Oriental theileriosis associated with a new genotype of Theileria orientalis in buffalo (Bubalus bubalis) calves in Uttar Pradesh, India.

Theileria orientalis is known to cause a benign infection in cattle and buffalo (Bubalus bubalis). However, the Ikeda and Chitose genotypes of the parasite cause lethal disease in beef and dairy cattle. Recently an outbreak of clinical oriental theileriosis occurred in buffalo calves in a Government Animal Husbandry and Agricultural Farm located in Uttar Pradesh, India. Examination of Giemsa stained thin blood smears revealed typical rod-shaped T. orientalis piroplasms in the erythrocytes. The clinical signs included pyrexia, nasal discharge, lacrimation, lethargy, inappetence and anaemia with varying degrees of paleness of the visible mucous membranes. Vascular congestion in internal organs, pulmonary emphysema and consolidation of lungs, focal areas of necrosis in the heart with mononuclear cell infiltration, focal mononuclear cell aggregation in the cortex and tubular degeneration of the kidney were significant necropsy findings. The T. orientalis major piroplasm surface protein (MPSP) gene was amplified by polymerase chain reaction (PCR) using specific primers. The nucleotide sequence analysis of the PCR product revealed 84.8% identity between the T. orientalis Uttar Pradesh isolate and other reference genotypes available in the public domain. Furthermore, the phylogenetic analysis of the MPSP gene sequence ratified that this is a new genotype of T. orientalis. This is the first report of a clinical outbreak of oriental theileriosis in Indian buffalo calves caused by a novel genotype of T. orientalis.

A Multi-copy Nucleic Acid-Based Diagnostic Test for Bovine Tropical Theileriosis.

BACKGROUND: Bovine tropical theileriosis (BTT) is a haemoprotozoan tick-borne disease that implicates huge losses to livestock in terms of considerable mortality and morbidity in tropical and subtropical regions of the globe. Currently available diagnostic methods have less specificity and sensitivity towards the detection of Theileria species. Therefore, an attempt was made to diagnose Theileria annulata by targeting a multi-copy gene, viz. mitochondrially encoded cytochrome b (MT-CYB) gene via polymerase chain reaction (PCR) in different agro-zones of India. METHODS AND RESULTS: 129 cattle blood samples were collected from major livestock rearing regions of India and processed for both molecular and microscopic techniques. Screening of Giemsa-stained thin blood smears was able to detect 14 samples (10.85%) as positive for T. annulata. However, the MT-CYB gene-based PCR assay detected 107 samples (82.94%) positive for T. annulata out of 129 samples. Furthermore, the MT-CYB gene-based PCR assay was standardized in terms of its sensitivity and specificity. Specificity of PCR assay was evaluated against other common haemoprotozoan parasites of tropical countries viz. Babesia bigemina, Anaplasma marginale and Trypanosoma evansi. The multi-copy MT-CYB gene-based PCR assay provided an optimum level of sensitivity (up to the level of 10 femtogram) and high specificity. Haematological examination (Hb, PCV and TLC) of 113 samples revealed significantly (p < 0.05) decreased Hb and PCV levels in positive animals in comparison with the control group of healthy animals. However, the control group had significantly higher (p < 0.001) TLC levels than the positive group. CONCLUSION: The MT-CYB gene-based PCR assay was found to be highly sensitive that can accurately detect the occurrence of T. annulata infection in carrier animals which are potential infection sources to healthier populations in naive demographic locations through infected ticks.

Development of a recombinant TaSP-based Dot-ELISA for detection of Theileria annulata infection in cattle.

