Molecular Detection of Coxiella burnetii in Vaginal Swab Samples from Sheep That Aborted

Introduction

Coxiella burnetii is an obligate intracellular Gram-negative bacterium with a worldwide distribution, with the exception of New Zealand. In humans, it is the causative agent of Q fever and coxiellosis in animals and can infect a wide variety of domestic animals, wild animals, marine mammals, birds, reptiles, and arthropods (Kazar, 2005; Sahu et al., 2020). Cattle, sheep, and goats are the main reservoirs and a frequent source for human infection. The presence of this pathogen within herds not only compromises the productive efficiency of the animals, but also represents a public health risk (Eldin et al., 2017; van den Brom et al., 2015).

The main routes of transmission are through contaminated aerosols, direct contact with vaginal secretions, feces, urine, contaminated placenta, and abortions tissues of infected animals. The ingestion of unpasteurized milk or cheese and transmission through tick bites are other routes of infection (Celina & Cerný, 2022).

Coxiellosis in Mexico is considered an exotic disease. However, in 2002, a seroprevalence study in ruminants suggested the presence of this microorganism (Salinas et al., 2002). Likewise, in Canada and the United States, C. burnetii has been detected in fluids and tissues of aborted sheep and goats, as well as in bovine milk samples (Hazlett et al., 2013; Olivas et al., 2016). In Latin America, there are reports of coxiellosis in Colombia, Chile, and Uruguay, detected in blood samples, milk, and bovine tissue, respectively (Cabrera Orrego et al., 2020; Cornejo et al., 2020; Macías-Rioseco et al., 2019). Furthermore, Brazil has detected C. burnetti in slaughterhouse animals (Mioni et al., 2020), humans with suspected dengue fever (França et al., 2022; Meurer et al., 2022), in aborted bovine fetuses (Mioni et al., 2022), milk (Mioni et al., 2019; Nascimento et al., 2021; Rozental et al., 2020), and in tissue samples from several domestic and wild animals (Mares-Guia et al., 2018), and French Guiana in the three-toed sloth (Davoust et al., 2014). The aim of the present study was to demonstrate the presence of C. burnetii DNA in vaginal swabs from sheep that aborted, as well as in semen from rams in Mexico.

Material and Methods

A total of 180 samples of vaginal swabs were obtained from herds located in five states of Central and Southern Mexico where abortions occurred: Aguascalientes (n: 45), Chiapas (n: 5), State of Mexico (n: 51), Morelos (n: 60), and Querétaro (n: 19). Samples were taken within the first 20 days postabortion. Additionally, 20 ram semen samples were obtained from the herd of State of Mexico. Vaginal swabs were transported in 2 mL of sucrose phosphate glutamate medium, at 4°C and finally stored at −80°C until processing. Semen samples were obtained by electroejaculation and transported at 4°C until processing.

All animals were subjected to vaginal or semen sampling according to the good veterinary practices. In Mexico, for this type of study, no ethical/welfare authority approval was required. The authors declare no mishandling or misuse of animals in this work.

Total DNA extraction from vaginal swabs and semen was performed with DNeasy Blood and Tissue kit (Qiagen, Ventura, CA. USA), following the manufacturer’s specifications. For detection of C. burnetii, primers IS1trg-f (5′-AGAATTTCTATTTTCAAAAAAAGGAGAAG-3′) and IS1trg-r (5′-CGGTTCAACAATTCGGTATACAAACAA-3′) that amplify a 605 bp product of the IS1111 insertion sequence, were used (de Bruin et al., 2011). C. burnetii DNA, donated by the Department of Animal Health of the University of Murcia, Spain, was used as a positive control.

Amplification products from three vaginal swab samples from Morelos, two samples from Queretaro, and one sample from the State of Mexico, as well as an amplification product of semen sample from the State of Mexico, were purified using a QIAquick Gel Extraction Kit (Qiagen, Ventura CA, USA) according to the manufacturers’ instructions and sequenced in both directions. The nucleotide sequence was performed using a Taq FS dye terminator cycle sequencing fluorescence-based sequencing. The obtained sequences were edited with MEGA program (version 10.1.7) and the consensus sequences homology search was performed in the GenBank database using the nucleotide BLAST tool at the NCBI website.

