Sources and Excerpts On The Use Of Canine Vaginal Culture
MYCOPLASMA INFERTILITY IN THE DOG: DIAGNOSIS AND TREATMENT
Donald H. Lein, DVM, PhD Director Diagnostic Laboratory Associate Professor Pathology and Theriogenology
New York State College of Veterinary Medicine, Cornell University P. O. Box 786 Ithaca, NY 14851
Mycoplasmas and ureaplasmas either alone, or comixed with bacteria are frequently opportunistic, developing long term carrier states on the mucosal membranes of the conjunctive, nasopharyngeal cavity and the urogenital tract with no clinical expression until the right conditions in the host defense system and numbers of organisms are present to invade the adult host and embryonal, fetal or neonatal tissue.
Canine Vaginal Flora
Patricia N. Olson, DVM, PhD St. Paul, Minnesota
Vaginal swabs are frequently obtained from bitches with signs of vaginal/vestibular inflammation in an attempt to correlate a causative agent with the reproductive disorder. Such correlation may be difficult because many causes of inflammation of the caudal reproductive tract do not involve an infectious etiology (de, conformation, foreign bodies). Additionally, since the canine vagina normally harbors microorganisms, merely isolating bacteria from the vagina does not constitute a basis for diagnosing disease of the caudal reproductive tract.
Breeders may also request pre-breeding vaginal cultures for their bitches. Such requests are probably not indicated unless the bitch has a history of reproductive disease or infertility.
COMPARATIVE CLINICAL AND DIAGNOSTIC ASPECTS OF HERPESVIRUS
INFECTIONS OF COMPANION ANIMALS WITH PRIMARY EMPHASIS ON THE DOG
James F. Evermann Department of Veterinary Clinical Medicine and Surgery and Washington Animal Disease Diagnostic Laboratory College of Veterinary Medicine Washington State University
Pullman, NA 99164
Infection and Disease.
Canine herpesvirus (CHV) infections may commonly occur throughout the life of the dog. The periods of greatest susceptibility to disease are those during pregnancy, neonatal development, and immunodeficiencies due to drug therapy, and other viral infections, i.e. canine parvovirus. The virus is spread predominately by areosol and direct contact of oral-nasal secretions. It can be spread venereally by infected male dogs, and it has been reported that CHV can be shed in semen. Female dogs are unusually susceptible to infection during the last 3 weeks of pregnancy, while puppies are unusually susceptible 3 weeks after whelping.
How common is the infection?
Prevalence of infection varies, from low risk* dogs (10-15%) to high risk dogs (85%)** due to the requirement for close contact for transmission.
Can CHV be eradicated from a particular dog/kennel?
Once infected by CHV, current thinking is that the dog remains infected for life. The infection may remain latent for the life of the animal or express itself later in life. Eradication not practical at this time.
Common modes of CHV spread
a. aerosol, oral-nasal secretions
b. venereal (rare)
*Single dog household
**Multiple dog facility, kennels, humane facilities, show dogs, etc.
Long-term study of aerobic bacteria in the genital tract of stud dogs.
L. Bjurnstrom and C. Linde-Forsberg, Am J Vet Res 53:670-673 (1992).
Bacterial samples were collected from the prepuce and semen of 15 stud dogs for 18 months. The mean pregnancy rate, litter size, and pup mortality for the bitches they had mated were all within normal limits. The most frequent bacteria isolated from the prepuce and semen were Pasteurella multocida, B-hemolytic streptococci, and Escherichia coli. Bacteria were transferred between the dog and bitch at mating but in this population of healthy dogs, the fertility of neither the dog nor the bitch was affected by the bacteria transferred.
Effects of ampicillin and trimethoprimsulfamethoxazole on the vaginal bacterial flora of bitches
Vaginal aerobic bacterial flora was studied in 5 healthy bitches before, during, and after a 10-day period of treatment with ampicillin and an equally long period of treatment with trimethoprimsulfamethoxazole. Blood variables and antimicrobial drug susceptibility also were studied. Bacteria were isolated from all bitches before the first treatment period. Bitches from which only a sparse number of bacteria were isolated had flora that varied from day to day.
In most instances when bitches were given an antibiotic to which their vaginal bacterial flora was susceptible, these bacteria were eradicated after only 1 day of treatment. This was true for pasteurellae, streptococci, and, in all but one case, Escherichia coli. Staphylococcus intermedius was more difficult to eradicate, and, although susceptible in vitro, it was unaffected by antibiotic treatment in 1 bitch and it took 7 days to eradicate in another. Eradication of aerobic bacteria in the vagina was total only in the bitch that had sparse flora from the beginning. Bacteria colonized within O (in 4/5 bitches) to 4 days after termination of treatment with ampicillin and within O (4/5 bitches) to 3 days for trimethoprim-sulfamethoxazole. Mycoplasmas emerged during and after both treatment periods, and E coli became apparent during treatment with trimethoprim-sulfamethoxazole. Because mycoplasmas may be genital pathogens in bitches and E cold is a common uropathogen, their appearance should be an argument against widespread use of antibiotics in healthy breeding bitches. Two bitches developed a vaginal discharge during treatment or shortly after. Blood variables did not change during the study, nor did antimicrobial drug resistance of the isolated bacteria.—B. Strom and C. Linde-Forsberg in Am J Vet Res 54 aug. 1993).