The study was conducted to develop and validate Dot-ELISA for the diagnosis of Theileria annulata infection in cattle using recombinant Theileria annulata surface protein (r-TaSP). The r-TaSP based indirect plate-ELISA was used as a reference test to compare the efficacy of the Dot-ELISA. The Dot-ELISA was optimized with 500 ng of antigen per dot, 1:150 dilution of serum and 1:1000 dilution of secondary antibody for positive and negative reaction. A total of 17 confirmed positive, 25 negative and 129 field sera samples were used to calculate the diagnostic accuracy of Dot-ELISA in comparison with indirect plate-ELISA. The diagnostic sensitivity and specificity of the Dot-ELISA was 95.8 per cent (95% CI, 93.1-97.2) and 80 per cent (95% CI, 48.1-96.2), respectively. The positive predictive value (PPV) of Dot-ELISA was 98.2 percent (95% CI, 95.5-99.7) and negative predictive value (NPV) was 61.6 percent (95% CI, 37-74). The positive and negative likelihood ratios were 4.79 (95% CI, 1.8-25.69) and 0.053 (95% CI 0.03-1.4), respectively. The Dot-ELISA showed moderate agreement (k value, 0.67, 95% CI, 0.36- 0.82) with indirect plate-ELISA. The developed Dot-ELISA is less expensive and convenient for the diagnosis of T. annulata infection in cattle under field conditions.

Molecular mechanism of synthetic pyrethroid and organophosphate resistance in field isolates of Rhipicephalus microplus tick collected from a northern state of India.

The frequently used chemical control method to manage Rhipicephalus microplus is limited by the emergence of resistance populations. Understanding of resistance mechanisms is essential to develop strategy for sustainable management. The present study was focused on working out the molecular mechanisms of resistance against synthetic pyrethroids (SPs) and organophosphates (OPs) in field isolates of R. microplus collected from six districts of Uttar Pradesh, India. Adult immersion test with discriminating concentrations (AIT-DC) was used to determine resistance status of isolates to SPs (deltamethrin, cypermethrin) and OPs (diazinon, coumaphos). All the six isolates were found resistant to SPs with resistance factor (RF) of 2.9-58.6 and to one of the OP compounds, diazinon having RF of 3.5-13.7 but susceptible to coumaphos (RF < 1.4). Three R. microplus genes, viz. para-sodium channel domain II S4-5 linker, carboxylesterase (372 bp) and acetylcholinesterase 2 (1692 bp) were sequenced and compared with respective sequences of reference susceptible IVRI-I, reference OP resistant population (IVRI-III), IVRI-IV and multi-acaricide resistant population (IVRI-V) of R. microplus. A C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to L64I amino acid substitution was detected in all six isolates. The G1120A mutation in the carboxylesterase gene could not be detected in any isolate. Five nucleotide substitutions viz., G138A, G889A, T1090A, C1234T and G1403A were identified in the acetylcholinesterase 2 gene leading to four amino acid substitutions. The findings of the study corroborate the role of mutation in sodium channel and acetylcholinesterase 2 genes in SP and OP resistance in this part of India.

Determination and validation of discriminating concentration of ivermectin against Rhipicephalus microplus.

Rhipicephalus microplus, the major cattle tick species of India is prevalent all over the country and causes huge economic loss directly or indirectly to the dairy industries. Chemical acaricides are playing an important role in managing tick infestations on livestock for many years and consequently, resistance to commonly used organophosphate (OP) and synthetic pyrethroid (SP) compounds has been reported. Subsequently, ivermectin (IVM) has been emerged as an alternative to manage OP and SP resistant ticks. However, with the increase of use during the last 5-8 years, there is a possibility of development of resistance and thus there is an urgent need to develop a robust resistance monitoring tool to safeguard the drug. Lethal concentrations for 50 and 95% mortality of treated ticks were determined to work out discriminating concentration (DC) in order to diagnose resistance in the field situation. The DC (2 x LC95) was determined as 93.54 ppm using an established reference susceptible IVRI-1 line of R. microplus adopting adult immersion test. For validation of DC, the resistance status was checked in seven tick isolates of R. microplus collected from northern and eastern regions of India. The RR50 and RR95 values of the field isolates against ivermectin were determined and were in the range of 1.56-8.25 and 1.93-27.58, respectively. All the collected isolates were found to have higher lethal concentration and resistance ratio in comparison to reference susceptible IVRI-1 tick line (LC50 = 21.68, LC95 = 46.77 ppm, RR = 1.0). Amongst the field isolates, the isolate collected from Fatehgarh Sahib district (FTG) of Punjab state showed highest RR50 of 8.25 indicating high level of resistance to IVM. The generated DC will be used for IVM resistance characterization of ticks infesting cattle in different parts of the country.

