Seasonal pattern in occurrence of rotavirus infection (RV) in diarrheic children, calves and piglets from Bareilly, India.

Rapid and reliable diagnosis for diarrhoeal disease is critically important for the differentiation of etiological agents and subsequent suitable treatment modalities. The objective of the study is to reveal the seasonal pattern in the occurrence of rotavirus in diarrheic children, calves and piglets from Bareilly, Uttar Pradesh, India. A total of 115 diarrhoeal samples were collected, out of which 51 were collected during post-monsoon/autumn (September 2018-November 2018) and 64 during the winter season (December 2018-February 2019). The samples were collected from children <5 years (n = 50), piglets <3 months (n = 35) and calves <6 months of age (n = 30). These samples were screened by ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) by targeting the VP6 gene of rotavirus A (RVA) and the two were compared. In RNA-PAGE 29.4% (5/17), 6.3% (1/16) and 0% (0/18) samples collected from children, calves and piglets, respectively were rotavirus positive during the autumn season while 45.5% (15/33), 21.4% (3/14) and 17.7% (3/17) samples in the winter season. In RT-PCR, 41.2% (7/17), 12.5% (2/16) and 0% (0/18) samples were rotavirus positive in the autumn season while 51.5% (17/33), 28.6% (4/14) and 29.4% (5/17) samples in winter season collected from children, calves and piglets, respectively. On statistical analysis, no significant difference between the season and number of positives in children and calves (p > 0.05) was observed, however in piglets significantly higher number of RVA positives were detected in the winter season than autumn (p < 0.01). The diagnostic test comparison of RNA-PAGE and RT-PCR showed no statistically significant difference in detecting the RVA positives (p > 0.05). Overall the percent positivity showed a seasonal pattern with higher positivity in winter as compared to autumn season.

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.

Molecular tools-advances, opportunities and prospects for the control of parasites of veterinary importance.

The recent advancement in genome sequencing facilities, proteomics, transcriptomics, and metabolomics of eukaryotes have opened door for employment of molecular diagnostic techniques for early detection of parasites and determining target molecules for formulating control strategies. It further leads to the introduction of several purified vaccines in the field of veterinary parasitology. Earlier, the conventional diagnostic methods was entirely based upon morphological taxonomy for diagnosis of parasites but nowadays improved molecular techniques help in phylogenetic study and open an another area of molecular taxonomy of parasites with high precision. Control measures based upon targeting endosymbionts in parasites like Dirofilaria immitis is also under exploration in veterinary parasitology. Metagenomics have added an inside story of parasites bionomics which have created havoc in human and animals population since centuries. Omics era is playing a key role in opening the new approaches on parasite biology. Various newer generations of safer vaccines like edible vaccines and subunit vaccines and diagnostic techniques based upon purified immunologically active epitopes have become commercially available against the parasites (helminths, protozoa and arthropod borne diseases). Nowadays, a transgenic and gene knock out studies using RNA interference and CRISPR are also helping in understanding the functions of genes and screening of target genes, which are not available before the advent of molecular tools. Molecular techniques had paramount impact on increasing the sensitivity of diagnostic tools, epidemiological studies and more importantly in controlling these diseases. This review is about the advancements in veterinary parasitology and their impact on the control of these pathogens.

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.