Molecular detection of Coxiella burnetii and Coxiella species in rats and chickens from poultry farms in North West Province, South Africa

Abstract Background Coxiella burnetii is a bacterial pathogen that causes query fever and coxiellosis in humans and animals, respectively. There is a scarcity of studies on the prevalence of C. burnetii infections in rats and chickens in South Africa. Objective The aim of this study was to determine the occurrence of C. burnetii in rats and chickens sampled from poultry farms in the North West Province of South Africa. Methods DNA was extracted from rodent kidneys (n = 68) and chicken faeces (n = 52). Two rodent pest species, namely Rattus rattus and Rattus tanezumi, were identified by analysis of CO1 gene sequences. Detection of C. burnetii was carried out using polymerase chain reaction assays targeting 23S rRNA, 16S rRNA and IS111 markers. Results C. burnetii was detected in 16.2%, 8.8% and 25% of R. rattus, R. tanezumi and chickens, respectively. Conclusions The findings in this study demonstrate that rodents and chickens are harbouring C. burnetii at sampled poultry farms. There should be frequent screening for C. burnetii in poultry operations. The likelihood of future transmission between rodents and chickens, including humans, also needs to be investigated.

Similarly, Coxiella-like bacteria are a group of bacteria that remain to be isolated and are characterized as phylogenetically close to C. burnetii (Rahal et al., 2020).Duron et al. (2015) published a recent study in which all C. burnetii strains were shown to descend from a Coxiella-like progenitor.It was, however, established that these strains pose a much lower infection risk to vertebrates than C. burnetii (Duron et al., 2015).Domestic ruminants, including sheep, goats and cattle, are thought to be the primary source of human infection outbreaks (Cabrera Orrego et al., 2020;Tawana et al., 2022).Other hosts, such as birds Vet Med Sci.2023;9:2185-2191.
Additionally, rodents contribute to the transmission of C. burnetii (Thompson et al., 2012).
Recently, Q-fever has gained attention due to a number of outbreaks in different countries, the majority of which have been linked to domestic animals (Simpson et al., 2018;Sprong et al., 2012).Farmers, veterinarians, abattoir workers and laboratory personnel culturing C. burnetii and, more importantly, working with C. burnetii-infected animals are all at risk of falling sick with Q-fever illness (Frean, 2022;Maurin & Raoult, 1999;Mioni et al., 2022).The first recorded cases of human Q-fever in South Africa were documented in 1950 (Gear et al., 1950).The endemic status of Q-fever in South Africa is not widely known, and this could be associated with the fact that the Animal Diseases Act 35 of 1984 does not recognize the illness as a controlled or a notifiable disease (Maurin & Raoult, 1999).Serological survey of Q-fever comprised few studies carried out in South Africa (De Boni et al., 2022;Donnelly et al., 2021;Gummow et al., 1987;Mangena et al., 2021).
There is a lack of data regarding molecular epidemiological studies of C. burnetii occurrence in rats and chickens in South Africa.The main aim of this study was to investigate the occurrence of C. burnetii infections in rat and chicken samples obtained from poultry farms in South Africa's North West Province.

Sample collection and DNA extraction
Samples were collected from chickens (n = 52) and rats (n = 68) at six commercial farms in Ngaka Modiri Molema District (26.0282Province, South Africa (Figure 1).Wild rats were captured within poultry farm enclosures and euthanized as previously described by Ramatla et al. (2019).Tissue samples were harvested from the rodent kidneys.
Fresh chicken faecal samples were collected in chicken enclosures within poultry houses.Genomic DNA was extracted from both rat tissues and chicken faeces using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following manufacturer's instructions.
Then, a second round of PCR was performed with primers Cox16SF2 (5′-TGA GAA CTA GCT GTT GGR RAG T-3′) and Cox16SR2, yielding 624-627 bp amplicons (Seo et al., 2016).Each PCR reaction included a total reaction volume of 25 μL containing 12.5 μL of a 2× DreamTaq Green Master Mix (0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP, 4 mM MgCl 2 and loading buffer) (Thermo Fisher Scientific, South Africa), 8.5 μL of nuclease-free water, 2.0 μL of the template DNA and 1.0 μL of each oligonucleotide primer.Pre-denaturation at 93 • C for 3 min; 30 cycles of denaturation at 93 • C for 30 s, annealing at 56 • C for 30 s, and polymerization at 72 • C for 1 min; and a final elongation step at 72 • C for 5 min.The PCR reactions were carried out on an Applied Biosystems ProFlex PCR System (Applied Biosystems, Singapore).

