339Chapter 38 Contagious Equine Metritis

38 

Contagious Equine MetritisMichaela Kristula

C H A P T E R 

Etiology

Contagious equine metritis (CEM) is a transmissible bacterial venereal disease of horses caused by Taylorella equigenitalis, a gram-negative coccobacillus.

Epidemiology

Taylorella equigenitalis is transmitted by either carrier stallions or mares directly during mating or by indirect genital contact with contaminated fomites (vaginal speculums, artificial vagina, or wash buckets).1 There is evidence that transmission of T. equigenitalis through the use of fresh cooled or extended semen (with antibiotics) from an infected stallion is possible2 but most likely infrequent.1 The risk of transmission of T. equi-genitalis through frozen semen has not been established.1

Contagious equine metritis was first reported in 1977 in Newmarket, England,3 and the disease spread rapidly among horses in Europe. Two carrier Thoroughbred stallions imported from France were thought to have introduced the disease into the United States in 1978,4 causing significant economic losses. Recently, pulsed-field gel electrophoresis (PFGE) confirmed

that the 1978 CEM outbreak was caused by the two imported carrier thoroughbred stallions, each carrying a different strain of T. equigenitalis.5 Further small-scale outbreaks of CEM were reported in 1979 in Trakehners in Missouri, in 1982 in thor-oughbreds in Kentucky, and in 2006 in Lipizzaners in Wiscon-sin.1 These outbreaks had minor economic consequences and were linked to imported carrier animals.1 In 2008, a U.S.-origin stallion undergoing routine testing for semen export cultured positive for CEM. An epidemiologic investigation by the U. S. Department of Agriculture Animal Plant Health Inspection Service (USDA-APHIS) identified 23 stallions and 5 mares as infected with CEM in the United States.6 Genetic profiles gen-erated by PFGE identified a single common strain in all the infected horses,5 and the suspected source of the outbreak was a stallion imported from Denmark in late 2000.5,6 A significant finding of the 2008 domestic CEM outbreak was that most of the carrier stallions identified were exposed to T. equigenitalis through contaminated fomites at semen collection centers in the United States. An Arabian stallion in California tested posi-tive for T. equigenitalis in 2010, with a PFGE strain not previ-ously seen in this country.5 The positive stallion was from Belgium but had been imported to the United States from the United Arab Emirates, a country not known to be affected by CEM.2,7 In July 2011, the USDA National Veterinary Services Laboratories confirmed that an Arabian stallion born in Arizona

340 Section 3 Bacterial and Rickettsial Diseases

and are most frequently isolated from the fossa glandis and urethral sinus. The organism can also be isolated from the distal end of the urethra, the prepuce, and surface of the penis.19 Schluter et al20 isolated T. equigenitalis from the urethra, testis, epididymis, and seminal vesicles of an infected stallion at post-mortem, suggesting T. equigenitalis can be transmitted through seminal fluid.

Clinical Findings

Infected stallions do not show clinical signs. Clinical signs in infected mares range from copious purulent vaginal discharge (Fig. 38-2) for up to 14 days after mating to shortened diestrous intervals unaccompanied by other clinical signs.18 Taylorella equigenitalis typically causes temporary infertility in the mare. The acutely infected mare does not usually conceive. If preg-nancy is successful, mares may still abort or produce foals at term that are also carriers. The variation in clinical disease in exposed mares is due to variation among strains of T. equigeni-talis.1 Bleumink-Pluym et al21 demonstrated biologic differences between strains of Taylorella, and proposed strains differed in pathogenicity. Some mares recover spontaneously, whereas a smaller percentage of mares become chronic carriers, with the bacteria localizing most often in the clitoral sinuses and fossa and occasionally in the uterus.1

Diagnosis

The bacteriologic culture method for identification of T. equi-genitalis is the “gold standard” method for preexport and pre-import certification and detection of the carrier state. Cultures must be placed in Amies transport media with charcoal (BBL CultureSwab Plus Amies, Becton, Dickinson and Company, Sparks, MD) and refrigerated (4° C-6° C) during transit to a

tested positive for T. equigenitalis with a strain not previously identified in the United States, and an investigation for its source is ongoing.7

