Serological Detection and Risk Analysis of Coxiella burnetii in Rodents of Punjab,Pakistan

Abstract

Introduction:

Coxiella burnetii, the causative agent of Q fever, remains poorly understood in Pakistan, despite its clinical relevance in both humans and ruminants. This study aimed to determine the seroprevalence of C. burnetii in rodents.

Methods:

Rodents were captured in urban settings across three districts of Punjab, Pakistan. A total of 300 serum samples were collected from rodents belonging to the Muridae family (n = 268) and the Sciuridae family (n = 32). Samples were screened for C. burnetii antibodies using an indirect enzyme-linked immunosorbent assay.

Results:

An overall seroprevalence of 12.7% (38/300) was observed, with a higher prevalence in males compared with females (p < 0.05). Using multiple logistic regression, age was identified as a potential risk factor for C. burnetii in rodents, with 14.1% (37/262) of adult rodents testing positive for C. burnetii antibodies, compared with a 2.6% (1/38) detection rate in juvenile rodents. Coxiella burnetii antibodies were detected in five rodent species, Tatera indica, Mus musculus, Millaria meltada, Rattus rattus, and Rattus norvegicus with seroprevalence ranging from 7.8% to 23.3%, depending on the species.

Conclusion:

This detection of C. burnetii in rodents residing in populated regions of Punjab, Pakistan indicates pathogen exposure. Additional studies, including molecular testing are needed to confirm their role as pathogen reservoirs.

Keywords

rodents seroprevalence ELISA

Introduction

During the past few decades, zoonotic diseases in mammalian hosts have notably increased compared with the past century. Rodents harbor the greatest diversity of zoonotic pathogens and represent nearly 42% of the global mammal population (Burgin et al., 2018; Ecke et al., 2022). They are known carriers of pathogens of medical and veterinary health importance such as Coxiella burnetii, Hymenolepis diminuta, Bartonella sp., and Rickettsia sp., and are responsible for multiple disease pandemics, including murine typhus, plague, and leishmaniasis (Rabiee et al., 2018). Previous research on rodents and pathogen transmission highlights the large risk to human and animal health (Foronda et al., 2015).

Q fever, an acute febrile disease caused by C. burnetii, is mainly transmitted to humans through inhalation of polluted aerosols (Sahu, et al., 2021). Other transmission routes have been described such as consumption of contaminated food, contact with infected feces or urine, or through infected tick vectors (Sahu et al., 2021). In humans, Q fever manifests with nonspecific symptoms such as flu-like illness with fever and pneumonia, which likely leads to underdiagnoses (Miller et al., 2021). Acute Q fever can progress into a more severe, chronic illness such as endocarditis or hepatitis (Anderson et al., 2013). Q fever is often asymptomatic in small ruminants but can lead to weak offspring and abortion (Eibach et al., 2012). Infection in ruminants can lead to economic losses and pose a risk for transmission to humans (Roest et al., 2011; Mangena et al., 2023).

Coxiella burnetii is present in a wide range of animal reservoirs, including rodents (Abdel-Moein and Hamza, 2018). Seropositivity has been reported for brown rats making them a potential reservoir of C. burnetii (Meredith et al., 2015b). Other studies have documented by serology and only one study by PCR detection a wider range of wildlife species that may serve as potential reservoirs of Q fever infection globally (Meerburg and Reusken, 2011; Meredith et al., 2015b). Especially when there is close contact between humans and animals, pathogens may be transmitted between different species (Meerburg et al., 2009). The exposure risk from rodents is likely compounded by rodent population growth and inhabitation of areas in close proximity to humans (Easterbrook et al., 2007; Kamani et al., 2018).

However, there are limited studies about C. burnetii infection in small mammals using serological tests and molecular techniques (Meredith et al., 2015b; Rozental et al., 2017). Existing enzyme-linked immunosorbent assay (ELISA) kits for serological detection of C. burnetii are planned for testing domestic ruminant samples and have not been validated for wild species such as rats and squirrels. Given that seroepidemiological research on C. burnetii antibodies in rodents in Pakistan is limited, this study aimed to identify the current prevalence of C. burnetii and associated risk factors in rodents using a custom ELISA kit in Punjab, Pakistan.

