Molecular identification of haemoparasites in animals using blood lysate PCR: a quick and inexpensive alternative to purified whole genomic DNA.

Haemoparasitic diseases constitute a significant constraint to economic livestock farming. Diagnostic techniques that are inexpensive, rapid, reliable, and precise are crucial for the management of diseases. In this context, PCR assays are very valuable yet expensive since the samples must be processed before being included in the PCR reaction. Accordingly, the goal of the current study was to lower the PCR costs without jeopardizing the assay's sensitivity and specificity. For that purpose, the alkaline solution was optimized for low cost and quick DNA extraction (blood lysate), and PCR reagents were modified for optimum reaction. In comparison to purified whole blood genomic DNA, the currently developed and optimized blood lysate method was found to be 95.5% less expensive, as well as being equally sensitive and specific for the molecular detection (PCR) of haemoparasites like Babesia, Theileria, Trypanosoma and rickettsiales in cattle, buffaloes, horses, and dogs. The assay was also demonstrated to be quick, less likely to cross-contaminate, and appropriate for use in laboratories with limited resources. Therefore, the currently developed and optimized blood lysate method could serve as a viable alternative to purified whole blood genomic DNA for molecular detection (PCR) of haemoparasites in animals particularly in resource-limited settings.

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.

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.

Haemato-biochemical characterization of fasciolosis in Gir cattle and Jaffrabadi buffaloes.

A total of 438 faecal samples of Gir cattle & Jaffrabadi buffaloes were examined by sedimentation technique and out of these, only 10 (2.28%) samples were found positive for Fasciola infection. Overall, the highest incidence of Fasciola infection was noticed in Jaffrabadi buffaloes (2.65%) than Gir cattle (1.72%) without significant difference (p > 0.05) between two species of animals. Haematological changes of Fasciola infected indigenous bovines showed the significant (p < 0.05) reductions in the mean values of haemoglobin (Hb), total erythrocytes count, packed cell volume, lymphocytes, and monocytes while total leucocytes count (TLC), mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, neutrophils, and eosinophils were increased significantly (p < 0.05). Biochemical analysis revealed hypoproteinemia, hypoalbuminemia as well as a significant increase in total bilirubin, Gamma-glutamyltransferase (GGT) and Alanine aminotransferase (ALT). The data presented here will not only help the epidemiologist but also help the field veterinarians in prognosis, diagnosis and planning the treatment and control strategies against Fasciola infection in bovines.

Study on prevalence of ancylostomosis in dogs at Anand district, Gujarat, India.

AIM: This study was undertaken to derive the prevalence rate of ancylostomosis in dogs by a collection of fecal samples from Anand district. MATERIALS AND METHODS: The fecal samples were collected from the dogs brought to the Hospital of Veterinary College (Teaching Veterinary Clinical Service Complex) and the surrounding areas of Anand district. On the day of collection, fecal samples were collected and brought to the Department of Veterinary Parasitology and processed for standard qualitative examination. The sedimentation technique was used to detect the presence of Ancylostoma spp. eggs in the samples. RESULT: The highest prevalence rate was observed in the month of May (36.66% fecal samples) and the lowest in the month of December (13.79% fecal samples) at Anand district. CONCLUSION: It can be concluded that heavy infection is present in Anand district especially in the season of summer followed by monsoon and the least in winter.