CULTURING THE STALLION

Culture samples should be taken from the unwashed, fully erect penis, the preejaculatory fluids, and an antiseptically collected semen sample. In the case of semen to be frozen, cultures should be taken from the extended semen just before being placed in the freezing straws. Fresh cooled semen should be cultured periodically, if not on each shipment, to ensure that the extender antibiotics have killed all bacteria. The culture swabs should be placed in a transport media such as Amies and kept cool while being transported to the laboratory without delay. In the case of fastidious bacteria such as T. equigenitalis, cultures should be immediately refrigerated and sent to the laboratory as quickly as possible (ideally less than 24 hours). The stallion is presented to an estrous mare to stimulate erection, and the penis is rinsed with warm water and dried with a paper towel (soap and disinfectants will lessen the chance of bacterial isolation). Culture swabs are obtained from the fossa glandis, the urethral orifice, and the skin of the penis and the preputial folds. By briskly rubbing the glands of the penis, a preejaculatory fluid sample can be obtained from the distal urethra. Following semen collection, a postejaculation urethral sample is obtained by grasping the penis above the glands and inserting a sterile swab just inside the urethral orifice. A swab is then taken of the fresh semen. While semen samples are unavoidably contaminated with bacteria from the penis and prepuce,⁶ the finding of large numbers of bacteria is highly suggestive of internal gland or urethral infection. This is particularly true if the semen isolates are inconsistent with the external genital cultures.^1,⁷

TAYLORELLA EQUIGENITALIS (CONTAGIOUS EQUINE METRITIS ORGANISM)

Contagious equine metritis (CEM) was first reported from Newmarket, England, in 1977, where it caused a very serious outbreak of equine metritis. The causative organism was identified as a gram-negative microaerophilic coccobacillus that was named Taylorella equigenitalis. One year later two stallions imported from France were found to be asymptomatic carriers after initiating an outbreak of CEM on several breeding farms in Kentucky.⁸ The significance of this outbreak is reflected by the imposed quarantine by the United States Department of Agriculture (USDA) that stopped the movement of all horses into and out of Kentucky until the disease was completely eradicated. A subsequent Code of Practice was developed by the State of Kentucky to prevent reintroduction of CEM. This code continues to be enforced 24 years later and has been adopted by several other states. The USDA also maintains stringent import requirements for mares and stallions desiring to enter the United States.⁹

Pathogenesis

The CEM organism (CEMO) is transmitted sexually between mares and stallions, but only the mare becomes infected. The stallion is considered an asymptomatic carrier. The organism may also be transmitted by contaminated instruments such as nondisposable vaginal speculums, rubber gloves, buckets, etc., and by contaminated frozen or fresh cooled semen.⁸ The organism is capable of eroding the uterine epithelium, causing severe endometrial damage. A purulent discharge is commonly seen for up to 2 weeks followed by gradual healing and elimination of the organism. Fertility is greatly reduced, and, while uncommon, abortions have been reported.^10,¹¹ Infected mares that carry to term have been known to have infected placentas.¹² The organism has been cultured from the vulva of fillies and the penis of colts from infected dams. These offspring are considered potential carriers of the CEMO.¹³ Recovery is usually associated with restored fertility, but a significant number of recovered mares become chronic carriers and are capable of spreading the disease sexually. The organism can be recovered from the endometrium, clitoral fossa, and sinus of carrier mares. In the case of the stallion, the organism can be found in several locations on the penis (fossa glandis, urethral orifice, the skin of the penis, the preputial folds) and in preejaculatory fluids. All isolates of CEMO have been pathogenic to Thoroughbreds.¹⁴

Diagnosis

Bacterial isolation and serologic examination are used to confirm a diagnosis of CEM. Serologic evaluation is limited to the mare because there is no immune response in the stallion. Test breeding healthy mares is also used to identify carrier stallions.¹⁵ This procedure is mandatory in the case of importing stallions into the United States. Following breeding, these mares are cultured and tested serologically at the appropriate time.

Culturing the organism from the stallion or the mare is the accepted method of diagnosing CEM.¹⁶ In the case of the stallion, the unwashed erect penis is swabbed at the fossa glandis, the urethral orifice, the skin of the penis, the preputial folds, and preejaculatory fluid. If semen is available, it is also cultured. The mare’s clitoral fossa and sinuses, as well as her endometrium, are swabbed. The culture swabs are placed in a transport media (Amies with charcoal) and refrigerated. They should be plated within 24 hours. The organism is fastidious, requiring special media and conditions for growth. Only a laboratory that is certified to culture T. equigenitalis should process samples. Suspect cases should be submitted to a reference laboratory for confirmation.¹⁷

The media of choice is chocolate agar with 10% horse blood and antibiotics to inhibit growth of contaminants. The plates are incubated at 37°C in 10% CO₂. Growth should occur by 48 hours, but negative samples should be held for 7 days. At 48 hours the colonies appear pinpoint, grayish, and smooth bordered. At 72 hours the colonies are glossy, raised, and have elevated opaque centers.¹⁸

Smears from acute cases stained with Gram’s or Giemsa reveal coccobacillus either free or inside of neutrophils. Diagnosis by this method should be confirmed by bacterial isolation.¹⁹

A complement fixation (CF) test is useful in monitoring mares bred to suspect stallions. This test is based on detecting antibodies resulting from exposure. The immune response requires 10 to 15 days after exposure before a positive test will occur, and then the antibodies are only reliable for 40 days. Therefore samples taken before 15 days post exposure may miss an infected mare, and those taken after 40 days may fail to identify a CEM carrier.^20,²¹ This test is routinely applied to mares between 15 and 40 days post breeding to imported stallions and to mares that follow the breeding of an imported mare.

A serum agglutination test (SAT) has been described that is reported to be very diagnostic in the acute phase of infection of the mare, but it also is not useful in the stallion.²²

Other serologic tests that have been developed for diagnosing CEM include an enzyme-linked immunosorbent assay (ELISA), a passive hemagglutination (PHA), and an immunofluorescence test. But the CF test remains the serologic test of choice in the United States to date (2003).