Materials and Methods

Study area

This study was conducted in three districts of Punjab, Pakistan: Pakpattan (30.2527°N, 73.1822°E), Okara (30.8138°N, 73.4534°E), and Kasur (31.0896°N, 74.1240°E). These three areas were selected based on the high density of livestock and rodents, livestock-human interface, and serological evidence of C. burnetii (Amin et al., 2022).

Sample collection

A total of 300 rodents were captured belonging to 6 species from July 01, 2019 to June 30, 2020. Villages within each of the three districts were randomly selected for the nonlethal collection of 100 rodent samples from each district. Sherman or iron mouse traps were prebaited with bread and grain before being set and left open for 3 days and checked daily. Blood was collected from rodents in a non-ethylenediaminetetraacetic acid (EDTA) test tube, centrifuged at 588g, and stored at −20°C until further processing (Meredith et al., 2015a). Species were morphologically identified using appropriate keys (Brooks et al., 1990). Data collected for each rodent included geographical location (district), sampling site, family, species, gender, urbanicity, age (adult vs. young), lice and/or tick infestation.

Serology

Antibody detection was performed using the commercial indirect ELISA IDEXX Q Fever Ab Test kit used for the diagnosis of Q fever in ruminants (IDEXX, Westbrook, ME, USA). The species-specific ruminant conjugate was replaced by a peroxidase-conjugated recombinant Protein G (Abcam, ab7460). Due to the limited availability of positive sera from rats, two sera from C. burnetii immunized mice and two sera from immunized rabbits were used as positive controls; preimmune sera were used as negative controls (BioGenes GmbH, Berlin, Germany). Protein G was serially diluted and tested with sera from mice, rabbits as well as the internal positive and negative controls supplied by the kit. The controls were validated and the S/P% calculated as described by the manufacturer. The internal controls and serum samples were correctly determined using a Protein G dilution of 1:10,000. Each run was validated using the internal controls supplied by the kit with Protein G as the conjugate. The serum was diluted at a ratio of 1:400, following the instructions of the manufacturer. The optical densities (ODs) of the controls and test samples were measured at a wavelength of 450 nm. The results were expressed as the percentage of the OD% value of the tested sample. Data were interpreted as per manufacturer instructions, where S/p values ≤30% were considered as negative and values ≥40% were considered positive.

Statistical analysis

The Statistical Package for Social Sciences, software version 21.0, was used for the calculation of descriptive statistics. Chi-square tests or Fisher’s exact tests were performed to evaluate the association of C. burnetii seroprevalence and risk factors, including study district (Kasur, Okara, Pakpattan), demographic characteristics, sampling sites, and rodent species. Logistic regression analysis was applied to determine associations between antibody status and associated risk factors, including Muridae family, species, district, urbanicity, sex, age, lice infestation, and tick infestation. A p-value < 0.05 was considered statistically significant.

Results

A total of 38 out of 300 rodent samples were seropositive for antibodies to C. burnetii, resulting in an overall seroprevalence of 12.7%. There was no significant association across study districts (p > 0.05): Kasur (9%), Okara (11%), and Pakpattan (18%) (Table 1). There was a significant link between rodent family and the prevalence of C. burnetii antibodies (Table 2). All positive samples were obtained from the Muridae family, while all samples from the Sciuridae family were negative; however, the number of samples tested in this group was only 32.

Table 1.

Coxiella burnetii Seroprevalence in Rodents from Districts in Punjab, Pakistan

Districts	Number of analyzed rodents (n)	Number of ELISA positive samples (%)	Chi-square value	p-value
Kasur	100	9 (9)	4.038	0.133
Okara	100	11 (11)
Pakpattan	100	18 (18)

ELISA, enzyme-linked immunosorbent assay.

Table 2.

