Seroprevalence and Molecular Evidence of Coxiella burnetii in Dromedary Camels of Pakistan

Coxiellosis is a zoonosis in animals caused by Coxiella burnetii. A cross-sectional study was conducted on 920 (591 female and 329 male) randomly selected camels (Camelus dromedarius) of different age groups from 13 districts representative of the three different ecological zones in the Province Punjab, Pakistan to determine the prevalence and associated risk factors of coxiellosis. The blood samples were collected and tested for anti-C. burnetti antibodies using indirect multispecies ELISA. Real-time PCR was used for the detection of C. burnetii DNA to determine the prevalence in heparinized blood pools. Out of 920 investigated camels, anti-C. burnetii antibodies were detected in 288 samples (31.3%) (95% CI: 28.3–34.4%). The highest (78.6%) and lowest (1.8%) seroprevalence were detected in Rahimyar Khan (southern Punjab) and in Jhang (central Punjab), respectively. Potential risk factors associated with seropositivity of the Q fever in camels included desert area (42.5%; OR = 2.78, 95% CI 1.12–3.21) summer season (35.7%; OR = 2.3, 95% CI: 1.31–3.2), sex (female) (39.1; OR = 2.35, 95% CI: 1.34–2.98), tick infestation (51.3%;OR = 2.81, 95% CI: 1.34–3.02), age (>10 years; 46.4%; OR = 1.56, 95% CI: 0.33–2.05) and herd size (38.5%; OR = 1.21, 95% CI: 0.76–1.54). Coxiella burnetii DNA was amplified in 12 (20%) and 1 (10%) of 60 ELISA-negative and 10 suspected camels, respectively. DNA could not be detected in ELISA positive blood pools. This study emphasizes the seroprevalence and associated risk factors of coxiellosis as well as its potential to spill over to animals and humans in contact with these camel herds.


INTRODUCTION
Coxiellosis (Q fever) is associated with ticks and is a neglected zoonosis at least in the developing countries caused by the intracellular γ-proteobacterial pathogen, Coxiella (C.) burnetii (1,2).
Coxiella-like bacteria and C. burnetii are closely related, they vary in their ecology, as illustrated by the differences observed in transmission routes and infectiousness. Recent investigations based on multilocus phylogenetic analyses and whole-genome sequencing data revealed that all known C. burnetii strains originated within the vast group of Coxiella-like endosymbionts and are the descendants of a Coxiella-like progenitor hosted by ticks (3). In this context, comparative genomic approaches will be highly valuable in enhancing understanding of the evolutionary ecology of both C. burnetii and Coxiella-like bacteria and in identifying genes involved in virulence and tick symbiosis.
Based on the structural variation in lipopolysaccharide (LP), two antigenic "phases" of the organism viz., phase I (virulent) and phase II (avirulent), exist (4). C. burnetii infection has been reported in humans, animals (both wild and domestic), and ticks (1,5,6). Domestic ruminants (sheep and goat) act as reservoirs for Coxiella and are usually incriminated as an origin of Q fever epidemics in humans (2,7). In humans, the symptoms of Q fever are non-specific; however, the acute disease manifests with fever, myalgia, and atypical pneumonia. The chronic infection develops following an acute course and may lead to endocarditis and vasculitis (8)(9)(10).
Livestock farmers, shepherds, veterinarians, abattoir workers, and laboratory technicians have a higher risk of C. burnetii infection (11,12). Q fever is often asymptomatic in the livestock; however, late abortion, stillbirth, and premature delivery can be seen in small ruminants (sheep and goats) whilst mastitis, metritis, and infertility are reportedly observed in cows (13). In ruminants, C. burnetii is shed in birth fluids, uterine discharge, placental tissues, milk, urine, feces, and semen. Although inhalation of C. burnetii is considered the major route of infection in humans; however, consumption of raw milk and milk byproducts, blood transfusion, transplacental infection, intradermal inoculation, and infection after contact with the infected animals' body secretions viz, urine, feces, and semen may occur (9). The involvement of sheep and goats in human outbreaks of Q fever is well-documented (14). However, the high prevalence of coxiellosis found in camels, raise the question of whether transmission of C. burnetii from camels to humans is possible (15).
Numerous sero-surveys of coxiellosis in camels have been conducted and reported as 66% positive in Egypt (16), 80% in Chad (17), 62% in Saudi Arabia (18), 71.2% in Algeria (19), 44% in Tunisia (20), 29% in Iran (21) and nearly 100 % in nomadic camels in southeastern Ethiopia (22). The studies published over the last 6 decades (23-28) display that Q fever has been a neglected zoonosis in Pakistan. To our best, serologic evidence of Q fever in Pakistani camels can be traced back to 1955 (23) but no epidemiologic data on the disease in camels was available later.
A variety of diagnostic tests are available for the diagnosis of C. burnetii infection in animals. Nevertheless, ELISA and PCR are believed to be among the most reliable methods for serological and molecular diagnosis, respectively (29). In view of the emergence of coxiellosis in camels and the scarcity of epidemiologic data in Pakistan, this study was designed to investigate sero-prevalence, and associated risk factors among one-humped (dromedary) camels (Camelus dromedarius) reared in Pakistan.