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.

Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus.

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6-99.4% and 98.2-99.7%) and among the tick species (77.3-99.3% and 85.1-99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5-89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2-53.4% and 72.0-74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p<0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.

Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus.

The problem of ticks and tick borne diseases is a global threat and growing reports of resistance to commonly used insecticides further aggravated the condition and demands for country specific resistance monitoring tools and possible solutions of the problem. Establishment of standard reference is prerequisite for development of monitoring tools. For studying possible role of different mechanisms involved in development of resistance in Rhipicephalus (Boophilus) microplus population and to develop newer drug to manage the problem of resistance, a deltamethrin exposed and selected tick colony, referred to as IVRI-IV, was characterized using reference susceptible IVRI-I tick line as control. The RF values of IVRI-IV ticks against deltamethrin, cypermethrin and diazinon were determined as 194.0, 26.6, 2.86, respectively, against adults. The esterase enzyme ratios of 2.60 and 5.83 was observed using α-naphthyl and ß-naphthyl acetate while glutathione S-transferase (GST) ratio was 3.77. Comparative analysis of IVRI-I and IVRI-IV carboxylesterase gene sequences revealed 13 synonymous and 5 non synonymous mutations, reported for the first time. The C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to leucine to isoleucine (L64I) amino acid substitution was also detected in the IVRI-IV population. In the present study, monitorable indicators for the maintenance of the reference IVRI-IV colony, the first established deltamethrin and cypermethrin resistant tick line of India, were identified.

Global gene expression profile of peripheral blood mononuclear cells challenged with Theileria annulata in crossbred and indigenous cattle.

Bovine tropical theileriosis is an important haemoprotozoan disease associated with high rates of morbidity and mortality particularly in exotic and crossbred cattle. It is one of the major constraints of the livestock development programmes in India and Southeast Asia. Indigenous cattle (Bos indicus) are reported to be comparatively less affected than exotic and crossbred cattle. However, genetic basis of resistance to tropical theileriosis in indigenous cattle is not well documented. Recent studies incited an idea that differentially expressed genes in exotic and indigenous cattle play significant role in breed specific resistance to tropical theileriosis. The present study was designed to determine the global gene expression profile in peripheral blood mononuclear cells derived from indigenous (Tharparkar) and cross-bred cattle following in vitro infection of T. annulata (Parbhani strain). Two separate microarray experiments were carried out each for cross-bred and Tharparkar cattle. The cross-bred cattle showed 1082 differentially expressed genes (DEGs). Out of total DEGs, 597 genes were down-regulated and 485 were up-regulated. Their fold change varied from 2283.93 to -4816.02. Tharparkar cattle showed 875 differentially expressed genes including 451 down-regulated and 424 up-regulated. The fold change varied from 94.93 to -19.20. A subset of genes was validated by qRT-PCR and results were correlated well with microarray data indicating that microarray results provided an accurate report of transcript level. Functional annotation study of DEGs confirmed their involvement in various pathways including response to oxidative stress, immune system regulation, cell proliferation, cytoskeletal changes, kinases activity and apoptosis. Gene network analysis of these DEGs plays an important role to understand the interaction among genes. It is therefore, hypothesized that the different susceptibility to tropical theileriosis exhibited by indigenous and crossbred cattle is due to breed-specific differences in the dealing of infected cells with other immune cells, which ultimately influence the immune response responded against T. annulata infection.

Evaluation of Kumaon hill goats for resistance to natural infection with gastrointestinal nematodes.