Association of Epidemiological Factors and Prevalence of C. burnetii Antibodies in Rodents

Variables	Number of analyzed rodents (n)	Number of ELISA positive samples (%)	Chi-square value	p-value
Family
Muridae	268	38 (14.2)	—	0.021
Sciuridae	32	0 (0)	—	0.021
Sex
Female	124	10 (8.1)	4.047	0.031
Male	176	28 (15.9)	4.047	0.031
Urbanicity
Rural	167	21 (12.6)	0.003	0.546
Urban	133	17 (12.8)	0.003	0.546
Age
Adult	262	37 (14.1)	—	0.063
Young	38	1 (2.7)	—	0.063
Lice infestation
Yes	7	1 (14.3)	—	1
No	293	37 (12.6)	—	1
Tick infestation
Yes	13	1 (7.7)	—	1
No	287	37 (12.9)	—	1
Sampling sites
Human settlements	109	11 (10.1)	2.55	0.467
Animal farms	83	9 (10.4)
Shops	51	8 (15.7)
Warehouses	57	10 (17.5)

— no chi-square value from the Fisher’s exact test as chi-square test assumptions were not met.

Bold values are statistically significant as designated by a p-value below .05 as per statistical analysis section of methods.

The seroprevalence in male rodents (28/176; 15.9%) was meaningfully higher (p < 0.05) compared with females (10/124; 8.1%). There was a minimal difference in C. burnetii seroprevalence in urban areas compared with rural areas, but this was statistically nonsignificant. Rodent age had a significant effect on disease prevalence (p < 0.05) with 37/262 (14.1%) seropositive adult rodents, compared with 1/38 (2.7%) seropositive young ones. Lice and tick infestation were not significantly associated with C. burnetii antibodies. The sampling site had a nonsignificant impact on the seroprevalence of Q fever. Between rodent species, there was a significant difference in seroprevalence (p < 0.05). In the current study, all species studied were seropositive except the Northern palm squirrel. Coxiella burnetii antibodies were found in five rodent species with the highest positivity of 23.3% in M. musculus, followed by 12.5% in R. rattus, 10.7% in T. indica, 8.6% in M. meltada, and 7.8% in R. norvegicus (Table 3). Rodent age was identified as a possible risk factor for Q fever seropositivity based on multiple logistic regression analysis (Table 4).

Table 3.

Association of Rodent Species and C. burnetii Seroprevalence in Three Districts of Punjab, Pakistan Using a Fisher’s Exact Test

Common name	Scientific name	Number of analyzed rodents (n)	Number of ELISA positive samples (%)	p-value
Indian gerbil	Tatera indica	56	6 (10.7)	0.010
House mouse	Mus musculus	86	20 (23.3)
Soft-furred rat	Millaria meltada	35	3 (8.6)
Northern palm squirrel	Funambulus pennantii	32	0 (0)
Black rat	Rattus rattus	40	5 (12.5)
Brown rat	Rattus norvegicus	51	4 (7.8)

Bold values are statistically significant as designated by a p-value below .05 as per statistical analysis section of methods.

Table 4.

Logistic Regression of Risk Factors Associated with C. burnetii Seroprevalence

			95% CI^a
Variables	p-value	Odds ratio	Lower	Upper
Family	0.998	0	0	—
Species	0.125	0.852	0.695	1.045
District	0.677	5.189	0	1.520
Urbanicity	0.689	1.169	0.543	2.516
Sex	0.050	2.177	0.99	4.787
Age	0.091	0.170	0.022	1.329
Lice infestation	0.689	0.637	0.07	5.778
Tick infestation	0.601	0.565	0.067	4.799

Confidence interval.

Discussion

Coxiella burnetii has been detected in livestock and humans in Pakistan, but few studies have focused on rodents, likely due to the lack of corroborated serological tests for this species (Greiner and Gardner, 2000). Previous studies have used ELISA tests designed for wildlife species where species-specific conjugated antibodies were not accessible and were substituted with conjugated Protein A or Protein G (Meredith et al., 2015b). The modified ELISA kit used in this study successfully detected C. burnetii antibodies in rodent serum samples.