Identification of Coxiella burnetii using the IS1111 gene
To further identify this bacterium, a species-specific conventional PCR targeting the IS1111 gene of C. burnetii was conducted with IS1 F (5′-CGC AGC ACG TCA AAC CG-3′) and IS1R (5′-TAT CTT TAA CAG CGC TTG AAC GTC-3′) primers, yielding 146 bp amplicons (De Bruin et al., 2011).The following PCR conditions were used: pre-denaturation for 15 min at 95 Note: From this table, based on the interpretation of the kappa value, there is no agreement in the observation of the samples that are positive for farms A and B. Furthermore, there is a slight agreement in observations of samples that tested positive for Coxiella from farms C, D and F whereas there is a fair agreement in observations from farm E.

Statistical analysis
Data from the samples that tested positive for different PCR detection

Rodent identification
Based on the CO1 gene analysis described in our previous study, a total of n = 68 collected rats were identified as Rattus rattus (69.1%) and Rattus tanezumi (30.9%) species (Ramatla et al., 2019).
On a heatmap (Figure 2), a total of 17 PCR amplicon sequences from Rattus spp.for 3 tested genes (IS111, 23S rRNA and 16S rRNA) clustered together and were considered true C. burnetii positive infections, highlighted by the red box.Other Coxiella spp.were assigned to samples that tested positive for both the 23S and 16S rRNA's but did not harbour the IS111 gene.Therefore, these samples were not classified as C. burnetii and are annotated within a green box.A total of 13 PCR amplicon sequences from chickens for 3 genes (IS111, 23S rRNA and 16S rRNA) clustered together on a heatmap and were considered true C. burnetii positive infections as indicated within the red box (Figure 3).

DISCUSSION
As C. burnetii is the causative agent of Q-fever, a global zoonosis, the detection of C. burnetii in rats and chickens is of particular significance (Filippitzi et al., 2017, Raele et al., 2018) (Mofokeng et al., 2022;Mtshali et al., 2015Mtshali et al., , 2017;;Wyk et al., 2022) and abattoir workers (De Boni et al., 2022) in South Africa.This study detected a total prevalence of 24.1% C. burnetii in chickens, which is higher than the prevalence reported in Japan but lower than in Iran, which were 4.2% and 17.2%, respectively, from egg samples using PCR (Rahimi & Doosti, 2012;Tatsumi et al., 2006).C. burnetii was detected in faecal samples from chickens and kidney tissues of rats in the current study indicating that pathogen is shed in faeces and/or urine.
The Q-fever agent can infect wild pigeons (Columba livia), and some studies have linked foci of human and animal coxiellosis to pigeons genes, whereas brown is the absence of these three genes.(Babudieri & Moscovici, 1952;Ebani & Mancianti, 2022;Lang, 1990).
Birds typically remove mites by preening, which exposes them to C. burnetii bacteria.Dermanyssus gallinae (the red poultry mite) can also encounter Coxiellae when using contaminated nesting materials like bird faeces (Ebani & Mancianti, 2022;Raele et al., 2018).Therefore, the detection of this pathogen in chicken faeces is not surprising, given that they can contract this bacterial infection from ingesting infected arthropod vectors or due to contact with contaminated faecal materials.
Coxiella spp.are exceptionally diverse and widespread in ticks but rarely detected in domestic animals (Seo et al., 2016).In this study, Coxiella spp.were detected in both rats and chicken samples by PCR using 23S rRNA and 16S rRNA markers.The percentage of positive samples in the current study was high in farms A (12.5%), D (11.7%) and C (10%), respectively.Elsewhere, other studies have used different PCR markers to detect Coxiella-like endosymbionts (CLE) in ticks collected from horses in South Korea (Seo et al., 2016) ticks collected from different animals (cattle, dogs and goats) and the environment in Zambia (Kobayashi et al., 2021) and from ticks collected from bovines in India (Rialch et al., 2022).
Study of seroprevalence on Coxiella in human was conducted by De Boni et al. ( 2022), where they found the seroprevalence was 33% from abattoir workers from Free State and Northern Cape provinces of South Africa.Other studies reported the exposure of abattoir workers to C. burnetii, via inhalation of aerosols or dust contaminated with the bacteria (Eldin et al., 2017;Porter et al., 2011).From a public health perspective, the detection of C. burnetii in chickens confirms the pathogen's excretion in the environment and indicates that chickens and rats must be considered reservoirs in South Africa.