Taylorella asinigenitalis, a new species of Taylorella, was iso-lated from donkey jacks in the United States in 1997 and 1998.8,9 In 1997, T. asinigenitalis (California strain) was isolated from a donkey jack in California.8,9 In 1998, T. asinigenitalis (Kentucky strain) was isolated from two donkey jacks, a stallion, and seven nurse mares bred to them in Kentucky.8-10 Experi-mental infection of mares was only possible with the Kentucky jack isolate of T. asinigenitalis not the California strain. Exposure to the Kentucky strain resulted in a clinical response and sero-conversion consistent with CEM,8 although mares naturally exposed to the Kentucky strain during the outbreak were not reported as developing any clinical signs.10 Further evidence of T. asinigenitalis was not found in the United States until 2011, when a strain of T. asinigenitalis was isolated from a miniature donkey undergoing export quarantine (P. Timoney, personal communication). Taylorella asinigenitalis was isolated from a stallion in Sweden in 200411; a stallion, mare, and 22 male donkeys between 1995 and 2008 in France12; and from 2 donkey jacks in Italy in 2008.13 The origin and prevalence of T. asinigenitalis in horses and donkeys is not known, but risk factors for transmission are likely similar to T. equigenitalis.10 More field studies are needed to establish the epidemiologic relationships between T. asinigenitalis in donkeys and horses.

Contagious equine metritis remains a clinically and eco-nomically important disease of horses.14 Although national and international control procedures have reduced the incidence of CEM,15,16 the disease has been detected in many countries, including Japan, Australia, most European countries, Iran, Turkey, and Brazil.17 Contagious equine metritis has the poten-tial to cause widespread outbreaks of short-term infertility in mares because of the increased international movement of stal-lions that occurs.14,17

Pathogenesis

In the mare, T. equigenitalis typically causes an intense neutro-philic endometritis (Fig. 38-1) that subsequently resolves with a subacute neutrophilic mononuclear endometrial response.8 Stallions are inapparent carriers of T. equigenitalis on their exter-nal genitalia.18 The bacteria may persist for an extended period

Figure 38-1 Purulent discharge and endometritis caused by T. equigenitalis in the uterus of a mare at postmortem. (Courtesy the Maryland Department of Agriculture.)

Figure 38-2 Purulent discharge caused by T. equigenitalis in a mare. (Courtesy Dr. Don Simpson.)

341Chapter 38 Contagious Equine Metritis

National Veterinary Services (NVSL)-approved laboratory22 within 48 hours, then inoculated onto Eugon agar (with 10% chocolated horse blood) and CEM-selective agar. The plates are incubated at 37° C (98.6° F) for at least 7 days in an atmosphere of 5% to 10% carbon dioxide. Bacteria from suspicious colonies that are gram-negative, react positive to oxidase and phospha-tase tests, and do not grow aerobically on blood agar are identi-fied as presumptive CEM bacteria by agglutination with rabbit serum. Confirmatory testing is performed at NVSL by the polyclonal fluorescent antibody test.22 Even with improved bac-teriologic techniques, false negatives are common, particularly in imported stallions.23-25 False-negative diagnoses are common because T. equigenitalis is fastidious and slow growing, and other bacteria that live in the genital tract of the horses overgrow T. equigenitalis despite inhibitors in the media.24

No serologic test is reliable for the diagnosis and control of CEM.26 The complement fixation test (CFT) for CEM is of value for detecting infections in acutely infected mares26,27 but is not reliable for identifying chronically infected mares.26,28 The CFT is of no value for identifying infected stallions because stallions do not develop a demonstrable serum anti-body response after exposure.26,27 To date, no serologic dif-ferences have been observed between different strains of T. equigenitalis.16

