed at the identification, molecular characterization, and risk factor assessment of Theileria infection among sheep of Haryana province, north India. A total of 402 blood samples were collected from three different climatic zones of Haryana from March 2020 to September 2021. Light microscopy of blood smears revealed Theileria spp. infection in 47.26% (n = 190), while 60.94% (n = 245) of blood samples were positive using nested PCR. Extensive molecular characterization of Theileria infection using four pairs of species-specific primers indicated the dominance of T. ovis (29.1%) followed by T. lestoquardi (12.69%), T. luwenshuni (5.97%) and T. annulata (1.49%). Mixed infection was detected in 11.69% of cases. Bidirectional sequencing and phylogeny further confirmed the presence of these four Theileria spp. in the investigated area under study. Hematology indicated a significant (p < 0.01) reduction in various haematological indices of animals infected with T. luwenshuni and T. lestoquardi compared to the healthy control group. Risk factors like age, sex, and zone were significantly associated with Theileria infection in sheep. The present investigation depicts the first comprehensive molecular report of ovine Theileria spp., which warrants further study to develop suitable control strategies against these haemoparasitic infections.

Salivary cell-free HSD17B1 and HSPA1A transcripts as potential biomarkers for estrus identification in buffaloes (Bubalus bubalis).

Estrus detection is a major problem in buffaloes because of the poor expression of estrus signs leading to low reproductive efficiency. Salivary transcripts analysis is a promising tool to identify biomarkers; therefore, the present study was carried out to evaluate their potential as estrus biomarkers. The levels of HSD17B1, INHBA, HSPA1A, TES transcripts were compared in saliva during estrous cycle stages [early proestrus (day -2, EP), late proestrus (day-1, LP), estrus (E), metestrus (ME) and diestrus (DE)] of cyclic heifers (n = 8) and pluriparous (n = 8) buffaloes by employing quantitative real-time polymerase chain reaction (qRT-PCR). The levels of HSD17B1 (EP/DE 1.46-2.43 fold, LP/DE 2.49-3.06 fold; E/DE 7.21-11.9-fold p < 0.01; ME/D 1.0-1.16 fold) and HSPA1A (EP/DE 0.93-2.39 fold, LP/DE 2.68-3.23 fold; E/DE 8.52-15.18 fold p < 0.01; ME/D 0.86-1.01 fold) were significantly altered during the estrus than other estrous cycle stages in both cyclic heifers and pluriparous buffaloes. Receiver operating characteristic curve analysis revealed the ability of salivary HSD17B1 (AUC 0.96; p < 0.001) and HSPA1A (AUC 0.99; p < 0.01) to differentiate E from other stages of the estrous cycle. Significantly higher levels of HSD17B1 and HSPA1A transcripts in saliva during the estrus phase suggest their biomarkers potential for estrus detection in buffaloes.

Development of a loop-mediated isothermal amplification assay based on RoTat1.2 gene for detection of Trypanosoma evansi in domesticated animals.

The early containment of trypanosomosis depends on early, sensitive, and accurate diagnosis in endemic areas with low-intensity infections. The study was planned to develop a simple read out loop-mediated isothermal amplification (LAMP) assay targeting a partial RoTat1.2 VSG gene of Trypanosoma evansi with naked eye visualization of LAMP products by adding SYBR® Green I dye. The visual results were further confirmed with those of agarose gel electrophoresis, restriction enzyme digestion of LAMP products with AluI, and sequencing of the PCR products using LAMP outer primers. The LAMP primers did not show cross reactivity and non-specific reactions with regional common hemoparasitic DNA revealing high specificity of the assay. The threshold sensitivity level of the LAMP assay was determined to be 0.003 fg compared to 0.03 fg RoTat1.2 amplified DNA fragments of T. evansi by PCR assay. Moreover, assessment of 500 blood samples collected from unhealthy domestic animals in field suspected for various hemoparasitic infections was carried out for the presence of T. evansi by microscopy, RoTat1.2 VSG PCR, and LAMP assay. LAMP could detect T. evansi in 36 samples, while PCR and microscopy could detect 33 and 12 samples, respectively. All the samples positive by microscopy and PCR were also confirmed positive by the LAMP assay. The current LAMP assay has appealing point of care characteristics to visually monitor the results, lessen the need of post DNA amplification procedure, and enable this method to be applied as a rapid and sensitive molecular diagnostic tool in under resourced laboratories and field setup.

