Serological and Molecular Investigations on Coxiellosis and its Relationship with Reproductive Disorders in Small Ruminants at Livestock Farms of Punjab, Pakistan

Abstract

Livestock raising is an important occupation for livelihood of rural poor in Pakistan and plays a vital role in poverty reduction. Coxiellosis is a disease caused by Coxiella burnetii and acts as a major trade barricade adversely affecting the productive and reproductive capabilities of the animal, and hinders with commercialization of animal products at local and international market level. This study was planned to conduct serological and molecular investigations on Coxiellosis and to identify any association of seropositivity against C. burnetii antibodies with sex, age, breed, species, parity, farm, district, lactational status, reproductive status (pregnant or non-pregnant), tick infestation, body condition and reproductive disorders in sheep and goats. A structured questionnaire was used to collect information about individual animal and general farm management. The sampling design was formulated considering an expected prevalence of 50%, confidence interval of 95%, and 5% desired absolute precision. A total of 1000 sera sample (500 from goats and 500 from sheep) were collected from animals maintained at nine different government livestock farms of Punjab. Firstly, all these samples were analyzed through Indirect-ELISA (IDEXX Q Fever, Coxiella burnetii, Antibody Test Kit) and then pools of seropositive, and suspected serum samples (29 pools) were investigated through real-time qPCR, using single copy isocitrate dehydrogenase (icd) gene, for detection of C. burnetii DNA. Additionally, 55 tick pools were also investigated through real-time qPCR, using multicopy IS1111 insertion element, for genomic detection of C. burnetii in these pools. The diagnostic work was carried out at National Reference Laboratory (NRL) for Q fever, Friedrich Loeffler Institute, Jena, Germany. Serological analysis revealed a prevalence of 15.6% (95% CI: 12.5-19.1) and 15.0% (95% CI: 12.0-18.4) in sheep and goats, respectively. Statistically, prevalence of C. burnetii antibodies in serum was non-significantly different (p=0.792, degree of freedom (df)=1, Chi-square (χ2) =0.069) between the two species. A significant association was found between seropositivity against C. burnetii antibodies and different variables like farm (p=0.000, df=8, χ2=141.869), district (p=0.000, df=6, χ22=49.689), breed (p=0.000, df=9, χ2=60.954), lactational status (p= 0.000, df=1, χ22=24.691), reproductive status (p= 0.008, df=1, χ2=7.023), ticks infestation (p=0.000, df=1, χ2=301.914), body condition (p=0.000, df=3, χ2=124.868) and reproductive disorders (p=0.000, df=4, χ2=133.984). However, seropositivity against C. burnetii infection was non-significantly associated with age (p=0.063, df=3, χ2=7.281), parity (p=0.838, df=2, χ2=0.353) and sex (p=0.302, df=1, χ2=1.064) of animal. Univariate analysis revealed a significant (p=0.031, df=1, χ2=4.668) difference in prevalence of C. burnetii DNA in tick pools of sheep and goats, however prevalence of C. burnetii DNA in serum pools was non-significantly (p=0.564, df=1, χ2=0.333) different between the two species. These findings revealed that C. burnetii infection is prevalent in small ruminants maintained at studied livestock farms, as well as in ticks. Further in-depth studies are required to explore its epidemiology more precisely in humans, ticks and various animal species.