Study Area
A cross-sectional sero-epidemiological survey was conducted from June 2018 to December 2019 in various districts of Punjab, Pakistan. According to Livestock Census, 13 camels were kept in prefectures from central, southern, and north-western parts of Punjab. Geographic coordinates, climatic conditions, and camel population of the selected districts are shown in Table 1.

Sampling Frame
The sample size was calculated by considering the expected disease prevalence up to 50% with a confidence interval (CI) of 99% and a desired absolute precision of 5%. The sample size was further expanded to improve the degree of accuracy and to cover the expected losses during handling and transportation from remote areas. A minimum of 897 samples were calculated to be needed for this survey. In practice, a total of 920 camels (591 females and 329 males) were randomly sampled from 13 districts of Punjab, Pakistan. Blood samples were drawn into a 4 mL, gelclot activator and EDTA coated vacuum vials, separately. The sera were harvested upon centrifugation and preserved (-40 • C) for further investigations. Meta data (age, breed, sex, body score, tick infestation, reproductive history/problems, and location, etc.) along with season and management/herd type were recorded on a questionnaire at the time of sampling. The animals were categorized into 3 different age groups; ≤ 5 years (n = 269), >5 to 10 years (n = 348) and >10 years of age (n = 303). For a random selection of herds and animals survey toolbox software was used (30).

Ethics' Statement
Blood samples were collected from camels as per the guidelines of the International Animal Care and Use Committee (IACUC) and after obtaining written consent from the owner of the camel. The study was approved by the Directorate of Graduate Studies following the ethical guidelines of the Institutional Biosafety Committee (IBC) and the Institutional Animal Care and Use Guidelines given in the Animal Care Handbook of the University of Agriculture, Faisalabad, Pakistan (31).

Serological Testing
Detection of anti-C. burnetii (against phase I and II antigens) antibodies were carried out by the commercially available indirect ELISA kit for the Q fever (ID Screen R Q fever indirect Multi-species ELISA, IDvet, Grabels, France) following the manufacturer's recommendations, and the results were expressed as optical density (OD) values. The absorbance was measured by an ELISA plate reader (Multiscan FC, Thermofisher Scientific, USA) at 450 nm. Sample/positive percentages (S/P%) for individual serum samples were calculated by using the following formula: [(OD sample-OD negative)/(ODpositive-ODnegative)] × 100. Samples were considered negative if they had S/P % ≤ 40%, doubtful for values between 40 and 50% and positive for S/P % > 50%. Any serum sample that was initially classified as "doubtful" was retested.

Molecular Investigation
Molecular testing was carried out on the blood pools (each pool comprised of five blood samples) from sero-negative (n = 60), positive (n = 11) and suspected (n = 10) camels using commercially available TaqMan-based real-time PCR assay (32). The DNA was eluted using a genomic DNA extraction kit (GeneJET Genomic DNA Purification Kit, Thermo Fisher, Germany), following the manufacturer's instructions, and quantified by Nanodrop 2000 spectrophotometer (Thermofisher Scientific, Germany) and stored at −40 • C until used. Extracted DNA was tested for C. burnetii DNA using realtime PCR kit (Liferiver TM Shanghai ZJ Bio-Tech Co., Ltd.) which is based on the fluorogenic 5 ′ nuclease assay according to the manufacturer's recommendations. The PCR reaction was performed on BIO-RAD CFX96 TM Real Time System (BIO-RAD Laboratories, Inc. USA) with the following protocol; First cycle at 37 • C for 2 min, second cycle at 94 • C for 2 min, followed by 40 cycles at 93 • C for 15 s and at 60 • C for 1 min. A sample was considered positive if the value of the threshold cycle (Ct) of the target gene was ≤38 (33). Both negative and positive controls were run in tandem with the samples (34).