Various polymerase chain reaction (PCR) tests have been developed to detect T. equigenitalis.23,29,30 Real-time PCR testing for the detection of T. equigenitalis shows great promise to improve the specificity, sensitivity, and speed of identification of T. equigenitalis compared to culture.30-32 Real-time PCR testing directly from genital swabs32 can discriminate T. equi-genitalis from T. asinigenitalis. Recent and future developments in molecular technology should enable epidemiologic analysis of different strains of T. equigenitalis and explain the patho-physiology and differences in virulence of different T. equigeni-talis strains.16

Import Requirements

Contagious equine metritis was classified as a reportable disease in the United States following the outbreak of CEM in 1978 in Kentucky. After a second outbreak of CEM in Missouri in 1979, the disease was eradicated from the United States by rigorous testing, treatment, quarantine, and surveillance of infected and exposed horses. Mandatory testing of imported mares and stal-lions for T. equigenitalis was implemented to prevent reintro-duction of CEM. Approximately 2500 horses are imported from CEM-affected countries annually and tested for CEM in U.S. quarantine stations.6

Currently, horses may be imported into the United States from CEM-infected countries after meeting preimport require-ments and testing negative for dourine, glanders, equine piro-plasmosis, and equine infectious anemia at the U.S. port of entry. Additionally, mares and stallions over 731 days of age are required to undergo further test procedures for CEM at a state-approved CEM quarantine facility.33 These tests are carried out by an accredited veterinarian under supervision by either state or federal officials.

At the CEM quarantine facility, swabs are taken from both the mare’s clitoral fossa (Fig. 38-3) and sinuses (Fig. 38-4) on three occasions, 3 days apart, for bacterial culture of T. equigeni-talis. On the third culture, the clitoral sinuses are flushed with a ceruminolytic agent (Cerumene, Evsco Pharmaceuticals, Buena, NJ) and 0.2% nitrofuracin solution (Equi-Phar Nitrofu-razone, Squire Laboratories, Revere, MA) and packed with 0.2% nitrofuracin ointment. The area is subsequently scrubbed with 4% chlorhexidine gluconate (Betasept, Purdue Frederick,

Figure 38-3 Location for culture of the clitoral fossa in the mare.

Clitoral fossa

Figure 38-4 Location of the clitoral sinuses for culture in the mare. Stretching of the clitoral fossa enabled the orifice of the median sinus to be visualized in the photograph.

Mediansinus

Rightlateralsinus

Leftlateralsinus

Norwalk, CT) and packed with 0.2% nitrofurazone ointment for 4 more days. Mares testing positive for CEM have the option of being returned to the country of origin or being treated.

For stallions, three separate swabs are obtained from the prepuce and surface of the penis (Fig. 38-5), the fossa glandis (Fig. 38-6), and the urethral sinus (Fig. 38-7). If the stallion is negative for T. equigenitalis, it is subsequently mated to two test mares and the stallion’s external genitalia are scrubbed for 5 days with 4% chlorhexidine gluconate and packed with 0.2% nitrofurazone ointment. Three sets of cultures are taken from the stallion’s test mare’s clitoral sinuses and fossa at 3, 6, and 9 days after mating. A CFT for CEM is performed on each test mare at 15 days after mating. Stallions testing positive for CEM have the option of either being treated, castrated, or returned to their country of origin.

In the 2008 domestic CEM outbreak, carrier mares and stallions were identified using the existing import regulations previously described and augmented by additional testing

342 Section 3 Bacterial and Rickettsial Diseases

Figure 38-7 A, Location of the urethral sinus for culture in the stallion. B, Location of the urethral sinus for culture in the stallion.

Urethral sinus

A B

Figure 38-6 Location of the fossa glandis for culture in the stallion.

Fossa glandis

Figure 38-5 Location for culture of the surface of the penis and folds of the prepuce in the stallion.

