Evaluation of associations among Coxiella burnetii and reproductive abnormalities in cats

Abstract

Objectives

Coxiella burnetii is an obligate intracellular bacterium that is found worldwide, is associated or suggested to be associated with reproductive abnormalities in a number of species including cats, and is the cause of Q fever in humans. In a previous study, C burnetii DNA was amplified from the uterine tissues of 8.5% of client-owned cats in the USA but reproductive history was unknown and histopathological examination was not performed. In this study, uterine tissues of 26 normal cats and 11 cats with histopathological evidence of uterine disease or other reproductive abnormalities were evaluated for the presence of C burnetii.

Methods

A PCR assay that amplifies the repetitive transposon-like region (Trans 1 and 2) and a PCR assay that amplifies the IS-1111-insertion sequence (IS-1111) were optimised and applied to the DNA extracts. The sensitivity threshold of both PCR assays was 12 pg/µl. Positive samples were evaluated for the presence of the organism using immunohistochemistry performed on paraffin-embedded tissue.

Results

Amplicons of the expected size developed in three samples (one from a cat with reproductive abnormalities) in the IS-1111 assay; however, there was not enough DNA for sequence analysis. Immunohistochemical analysis was used to further evaluate these three samples and was negative for C burnetii. While C burnetii could not be confirmed by sequence analysis or immunohistochemistry, the PCR positive prevalence rate (8.1%) was similar to that published previously.

Conclusions and relevance

Biosafety precautions should be taken when working with cats that are aborting or parturient. Further research should be performed to evaluate the role that C burnetii may play in reproductive abnormalities in cats.

Introduction

Q fever is a worldwide zoonotic disease caused by Coxiella burnetii, an obligate intracellular gram-negative bacterium. ¹ In humans with symptomatic disease, patients are usually febrile with mild flu-like symptoms. ² Farm animals are traditionally associated with transmission of disease to humans primarily through inhalation of the organism, as it is passed in very high concentrations in parturient secretions.^1,2 Direct and indirect contact with parturient cats has also been demonstrated as a potential source of Q fever outbreaks.^3,4 Domestic animals with C burnetii are usually subclinically infected, but the organism is known to cause abortion and stillbirths in a variety of species, including horses, cattle, sheep and goats.^2,5 Coxiellosis-associated abortion has been documented in cats; however, the organism has also been isolated from cats after normal parturition. ⁶ In a previous study, our laboratory showed the prevalence of C burnetii DNA in uterine tissues of client-owned cats to be 8.5%. ⁷ In that pilot study, all of the cats were believed by the owners to be normal and the authors did not attempt to associate C burnetii with clinical or histopathological findings.

Reproductive tract diseases occur in cats, although relatively few data regarding rates of pregnancy loss are available and frequently the cause of stillbirth or abortion is not determined. ⁸ Aetiologies of pregnancy failures can be classified as either infectious or non-infectious. Non-infectious causes include nutritional deficiencies, endocrine failures and endometrial diseases, while infectious causes involve viral (panleukopenia virus, feline leukaemia virus, feline rhinotracheitis virus), bacterial (Brucella species, Escherichia coli, Streptococcus species) or rickettsial infections like C burnetii.^8,9 Transmission of C burnetii to animals may occur by infected ticks, consumption of infected placentas or milk, aerosols and close contact. ¹ Diagnosis of C burnetii infection is made by serological testing, but the presence of antibodies does not distinguish previous exposure from current infection. ¹⁰ Isolation of the organism is not routinely performed because it is difficult, time consuming and, owing to its high virulence and zoonotic potential, requires confined biosafety level 3 laboratories to cultivate the organism.^1,10 As C burnetii can be difficult to identify histopathologically, is known to infect cats and is associated with reproductive problems in other species, we believe that it is reasonable to consider this agent a potential cause of disease in cats and to evaluate additional cats using molecular methods.

The purpose of the study was to determine if C burnetii DNA is associated with inflammatory lesions of the uterus or reproductive abnormalities in cats. To explore this hypothesis, we performed C burnetii PCR assays on the reproductive tissues of cattery cats and then assessed for associations with histopathological findings and clinical history.^7,11,12

Materials and methods

Samples

In a previous study assessing for associations among reproductive abnormalities and Tritrichomonas foetus infection, uterine tissues from 34 cats were examined histopathologically and for the presence of T foetus DNA by PCR assay. ¹¹ Of these 34 cats, eight cats had histopathological evidence of uterine disease but T foetus was not linked to reproductive problems. ¹¹ The total DNA extracts obtained from the uterine tissues of each cat were stored at −80°C for 4 years prior to use in this study. Our collaborator (JG) provided the histopathological reports, the reproductive histories for these cats and their catteries, and shipped an aliquot of total DNA from each cat to the Center for Companion Animal Studies by overnight express on dry ice. Three additional DNA extracts were included in the shipment, all were obtained from separate pyometra cases.

PCR assays

Two PCR assays were used in this study. In the first PCR, the primers targeted the repetitive transposon-like region (Trans 1 and 2). In the second PCR, a combination of primers targeting the IS-1111 insertion sequence (IS-1111) transposase elements in the genome of C burnetii and their surrounding sequence were used. Both PCR assays were performed following published protocols, with minor modifications.^7,12,13 In the transposon PCR, reactions were amplified using a Mastercycler EP gradient S thermal cycler (Eppendorf North America). In the IS-1111 PCR, the reaction was performed using 12.5 µl/reaction commercial master mix (Hot Start Qiagen Master Mix), 7.5 µl water, 1 µl of each primer and 3 µl of DNA. Amplification of DNA consisted of an initial denaturation at 95°C for 15 mins, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 59°C for 45 s, extension at 72°C for 45 s and a final extension at 72°C for 7 mins.