Bacteria And Infertility In A Bitch
The fact first: Brucella canis is the only bacterium known to be a specific cause of infertility in a bitch. That said, what can be said about all the other kinds of bacteria that are normally found in the vagina of dogs? Although the canine uterus itself is normally sterile, the so-called vaginal bacterial flora includes many kinds of aerobic (requiring oxygen to survive and multiply) and anaerobic (requiring no or little oxygen). Some experts say that some of these microorganisms can also cause infertility and other reproductive problems. However, Dr. E. van Duijkeren of the University of Utrecht, The Netherlands, reports that the bacteria actually isolated from bitches with reproductive disorders do not differ significantly from those found in healthy bitches. Nevertheless, many owners of stud dogs request a negative vaginal culture before accepting a bitch for mating, even though no correlation can be made between any organism (other than Brucella canis) and infertility.
On closer inspection, says this veterinarian, the case is not so clear-cut. If bacteria other than Brucella canis exist in large enough quantities, they may possibly cause infertility. Furthermore, the normal inhabitants of the vagina may become pathogens and then lead to infertility if there is a breakdown in the bitch's immunity. Bacteria may even enter the uterus during estrus and lead to an inflammatory process and endometritis and subsequent infertility. These considerations lead this expert to conclude that it is "not reasonable" to refuse a bitch for mating if bacteria that are part of the normal growth in the vagina are detected by tests.
Should antibiotics be used to treat an infertile bitch? This is another debatable issue, says Dr. Duijkeren, especially because there is little knowledge about the effects of antibiotic therapy on either the existing bacteria in the vagina or the fertility of the bitch. Some antibiotics are known to have a possibly detrimental effect on the fetus (a teratogenic effect, in technical terms) if the bitch is treated during preg
nancy. All of which brings us back to the fact: Brucella canis is the only bacterium known to cause infertility. Further research is needed to settle these other issues.
Questions from Our Readers
Q: I recently bought a nine-month-old bitch and learned soon afterward that she had canine Mycoplasma. Can you tell me something about bacteria of the Mycoplasma family, particularly about whether they can lead to infertility in a bitch?
A: Bacteria of the Mycoplasma family can be found as normal inhabitants of the reproductive tracts of normal male and female dogs. That is, bacteria including Mycoplasma are present in the vagina of normal female dogs and in the prepuce (foreskin) and urethra of normal male dogs. These areas of the reproductive tract are open to the environment, and will contain bacteria. However, there are areas within the reproductive tract, such as the uterus or testes, which normally should not contain any bacteria. Occasionally, bacteria will move from the vagina or prepuce into these deeper reproductive organs. If bacteria successfully invade these areas, the dog usually shows signs of infection, such as fever or discharges from the reproductive tract, and infertility may result. In such cases, antibiotic therapy is recommended for treatment of infection.
It is difficult to obtain culture samples from the deeper reproductive organs, but not difficult to obtain samples from the vagina or prepuce. The presence of bacteria in cultures of the vagina or prepuce does not necessarily indicate that the vagina or prepuce is diseased or that other parts of the reproductive tract contain these bacteria. Therefore, results of vaginal or preputial cultures are considered along with other important criteria, such as reproductive history, physical examination findings, and other laboratory test results before the veterinarian arrives at a treatment decision.
The Use and Misuse of Vaginal Cultures
Whenever inflammatory disease of the canine reproductive tract is diagnosed, the veterinarian must attempt to determine if the inflammation is resulting from an infectious process. Frequently, vaginal cultures are obtained in an attempt to correlate various reproductive disorders (inflammation of the clitoral fossa, vestibule, vagina, cervix, or uterus; pregnancy failure; neonatal deaths) with specific infectious agents. Since a variety of microorganisms are present in the vaginas of bitches with and without reproductive diseases, it is often difficult to associate disease with a specific isolate.
Approximately 60 per cent of normal bitches harbor aerobic bacteria in the cranial vagina (Olson et al., 1986, In: Current Therapy in Theriogenology, W.B. Saunders, Co., pp 469-475). Approximately 90 per cent of normal bitches harbor aerobic bacteria in the caudal vagina. Therefore, merely isolating bacteria does not constitute a basis for incriminating the isolate(s) with the reproductive disease. Frequently, bacteria may be very abundant during proestrus and estrus (Allen and Dagnall, J. Small Anim Pract 23:325, 1982; Baba et al., Am J Vet Res 44:606, 1983). Conversely, organisms known to cause infertility, abortion, stillbirths, or neonatal deaths (canine herpesvirus, Brucella canis, minute virus of canines, mycoplasma/ureaplasma) are difficult to isolate with routine culturing procedures. Hence, a negative vaginal aerobic culture does not ensure that a bitch is free of organisms which are associated with reproductive pathology.
