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.

Seasonal incidence of parasitic diseases in bovines of south western Gujarat (Junagadh), India.

The current study was conducted to investigate the incidence of parasitic diseases in bovines which were sick and brought at veterinary hospital for treatment. A total of 366 samples were investigated from cattle (n = 175) and buffaloes (n = 191) presented at Teaching Veterinary Clinical Complex (TVCC), Veterinary College, Junagadh, Gujarat during January to December 2014. Examination of Giemsa-stained peripheral blood smears exhibited that 58.6 % of cattle and 41.2 % of buffaloes were infected with haemoparasites comprising Babesia bigemina, Theileria annulata, and Anaplasma marginale @ of 54.0, 3.4 and 1.1 in cattle and 38.8, 1.2 and 1.2 percent in buffaloes, respectively. The incidence of total haemoparasites and B. bigemina infections was significantly higher (p < 0.05) in cattle whereas, incidence of haemoparasites were recorded significantly higher in the month of July to November (i.e., rainy and autumn) in both cattle and buffaloes, respectively (p < 0.01 and p < 0.001). Coprological examination revealed that the overall incidence of gastrointestinal (GI) parasitic infection was 45.5 % in cattle and 43.4 % in buffaloes. The incidence of individual parasite was 11.4, 1.1, 2.3, 4.5, 1.1, 3.4, 2.3 and 19.3 in cattle and 4.7, 0.9, 0.0, 2.8, 0.9, 5.7, 0.0 and 28.3 % in buffaloes for Eimeria spp., Trichuris spp., Toxocara vitulorum, Strongyle, Fasciola spp., amphistomes, Schistosoma indicum and Buxtonella sulcata, respectively which differ insignificantly (p > 0.05). Seasonal prevalence of GI parasites was highest in summer in both cattle and buffaloes, the data being statistically non-significant (p > 0.05). However, the incidence of B. sulcata in both cattle (19.3 %) and buffaloes (28.3 %) was higher in comparisons to other GI parasites. The present investigation emphasized that B. bigemina and B. sulcata are the most important parasites of bovines of this region.

Anti cancerous efficacy of Ayurvedic milk extract of Semecarpus anacardium nuts on hepatocellular carcinoma in Wistar rats.

The objective of the study was to determine the anticancerous efficacy of Ayurvedic preparation made of Semecarpus anacardium (SA) nuts. Five groups of rats were used for the study. Group I served as water control. Hepatocellular carcinoma (HCC) was induced in groups II, III and IV animals using N-nitrosodiethylamine as inducing agent followed by phenobarbitone as promoter for 13 weeks. Group-II animals were kept untreated as hepatocellular carcinoma control. Group-III animals were treated with Ayurvedic milk extract of Semecarpus anacardium nuts at dose mentioned in Ashtangahridaya, an authentic book of Ayurveda for 49 days and group-IV animals were treated with doxorubicin as reference drug at dose of 1mg/kg twice a week for 7 weeks. Group V animals were kept as drug (SA nut milk extract) control for studying the effect of nut milk extract on normal rats. After 154 days of experiment, all animals were subjected to screening for HCC by estimation of liver enzymes, HCC marker (alpha-2 macroglobulin) and histopathology. Both liver enzymes and HCC marker were increased in hepatocellular carcinoma control along with neoplastic changes in liver and were decreased in Semecarpus anacardium nut milk extract treated group. The Ayurvedic drug showed positive correlation with the action of doxorubicin. This study demonstrated the efficacy of Semecarpus anacardium nut milk extract for the treatment of hepatocellular carcinoma either alone or along with chemotherapy.