Contagious equine metritis

Introduction

There are two known species within the genus Taylorella, T. equigenitalis which causes contagious equine metritis (CEM) and T. asinigenitalis, a closely-related species found mainly in donkeys, but which is currently considered non-pathogenic with natural infection.³⁹

Contagious equine metritis is a true venereal disease naturally transmitted to naïve mares at the time of mating by chronically infected, asymptomatic carrier stallions. A hallmark of the South African outbreak in 2011³⁶ and similarly in other recently reported outbreaks from the USA¹⁴ and the UK,⁴⁵ was the horizontal transmission of T. equigenitalis by unknown fomites to other stallions associated with an equine artificial breeding centre. These reports highlight the need for vigilance with assisted reproductive techniques that are currently commonplace in most equine breeds. Metritis is a misnomer for this disease in that it actually causes a transient endometritis in mares, usually resulting in temporary infertility. Most mares clear themselves of infection, however up to 25 per cent of infected mares may become chronic carriers.²⁹ An outbreak of CEM in an industry reliant solely on natural mating, such as the Thoroughbred industry has the potential for severe economic repercussions. The costs associated with CEM are not only related to the direct losses as a result of infertility, but also due to significant indirect costs in terms of quarantine and surveillance measures, those accrued by outbreak management and treatment protocols as well as international trade and movement restrictions.⁴⁷ As a consequence, CEM is possibly the most highly regulated bacterial equine disease in the world being a World Organisation for Animal Health (OIE)-notifiable disease and is considered part of veterinary certification for international trade purposes.⁴⁷

The causative agent was first isolated in Newmarket, United Kingdom in May 1977,^{8, 44} suspected to have been introduced via Ireland from two Thoroughbred mares originating from France.^{13, 29} The organism was first described as Haemophilis equigenitalis⁵² but was later renamed Taylorella equigenitalis.⁵⁰ By the end of the 1977 breeding season, T. equigenitalis had been diagnosed on 29 Thoroughbred studs in the Newmarket area, with 23 stallions and approximately 200 mares affected.^{13, 29} This resulted in closure of the National Stud and other stud farms in the Newmarket area, resulting in massive losses in both stud fees and foal sales and a reported decrease in the foaling rate to 42 per cent from the 72 per cent recorded in the previous year.²⁹ Despite rapid imposition of a ban on the importation of horses from the UK and Europe, T. equigenitalis was confirmed in Kentucky (USA) in 1978,⁵¹ and subsequently from Missouri and Canada in 1979.¹³  The 1978 outbreak was estimated to have cost the Kentucky Thoroughbred breeding industry a million dollars a day with an estimated cost of 13.5 million dollars for eradicating T. equigenitalis from the USA.^{26, 55}

Following the first reported cases of CEM in the UK and Ireland, the disease has been found to have a worldwide distribution and has been confirmed in various countries including several within Europe, the USA, Australia, Japan and South Korea^{6, 19, 21, 26, 29, 43, 49, 51, 55} with the most recent outbreaks being reported from the USA¹⁴ and the UK.⁴⁵

South Africa was considered free of CEM until May 2011 when an outbreak was confirmed and reported to the OIE.³⁶ Epidemiological evidence initially suggested that T. equigenitalis had entered South Africa via importation of a Warmblood stallion from Germany. A nationwide stallion-screening programme, traceback of the exposed mares and their resultant offspring and further investigation of a sub-population focus in the South African Lipizzaner population, however, revealed that T. equigenitalis was most probably first introduced into South Africa at least as early as 1996 by importation of Lipizzaner stallions from Europe. Thereafter the organism had unwittingly been maintained within this closed population. The later introduction of assisted reproductive techniques by the South African Lipizzaner Centre’s breeding management, allowed for dissemination of this organism by fomite transmission to the wider Sport horse population where it was maintained for several years until the identification in 2011 of the index case.