Surface of penis

Folds of prepuce

requirements.2 Most of the domestic carrier stallions (20 of 23) were identified on prebreeding cultures,1,2 although only 18 out of 23 were identified on the first culture. Three of the 23 carrier stallions were found positive by test breeding susceptible mares followed by bacteriologic culture and serology of the test mares.2 This contrasts with data on imported carrier stallions from U.S. quarantine stations, where a smaller percentage were identified on prebreeding cultures.1,25 However, in both U.S. quarantine stations and the 2008 domestic CEM outbreak, the test breeding of stallions to test mares has been critical for the detection of carrier stallions negative on bacterial culture for T. equigenitalis.2,25 More research is needed to determine the value of validating a PCR test and incorporating the PCR test into the current regulatory requirements.2

In 2010, the USDA tested 292 stallions for CEM.2 The stal-lions were either actively breeding or previously imported into the United States.2 None of the 292 stallions tested positive for T. equigenitalis.2 Based on the resolution of the 2008 domes-tic CEM outbreak and the negative testing results of the 292

stallions, the USDA declared the United States free of T. equi-genitalis in December 2010.2 The United States could not main-tain the 12-month CEM-free status because in 2011 the USDA reported the Arabian stallion from Arizona as positive for CEM. Because of the domestic outbreaks of CEM, many countries no longer recognize the United States as CEM-free and have placed restrictions on U.S. equine imports.6

Despite requiring preimport testing for CEM in CEM-affected countries of origin, at least 28 CEM positive horses have been identified during quarantine in the United States during the past 10 years.6 As a result of the domestic CEM incidents in 2006 and 2008, APHIS is in the process of strengthening the CEM regulations by amending the regula-tions regarding the importation of horses from countries affected with CEM. This includes incorporating additional cer-tification requirements for imported horses 731 days of age (2 years of age) or less and adding new testing protocols for horses over 731 days of age.6 Additionally, the rules will be strengthened for importing Spanish Purebred horses,

343Chapter 38 Contagious Equine Metritis

sinuses are flushed with a ceruminolytic agent and 0.2% nitro-furacin solution and packed with 0.2% nitrofurazone ointment. The clitoral sinuses and fossa are subsequently scrubbed for 4 more days with 4% chlorhexidine gluconate and packed with 0.2% nitrofurazone ointment. The external genitalia of the infected stallion are scrubbed for 5 days with 4% chlorhexidine and packed with 0.2% nitrofurazone ointment. Both treated stallions and mares are retested for CEM no less than 21 days after completion of treatment as previously described.

The USDA treatment protocols are not always effective, and a more aggressive combination of both topical and systemic antibiotics is required to treat some CEM-positive stallions and mares.20,25,36,37 A treatment regimen with either oral trimethoprim-sulfamethoxazole 30 mg/kg twice daily (Mutual Pharmaceutical, Philadelphia) or an antibiotic choice based on sensitivity, along with cleaning of external genitalia (as previ-ously described) and packing with 1% silver sulfadiazine cream (Thermazene, Kendall, Mansfield, MA), is recommended.25 The addition of local antibiotics to the uterus of an infected mare may also be beneficial.

During the 2008 domestic CEM outbreak, 3 of 23 carrier stallions and 1 of 5 carrier mares remained culture positive after initial treatment following the USDA treatment protocol.2 The treatment was switched to topical 1% silver sulfadiazine, and all horses were confirmed culture negative.2

Prevention and Control

Stringent control methods and regulations for horse movement continue to be necessary to minimize and control the spread of CEM. Control measures are based on taking bacterial swabs prior to breeding to establish freedom of disease and using strict hygiene measures during breeding activities. Specifically, dispos-able gloves should be worn and changed between horses, equip-ment should be either disposable or sterilized between horses, and clean water should be used for each horse. Although there is an increasing awareness of CEM in the United States, pre-breeding cultures for CEM are not routinely requested except by organizations engaged in the collection of stallions for arti-ficial insemination.

With the estimated annual market of export of U.S. horses valued at $300 million,14 the United States seeks to regain its CEM-free status to both facilitate international trade in horses and protect the U.S. domestic equine population and industry.

Public Health Considerations

There is no evidence that T. equigenitalis affects humans.38

The complete reference list is available online at www. expertconsult.com.

