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CASE NOTES


Advances in diagnosing Chlamydia

Cheryl Jenkins and Pedro Pinczowski, NSW Department of Primary Industries, Biosecurity & Food Safety, EMAI, Menangle, NSW

Introduction

Chlamydia infections have traditionally been difficult to diagnose in the veterinary setting. Due to the intracellular nature of these organisms, cultivation can only be achieved in cell culture systems limiting the range of diagnostic tests available. Many early serological and molecular tests that were developed for the detection of Chlamydia spp. lacked sensitivity and specificity. Recent advances in genome sequencing have improved our understanding of genetic diversity within the Chlamydiales and enabled the development of highly specific and sensitive assays for a number of Chlamydia species. These improvements in molecular diagnostic testing are assisting to expand the known host range and the range of disease syndromes caused by Chlamydia species. The Elizabeth Macarthur Agriculture Institute (EMAI) has recently introduced quantitative PCR (qPCR) tests and immunohistochemical (IHC) stains to assist in the diagnosis of infection with Chlamydia psittaci, Chlamydia pecorum and Chlamydia abortus. In recent years, these tests have assisted in the exclusion of the exotic pathogen C. abortus and aided abortion investigations in sheep and horses. Whole genome sequencing is also being applied in some cases to better understand the strains involved in unusual cases.

C. psittaci and equine reproductive loss

An investigation into a potential chlamydial cause of equine reproductive losses was precipitated when several cases of psittacosis were diagnosed in veterinary students who had attended a horse abortion in Wagga Wagga (Chan et al., 2017). Quantitative PCR was used to demonstrate that the equine placenta associated with the human cases contained high loads of C. psittaci particles (2 × 106 genome copies/µL of placental DNA extract), a level equivalent to the chlamydial loads seen in birds with psittacosis. A cluster of equine abortion cases was subsequently investigated in the Hunter Valley Thoroughbred breeding area to determine if C. psittaci could be associated not only with zoonosis but also with equine reproductive losses (Jenkins et al., 2018). A total of 199 cases were examined across 54 properties consisting of abortions and neonatal foal deaths. Other potential causes of abortion were also examined including equine herpesvirus-1 (EHV-1), equine amnionitis foetal loss (EAFL), leptospirosis, coxiellosis and C. abortus. General aerobic culture for ascending bacterial infection was also attempted. No significant findings were observed on bacterial culture or using PCR tests for Leptospira, Coxiella or C. abortus. Of the cases examined, 4.5% tested positive for EHV-1 while only one case was confirmed as EAFL on histopathology (Begg et al., unpublished data). In contrast, a total of 22% of cases returned positive results for C. psittaci with positive cases detected on 21/54 properties, implicating C. psittaci in a large proportion of the abortion cases (Jenkins et al., 2018).

Begg et al., are in the process of providing a detailed description of the pathological features of chlamydial equine abortion; however, placentitis, amnionitis and vasculitis were major features of C. psittaci abortion cases, while full term foals frequently presented with respiratory distress and pneumonia. To link the presence of the agent with the pathology observed, 21 cases from the Hunter Valley epizootic, with tissues deemed suitable for IHC staining, were stained for the presence of chlamydial lipopolysaccharide (LPS). The presence of the pathogen was confirmed in placental lesions, lung tissue, and in some liver and spleen samples using Chlamydia IHC, although it wasn’t detectable in thymus samples. These results aligned with those from qPCR which demonstrated the highest loads of C. psittaci in placental and lung tissues and lower loads in the liver, spleen and thymus (Begg et al., in preparation). Placenta and lung samples are therefore the preferred samples for diagnosis of C. psittaci equine abortion.

Testing of 600 archival samples (collected between 1994 and 2019) from equine abortion cases across Australia indicated that C. psittaci was present in 6.5% of cases (3.9% in NSW), suggesting C. psittaci has been an under-diagnosed cause of equine reproductive losses in Australia for at least 25 years (Akter et al., 2021). It also raises questions about the environmental triggers for outbreaks of disease such as those seen during the Hunter Valley epizootic. Spill over of C. psittaci from Australian native parrots has been proposed as the likely source of infection based comparisons of genome sequences from horse and bird strains (Jenkins et al., 2018, Akter et al., 2021). Abortion cases also occur predominately in the winter months (Anstey et al., 2021).

Because of the high value of Thoroughbred foals and the public health issues surrounding potential transmission of C. psittaci from horses to humans, early diagnosis of infection has been a key aim of the Thoroughbred industry. Recently, point-of-care testing was developed for the detection of C. psittaci in equine tissue swabs to complement routine laboratory testing. Testing involves loop-mediated isothermal amplification (LAMP) and can be performed in under an hour with a pink to yellow colour reaction indicating a positive result (Jelocnik et al., 2017). Laboratory-based testing also continues to evolve with a multiplex qPCR under development for use in equines for the simultaneous detection/exclusion of Chlamydia genus, C. psittaci and C. abortus.