The current gold standard recognized by the OIE for identification of T. equigenitalis is bacterial culture. Complicating this however, is the organism being characterized during bacterial culture as fastidious, slow growing and rapidly overgrown by other bacteria present in the reproductive tract of horses.^{5, 12, 34} These characteristics decreases the sensitivity of this method. Real time polymerase chain reactions (RT- PCR) methods have subsequently proven to be more robust assays with increased sensitivity and specificity and RT-qPCR is currently the method of choice for screening for T. equigenitalis in many countries including South Africa.³⁷

Aetiology

Taylorella equigenitalis is a non-motile, microaerophilic Gram-negative, frequently pleiomorphic bacterium^{44, 46, 52, 55} with fastidious culture requirements. It is sensitive to a broad range of antimicrobials and disinfectants.^{47, 52, 55} Two biotypes (strains) are recognised based on their sensitivity or resistance to streptomycin, ^{34, 52} and all strains of T. equigenitalis isolated in the UK, Australia and Ireland have been streptomycin-resistant.⁵⁸ Given the relative ease with which the organism may acquire streptomycin resistance, and based on the isolation of streptomycin-sensitive strains of the organism from several European countries, it is likely that CEM originated in these countries and was introduced into the equine populations of other countries following the importation of unapparent carrier animals.⁵⁸

In 1997²⁰ and 1998,²³ three atypical isolates were obtained from donkey jacks in California (UCD-1T) and Kentucky (UK-1 and UK-2) respectively. Sequence analysis of the 16S rDNA (97.6 per cent  similarity), DNA-DNA hybridization studies (23 per cent similarity) and G + C composition analysis of the genomic DNA between T. equigenitalis and these three donkey isolates confirmed that the isolates were closely related but not identical to T. equigenitalis strains³⁴ and a new species of the genus Taylorella¸ T. asinigenitalis was proposed.²⁰ This new species had slower growth rates and a weaker positive reaction on immunofluorescent antibody testing than T. equigenitalis. Mares bred either naturally (Kentucky) or artificially (California) to the infected jacks did not develop clinical signs of disease, although the micro-organism was cultured from the mares bred by natural service.²⁰ Further studies reported that intrauterine inoculation of mares with the Kentucky strain of the atypical isolate resulted in transient clinical signs of an abnormal vaginal discharge, cervicitis and shortened oestrous cycles.²³ These mares responded several days later and with less intensity than mares inoculated with classic T. equigenitalis isolates.

Epidemiology

Taylorella equigenitalis is a venereally-transmitted organism spread directly or indirectly during natural mating or artificial insemination with fresh, cooled or extended semen collected from a carrier stallion.⁵⁵ An important trend of increasing relevance especially in recent outbreaks has been indirect transmission by fomites, including: (i) during assisted reproductive procedures and associated equipment e.g. semen collection assisted by breeding phantoms and artificial vaginas; (ii) during gynaecological examination of the mare, e.g. examination gloves, tail bandages, specula; and (iii) housing and routine management e.g. grooming equipment, buckets, bedding.^{1, 7, 47, 55}

Taylorella equigenitalis has been reported to persist in the reproductive tract of pregnant mares without appearing to compromise the health of the unborn foal. Such foals may apparently be born T. equigenitalis-positiveon their external genitalia, or may be exposed shortly after birth through contact with infected placenta, foetal fluids or the contaminated clitoral area of the mare at foaling.^{29, 55, 58}

The risks of transmission of T. equigenitalis using fresh, chilled or frozen thawed semen are currently undefined. Antibiotic-containing semen extenders did reduce the presence of T. equigenitalis compared to extenders containing no antibiotic in one study.⁴² However, in the South African context, there was proven transmission of T. equigenitalis to a mare using artificial insemination with chilled semen processed and transported in antibiotic-containing extender. The risks associated with cryopreserved semen have not yet been established, although they are believed to be minimal.⁵⁵  Repeated freeze- (-80°C) thaw cycles had a minimal effect on survival of T. equigenitalis, however dilution of semen with a semen extender containing antimicrobials (amikacin & penicillin G potassium) prevented development of CEM in mares inseminated with either T. equigenitalis-contaminated extended or cryopreserved semen.²⁵ Taylorella equigenitalis was isolated by bacterial culture and nucleic acid was identified by RT-qPCR in 2015 from both a frozen (-80°C) raw semen sample (obtained in 1996)³⁸ as well as from cryopreserved extended semen (frozen in 2008) (A Guthrie, Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, South Africa, personal communication). However, the potential for disease transmission from this contaminated semen is currently unknown and warrants further investigation.