Pentaplex PCR assay for rapid differential detection of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi in cattle.

A multiplex PCR (mPCR) assay for simultaneous detection and differentiation of four major haemoparasites in crossbred cattle was established using parasite specific genomic DNA and four sets of primer pairs targeting AMA-1, Tams1, MSP5 and VSG genes of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi generating precise amplicons of 448, 156, 382 and 110 bp, respectively. An internal amplification control, 202 bp bovine ß-casein gene fragment, was simultaneously amplified with four target genes to avoid false-negative results. The sensitivity of mPCR was 3.44 × 102, 5.9 × 103, 2.88 × 102 and 3.3 × 103 copies for B. bigemina, T. annulata, A. marginale and T. evansi, respectively. mPCR of cattle clinical samples (n = 516), suspected for haemoparasites, revealed single haemoparasitic infection in 279 (54.06%) cases, whereas mixed infection was recorded in 54 (10.46%) samples. In clinical samples, coinfection with T. annulata and A. marginale was the most common. The findings of mPCR were consistent with uniplex PCR under field conditions except for subtle variations in A. marginale infection. Overall, the mPCR assay represents an economical, reproducible and robust diagnostic tool for concurrent detection of cattle haemoparasites and large scale epidemiological studies.

Molecular detection and characterization of prevailing Theileria equi genotype in equine from northern India.

Equine piroplasmosis caused by Theileria equi is a febrile, tick-borne disease of equids. However, there is limited literature about the genotyping of T. equi in India. Blood samples were collected from 202 horses and subjected to microscopy and PCR to detect T. equi. Initially, a universal screening primer pair targeting 18S ribosomal RNA genes common for Babesia caballi and T. equi was employed to amplify the DNA of both parasites. Thereafter additional primers were employed for species-specific detection resulting in amplification of approximately 435 bp specific for T. equi. T.equi was detected in 9.9% and 20.79% of horses screened by microscopy and PCR, respectively. The representative samples confirmed positive by PCR were sequenced, submitted to NCBI (OR651254, OR687254, OR685656, OR650830, OR650834), and used for genotype characterization and phylogenetic analysis. Employing Genetool and MEGA X software, the T. equi Indian isolates and across the globe were compared, and the results demonstrated 99.05-100% and 95.86-100% homologies, respectively. All the T. equi Indian isolates belonged to genotype A. Phylogeny based on the EMA-1 gene of five isolates (OR731831, OR731833, OR731829, OR731830, OR731832) were also characterized by sequencing and support the previous findings. Genotypes C and D, as well as genotypes B and E, exhibited lower levels of evolutionary divergence compared to other genotypes. The EMA-1 gene exhibited limited diversity and might not be the most suitable target for assessing variability within T. equi populations. The findings also reveal a significant association (p < 0.01) between T. equi infection and the presence of ticks.

Local and systemic inflammatory response to the intrauterine infusion of enzymes during estrus in water buffaloes with subclinical endometritis.