Chlamydia pecorum abortions in sheep

Chlamydia pecorum is a common cause of polyarthritis and conjunctivitis in sheep and a common inhabitant of the ovine gastrointestinal tract. Association of this organism with sporadic ovine abortions has only been rarely reported and, therefore, published descriptions of naturally occurring infections with foetoplacental lesions have been lacking. We recently described severe necrosuppurative placentitis with vasculitis, and foetal pyelonephritis and enteritis in late-term abortions of maiden ewes in a Northwest NSW flock (Westermann et al., 2021). Chlamydial infection was detected using a Chlamydia genus-specific qPCR (quantitative polymerase chain reaction) on tissue extracts from 3 foetuses. C. pecorum was identified in, and C. abortus excluded from, and all tissue extracts using targeted qPCR assays. Other potential abortigenic agents including Leptospira, Coxiella, Campylobacter and Toxoplasma were excluded using a combination of molecular, serological and culture-based methods. The C. pecorum qPCR revealed high loads of C. pecorum in all 3 foetuses, suggesting that this organism was the likely causative agent. The presence of viable C. pecorum in foetal samples was also demonstrated by replication of the organism in cell culture. IHC targeting the chlamydial LPS showed strong positive immunolabelling of foetoplacental lesions confirming C. pecorumas a cause of ovine abortion and providing the first descriptions of associated foetoplacental lesions in naturally infected sheep.

Subsequent qPCR testing of placental and foetal tissue samples from a further five properties across NSW experiencing ovine abortion events demonstrated that C. pecorum abortion may be more common than initially believed. On four out of five of the properties, the diagnosis of C. pecorum abortion was confirmed by necropsy and pathology findings (Watt et al., 2021). Placental tissue often contained the very high loads of C. pecorum and PCR testing of various foetal tissues indicated that loads of C. pecorum were generally highest in abomasal fluid which is consistent with usual niche of C. pecorum (the gastrointestinal tract). Placental tissue or abomasal contents are therefore the preferred tissues to aid diagnosis of C. pecorum abortion.

Abortions caused by C. pecorum have also recently been reported in Western Australia (Clune et al., 2021) suggesting that C. pecorum abortions may have been under-diagnosed in Australia. Improvements to molecular diagnostics and strain typing methods are undoubtedly enhancing disease detection and tracing. In both the NSW and WA case reports, multilocus sequence typing (MLST) has identified sequence type (ST) 23 as the sequence type of C. pecorum causing abortion cases (Westermann et al., 2021, Clune et al., 2021). ST23 has been associated with a range of syndromes in different hosts. Recent efforts to sequence the genome of the NSW abortigenic strain at EMAI have also revealed that this strain shares a high degree of similarity with C. pecorum strains causing sporadic bovine encephalomyelitis (SBE) in cattle. Despite this, the abortigenic strain displays some unique genetic features which may assist in explaining the tropism for placental tissue (Jenkins et al., accepted).

Conclusion

Chlamydia species are increasingly being detected in association with new disease presentations and in new hosts. Recent work conducted at EMAI has resulted in improved diagnostic methods which have demonstrated the importance of these species in abortion cases, therefore infection with Chlamydia should be considered as a differential diagnosis in future cases where the cause of abortion is otherwise inapparent.

References

  1. AKTER, R., SANSOM, F. M., EL-HAGE, C. M., GILKERSON, J. R., LEGIONE, A. R. & DEVLIN, J. M. 2021. A 25-year retrospective study of Chlamydia psittaci in association with equine reproductive loss in Australia. J Med Microbiol, 70
  2. ANSTEY, S., LIZARRAGA, D., NYARI, S., CHALMERS, G., CARRICK, J., CHICKEN, C., JENKINS, C., PERKINS, N., TIMMS, P. & JELOCNIK, M. 2021. Epidemiology of Chlamydia psittaci infections in pregnant Thoroughbred mares and foals. Vet J, 273, 105683
  3. CHAN, J., DOYLE, B., BRANLEY, J., SHEPPEARD, V., GABOR, M., VINEY, K., QUINN, H., JANOVER, O., MCCREADY, M. & HELLER, J. 2017. An outbreak of psittacosis at a veterinary school demonstrating a novel source of infection. One Health, 3, 29-33
  4. CLUNE, T., BESIER, S., HAIR, S., HANCOCK, S., LOCKWOOD, A., THOMPSON, A., JELOCNIK, M. & JACOBSON, C. 2021. Chlamydia pecorum detection in aborted and stillborn lambs from Western Australia. Vet Res, 52, 84
  5. JELOCNIK, M., ISLAM, M. M., MADDEN, D., JENKINS, C., BRANLEY, J., CARVER, S. & POLKINGHORNE, A. 2017. Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens: Chlamydia psittaci and Chlamydia pecorum. Peer J, 5, e3799
  6. JENKINS, C., JELOCNIK, M., MICALLEF, M. L., GALEA, F., TAYLOR-BROWN, A., BOGEMA, D. R., LIU, M., O'ROURKE, B., CHICKEN, C., CARRICK, J. & POLKINGHORNE, A. 2018. An epizootic of Chlamydia psittaci equine reproductive loss associated with suspected spillover from native Australian parrots. Emerg Microbes Infect, 7, 88
  7. JENKINS, C., JELOCNIK, M., ONIZAWA, E., MCNALLY, J., COILPARAMPIL, R., PINCZOWSKI, P., BOGEMA, D. R. & WESTERMANN, T. W. 2021. Chlamydia pecorum ovine abortion: associations between maternal infection and perinatal mortality. Pathogens. (Accepted)
  8. WATT, B., MCNALLY, J., BOURKE, J., CAVANAGH, C., MASTERS, A., MCCARTHY, T., JENKINS, C. & STAPLES, P. 2021. Chlamydia pecorum abortion in five widely dispersed NSW ewe flocks. www.flockandherd.net.au
  9. WESTERMANN, T., JENKINS, C., ONIZAWA, E., GESTIER, S., MCNALLY, J., KIRKLAND, P., ZHANG, J., BOGEMA, D., MANNING, L. K., WALKER, K. & PINCZOWSKI, P. 2021. Chlamydia pecorum–Associated Sporadic Ovine Abortion. Veterinary Pathology, 58, 114-122

 


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