Thoroughbred horses, and temporarily imported competition and entertainment horses.6

To increase the likelihood of detecting CEM in imported mares, the following amendments to the regulations are likely to be adopted6: (1) an additional culture sample from either the distal cervix or the endometrium will be required on the third set of samples and (2) a CFT will be required on arrival at a state’s CEM quarantine facility. In addition, the time frame to complete sampling will be extended to a 12-day period with no less than 72 hours between each set of samples.

To increase the likelihood of detecting CEM in imported stallions, the following amendments to the regulations are likely to be adopted6: (1) an additional culture site from the stallion’s distal urethra will be required (Fig. 38-8), (2) an additional culture sample from either the distal cervix or the endome-trium will be required on the third set of samples from each test mare, (3) the CFT on the test mares will be required on day 21 instead of day 15 after breeding. In addition, the allow-able time frame to complete all three culture sets on the test mares will be extended to a 12-day period, with specimens collected anytime between the third and fourteenth day after breeding and a minimum of 72 hours between each set.

Therapy

Although isolates of T. equigenitalis are distinguished by their variable sensitivity to streptomycin, the bacteria is sensitive to most common antibiotics such as penicillin, ampicillin, tet-racycline, and trimethoprin-sulfamethoxazole.34,35 The USDA guidelines for treatment of CEM-infected mares and stallions are the same as the prophylactic treatment schedule outlined for routine testing for CEM. For an infected mare, the clitoral

Figure 38-8 Location of the terminal urethra—proposed new culture site for the stallion.

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11. Baverud V, Nystrom C, Johansson KE: Isolation and iden-tification of Taylorella asinigenitalis from the genital tract of a stallion, first case of a natural infection. Vet Microbiol 116:294–300, 2006.

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21. Bleumink-Pluym NMC, Werdler MEB, Houwers DJ, et al: Differences between Taylorella equigenitalis strains in their invasion of and replication in cultured cells. Clin Diagn Lab Immunol 3(1)47–50, 1996.

22. United States Department of Agriculture, Animal and Plant Health Inspection Service: Veterinary Services Memoran-dum No. 558.2: Approval of Laboratories to Conduct the Diagnostic Procedures for CEM, Fort Collins, CO, 2001.

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29. Anzai T, Eguchi M, Sekizaki T, et al: Development of a PCR test for rapid diagnosis of contagious equine metritis. J Vet Med Sci 61(12):1287–1292, 1999.

30. Premanandh J, Wernery GU, Sasse J: Evaluation of a newly developed real-time PCR for the detection of Taylorella equigenitalis and discrimination from T. asinigenitalis. Vet Microbiol 95:229–237, 2003.

31. Ousey JC, Palmer R, Cash RSG, et al: An investigation into the suitability of a commercial real-time PCR assay to screen for Taylorella equigenitalis in routine prebreeding equine genital swabs. Equine Vet J 41:878–882, 2009.

32. Wakeley PR, Errington J, Hannon S, et al: Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylo-rella asinigenitalis. Vet Microbiol 118:247–254, 2006.

33. Zirkle, E: Contagious equine metritis quarantine facility protocols and procedures, Trenton, 1998, New Jersey Department of Agriculture, Division of Animal Health.

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35. Sugimoto C, Isayama Y, Kashiwazaki M, et al: Susceptibility of Haemophilus equigenitalis, the causal agent of contagious equine metritis to 31 antimicrobial agents. Nat Inst Anim Health Q 21(4):159–162, 1981.

36. Andresen O: Contagious equine metritis. Treatment of infections with Taylorella equigenitalis in mares and stal-lions. Norsk Veterinaertidsskrift 99(3):193–198, 1987.

37. Lorin D, Prilhofer K, Arbeiter K: Detection of contagious equine metritis in Austria. Wien Tierarztliche Monats-schrift 71(3):81–85, 1984.

38. United States Department of Agriculture, Animal and Plant Health Inspection Service: Questions and answers: Contagious Equine Metritis 2009. Available at http://www.aphis.usda.gov/publications/animal_health/content/printable_version/faq_CEM09.pdf.

343.e1Chapter 38 Contagious Equine Metritis