The present study deals with the investigation of different degrees of genetic resistance/resilience of Uttarakhand hill goats to natural infection with gastrointestinal nematodes in order to introduce into breeding schemes. Animals were naturally infected with Haemonchus contortus, Teladorsagia circumcincta, Oesophagostomum spp. and Trichostrongylus spp. Faecal egg counts (FEC) were carried out every month for a period of 1 year and blood samples were collected every third month for the determination of indicator traits such as FEC, packed cell volume (PCV) and haemoglobin (Hb). The mean egg per gram (EPG), PCV and Hb were 1,579.6 ± 346, 35.12 ± 1.1 and 8.7 ± 0.2, respectively. The goats were divided into three groups (<800, 801-2,000 and >2,000) based on EPG. The EPG showed a negative correlation with both Hb and PCV (P < 0.01). Therefore, it was concluded that the Hb and PCV value would decrease, if EPG increases.

Prophylactic potential of resiquimod against very virulent infectious bursal disease virus (vvIBDV) challenge in the chicken.

The study evaluated the prophylactic potential of resiquimod (R-848), a synthetic TLR7 agonist, against very virulent infectious bursal disease virus (vvIBDV) infection in chicken. Specific pathogen free White Leghorn chicks of three week age were treated with R-848 (50µg/bird, intramuscular) or PBS (n=26/group). Twenty four hour later, half of the birds from each group were challenged with 10(5) ELD50 of vvIBDV and observed for 10days. To understand the effect of R-848, immune response genes such as interferon (IFN)-ß, IFN-γ, IL-1ß, IL-4, iNOS and TLR7 were analyzed at 24 and 48h post-challenge in PBMCs ex vivo by real-time PCR (n=6/group). On day 4 post-challenge, representative birds (n=3/group) were sacrificed to study the bursal damage and IBDV antigen clearance. Immunosuppression was assessed by antibody response against live Newcastle disease virus (NDV) vaccine, which was administered on day 10 post-challenge. R-848 pre-treatment significantly upregulated the transcripts of each immune response gene studied (P<0.05). There was 50% mortality on vvIBDV challenge in control birds, while it was only 20% with R-848 group. R-848 pre-treatment reduced the bursal damage as indicated by lower bursal lesion score in histopathology, reduced IBDV antigen signal in immunohistochemistry and improved antigen clearance in agar gel immunodiffusion test. Further, it protected significantly against vvIBDV induced immunosuppression as indicated by HI antibody titre. It is concluded that pre-treatment of R-848 conferred partial protection from mortality and bursal damage while complete protection against immunosuppression in chicken when challenged with vvIBDV, which could be due to the upregulation of immune response genes.

No change in mRNA expression of immune-related genes in peripheral blood mononuclear cells challenged with Theileria annulata in Murrah buffalo (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) act as carrier to Theileria annulata and show less clinical sign of tropical theileriosis as compared to indigenous and exotic cattle. Differential expression of immune-related genes such as major histocompatibility complex, class II, DQ alpha 1 (MHC-DQα), signal-regulatory protein alpha (SIRPA), prion protein (PRNP), Toll-like receptor 10 (TLR10), c-musculoaponeurotic fibrosarcoma oncogene homolog (cMAF) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) genes influence host resistance to this disease in exotic, crossbred and indigenous cattle. In the present study we examined the differential mRNA expression of the abovesaid immune-related genes in response to T. annulata infection in buffaloes. Peripheral blood mononuclear cells (PBMCs) harvested from blood samples of buffaloes were challenged with ground-up tick supernatant carrying T. annulata sporozoites in vitro. After 48h of in vitro challenge qPCR was employed to measure the relative mRNA expression of MHC-DQα, SIRPA, PRNP, TLR10, cMAF and MAFB genes in infected and control PBMCs. In the current study, the selected genes showed no change in mRNA expression after T.annulata infection which indicates that they have little role in providing host resistance to theileriosis in buffaloes.

Esterase mediated resistance in deltamethrin resistant reference tick colony of Rhipicephalus (Boophilus) microplus.