Survival of T. equigenitalis outside the body is reportedly brief, as the organism is susceptible to many disinfectants, ultraviolet light, high temperatures and reduced humidity.^{47, 55} The organism does, however, survive as a surface contaminant associated with various fomites if environmental conditions are favourable, such as those associated with stallion handling and breeding activities.^{54, 55}

Pathogenesis and clinical signs

Clinical signs seen in naïve mares exposed to T. equigenitalis vary significantly from overt signs of the disease to a subclinical infection. Mares typically show a muco-purulent vaginal discharge and irregular interoestrus period associated with acute endometritis and early embryonic death. The incubation period varies from 2-12 days.^{28, 58} Mares may suffer from endometritis, cervicitis or vaginitis, or may show no clinical signs.^{14, 29, 44, 46, 54, 58} A study of the kinetics of invasion and replication of four strains of T. equigenitalis report a marked difference with in vitro invasion of and intracellular replication in equine dermal cells amongst the four strains. Two early isolates were considered highly contagious and showed high invasion levels in the study. There was also considerable variation in intracellular replication ability within each group, leading to the observation that the milder clinical signs of CEM observed in Thoroughbreds since the disease was first reported may be associated with the loss of intracellular reproducibility.⁴

Most mares rid themselves of infection and develop a short-lived humoral antibody response. Subsequent reinfection does not appear to result in such severe clinical signs.^{29, 55, 57, 58} It has however been reported that up to 20-25 per cent of aymptomatically infected mares may attain carrier status lasting for many months or even years.^{29, 34, 54, 55, 58} Most commonly, carrier mares harbour the organism in the clitoral fossa and sinuses, although (rarely) the organism may be carried in the endometrium. Rarely, abortions have been reported.⁵⁵ A difference in the distribution of T. equigenitalis between the initial infection and subsequent carrier status has been reported; a detection rate of 93 per cent (clitoral) and 31 per cent (cervix) was reported in chronically affected mares versus 69 per cent (clitoral) and 84 per cent (cervix) in acutely infected mares.⁶⁰

Stallions are subclinical carriers of the organism and this smegma-associated commensal merely colonises the predilection sites of the external genitalia mucosal surfaces without eliciting an immune response or clinical signs. The organism shows a tropism for the urethra, the urethral sinus and the lamina interna.^{14, 26, 29, 44, 54, 58} Affected stallions are the principal source of infection and the carrier status may persist for months or years.^{47, 58}

Diagnosis

The current gold standard test for T. equigenitalis recognized by the OIE is bacterial culture according to the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Chapter 2.5.1, Contagious Equine Metritis version adopted 20/10/2011). Swabs are obtained in stallions from the urethra, urethral fossa and lamina interna, and in mares from the non-pregnant endometrium, clitoral sinus (using a paediatric swab) and clitoral fossa. The swabs are transferred in charcoal Amies medium on ice to reach the laboratory within 24 hours. The closely-related organism, T. asinigenitalis has similar colony morphology and can be confused with T. equigenitalis.²³

Due to the associated difficulties in isolating T. equigenitalis and the discovery of the morphologically similar isolate, T. asinigenitalis, various RT-qPCR tests have been developed in order to rapidly identify T. equigenitalis and to discriminate it from T. asinigenitalis.^{2, 5, 12, 27, 41, 43, 59, 61} These RT-qPCRassays are rapid and have increased specificity and sensitivity for the detection of T. equigenitalis when compared to culture.^{2, 5, 43} Comparatively, qPCR shows greater practicality, quicker turnaround times and lower costs than bacteriology. Plain swabs are taken from the same predilection sites as for culture, however, there are fewer limiting constraints from the sample-taking viewpoint including media, temperatures and transfer interval.

Serology has limited application in the detection of T. equigenitalis-affected animals, with sero-conversion being reported as a transient feature associated only with the acute phase of endometritis in the mare, and being absent in stallions. Antibody titres rise from 7 days post exposure to reach a peak at three weeks, before declining at six to 10 weeks.^{29, 54} In certain countries, serology continues to be applied in post-treatment quarantine test-breeding protocols in stallions and mares.^{14, 29, 56}

Various authors^{1, 3, 22, 30-33, 40, 48, 49, 53} have used gel electrophoresis to further discriminate between different strains of T. equigenitalis as well as T. asinigenitalis in order to elucidate the source and trace the epidemiology of outbreaks.

More recently, multilocus sequence typing (MLST) for the Taylorella genus has been developed using seven housekeeping genes that were well conserved across both Taylorella species.¹¹ Multilocus sequence typing is more discriminatory when compared to other forms of molecular typing such as PGFE and the strain types can be compared using the Taylorella database http://pubmlst.org/taylorella/. When MLST was performed on 163 strains collected in several countries over 35 years (1977-2012), this revealed 39 sequence types (ST) which could then be grouped into clonal complexes. Further investigation of the different clonal complexes revealed that a single complex contained isolates from the first CEM outbreaks that occurred almost simultaneously in several countries (UK, USA and Australia) in the late 1970s suggesting that these strains originated from a single isolate of unknown origin. The recent identification of T. equigenitalis strains in France belonging to this same complex from mares showing few or no clinical signs, suggests that the initially virulent strain may have lost some of its pathogenicity in favour of its persistence. Most clonal complexes contain strains that originate from a single country, suggesting that the regulatory measures instituted by countries to prevent the spread of CEM, have been generally effective and it is important to maintain these measures in order to prevent the spread of more virulent strains of CEM.