Our objective was to determine the effects of intrauterine infusion of proteolytic enzymes in buffaloes with subclinical endometritis (SCE) at estrus on the resolution of endometrial inflammation and reproductive performance. Buffaloes at spontaneous estrus (E1) were screened for SCE by endometrial cytology to identify SCE (≥5% PMN, n = 22) and non-SCE (<5% PMNs, n = 14) animals. All buffaloes underwent uterine ultrasonographic examination, low volume uterine lavage (cytokines and acute phase proteins) and blood sampling (cytokines and acute-phase proteins) at E1. On the same day (E1), SCE buffaloes were randomly selected either for intrauterine infusion of proteolytic enzymes (ENY, n = 11) or saline (PC, n = 11). Buffaloes without SCE were kept as untreated control (NC; n = 14). All buffaloes were re-examined and re-sampled during subsequent estrus (E2), inseminated during the following estrus (E3), and assessed for fertility related outcomes. Proteolytic infusion resulted a reduction in uterine PMN (P < 0.01) in SCE buffaloes. The concentrations of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in uterus, and TNF-α and IL-10 in serum were higher (P < 0.01) at E1 in buffaloes with SCE (PC and ENY) compared to NC. After treatment, uterine IL-1ß and TNF-α (P = 0.02), and serum TNF-α and IL-10 were lower within the animals of ENY group (P < 0.01). Before treatment, buffaloes with SCE had higher concentrations (P < 0.01) of serum and uterine amyloid-A and haptoglobin, which decreased (P < 0.01) after treatment in the ENY group. None of the fertility outcomes differ between the treatment groups. In conclusion, intrauterine infusion of proteolytic enzymes reduced endometrial inflammation; however, did not improve reproductive outcomes.

Molecular Detection and Differentiation of Different Theileria Species in Naturally Infected Goats Using Nested PCR-RFLP: A First Report from Northern India.

PURPOSE: Theileriosis is an economically important tick-borne pathogen with a serious impact on livestock health and productivity. Despite the fact that bovine theileriosis has been widely investigated, there exists a paucity of information on these infections in small ruminants, especially in India. The present study was carried out to detect and differentiate different Theileria spp. in goats using nested PCR RFLP. METHODS: Blood samples and ticks were collected from 405 goats in various agro-climatic zones of Haryana state, India. The blood samples were screened by microscopy, nested PCR-RFLP, and sequence analysis. The nested PCR-RFLP was performed with four restriction enzymes viz., Hpa II, Bsh 1285I, Hae II and Rsa I. Six nested PCR amplicons with different RFLP patterns were sequenced and submitted to NCBI (OM666861, MZ220430, OM666628, MZ220437, OM666637, OM721806). RESULTS: Microscopy revealed 18.27% (n = 74) infection with Theileria spp., while 33.58% (n = 136) of blood samples were confirmed positive by nested PCR. Out of 136 positive samples, 43.38% (n = 59), 11.02% (n = 15) and 20.58% (n = 28), were positive for T. ovis, T. lestoquardi and T. luwenshuni (Theileria sp. China 1), respectively. Mixed infection was detected in 25% (n = 34) cases. Based upon Hpa II digestion pattern, 13 samples with T. lestoquardi and T. ovis, and 21 samples with T. ovis and T. luwenshuni were detected. Sequence study further confirmed their identity. The majority of ticks collected from goats were identified as Rhipicephalus spp., Hyalomma anatolicum and Hemaphysalis spp. CONCLUSION: This study represents the first confirmed molecular report of goats infected with T. ovis, T. lestoquardi, and T. luwenshuni from northern India.

18S rRNA Gene-Based Piroplasmid PCR: An Assay for Rapid and Precise Molecular Screening of Theileria and Babesia Species in Animals.