Monitoring of acaricide resistance is considered as one of the important facets of integrated tick management. In an attempt of development of resistance monitoring indicators, in the present study two reference tick lines of Rhipicephalus (Boophilus) microplus maintained in the Entomology laboratory, Indian Veterinary Research Institute (IVRI), Izatnagar, India, were studied to determine the possible contributing factors involved in development of resistance to deltamethrin. Electrophoretic profiling of esterase enzymes detected high activities of EST-1 in reference resistant tick colony designated as IVRI-IV whereas it was not detectable in reference susceptible IVRI-I line of R. (B.) microplus. Esterases were further characterized as carboxylesterase or acetylcholinesterase based on inhibitor study using PMSF, eserine sulphate, malathion, TPP and copper sulphate. It was concluded that an acetylcholinesterase, EST-1, possibly plays an important role for development of deltamethrin resistance in IVRI-IV colony of R. (B.) microplus.

Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken.

Resiquimod (R-848), an imidazoquinoline compound, is a potent synthetic Toll-like receptor (TLR) 7 agonist. Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P ≤ 0.01) as well as CD4(+) and CD8(+) cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 µg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-ß, IFN-γ, IL-1ß, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes.

Survey of acaricides resistance status of Rhipiciphalus (Boophilus) microplus collected from selected places of Bihar, an eastern state of India.

Monitoring acaricide resistance in field ticks and use of suitable managemental practices are essential for controlling tick populations infesting animals. In the present study, the acaricide resistance status in Rhipicephalus (Boophilus) microplus ticks infesting cattle and buffaloes of five districts located in the eastern Indian state, Bihar were characterized using three data sets (AIT, Biochemical assays and gene sequences). Adult immersion test (AIT) was adopted using seven field isolates and their resistance factor (RF) was determined. Six isolates (DNP, MUZ, BEG, VSH, DRB and SUL) were found resistant to both deltamethrin and diazinon and except VSH all were resistant to cypermethrin. One isolate (PTN) was susceptible with a RF below 1.5. To understand the possible mode of resistance development, targeted enzymes and gene sequences of the para sodium channel and achetylcholinesterase 2 (AChE2) were analyzed. The esterase, monooxygenase and glutathione-S-transferase (GST) activity of reference susceptible IVRI-I line was determined as 2.47 ± 0.007 nmol/min/mg protein, 0.089 ± 0.0016 nmol/mg of protein and 0.0439 ± 0.0003 nmol/mg/min respectively, which increased significantly in the resistant field isolates. However, except esterases, the fold increase of monooxygenase (1.14-2.27 times) and GST (0.82-1.53 times) activities were not very high. A cytosine (C) to adenine (A) nucleotide substitution (CTC to ATC) at position 190 in domain II S4-5 linker region was detected only in one isolate (SUL) having RF of 34.9 and in the reference deltamethrin resistant line (IVRI-IV). However, the T2134A mutation was not detected in domain IIIS6 transmembrane segment of resistant isolates and also in reference IVRI-IV line despite of varying degree of resistance. The flumethrin specific G215T and the recently identified T170C mutations were also absent in domain II sequences under study. Four novel amino acid substitutions in AChE2 gene of field isolates and in organophosphate (OP) resistant reference IVRI-III line were identified which can possibly have a role in resistance development.

Administration of TLR7 agonist, resiquimod, in different types of chicken induces a mixed Th1 and Th2 response in the peripheral blood mononuclear cells.

This study evaluated the variation in immune response in peripheral blood mononuclear cells (PBMCs) of broiler, White Leghorn (WL) and Kadaknath breeds of chicken following administration of TLR7 agonist, resiquimod (R-848). Expression of different immune related genes viz., interferon-ß (IFN-ß), IFN-γ, IL-1ß, IL-4, TLR7 and iNOS was assessed by quantitative real time PCR over a period of 24 h. The results indicated that there was a significant up-regulation in the relative expression of immune response genes post R-848 administration (P < 0.01). In conclusion, the transcriptional expression of IFN-ß, IFN-γ, IL-1ß, IL-4, iNOS and TLR7 genes in the PBMCs was significantly up-regulated over 24 h in broiler, WL and Kadaknath breeds of birds after the administration of R-848. Overall, R-848 induced a mixed Th1 and Th2 response in PBMCs of chicken origin ex vivo.

