Prof. Dr. Ivaylo Chenchev, PhD, DVSc Legislative background of horse infectious disease surveillance principles in EU with particular reference to infectious metritis Prof. Dr. Ivaylo Chenchev, PhD, DVSc National Diagnostic and Research Veterinary Medical Institute, Bulgaria

CONTAGIOUS EQUINE METRITIS Contagious equine metritis is an inflammation of the endometrium of mares caused by Taylorella equigenitalis, which usually results in temporary infertility. It is a nonsystemic infection, the effects of which are restricted to the reproductive tract of the mare.

CONTAGIOUS EQUINE METRITIS In general the clinical signs are a slight to copious mucopurulent vaginal discharge and a variable cervicitis and vaginitis. Recovery is uneventful, but prolonged asymptomatic carriage is established in a proportion of infected mares.

CONTAGIOUS EQUINE METRITIS Taylorella equigenitalis is most frequently transmitted by sexual contact with carrier stallions, which are always asymptomatic and in which the principal sites of T. equigenitalis colonisation are the urogenital membranes (urethral fossa, urethral sinus, urethra and penile sheath). The sites of persistence of T. equigenitalis in the mare are urogenital membranes, principally in the clitoral sinuses and fossa and very infrequently in the uterus. Foals born of carrier mares may also become carriers. The organism can infect equid species other than horses, e.g. donkeys.

CONTAGIOUS EQUINE METRITIS Contagious equine metritis was first described in the United Kingdom in 1977, after which it was diagnosed in a number of countries world-wide. It first presented as disease outbreaks characterised by a mucopurulent vaginal discharge originating from inflammation of the endometrium and cervix, resulting in temporary infertility.

CONTAGIOUS EQUINE METRITIS Serum antibody persists for 3–7 weeks after infection, but often it is not detectable for up to 15–21 days after recovery from acute infection in the mares. Most mares recover uneventfully, but some may become carriers of T. equigenitalis for many months. Many primary cases of infection with T. equigenitalis in the mare are subclinical, and a frequent indicator of infection is a mare returning in oestrus prematurely after being bred to a putative carrier stallion.

CONTAGIOUS EQUINE METRITIS Carrier mares and stallions act as reservoirs of T. equigenitalis, but stallions, because they mate with numerous mares, play a much more important role in transmission of the bacterium. The urogenital membranes of stallions become contaminated at coitus, leading to a carrier state that may persist for many months or years. Other sites of the horse’s body are not known to harbour T. equigenitalis. Most carrier mares are clitoral carriers of T. equigenitalis. Taylorella equigenitalis can cause abortion in the mare but this is a rare occurrence.

CONTAGIOUS EQUINE METRITIS Prior infection and vaccination are not fully protective, and failure of antibody to persist has meant that control of infection has relied entirely on prevention of transmission through the detection of T. equigenitalis on swabs of urogenital membranes. In spite of difficulties in culturing T. equigenitalis, screening mares and stallions prior to and while on the stud farm has successfully eliminated the disease from thoroughbred horses in countries using a voluntary code of practice.

Proposed minimum standards Housed in individual stalls One empty stall between CEM quarantine horses and other horses – prevent direct contact Groups of horses (Gypsies) can be kept in same paddock together (housed together before shipped) Test mares separated after breeding

Helpful farm policies Individual water and feed buckets cleaned daily Isolation barn for sick horses Monitor for feed intake and manure Temperature daily Concerns: EHV1, Strangles, Influenza Facilities should not harm horses

Mares and Stallions One person to hold mare One person to hold tail One person to handle stallion One person to handle test mares One person to assist breeding

Import mare procedures 3 sets of clitoral sinus and fossa cultures Interval – 3 days between cultures Current regulations - 1, 4, and 7 days of a 7 day period Complete cultures by 12 days after 1st culture

How to get the swab from mare

Stallions – swab sample – urethral sinus

Stallions – swab sample – urethral sinus

Stallions – swab sample - Fossa

Stallions – swab sample - prepuce

Stallions – swab sample - prepuce

Stallion – swab sample – terminal urethral

Swab (Culture) sample handling Special media – Amies charcoal media Refrigerate right away (4º C) Needs to arrive to lab within 48 hours Approved lab Cultures read at 7 days after plating Proposed clarification – read by hours -168 hrs vs 7 days

Test breeding of stallions After negative cultures – at least 7 days Breed stallion to two qualified test mares Clean vulva and place tail bandage on test mare Tease stallion before breeding to see if test mare is in heat

Test mare qualification 3 negative sets of sinus and fossa cultures with at least 3 days between cultures Negative CF test for CEM Vaccinate test mares for EVA Negative Coggins test (EIA)

Positive stallions Positive – Either initial culture, culture in test mares or positive CF in test mares Same procedures as described for stallions after breeding Treatment Wait 21 days after last treatment Repeat initial culture protocol and procedures

Positive mares Positive culture or CF test in test mares Same as previously described for import mares after 3rd culture Treatment Wait at least 21 days Repeat qualification protocols Proposed – Identify and don’t use positive test mares

Positive test mares - discharge

Example Treatment protocols – for positive mares Sulfamethoxazole Trimethoprim (TMS) – Systemic (30mg/kg PO) 5 days 1500 mg Gentamicin + 20 cc 8.4% Sodium Bicarbonate + 25 cc saline – IU for 3 days Flush beans (Cerumene®) & sinuses (4% Chlorhexidine) day 1 Scrub area for 2-5 days – 4% Chlorhexidine Pack area with 1% Silver Sulfadiazine 5 days

After Treatment protocols Wait 21 days Repeat initial protocols – include intra-uterine in one set of the 3 cultures Test mares – negative CF

Example treatment protocol for positive stallion TMS – 30mg/kg PO for 7 days Scrub penis for 10 days with 4% Chlorhexidine scrub Pack penis for 10 days with 1% Silver Sulfadiazine Cream Wait 21 days before culturing Culture at 21, 28 and 35 days after treatment Breed two test mares and follow previously stated protocols – starting over

CONTAGIOUS EQUINE METRITIS IDENTIFICATION OF THE AGENT Taylorella equigenitalis is a Gram-negative, nonmotile, bacillus or cocco-bacillus that is often pleomorphic (up to 6 μm long) and may exhibit bipolar staining. It is catalase positive, phosphatase positive, and strongly oxidase positive. If a slow-growing organism is isolated that fits the description for cellular morphology and that is strongly oxidase positive, it should be tested for reactivity with T.-equigenitalis-specific

Agglutination method A latex agglutination kit is available commercially for the antigenic identification of T. equigenitalis. It is based on polyclonal antibodies. This is widely used by routine testing laboratories for the confirmation of the identity of colonies growing on selective medium that give a biochemical reaction consistent with T. equigenitalis. It should be emphasised that it will not necessarily distinguish strains of T. equigenitalis from T. asinigenitalis.

PCR method A PCR method has been used for detecting T. equigenitalis and was compared with culture methods. It was highly specific and was able to detect very small numbers of T. equigenitalis PCR is more sensitive than culture for the detection of T. equigenitalis from genital swabs of horses in the field. Real-time PCR is more useful and it uses directly on genital swabs and compared with culture. This PCR has advantage of speed of result and also differents T. equigenitalis from T. asinigenitalis.