PURPOSE: The parasites of genera such as Babesia and Theileria are called piroplasmids due to the pear-shaped morphology of the multiplying parasite stages in the blood of the vertebrate host. Because of the enormous number of parasite species and the challenges of multiplex PCR, initial screening of samples using piroplasmid-specific PCR may be a more cost-effective and efficient technique to identify parasite species, especially during epidemiological studies. Accordingly, 18S rRNA PCR was standardized and optimized on common piroplasmids of different animals like cattle, buffaloes, sheep, goats, dogs, horses, and leopards. METHODS: Bloods samples from 1250 animals were collected from different animals in Junagadh district of Gujarat, India. 18S rRNA PCR was standardized and optimized as a primary method for molecular screening of piroplasms in domestic and wild animals. The method was checked for its analytical sensitivity and specificity. Parasite species-specific PCR and sequencing was used to validate the test. Moreover, in-silico restriction enzyme (RE) analysis was also done to assess its applicability in PCR-RFLP. RESULTS: Piroplasm infections were recorded in 63.3% of animals in Junagadh. The 18S rRNA PCR detected the piroplasmid DNA in as low as 39 picograms (pg) of whole blood genomic DNA isolated from microscopically Theileria positive blood samples and no reactivity was recorded from common but unrelated haemoparasites viz., Trypanosoma evansi, Hepatozoon spp., Anaplasma spp., and Ehrlichia canis was observed. The 18S rRNA PCR assay findings were confirmed by species-specific PCR and sequencing. Analysis of different sequences generated using 18S rRNA PCR revealed that the amplicon size of Babesia spp. is nearly 400 bp (393-408 bp) whereas Theileria spp. were more than 400 bp (418-424 bp). The percentage of sequence divergence among Babesia and Theileria spp. was 7.3-12.2% and 0.7-12.2%, respectively. In-silico restriction enzyme (RE) analysis reveals the presence of at least one site for a commercially available RE in 18S rRNA fragments of every parasite, which can differentiate it from its congeners. CONCLUSIONS: The presented universal oligonucleotide-based PCR assay provides a highly sensitive, specific, cost-effective, and rapid diagnostic tool for the initial screening of piroplasmids infecting domestic and wild animals and is potentially helpful for large-scale epidemiological studies.

Evaluation of the Immunoprotective Potential of Recombinant Paraflagellar Rod Proteins of Trypanosoma evansi in Mice.

Trypanosomosis, caused by Trypanosoma evansi, is an economically significant disease of livestock. Systematic antigenic variation by the parasite has undermined prospects for the development of a protective vaccine that targets the immunodominant surface antigens, encouraging exploration of alternatives. The paraflagellar rod (PFR), constituent proteins of the flagellum, are prominent non-variable vaccine candidates for T. evansi owing to their strategic location. Two major PFR constituent proteins, PFR1 (1770bp) and PFR2 (1800bp), were expressed using Escherichia coli. Swiss albino mice were immunized with the purified recombinant TePFR1 (89KDa) and TePFR2 (88KDa) proteins, as well as with the mix of the combined proteins at equimolar concentrations, and subsequently challenged with virulent T. evansi. The PFR-specific humoral response was assessed by ELISA. Cytometric bead-based assay was used to measure the cytokine response and flow cytometry for quantification of the cytokines. The recombinant TePFR proteins induced specific humoral responses in mice, including IgG1 followed by IgG2a and IgG2b. A balanced cytokine response induced by rTePFR 1 and 2 protein vaccination associated with extended survival and improved control of parasitemia following lethal challenge. The observation confirms the immunoprophylactic potential of the covert antigens of T. evansi.

High seroprevalence of Toxoplasma gondii in goats in Jharkhand state of India.

Toxoplasmosis, caused by Toxoplasma gondii, is an important food borne zoonosis worldwide. Although goat meat constitutes an important dietary protein source, improperly cooked meat is a potential source of infection to humans. Data on prevalence of toxoplasma in goat is scanty from India. Serological detection is the practical option for prevalence studies on T. gondii, as no biological stage of the parasite is present in the clinical materials from the intermediate hosts. The present study was undertaken in the Jharkhand state of India which is largely inhabited by economically weaker aborigine population, who depend largely on animal husbandry for livelihood. A total of 445 serum samples were collected for testing, which represented goats under intensive and free range system of rearing. T. gondii specific IgG antibodies were detected in 42.47% (n = 189) samples by rSAG1 based indirect ELISA. The seroprevalence data were analyzed in respect of age, sex, breed of the goats and altitude of the study area as well as rearing conditions of the animals to establish correlation, if any. Though age and sex of the animals had a direct correlation with infection, the same could not be established with the other factors. The sensitivity and specificity of the diagnostic ELISA were compared with IFAT, as well as with a commercially available ELISA kit. The rSAG1-ELISA had 92.66% sensitivity and 90.67% specificity with a positive predictive value of 86.77% and negative predictive value 94.92% when compared with IFAT, whereas when compared with the commercial ELISA kit, 87.50% sensitivity and 90.91% specificity with a positive predictive value of 91.30% and negative predictive value 86.96% were observed. Inter rater agreement (kappa) was calculated. rSAG1-ELISA showed good agreement with IFAT (kappa = 0.824) and commercially available ELISA Kit (kappa = 0.783). Receiver Operating Characteristics (ROC) curve analysis, revealed a larger area under curve (AUC) of 0.99 (95%CI, 0.97-1.0) when compared with IFAT as gold standard and a highest relative sensitivity 91.30 (95% CI 72-98.3) and specificity 1.0 (95% CI 85.2-100) for the cut off value of 0.6005. The present study revealed high seroprevalence of T. gondii in goats from Jharkhand, which has public health significance.