The mRNA expression of immune-related genes in crossbred and Tharparkar cattle in response to in vitro infection with Theileria annulata.

Tropical theileriosis is a major protozoan disease of cattle and is associated with high rates of morbidity and mortality. Indigenous cattle (Bos indicus) are less affected by this disease than exotic and crossbred cattle. Genetic basis of resistance to tropical theileriosis in indigenous cattle is not well studied. Recent reports suggest that number of immune response genes expressed differentially in exotic and indigenous breeds play an important role in breed specific resistance to tropical theileriosis. Such studies comparing expression of these genes in crossbred cattle and indigenous cattle are lacking. The present study compares the mRNA expression of immune-related genes in response to Theileria annulata infection in indigenous and crossbred cattle. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of indigenous (Tharparkar) and crossbred (HF/BS/Jersey × Hariana) cattle and challenged with prepared ground-up tick supernatant carrying Theileria annulata sporozoites in vitro. qPCR was employed to measure relative mRNA expression of toll-like receptor 10 (TLR10), signal-regulatory protein alpha (SIRPA), MHC class II DQα (BoLA-DQA), musculoaponeurotic fibrosarcoma (MAF) and prion protein (PRNP) genes in infected and control PBMCs from crossbred and indigenous cattle. On the basis of comparative fold change analysis, significant up-regulation in SIRPA, PRNP and MHC DQα genes and significant down-regulation in TLR10, cMAF and MAFB genes in crossbreds as compared to indigenous cattle was observed. Results of the present study suggest that breed specific differential expression of the genes under study may contribute to the breed specific resistance to Theileria annulata infection in indigenous cattle compared to crossbred cattle.

Babesia bigemina infection in yak (Poephagus grunniens L.): molecular detection and characterization.

Yaks contribute significantly in the Himalayan high land economy. Specific information on prevalence of babesiosis in yaks is lacking. A fast and reliable PCR assay targeting Babesia bigemina small subunit ribosomal RNA sequence (SS rRNA) was laboratory standardized for molecular detection of B. bigemina in yaks. Restriction digestion of the PCR amplified 675 bp target sequence with Vsp I confirmed the prevalent species of Babesia as B. bigemina. Nucleotide sequencing and phylogenetic analysis of PCR amplified 675 bp SS rRNA sequence revealed a close genetic relationship with other bovine isolates of B. bigemina. A PCR based survey involving 94 blood samples of yak from the National Research Centre on Yak, Dirang, Arunachal Pradesh detected infection in 5.32% of yak blood samples, which was significantly higher in comparison to microscope based detection of infection in 2.13% blood smears. This is the first report on sensitive PCR based detection of B. bigemina infection in yaks and PCR-RFLP and nucleotide sequence analysis based molecular characterization of the B. bigemina isolated from yaks.

Molecular characterization of surface antigen 3 (SAG3) gene of Toxoplasma gondii RH-IVRI strain.

Toxoplasmosis, caused by Toxoplasma gondii, is a zoonotic disease and is responsible for abortions, stillbirth, and neonatal complications in livestock, especially in sheep, goats, and pigs. The molecular characterization of the parasite having global distribution is considered important to delineate the phylogenetic relationship among different isolates/strains of the parasite. The present communication deals with the molecular cloning and sequence analysis of the 1,158 bp entire open reading frame of surface antigen 3 (SAG3) of T. gondii RH strain being maintained at the Indian Veterinary Research Institute (IVRI). The sequence comparison analysis revealed 99.9 % homology with the published sequence of T. gondii RH strain, with a single substitution of guanine 'G' instead of adenine 'A' at the 397th position of SAG3 sequence. The substitution of single nucleotide consequently resulted in the change of one amino acid residue of aspartic acid (D) instead of asparagine (N) present in the published sequence of RH strain. This denotes that the SAG3 gene of this RH strain has not undergone a major change in its molecular conformation even after repeated passage in mice for more than a decade at IVRI. The finding is important from the molecular phylogeny point of view.

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata.

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.