Optimized two-step gradient PCR for efficient clinical screening of vector-borne hemoparasites in goats

Vector-borne pathogens pose a significant threat to the optimal production performance of farm animals in tropical endemic regions like South India. Early clinical diagnosis and timely administration of targeted chemotherapy are crucial for developing effective control strategies against these vector-borne diseases (VBDs). Although arthropod vectors transmit various pathogens, the major VBDs affecting goats can be categorized into hemotropic bacteria (Anaplasma/Ehrlichia and Mycoplasma spp.) and hemoprotozoans (Theileria, Babesia, Hepatozoon, and Trypanosoma spp.). The simultaneous detection of these six genera typically requires multiple PCR protocols with entirely different thermal conditions, and attempts to standardize multiplex PCR often fail due to this variation. We developed an optimized screening method using the gradient function of a PCR thermocycler, enabling simultaneous genus-specific detection. Blood DNA was extracted, and validated primers from the literature were applied in a two-step process: initial screening with universal primers (16S, 18S, apicomplexan rRNA), followed by genus-specific PCR. The gradient function of the thermocycler was employed in both steps to accommodate varying annealing temperatures, enabling simultaneous amplification of target sequences from different genera within the same PCR run. This approach reduces testing time, enhances detection in clinical samples, and facilitates precise, narrow-spectrum therapy, thereby minimizing treatment failures and mitigating the risk of drug resistance. This protocol provides a rapid and broadly adaptable diagnostic tool for the simultaneous detection of multiple hemoparasite genera, supporting timely, targeted treatment and improved management of VBDs in clinical settings.