Statistical Analyses
Univariate and multivariate analyses were conducted to determine the association of the risk factors with the seroprevalence. Variables kept in the initial model: district zone, season, breed, age, sex, body condition score (BCS), herd size, husbandry system, reproductive disorder history, and bioclimatic zones (p < 0.2 in the univariable analysis). BCS, herd size, season, breed, and husbandry system were removed in subsequent steps (p > 0.05). A p ≤ 0.05 was considered as a level of significance. A backward stepwise approach was used for the binary logistic regression analysis (35). All variables with a p < 0.2 in the initial bivariable analysis, were used to construct a multivariable analysis. Based upon likelihood ratio tests, variables were removed one by one to construct a logistic regression model. Outliers were identified at the 0.5 cut-off point by observing the Hosmer-Lemeshow test, Nagelkerke R square, and residual statistic values used to assess the model-fitness (36). The statistical analysis was conducted using the IBM SPSS Statistics (IBM Corporation, Armonk, NY, USA). The maps were generated by using ArcGIS (ESRI, Redlands, CA, USA).

Seroprevalence of C. burnetii and Univariable Analysis
An overall sero-prevalence of 31 Table 2). The geographical distribution of the seroprevalence coxiellosis in sampled districts of Punjab, Pakistan is shown in Figure 1.
Significantly higher seroprevalence (p < 0.05) was found in summer 35

Real-Time PCR
The rate of DNA detection differed significantly among blood pools of camels with different serologic statuses i.e., seronegative, doubtful, and sero-positive. Real-Time PCR demonstrated infection rate in sero-negative and doubtful pools at 20 and 10%, respectively. C. burnetii DNA could not be detected in the blood pools of sero-positive (n = 11) camels ( Table 5).