Both PCR amplifications included negative (sterile water) and positive (C burnetii DNA) controls. Positive control DNA for C burnetii was extracted from commercially available immunofluorescence assay slides with both phase 1 and phase 2 antigens (Focus Diagnostics). DNA was eluted from the slides with sterile phosphate-buffered saline and purified using a QIAamp DNA blood kit (QIAGEN) and stored at −20°C until use.

A 10 µl sample of each PCR product was analysed by gel electrophoresis on a 1.5% Tris Borate EDTA agarose gel run at 120 V for 45 mins, stained with 2 µl loading dye (EZ-VISION; AMRESCO LLC), visualised with an ultraviolet transilluminator and photographed.

Immunohistochemical analysis

Samples positive for C burnetii DNA were evaluated for the presence of the organism using immunohistochemistry performed on paraffin-embedded tissue using a previously published protocol. ¹⁴

Results

C burnetii DNA was not amplified from any of the samples using the Trans 1 and Trans 2 primers. ⁷ A band consistent with the size of the C burnetii DNA PCR product was amplified from 3/37 samples using the IS1 and IS9 primers;^12,13 however, there was not enough DNA to submit for sequence analysis (Figure 1). The expected 687 base pair (bp) and 600 bp products were obtained from amplification of the C burnetii control DNA.^7,12,13 No product was amplified using the negative (sterile water) control template. The sensitivity threshold of the PCR assays for the Trans 1, Trans 2, IS1 and IS9 were obtained from the 10⁻² dilution (12 pg/µl) of the C burnetii-positive control DNA. The three samples with amplicons consistent with the size of C burnetii were immunohistochemically negative for the presence of C burnetii bacteria. ¹⁴

Figure 1

Gel electrophoresis image of one positive cat with negative and positive controls in the PCR assay targeting the IS-1111 insertion sequence (IS-1111). 1 = 100 base pair ladder; 10 = positive sample; 18 = negative control; 20 = positive control; 2–9 and 11–16 = negative samples; 17, 19 = blank lanes

The first cat positive for C burnetii DNA by the IS-1111 PCR was an 18-month-old Tonkinese from Colorado, with a pyometra and history of abortion and early kitten death. The second was a 7-year-old Ragdoll from New York with normal uterine histopathology and history of stillborn kittens and early kitten death. The third was a 3-year-old Ragdoll from Ohio with normal uterine histopathology and no reproductive abnormalities. ¹¹

Discussion

C burnetii is a widely distributed zoonotic infectious agent that is known to infect a variety of domestic animal species, including cats. While it has been suspected as a cause of abortion in cats, ⁶ to our knowledge, this is the first study to document and compare the presence of C burnetii DNA in uterine samples from cats with or without clinical and histopathological evidence of reproductive abnormalities. ¹¹ Three of 37 samples were positive for C burnetii DNA by the IS-1111 PCR and there was no evidence for false-positive results based on appropriate performance of all control wells. However, results were not reproducible and sequencing could not confirm the positives. In addition, C burnetii could not be documented in the three PCR-positive tissues by immunohistochemistry. Therefore, these results should be interpreted cautiously. However, if the samples were truly positive for C burnetii DNA, the 8.1% prevalence rate was similar to a previous report. ⁷ Of these three cats, only one had histopathological evidence of an inflammatory disease of the uterus (pyometra). However, two of the cats had a history of abortion, stillborn kittens or early kitten death. ¹¹

Failure to confirm the presence of C burnetii by genetic sequencing or immunohistochemistry in these cats could be owing to a true lack of infection. While the cats sampled represented a wide geographic distribution, midwestern and western states report higher annual incidence rates of Q fever than other areas of the USA. ⁶ Two of the three cats found positive for C burnetii DNA by the IS-1111 PCR assay were from midwestern or western states (Ohio and Colorado, respectively). Because infection is acquired by infected ticks, aerosols or ingestion of contaminated milk or placentas, a cattery environment may not have the same exposure to this infectious agent.^1,6 Failure to confirm the presence of C burnetii in the reproductive tract specimens could also be attributed to timing of sample collection. In a previous study that followed 24 dairy cows that had experienced an abortion and were shedding C burnetii in vaginal mucosa at the time of abortion, only one-third of the cows were still shedding at day 14. ¹⁵ It is known that C burnetii can be cultured from the uterus of cats for 10 weeks after parturition. ⁷ The time period between elective ovariohysterectomy and last parturition in these cats is unknown.

The failure of obtaining genetic sequencing and immunohistochemistry to confirm the suspected C burnetii infection may also be related to presence of only a few organisms in the DNA extractions that were at the limit of detection of the PCR assay. The diagnostic sensitivity of both PCR assays was 12 pg/µl. It is possible that the cats were shedding a number of organisms below this level. In one study, a breeding queen demonstrated a strong seropositivity via complement fixation test, indirect immunofluorescence assay and enzyme-linked immunosorbent assay but PCR analysis of the reproductive tract was negative for C burnetii. ¹⁶ Serum samples for these 37 cats were not available in this study, but C burnetii serology should be considered for use in future prospective studies. It is also possible that C burnetii DNA had begun to decay as the total DNA samples had been stored at −80°C for 4 years. ¹¹

Conclusions

These results suggest that further studies to evaluate the role of C burnetii in reproductive abnormalities in cats may be warranted and that biosafety precautions such as wearing gloves and a mask should be considered while attending to aborting or parturient cats and neonatal kittens.