Results and Discussion

Isolation of C. burnetii is difficult, laborious, and requires a biosafety level 3 laboratory. Culture can be performed using laboratory animals, embryonated eggs, and diverse cell cultures (Sahu et al., 2020; Eldin et al., 2017). Another alternative for the diagnosis of C. burnetii includes serological tests such as ELISA, complement fixation, and immunofluorescence. These serological methods use LPS or membrane proteins as antigen (Imbert & La Scola, 2006; Muleme et al., 2016). However, molecular methods are faster, more sensitive, and specific; among the most frequently used genetic markers are genes icd, com1, sod and the insertion sequence IS1111 (de Bruin et al., 2011). The most widely used genetic marker is IS1111, which has proven to be the most sensitive and specific; it has the characteristic of being present in multiple copies within the microorganism’s genome (Madariaga et al., 2003; van den Brom et al., 2015).

In the present work, total DNA was obtained from 180 vaginal swab samples and from 20 semen samples, and the identification of C. burnetii was performed by endpoint PCR using IS1111 insertion sequence as molecular marker. Out of these, 110 (110/180: 61%) vaginal samples and 12 (12/20: 60%) semen samples were positive for C. burnetii. The state with the highest percentage of abortions positive for Coxiella was Queretaro (17/19: 89%), followed by Morelos (50/60: 83%), the State of Mexico (27/51: 52.9%), Aguascalientes (15/45: 33.3%), and Chiapas (1/5: 20%).

The amplification fragments from three different areas were sequenced in both directions and a consensus sequence was compared with GenBank databases. Sequences showed 100% homology with C. burnetii MSU Goat Q177 (CP018150.1), K2Q2 (CP107268.1), and AuQ31 (KT954146.1) strains, among others. The complete sequences were deposited in the GenBank under accession numbers: MT070965, MT536351, and MT536352 (vaginal swabs from Morelos), MT536354 and MT536355 (vaginal swabs from Queretaro), MT536356 (vaginal swabs from State of Mexico), and MT536353 (semen sample from State of Mexico).

In general, our results show 60% of the vaginal or semen samples were positive for the identification of Coxiella. However, depending of the area, this percentage was as high as 89% or as low as 20%. Different percentages have also been reported worldwide. In Egypt, C. burnetii was identified in 100% (21/21) sheep vaginal swabs using the same molecular marker (Selim et al., 2019). However, in the Netherlands, C. burnetii was detected in 47% (173/368) of the vaginal exudates of sheep, goats, and cows (de Bruin et al., 2011); in France, it was identified in 33.33% (6/18) samples of vaginal exudates from sheep (Berri et al., 2000); in Greece, the bacterium was identified in 24.44% (11/45) samples from aborted sheep fetuses (Chochlakis et al., 2018); in Italy, it was identified in 21.5% (81/376) of aborted sheep fetuses (Parisi et al., 2006); in Portugal, it was detected in 10.53% (8/76) of tissues from fetuses and sheep; and in India, it was identified in 7.3% (9/123) of vaginal swabs from sheep (Cumbassá et al., 2015; Gangoliya et al., 2019).

Our work reports a higher percentage of positive samples in relation to vaginal swab samples from other countries; this difference can be attributed to the type of samples we worked with, as we used vaginal swab samples obtained from sheep within 20 days after they had abortion, time when the bacteria is excreted in large number in vaginal secretions, contaminated placenta, and abortions tissues, being this the main route of excretion, in contrast to cows and goats, in which the main route of excretion is milk (Berri et al., 2007; van den Brom et al., 2015).

Our results are more similar to those obtained in Canada, where 69% (113/163) of the samples from sheep fetuses that suffered abortion were positive (Hazlett et al., 2013). There are some reports demonstrating that C. burnetii is distributed in herds of sheep and goats from Mexico and North America with similar percentages. On the contrary, in Chile, the percentage of positive detected cases is very low compared with our results, as only 2.1% (2/105) of the cow’s milk samples were positive (Cornejo et al., 2020). A recent work from our group reported C. burnetii in 82.35% (170/140) vaginal swab samples from Mexican goats that aborted (Flores-Perez et al., 2022).

C. burnetii is shed by ruminants mainly through the placenta, vaginal discharge, milk, feces, urine, and semen (Guatteo et al., 2006). There are very few reports of C. burnetii in semen; one of them in 15% (9/57) bulls in Poland and another in 37.5% (8/3) ram semen in Spain (Kruszewska & Tylewska-Wierzbanowska, 1997; Ruiz-Fons et al., 2014). Our results (60% positive ram semen cases) show that C. burnetii can be eliminated via semen. Therefore, this route can probably play an important role in transmission of infection in herds either through natural mating or through artificial insemination. The increased role of molecular tools for epidemiological studies has demonstrated to be of great value both in the identification of animals seeding and tracking back the disease, especially when followed by genotyping methods.

Conclusion

Our work demonstrated the presence of C. burnetii DNA in vaginal swabs from sheep that aborted, as well as in ram semen, suggesting that this microorganism is distributed in high percentages in the regions covered in this study in Mexico.