DISCUSSION
The purpose of the present study was to determine the seroprevalence of anti-C. burnetii antibodies and associated risk factors in the dromedary camel population of Punjab, Pakistan. To the best of our knowledge, this is the first comprehensive investigation on coxiellosis in camels of Pakistan. This study on coxiellosis in camels revealed a significantly high prevalence. Out of 920 sampled camels, 288 (31.3%) were seropositive. These findings are similar to those of studies from Iran where seroprevalences varied from 10.7 to 29 % (21, 37). However, this prevalence was lower than that observed in Saudi Arabia (62%) (18), Egypt (66%) (16), and Chad (80%) (17). This might be due to that those camels had the highest off all ruminants (38,39). The higher prevalences of coxiellosis in camels may be due to genetic susceptibility of camels to C. burnetii infection (22) or predilections of tick vectors to camels. Seropositivity differs significantly (p < 0.05) among different districts of Punjab, Pakistan, with peak prevalence (78.6%) at Bahawalnagar and the lowest (1.8%) at Jhang, which might be attributed to prevailing climatic conditions, hygienic measures, and management practices. Although, antibodies against coxiellae were detected throughout the year in the present study; however, a peak of detection was observed in Summer (35.7%). This is in close agreement with the previous study conducted by Danish investigators on dairy cows where it was demonstrated that the cows were at a higher risk of C. burnetti in summer (40). However, the results of this study are in contrast with those from France, where the human infection is associated with the lambing season in October and November (5).
This study documented that seroprevalence of C. burnetii among dromedaries was having a significant positive association with age as the prevalence in aged camels was higher (46.5%) than in younger ones (14.9%). The probable reason might be that the older animals have an extended duration of exposure to the pathogens in the environment causing higher probability of infection than that in the young stock. This observation agrees with previous studies (19,41) which presented that the seropositivity of C. burnetii increases with age. In a similar pattern, seropositivity in the domestic animals (cattle, sheep, and goat) upsurges with the age (22,42,43). The same is correct in the case of humans where the prevalence of C. burnetii (Q fever) increases with the advancement in age (44,45).
The sex of the animals was one of the dominant risk factors. Female camels were more often positive (39.1%) when compared to males (17.33%). These results are concomitant with those of previous studies (19,21). Higher susceptibility of females, particularly aged females, might be due to the predilection of C. burnetii to the placenta, udder and other reproductive tissues (46). These tissues can carry up to one billion organisms per gram (47).
The results of the current study revealed a statistically significant association (p < 0.05) between tick infestation and the existence of anti-C. burnetii antibodies which are in agreement with those reported elsewhere endorsing the vital role of ticks in the maintenance and spread of C. burnetii infection among animals and humans (9,48,49).
In this study, the herd size was another potentially associated risk factor for seropositivity of C. burnetii being higher among dromedary camels belonging to smaller herds (38.5%). This is in contradiction with the precedent studies (19,21,43) documenting higher seroprevalence in the larger herds. Our finding of a higher prevalence rate in small-sized herds can be elucidated by the reality that camels belonging to small herds were restricted in close sheds and hence, more prone to inhalation of the infected aerosol (50).
The husbandry system associated significantly with the seropositivity i.e. intensive husbandry system showed higher seropositivity compared to an extensive and semi-intensive system ( Table 2). This might be due to a higher risk of contact between the animals e.g., common watering points were found to be a source of brucellosis transmission in camels of Muzaffargarh (51). The bioclimatic zone is one of the significant risk factors for seropositivity. Seroprevalence was higher in dromedary camels belonging to the desert areas. This finding with those results described in Iran demonstrates a high seroprevalence in the desert and blowy areas (21). Thus, camelids from desert areas are more prone to aerosol transmission due to the frequency of storms in desert areas.
The breed was a risk factor in univariate analysis, which is in close agreement with the previous reports that recognized the breed as a risk factor for coxiellosis in cattle and sheep (40,49). There is no association between a history of abortion and seropositivity observed in the current study. This finding is in agreement with previous findings in Saudi Arabian (18). Such an association has been discussed repetitively in the literature for other animal species as well (49). Unexpectedly the husbandry system was not statistically significant for seropositivity.
Additional risk factors like ruminant contact, contact with other camel herds, purpose, and reproductive disorders possibly be associated with the seroprevalence of C. burnetii were not found significant in the current study. Contribution to other ruminants was not significant in this study. This agrees with previous findings in Algeria (19). In the current work, no significant association was observed with seropositivity. This is in contrast with previous findings from Tunisia which reported  high seroprevalence in camels intended for meat production (20). Reproductive disorders potentially associated with seropositivity were not statistically significant in the present study; however, these findings are not consistent with the earlier reports (49, 52-54) describing a higher prevalence associated with the reproductive disorders.
Other factors e.g. body score (BCS), ruminant contact, contact with other camel herds, and purpose were not found significant in the current study. This agrees with previous findings in Algeria (19). This is in contrast with previous findings from Tunisia which reported high seroprevalence in camels intended for meat production (20).
The overall prevalence of C. burnetii DNA in the camel blood samples was 16.05 % which is in close agreement with the previous findings in Iran (37). These results indicated that new infections play an important role in camels in Punjab, Pakistan as the agent is no longer present in the blood in which antibodies have been formed.

CONCLUSIONS AND RECOMMENDATIONS
The findings of this study indicated that coxiellosis is prevalent in clinical and/or subclinical forms in the camel population of different agro-geo-climatic zones of Punjab, Pakistan. Camelids are likely to play a significant role in the epidemiology of Q fever among the human population in Pakistan and contiguous countries. Epidemiology of C. burnetii involves many risk factors, like age, herd size, season, sex, exposure to ticks, and bioclimatic zone while developing causal models for the disease occurrence and distribution. Coxiellosis is commonly asymptomatic; yet results in serious health problems in humans, besides reproductive issues and financial losses in animals. In brief, the presence of C. burnetii in dromedary camels is alarming and must be considered while developing control strategies. C. burnetii is a major source of infection for humans and animals.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

ETHICS STATEMENT
The animal study was reviewed and approved by Blood samples were collected from camels as per the guidelines of the International Animal Care and Use Committee (IACUC) and after obtaining the written consent from the owner of the camel.

ACKNOWLEDGMENTS
All colleagues from the Departments of Parasitology and Clinical Medicine and Surgery, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan are highly thanked for their help at any level at any case during conduction of this study.