Cost-effective and efficient diagnosis of bovine theileriosis in a low-income setting.

Sample pooling combined with conventional polymerase chain reaction (cPCR) is a rapid and cost-effective approach for herd-level detection of Theileria spp., particularly in resource-limited settings. Bovine theileriosis is a tick-borne protozoan disease causing major economic losses to livestock production worldwide. In Sri Lanka, particularly in Jaffna, Theileria infections threaten cattle productivity, highlighting the need for affordable and sensitive surveillance tools. This study evaluated pooled cPCR for herd-level detection of Theileria spp. in cattle from three farms in Jaffna. Sixty blood samples (27, 26, and 7 animals per farm) and 27 ticks were collected. Giemsa-stained blood smears and individual cPCR analyses were performed. Then, three pools were prepared, taking 200 µL from each sample (one pool per farm) and analysed using cPCR targeting the major piroplasm surface protein (MPSP) gene. Microscopy detected a prevalence of 50%, whereas cPCR detected 81.7% (χ² = 13.374, p = 0.0003). Both Theileria orientalis (types 5 and 7) and Theileria sp. Yokoyama were identified. Pooling before cPCR and genomic DNA extraction reduced diagnostic costs by approximately 12-fold. Tick identification revealed predominance of Haemaphysalis bispinosa (88%). No significant associations were observed between infection prevalence and age, sex, or farm location. Despite widespread infection, most cattle were clinically healthy, indicating extensive subclinical circulation and possible endemic stability. Contrary to expectations, pooling samples from up to 27 cattle remained effective in herd-level detection of Theileria spp. despite low-parasitaemia levels. These findings demonstrate that pooled cPCR is a reliable and economical strategy for large-scale surveillance of bovine theileriosis in endemic, high prevalent and resource-limited settings.