Insights into animal reservoir hosts for the circulation of Leishmania parasite in the sustainable phase of National Kala-azar Elimination Programme in India

India, currently in the sustained phase of visceral leishmaniasis elimination, continues to face challenges due to reports of indigenous cases from emerging foci, detection of parasitic DNA in multiple sand fly species, and the potential involvement of animal reservoirs in disease transmission. Although Leishmania donovani has traditionally been regarded as anthroponotic, domestic animals may play an under-recognized role in maintaining transmission. A multicentric study was conducted across seven districts in endemic (Bihar, Jharkhand, West Bengal) and emerging foci (Kerala, Tamil Nadu) states. A total of 782 domestic animals (dogs, goats, cattle, and cats) were screened using rK39 serology, microscopy, and quantitative PCR targeting Leishmania kinetoplast DNA. Species confirmation was performed by ITS1-PCR and sequencing. Sand flies were examined for natural infection, and COI barcoding was used for blood-meal analysis. Amastigotes were microscopically confirmed in canine samples. Overall, out of 782 domesticated animals screened, 98 animals were rK39 seropositive. Leishmania DNA was detected by qPCR in 129 animals, of which 81 were confirmed by ITS1-PCR across dogs, goats, cattle, and one cat; 21 samples were sequenced and identified as L. donovani . Natural infection with L. donovani was also confirmed in Phlebotomus argentipes , which also feeds on domesticated animals like cattle. These findings provide molecular and serological evidence of L. donovani infection in multiple domestic animal species, suggesting the potential involvement of a zoonotic transmission cycle. Sustained success of India's kala-azar elimination program will require holistic integrated surveillance systems that include domestic animal reservoirs in addition to human and vector populations. • Molecular evidence of Leishmania donovani infection in multiple domestic animals. • ITS1-PCR and sequencing confirmed L. donovani in animal hosts. • Natural infection detected in Phlebotomus argentipes sand flies. • A possible cryptic zoonotic transmission cycle in India.