Leptospirosis is an important zoonotic disease. It affects a wide range of mammals, fish and even a few reptiles. It is caused by Leptospira interrogans, having more than 250 serovars, distributed geographically throughout the world. In horses, Leptospira interrogans causes liver and renal abnormalities, ERU, and reproductive disorders in mares like abortion, perinatal death and still birth. It is transmitted to human beings, working with live or dead tissue of infected horses and through surfaces contaminated with urine of carrier or infected animals. In humans, it causes influenza like illness and death in severe cases. Serological testing, bacterial culture and molecular techniques are used for the diagnosis of disease. This study was aimed at estimating the seroprevalence of Leptospira spp. in horses and humans of three climatically distinct regions of Punjab, Pakistan. Furthermore, molecular biology techniques were employed for the confirmed diagnosis of equine leptospirosis and therapeutic efficacy of ampicillin and adhatoda vasica was analyzed against disease. It was the very first study in Pakistan conducted to explore equine leptospirosis in the country.
During this study, 384 horse blood samples and epidemiological data were collected from three climatically distinct regions, viz;Rawalpindi, Lahore and Bahawalpur (128 from each study area) and were subjected to ELISA to determine seroprevalence of Leptospira. Results showed overall prevalence of 33.85% in Punjab with highest prevalence in Rawalpindi (40.62%) which experienced highest rainfall, followed by Lahore (38.28%), and least in Bahawalpur (22.65%). Risk factor analysis showed that age, gender, living area, herd size, water source, exposure to rodents and floods, feeding practices and usage of animals were found significantly associated with the disease. To study the seroprevalence of human leptospira, 360 human blood samples were collected (120 from each study area). Epidemiological data on pre-structured questionnaire
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were collected from all the participants of study. All the samples were subjected to ELISA and results showed overall prevalence of 40.83%, with highest seroprevalence in Rawalpindi (50.83%), followed by Lahore (38.28%) and least in Bahawalpur (27.50%). Age, gender, occupational and living area, water recreational activities, occupation, exposure to floods, educational status and history of wound were significantly associated risk factors while use of PPE during work was deterrent.
During this study, 65 ELISA positive horse samples were subjected to molecular biology diagnostic technique PCR for the molecular characterization of equine leptospirosis in country. After DNA extraction, PCR was performed using primer sets specific for 16S rRNA gene, which yielded a fragment of length 306bp after gel electrophoresis. Out of 65 tested samples, 20 samples (30.76%) were PCR positive and was further sequenced and phylogenetic tree was constructed. Dendogram showed the sequenced samples were related to pathogenic Leptospira interrogans, revealing potential of 16S rRNA primer sets for the detection of eqine leptospirosis in country. Dendogram further showed closed resemblance of analyzed samples with serovar Icterohemmorhagae, Australis and Autumunalis which are dominant serovars in India, Iran and China, the neighboring countries of Pakistan. Therapeutic efficacy of ampicillin and AV was studied by analyzing the hematology, liver function test, renal function tests and serum mineral levels at day 0 (pre-treatment), 7, 21 and 35 (post-treatment). Results showed that all the tested parameters were changed significantly during infection and significant improvement was observed after treatment. Ampicillin was instrumental in revealing hematological abnormalities while AV played important role in normalizing the liver and renal insufficiency. After treatment ampicillin treated 58.33% of animals and AV treated 41.66% of animals.
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This first ever study of equine leptospirosis in country uncovers the high prevalence rates in horses and humans and raises a need for control strategies to prevent the transmission and spread of the disease. It also highlights the potential of molecular biology techniques for the confirmed diagnosis of equine leptospirosis and explores options for designing better specie specific treatment regimes for the disease.