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.

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.

Design and Assessment of a Novel In Silico Approach for Developing a Next-Generation Multi-Epitope Universal Vaccine Targeting Coronaviruses.

In the past two decades, there have been three coronavirus outbreaks that have caused significant economic and health crises. Biologists predict that more coronaviruses may emerge in the near future. Therefore, it is crucial to develop preventive vaccines that can effectively combat multiple coronaviruses. In this study, we employed computational approaches to analyze genetically related coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, focusing on the spike glycoprotein as a potential vaccine candidate. By predicting common epitopes, we identified the top epitopes and combined them to create a multi-epitope candidate vaccine. The overall quality of the candidate vaccine was validated through in silico analyses, confirming its antigenicity, immunogenicity, and stability. In silico docking and simulation studies suggested a stable interaction between the multi-epitope candidate vaccine and human toll-like receptor 2 (TLR2). In silico codon optimization and cloning were used to further explore the successful expression of the designed candidate vaccine in a prokaryotic expression system. Based on computational analysis, the designed candidate vaccine was found to be stable and non-allergenic in the human body. The efficiency of the multi-epitope vaccine in triggering effective cellular and humoral immune responses was assessed through immune stimulation, demonstrating that the designed candidate vaccine can elicit specific immune responses against multiple coronaviruses. Therefore, it holds promise as a potential candidate vaccine against existing and future coronaviruses.

Seroprevalence of toxoplasmosis in humans and animals in Pakistan: a systematic review and metadata analysis.

OBJECTIVE: To review the seroprevalence of toxoplasmosis in Pakistan. METHODS: The systematic review comprised search on Science Direct, Google Scholar, PubMed and Scopus databases for studies related to the seroprevalence of toxoplasmosis in Pakistan published between 2006 and 2020 which used serological diagnostic tests to detect Toxoplasma gondii. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used throughout the review and statistical analysis was done using forest plot and random effect model. RESULTS: Of the 7093 human studies initially found, 20(0.28%) were reviewed. Of the 16,432 animal studies, 16(0.09%) were selected for detailed review. The pooled seroprevalence of toxoplasmosis in humans, calculated in this review was found as (76%) (95% confidence interval: 69-83%). Seroprevalence of human toxoplasmosis was higher in Khyber Pakhtunkhwa (31.7%) than Punjab (20.4%). Pooled seroprevalence in animals calculated in this review was found as (69%) (95% confidence interval: 64-74%). Seroprevalence in animals was higher in Khyber Pakhtunkhwa (44.7%) than Punjab (29.4%). CONCLUSIONS: The seroprevalence of toxoplasmosis in both humans and animals should be studied it other parts of Pakistan as well.