The seroprevalence of C. burnetii detected in our study was 12.7%, which was lower than the 18% seroprevalence in rodents from another study in Pakistan (Ahmed, 1987). Globally, this was lower than studies on rodents conducted in East Asia, Europe, Somalia, and Zambia where the prevalence ranged from 17.3% to 21.9% with the exception of Zambia where it was 45%. (Pittermannova et al., 2021; Zakovska et al., 2021; Chitanga et al., 2018; Mccaughey et al., 2010; Textoris et al., 2010; Reusken et al., 2011). Whereas other studies, on rodents from California, the Netherlands, Central Africa, and Norway had a lower seroprevalence between 3% and 6.7% (Riemann et al., 1979; Reusken et al., 2011; Pascucci et al., 2015; Mangombi et al., 2021; Abdel-Moein and Hamza, 2018). Based on C. burnetii antibody detection rates, these rodents are likely a reservoir for these pathogens in Pakistan (Meerburg and Reusken, 2011). Within Pakistan, there have been a few C. burnetii seroprevalence studies on humans with detections ranging from 26.8% in symptomatic patients to 8.4% in pregnant women (Ahmed, 1987; Ali et al., 2022). Several studies have been conducted in small ruminants, goats and sheep. In Punjab, 11.3 − 15.3% of small ruminants had positive serological detections of C. burnetii (Amin et al., 2022; Ullah et al., 2019). Global variations in C. burnetii prevalence may be influenced by climate conditions and differences in rodent species. Climate conditions impact pathogen survival, vector dynamics, and rodent distribution (Celina and Cerny, 2022; Guatteo et al., 2011), whereas rodent species can have various competencies for pathogens and differing interactions with livestock and humans (Eldin et al., 2017).

While there is global variation, there are also regional differences within Pakistan with the highest prevalence in this study detected in rodents from Pakpattan (18%), followed by Okara (11%) and Kasur (9%). This differs from the serological detection in small ruminants, where Pakpattan had 11–14% positivity, Okara had 8.7–9% positivity, and Kasur had 14% positivity (Amin et al., 2022; Amin et al., 2024). This discrepancy may be due to differing exposures of rodents and livestock to C. burnetii in these districts including habitat, distribution, and animal management (Selemani et al., 2024; Nthiwa et al., 2019). Pakpattan has an overcrowded population of rodents due to improper storage of grains that can provide suitable habitats for rodent breeding (Shukla et al., 2025). These conditions can lead to the proliferation of rodents at the livestock-human interface and increase the risk of cross-species transmission (Dobigny and Morand, 2022). These regional differences are contrary to a comparative analysis in the Atlantic archipelagos that showed a similar prevalence of C. burnetii between the two archipelagos (Foronda et al., 2015). There may be unmeasured factors including livestock density, grain storage, and other interactions that led to the differences in seroprevalence rates by district (Shepon et al., 2023; Brown and Henry, 2022).

There is contradictory evidence of the impact of sex on C. burnetii infection. One study found a greater vulnerability of C. burnetii infection in male versus female individuals based on studies of human samples and laboratory mice (Textoris et al., 2010). Similarly, our study shows higher seroprevalence rates in males compared with females. While other studies have shown a nonsignificant relationship between C. burnetii infection and animal sex (Liu et al., 2013; Abdel-Moein and Hamza, 2018). A higher seroprevalence of C. burnetii was detected in adults compared with young rodents. Mature rodents cover a greater area increasing their infection likelihood due to environmental exposures (Liu et al., 2013). There was a higher seropositivity of C. burnetii observed in rodents captured from warehouses (17.5%), followed by shops (15.7%), while a lower rate was observed in animal farms (10.4%) and human settlements (10.1%). The availability of surplus food in warehouses could explain the higher seropositivity.