Whole genome sequencing (available on GenBank) of T. equigenitalis and T. asinigenitalis has clarified that the genus Taylorella falls under the family Alcaligenaceae within the order Burkholderiales and has resulted in identification of factors potentially involved in the pathogenicity and host colonization of Taylorella genus members.^{15, 16, 17, 18, 38}

Control

Contagious equine metritis is a controlled animal disease in South Africa in terms of the Animal Diseases Act (Act 35 of 1984). Initial legislation required that infected animals be euthanased in the case of mares or that stallions must be castrated or euthanased. Following the 2011 South African outbreak of T. equigenitalis, The Director: Animal Health issued a derogation that allowed for the treatment of infected equines at an approved isolation facility by an authorised person provided the procedural manuals were complied with in full.⁹

Stallions, colts and geldings need to be sedated prior to commencement of daily treatment. Initially, the prepuce and exteriorised penis must be thoroughly irrigated with a topical surfactant solution (5 per cent docusate sodium) and then cleaned with 2 per cent chlorhexidine gluconate scrub. Plain swabs dipped in chlorhexidine gluconate scrub can be used to clean out the urethral sinus and fossa. The penis and prepuce are then dried with paper towel and a topical application of either 0.2 per cent nitrofurazone or 1 per cent silver sulphadiazine is liberally applied.²⁴

Similarly in mares, the vestibulum, clitoral fossa and clitoral sinuses are flushed with the 5% docusate sodium and then cleaned with the 2 per cent chlorhexidine gluconate scrub taking particular care to clean the clitoral fossa and sinuses with swabs dipped in chlorhexidine solution. The area must then be thoroughly dried before 1 per cent silver sulphadiazine is liberally applied.

Based on experience gained during the 2011 South African outbreak, the addition of a dry cow intramammary antimicrobial ointment applied specifically to the urethral sinus and fossa in stallions and the clitoral sinuses in mares appeared to hasten treatment end point.³⁵ The recommended duration of treatment in male equids is nine days and in mares, five days, however, in-treatment swabbing for qPCR assay prior to commencing each daily treatment allows for accurate determination of treatment end point.²⁴

Strict adherence to biosecurity principles includes wearing protective clothing, double gloving for treatment and correct disposal and incineration of all associated consumables, is crucial to prevent contamination of the environment. Similarly, effective disinfectants must be used on all hands and any equipment such as tack that comes into contact with an infected animal.

Due to the worldwide regulation of T. equigenitalis, strictly-defined point of exit and entry controls are stipulated by most countries, including pre- and post- arrival quarantine and swabbing for culture and, or qPCR.

Following the 2011 South African outbreak, a national stallion screening programme was instituted which required all breeding stallions to be tested and confirmed negative for T. equigenitalis on qPCR assay of swabs obtained from the prescribed sites prior to breeding or assisted reproductive techniques.¹⁰ Only foals born to stallions with a valid CEM-free certificate for that year could be registered with their relevant breed society. Current legislation requires two-yearly testing of breeding stallions, however, within the Thoroughbred breeding industry, annual testing is still mandatory according to the South African Thoroughbred Breeders Association. The future application of annual testing of Thoroughbred mares for venereally transmitted bacterial pathogens including T. equigenitalis is currently under discussion.

Should an animal test positive by testing using qPCR, both the animal and facility on which it is resident will be placed under immediate quarantine. Samples taken from the prescribed sites for qPCR and confirmatory culture are obtained and if positive, the animal will be treated according to the prescribed manual.¹⁰

In some countries, post-arrival test breeding is still required before a stallion is certified free of T. equigenitalis. This requires the stallion (or colt) is kept in post-arrival quarantine, test-bred to two certified CEM-free mares, with swabs taken at prescribed intervals from the mares from the predilection sites for bacterial culture and blood samples are taken for serology. Only after all tests have come back negative, may the animal be released from quarantine.⁵⁶ The act of natural mating is thought to agitate the organism free from its predilection sites on the penis and result in increased sensitivity of the culture assay. In future, the recognition that sensitive and specific PCR assays are available may make this culture-based testing method obsolete.

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