Newly optimized ELISA kit and LAT reveal significantly higher seroprevalence in sheep raised in agro-ecological zone as against range-ecological zone, with a significant association of meteorological parameters
Authors: Rahman Sarfraz ur , Akbar Haroon , Shabbir Muhammad Zubair , Ullah Ubaid , Rashid Muhammad Imran 
Published: December 2023

Background
Toxoplasma gondii is a zoonotic and foodborne intracellular parasite capable of inducing congenital infections, stillbirths and abortions in humans and animals, globally. The consumption of undercooked or raw mutton is “one of the vital risks” for acquiring toxoplasmosis: an asymptomatic condition in healthy persons, while life-threatening in immunodeficient individuals like "HIV/AIDS" patients.

Objectives
The current study has multiple objectives: to optimize a newly ELISA kit for Sheep, to find out the seroprevalence of ovine toxoplasmosis of two ecological zones of the Punjab, Pakistan through LAT and newly Optimized Sheep ELISA kit, to do the comparison of efficacies of various tests (LAT with newly Optimized ELISA kit and newly Optimized ELISA kit with commercial ELISA kit) and to determine the different meteorological parameters as the risk factors for T. gondii infection in sheep.

Methods
A cross-sectional study was conducted on 400 sheep sera, 200 were collected from sheep raised on open grazing system by local farmers in the adjoining areas of Civil Veterinary Dispensaries (CVDs) of range-ecological zone i.e. tehsil Kot Chutta (Dera Ghazi khan). Similarly, the remaining 200 were collected from agro-ecological zone i.e. tehsil Sharaqpur (Sheikhupura), to evaluate the comparative efficacy of LAT with optimized ELISA kit and newly optimized ELISA kit with commercial ELISA kit.

Findings
The newly ELISA kit optimized against a commercial ELISA kit was found to have 100% sensitivity, 97.6% specificity with 98% Positive Predictive Value, 100% Negative Predictive Value, Cut off value = 0.505, 28.28 LR+, 0.0104 LR-, and 2719.23 DOR. Seroprevalence of toxoplasmosis was detected significantly (P < 0.01; χ2) higher in Sharaqpur (44.5% by LAT; 35.5% by ELISA) as compared to that in Kot Chutta (39.5% by LAT; 31% by ELISA). The highest seroprevalence was seen in the sheep of the 1–2 years age group (P < 0.01; χ2), whereas the lowest in the oldest animals (≥ 4 years). Investigation of meteorological data of both the regions reveals that the zone with higher seroprevalence has relatively higher rainfall, higher humidity, lower environmental temperatures, and higher altitude as the critical factors, potentially behind the significant difference seen in seroprevalence level. The partial correlation of both tests (newly optimized ELISA kit and LAT) was 0.991 at maximum temperature in Sharaqpur while it was 0.981 in Kot Chutta.

Interpretation
A novel significant correlation was found between the meteorological parameters (relative humidity, minimum, maximum, and average temperatures) divided into yearly units of both the ecological zones, and year-wise seroprevalence (birth years of age-wise groups) of the corresponding regions. We hypothesize that such environmental conditions increase the risk of toxoplasmosis in grazing sheep, owing to a more favorable environment for coccidian oocyst survival. The ELISA kit optimized in this study will be helpful for the detection of seroprevalence of ovine toxoplasmosis in other ecological zones of Pakistan as well as of any other country in the world. More studies are recommended involving regions from other ecological zones of Pakistan to further explore the seroprevalence of ovine toxoplasmosis and to ratify the novel correlation of meteorological parameters with seroprevalence.