PCR-RFLP based genotyping of Indian isolates of Toxoplasma gondii.

Toxoplasma gondii is an apicomplexan parasite capable of infecting a wide variety of warm-blooded animals, including birds and humans and is zoonotically important too. Felidae serve its definitive hosts and most infections are inoccous while in various intermediate hosts (e.g. sheep), it is responsible for abortion, still births. Humans which are immune compromised are also susceptible to toxoplasmosis. Most of the epidemiological studies have revealed it to be belonging to three clonal types with exceptions in South Africa having atypical isolates. Current genotyping was carried out at 11 genetic loci (SAG1, 5'-SAG2, 3'-SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358 and PK1) using multiplex-nested polymerase chain reaction restriction fragment length polymorphism (Mn-PCR-RFLP). SAG1, alt SAG2, SAG3, BTUB, GRA6, C22-8, C29-2, L358 and PK1 could differentiate our strain/isolates as type I (T. gondii RH) and type III (T. gondii isolates from Chennai and Izatnagar). 5'SAG2 and 3'SAG2 in combination confirmed these as above mentioned genotypes. Further, the T. gondii RH was assigned Toxo DB#10 and local isolates of T. gondii were assigned Toxo DB#2. The present study is the first report on existence of Type III T. gondii lineage from animal population of Indian subcontinent based on PCR-RFLP.

Traditional versus molecular based detection of a rare occurrence of babesiosis in Gir calf and its therapeutic management.

A 30-day old Gir calf was brought to Veterinary Polyclinic with symptoms of high fever, dullness, dyspnea, pale mucus membrane and haemoglobinuria. Blood sample was collected and microscopic examination of thin blood smear confirmed the case of acute babesiosis. It was further confirmed by polymerase chain reaction that amplified an approximately 410 bp portion of the ssu-rDNA of Babesia spp. The calf was managed with diminazene aceturate @5 mg/kg (Berenil) intramuscularly followed by supportive therapy including intravenous infusions. The present study reports a rare case of bovine babesiosis, its clinical variants, diagnosis, hematology and therapeutic management.

Important hemoprotozoan diseases of livestock: Challenges in current diagnostics and therapeutics: An update.

Hemoprotozoan parasites pose a serious threat to the livestock population in terms of mortality, reduced milk yield and lowered draft power. Diagnosis of these diseases often poses a challenging task. Needless to say that impact of disease in health and productivity is huge though a fair economic assessment on the quantum of economic loss associated is yet to be worked out from India. The diagnosis of hemoprotozoan infections largely depends on various laboratory-based diagnostic methods as the clinical manifestations are often inconspicuous and non-specific. Traditional diagnostic methods rely on microscopical demonstration of infective stages in blood or tissue fluids. However, it is laborious, lesser sensitive, and cannot differentiate between morphologically similar organisms. Recent development in the technologies has opened new avenues for improvement in the accurate diagnosis of parasitic infections. Serological tests are simple, fast but lack specificity. With advent of molecular techniques, as DNA hybridization assays, polymerase chain reaction and its modifications ensure the detection of infection in the latent phase of the disease. Nucleic acid-based assays are highly sensitive, free from immunocompetence and can differentiate between morphologically similar parasites. With the advent of newer diagnostics complemented with traditional ones will be of huge help for targeted selective treatment with better chemotherapeutic agents.