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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.

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.

Aim: The aim was to study the seroprevalence and efficacy of the different serological tests used for detection of antibody against Brucella species in small ruminants of Banaskantha district of North-Gujarat., Materials and Methods: Total 1000 serum samples comprising of 485 from sheep and 515 from goat tested for detection of antibodies against the Brucella species by three different serological tests viz., Rose bengal plate test (RBPT), Standard tube agglutination test (STAT), and Indirect Enzyme-linked immunosorbent assay (I-ELISA)., Results: The seroprevalence of brucellosis in small ruminants was 11.30%, 11.10%, and 8.80% by RBPT, STAT, and I-ELISA, respectively. The seroprevalence of brucellosis was found to be higher in sheep than goats. The sensitivity of RBPT was found slight more than STAT, but the specificity of both tests was same. In this study, the overall agreement of RBPT and STAT with I-ELISA was found 92.50% and 92.30% in small ruminants, respectively., Conclusion: I-ELISA was a better serological test as compared to RBPT and STAT in the sense of sensitivity, specificity, and rapidity and it could be advocated for screening of brucellosis in sheep and goats.