CONCLUSION
This study has used PCR and sequencing to detect the occurrence of C. burnetii and Coxiella spp.from wild rat tissue and chicken faecal samples.These wild rats roam around within chicken houses and surroundings and potentially provide a good opportunity for environment-rat-poultry interaction.Future studies should investigate the transmission cycle of C. burnetii and characterize other Coxiella spp.occurring in this rat-chicken environment.Ectoparasites such as mites and fleas could potentially infest rats and should be investigated as to whether they also harbour Coxiella spp.To the best of our knowledge, this is the first molecular detection of C. burnetii and Coxiella spp.
from chicken and rat samples in South Africa.
. A nested PCR (nPCR) was used to amplify the 16S rRNA and 23S rRNA gene markers of Coxiella spp.First-round PCR was performed using the primers Cox-23SF1 (5′-GCC TGC GAW AAG CTT CGG GGA G-3′) and Cox-23SR2 (5′-CTC CTA KCC ACA SCT CAT CCC C-3′) which produced 694-1188 bp amplicons.The second round of PCR used Cox-23SF2 (5′-GAT CCG GAG ATW TCY GAA TGG GG-3′) and Cox-23SR1 (5′-TCGYTCGGTTTCGGGTCKACTC-3′) primers, which yielded 583-F I G U R E 1 Map of Africa showing the Mahikeng sampling area in the North West Province of South Africa.Source: This figure is adapted from a version published previously methods were analysed on Microsoft Excel Professional 2016 (Analysis Tool Package).Positive samples were summarized as percentages and tested at 95% confidence intervals of the mean.Cohen's weighted kappa (κ) was used to measure rates of agreement between samples that was positive across different farms with the α value that was set at 0.05, whereby κ values ≤0 as indicating no agreement and 0.01-0.20 as none to slight, 0.21-0.40 as fair, 0.41-0.60 as moderate, 0.61-0.80 as substantial and 0.81-1.00as almost perfect agreement(McHugh, 2012).Heatmap analyses of 23S rRNA, 16S rRNA and IS111 markers for distinguishing between C. burnetii and Coxiella-Like species from Rattus spp.and chickens were constructed using ChiPlot with default settings (http://chiplot.online/).

F
Heatmap showing analyses of 23S rRNA, 16S rRNA and IS111 markers for distinguishing between Coxiella burnetii and Coxiella spp.from Rattus spp.The dark blue colour represents the presence of Coxiella spp., grey absence of Coxiella spp., whereas the light blue colour represents the presence of Rattus rattus and the light grey represents the presence of Rattus tanezumi.Samples within the red box are confirmed as C. burnetii, whereas green box as Coxiella spp.

F
The clustering map showing the distribution of 23S rRNA, 16S rRNA and IS111 genes for detection of Coxiella burnetii and Coxiella spp. in chickens.Samples within red box are confirmed as C. burnetii.The blue colour represents the presence of 23S rRNA, 16S rRNA and IS111 Number of Coxiella burnetii positive samples from rat and chicken samples at six different farms.
• C; 35 cycles of denaturation for 30 s at 95 • C, annealing for 30 s at 60 • C and polymerization for 1 min at 72 • C; and a final elongation step for 10 min at 72 • C. The PCR products were resolved on a 1.5% (w/v) agarose gel stained with 0.001 g/mL ethidium bromide and visualized under ultraviolet illumination using the ENDURO (Labnet International Inc., USA).
Coxiella spp.were also identified by 23S rRNA and 16S rRNA markers.Data from 2021 published by Mangena et al. (2021) carried out on cattle, sheep and pigs from the Free State Province reported an overall C. burnetii prevalence of 6.9%.Other studies have detected C. burnetii in ticks collected from dogs, cattle, goats, sheep, reptiles . The current study provides the occurrence of C. burnetii infections in two different hosts, 25% (17/68) and 24.1% (13/55) in Rattus spp.and chickens, respectively, by IS111 markers.