title: Evidence of Coxiella burnetii in Punjab province, Pakistan.
abstract: Coxiella burnetii causes query (Q) fever, an important zoonotic disease with worldwide significance. The role of environment in the ecology of C. burnetti, and its influence on seroconversion in animals has not been elucidated in Pakistan. We carried out a cross-sectional study in Punjab province to (1) determine the prevalence and distribution of C. burnetii in soil using an ISIIII gene-based real time-polymerase chain reaction (RT-PCR) assay, (2) analyze association between the occurrence of C. burnetii in soil and its predictors i.e. soil characteristics (macro- and micro-nutrients) and several likely risk factors including the seroconversion in small ruminants at places where its genome had or had not been detected, and (3) predict homology and genetic diversity of the identified strains using sequences originated from different hosts worldwide. A total of 2425 soil samples from nine districts of Punjab province were processed. C. burnetii DNA was detected in 47 samples (1.94%, 95% CI: ±0.55) originating from 35 villages of studied districts (7.22%, 95% CI: ±2.30). The highest prevalence was found in Attock (7.11%, 95% CI: ±3.36), followed by Lahore (4.83%, 95% CI: ±3.49), Sahiwal (4.70%, 95% CI: ±2.6), Dera Ghazi Khan (2.33%, 95% CI: ±2.02), Faisalabad (1.35%, 95% CI: ±1.18) and Sheikhupura (0.68%, 95% CI: ±0.94). The odds of detecting bacterial DNA in soil was increased with a unit increase in organic matter [2.511 (95% CI: 1.453-4.340), p=0.001] and sodium [1.013 (95% CI: 1.005-1.022), p=0.001], whereas, calcium [0.984 (95% CI: 0.975-0.994), p=0.002] and potassium [0.994 (95% CI: 0.990-0.999), p=0.011] had protective effect where a unit increase in each analyte decreased odds for its occurrence by 1.0% approximately. Likewise, for categorical variables (risk factors), the odds of detecting C. burnetii were higher at locations >500m away from a main road [1.95 (95% CI: 1.06-3.78), p=0.04]. The enzyme-linked immunosorbent assay (ELISA) revealed an increased prevalence of antibodies in sheep (17.9%, 95% CI: ±5.54) compared with goats (16.4%, 95% CI: ±4.34). When determining the association between soil DNA and C. burnetii antibodies in small ruminants, the odds of detecting these antibodies were significant in sheep at the livestock barns [2.81 (95% CI: 1.20-7.37), p=0.02]. The IS1111 gene-based sequence analysis revealed a clustering of the DNA into two distinct groups with much genetic divergence (0.76-68.70%): the first group that contained sequences from Lahore district clustered with human and buffalo origin isolates, whereas the second group that contained the sequences from the remaining study districts clustered with goat-, rodent- and human-origin isolates. This study provides the first evidence of the presence of C. burnetii in the environment in Punjab province, Pakistan. Future studies are needed to ascertain the bacteria's molecular epidemiology over a wide geographical area, type the isolates, and evaluates the potential risks to human populations, particularly farmers and veterinarians.
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan. Electronic address: shabbirmz@uvas.edu.pk.
authors.forename: Muhammad Zubair
authors.initials: MZ
authors.lastname: Shabbir
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Sidra
authors.initials: S
authors.lastname: Akram
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Zia Ul
authors.initials: ZU
authors.lastname: Hassan
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Kashif
authors.initials: K
authors.lastname: Hanif
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Masood
authors.initials: M
authors.lastname: Rabbani
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Javed
authors.initials: J
authors.lastname: Muhammad
authors.affiliation: University of the Punjab, Lahore 54000, Pakistan.
authors.forename: Muhammad Hamid
authors.initials: MH
authors.lastname: Chaudhary
authors.affiliation: Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan.
authors.forename: Tariq
authors.initials: T
authors.lastname: Abbas
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Muhammad Taslim
authors.initials: MT
authors.lastname: Ghori
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Haroon
authors.initials: H
authors.lastname: Rashid
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Tariq
authors.initials: T
authors.lastname: Jamil
authors.affiliation: Quaid-i-Azam University, Islamabad 44000, Pakistan.
authors.forename: Zia-Ul-
authors.initials: ZU
authors.lastname: Islam
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Haisem
authors.initials: H
authors.lastname: Rasool
authors.affiliation: Quaid-i-Azam University, Islamabad 44000, Pakistan.
authors.forename: Asghari
authors.initials: A
authors.lastname: Bano
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Arfan
authors.initials: A
authors.lastname: Ahmad
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Muhammad Asad
authors.initials: MA
authors.lastname: Ali
authors.affiliation: University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.
authors.forename: Tahir
authors.initials: T
authors.lastname: Yaqub
authors.affiliation: The Pennsylvania State University, University Park, 16802, USA.
authors.forename: Walt
authors.initials: W
authors.lastname: McVey
authors.affiliation: The Pennsylvania State University, University Park, 16802, USA.
authors.forename: Bhushan M
authors.initials: BM
authors.lastname: Jayarao
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D016997:Coxiella burnetii / Q000235:genetics* / Q000302:isolation & purification; D003430:Cross-Sectional Studies; D004269:DNA, Bacterial / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D015511:Goat Diseases / Q000453:epidemiology*; D006041:Goats; D010154:Pakistan; D015995:Prevalence; D011778:Q Fever / Q000453:epidemiology / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction; D012418:Ruminants; D016036:Seroepidemiologic Studies; D012756:Sheep; D012757:Sheep Diseases / Q000453:epidemiology*; D012988:Soil Microbiology
chemical_list: D000907:Antibodies, Bacterial; D004269:DNA, Bacterial
keywords: Coxiella burnetii; IS1111 gene; Pakistan; Risk factor analysis; Small ruminants; Soil