Coxiella burnetii antibodies have been found in 35 species of rodents belonging to 26 different genera (Meerburg and Reusken, 2011). In this study, C. burnetii antibodies were detected in five out of six rodent species in the study region of Pakistan. Our study revealed that M. musculus had a significantly higher C. burnetii seroprevalence (23.3%) compared with other rodent species, which differs from another study where R. norvegicus and R. rattus had the highest seroprevalence of 53.3% and 31.1%, respectively (Ahmed, 1987). The higher seroprevalence detected in M. musculus compared with other species was likely due to the dense population of this species in the study areas. The genus Rattus is a known reservoir and has been reported as seropositive for C. burnetii in studies performed in Europe (González Barrio et al., 2021). This aligns with our study that detected C. burnetii seroprevalence of 7.8% and 12.5% in R. norvegicus and R. rattus, respectively. Compared with Karachi, Pakistan which reported a higher seroprevalence among the Rattus genus (53.3% of R. norvegicus and 31.1% of R. rattus) (Ahmed, 1987). Variation in seroprevalence by rodent species may be attributed to site-wise differences in rodent populations, testing method, contact with C. burnetii carrier animals, geographical surroundings, home range size, and habitat type (Lambin et al., 2000; Heyman et al., 2009; Meredith et al., 2015b). Further studies are needed in Pakistan for comprehensive evidence on potential hotspots of Q fever transmission by rodents.

Conclusion

The seroprevalence of Q fever had higher antibody detection rates in M. musculus and R. rattus, in Punjab, Pakistan. Moreover, rodent family, sex, and age were associated with previous C. burnetii exposure and warrant further investigation to inform control measures for rodents throughout Pakistan, especially in high-risk areas. Future studies that incorporate molecular testing are needed to elucidate the role of rodents as potential reservoirs of C. burnetii.

Acknowledgments

The authors acknowledge the support of the Federal Foreign Office, Germany.

Authors’ Contributions

Conceptualization (Ideas; formulation or evolution of overarching research goals and aims.) F.A. and S.A.: Data curation (Management activities to annotate (produce metadata), scrub data and maintain research data (including software code, where it is necessary for interpreting the data itself) for initial use and later re-use.) F.A., S.A., A.A., and N.G.C.: Formal analysis (Application of statistical, mathematical, computational, or other formal techniques to analyze or synthesize study data.) F.A., N.G.C., T.L., M.E.F., and I.G.R.: Funding acquisition (Acquisition of the financial support for the project leading to this publication.) H.N. and S.A.: Investigation (Conducting a research and investigation process, specifically performing the experiments, or data/evidence collection.) K.M. and I.G.R.: Methodology (Development or design of methodology; creation of models.) K.M.: Project administration (Management and coordination responsibility for the research activity planning and execution.) S.A.: Resources (Provision of study materials, reagents, materials, patients, laboratory samples, animals, instrumentation, computing resources, or other analysis tools.) H.N., K.M., and S.A.: Software (Programming, software development; designing computer programs; implementation of the computer code and supporting algorithms; testing of existing code components.) S.A.: Supervision (Oversight and leadership responsibility for the research activity planning and execution, including mentorship external to the core team.) K.M. and S.A.: Validation (Verification, whether as a part of the activity or separate, of the overall replication/reproducibility of results/experiments and other research outputs.) K.M. and S.A.: Visualization (Preparation, creation and/or presentation of the published work, specifically visualization/data presentation.) S.A., T.L., and A.A.: Writing—original draft (Preparation, creation and/or presentation of the published work, specifically writing the initial draft (including substantive translation).) F.A., S.A., N.G.C., and M.E.F.: Writing—review & editing (Preparation, creation and/or presentation of the published work by those from the original research group, specifically critical review, commentary or revision—including pre- or post-publication stages.) F.A., S.A., H.N., K.M., N.G.C., and M.E.F.

Footnotes

Author Disclosure Statement

The authors declare no conflict of interest.

Funding Information

This research was funded by the German Federal Foreign Office, funded project “Building a network of laboratories in Pakistan to enhance biosafety and biosecurity in Pakistan (AA-OR12-370:43 BIOS FLIPAK.)”

Ethical Approval

This study was conducted after the approval of the Ethical Review Committee for Animals of the University of Veterinary and Animal Sciences, Lahore, Pakistan (068/IRC/BMR, dated 8 October 2019), respectively. The handling of the animals for sampling purposes was done according to institutional rules.

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