JAVMA (172:708 ,1978.)
MYCOPLASMA -UREAPLASMA - ROLE IN REPRODUCTIVE DISORDERS
Mycoplasma and ureaplasma are among the smallest free living microorganisms. The family Mycoplasmatales is divided into two genera, Mycoplasma and Ureaplasma. Similar to many other microorganisms, mycoplasma and ureaplasma may be normal inhabitants of the nasopharyngeal, ocular, and urogenital mucosa of the dog, with no manifestations of clinical disease. In one study, mycoplasma was isolated from the vagina of 6/20 (30%) of normal adult bitches and from the prepuce of 7/20 (35%) of normal adult male dogs (Ling and Ruby, Am J Vet Res, April, 1978; 695). Eleven mycoplasma species, 4 serological groups of ureaplasma, and unclassified mycoplasma have been reported as mycoplasma flora in the dog (Rosendal, JAVMA 1982; 180:1212; Lein, Proceedings SFT, September 1989, 307).
Merely isolating mycoplasma/ureaplasma from swabbings of the vulva, vestibule, vagina, prepuce, or urethra does not indicate reproductive disease nor warrant therapy. Attempting to isolate the organism from normal bitches and male dogs as part of a prebreeding examination, is not indicated or necessary. In fact, veterinarians frequently fail in attempting to eliminate mycoplasma from the urogenital mucosa by administering various antimicrobials. Frequently, when therapy is discontinued, the normal flora (including mycoplasma) quickly re-establishes. Additionally, since most laboratories do not speciate mycoplasma, little information is currently available which associates certain species with reproductive disease. Therefore, a "positive" culture without clinical signs is very difficult to evaluate.
Although mycoplasma and ureaplasma can be considered part of the normal mucosal flora of dogs, these microorganisms have also been associated with urogenital disease. Mycoplasma has been associated with urinary tract disease. Clinical signs of urinary tract infection were noted in 20 of 31 dogs in which mycoplasmas were isolated in pure culture from urine obtained via cystocentesis, and numbers of WBC in the urine sediment were above the reported normal range in 22 of 25 urine specimens from those 20 dogs (Jang et al., JAVMA, 1984, 185:45). Ureaplasma urealyticum reportedly persists for at least 21 days in the urine of dogs with experimentally induced hydronephrosis (J Urol, 1989; 141:1437). Mycoplasma and ureaplasma are also associated with disease of the lungs, joints, mammary glands and eyes.
Bacteriological Studies of Reproductive Disorders of Bitches
G.W. Osbaldiston, BVSc, PhD
The vaginal bacteriafloras of 50 healthy bitches in anestrus and 297 bitches in which bacterial infection may have been involved in reproductive disorders were studied. In addition, bacterialflores of uterine specimens prom 64 bitches with endometrial hyperplasia and of 30 milk samples from bitches with clinical evidence of mastitis were studied. The results suggested that the recovery of anaerobic organisms from infections of the vagina and uterus may be more frequent than is generally recognized. Bacterialfloras isolated from the vaginas of healthy bitches in anestrus and bitches with vaginal and uterine disorders were similar. Many of the organisms were potential canine pathogens, but their association with vaginal or uterine infection could not be established in the dogs studied In vitro sensitivity studies showed that most bacterial isolates were sensitive to chloramphenicol and gentamicin. The exception was Pseudomonas which was resistant to most agents.
Meatal Recolonization In Bitches
In the normal canine urethra, indigenous bacteria inhibit establishment of certain foreign bacterial species, and hormones enhance the susceptibility to colonization. These were the findings of a study reported by Dr. Frank Hinman, Jr. of the University of California School of Medicine, San Francisco.
The normal canine urethrovaginal flora was determined by serial cultures. It consisted principally of Staphylococcus epidermidis, Corynebacterium, and Acinetobacter-Haemophilus. These particular organisms not only occur more frequently, but also appear in higher numbers, as shown in Table 1. Estrus produced a selective overgrowth of beta-Streptococcus and Acinetobacter-Haemophilus (Table 2).
A normal canine transient, Escherichia coli, and a normal resident, Corynebacterium, persisted only for a short time (mean, 4.5 days) unless inoculated during estrus, when they remained for a mean period of 70 days. In contrast, human E. coli and Pseudomonas persisted only a short time during anestrus (4.8 days) or during estrus (8.5 days).
The commensals had returned fully by 14 days after bacterial sterilization, while those in estrus over-responded.
In contrast to antibacterial therapy, immunosuppression promoted colonization by a foreign bacterium, which apparently was able to become established permanently.
J. Urology 118:559-563; oct. 1977.
Vaginal And Uterine Microflora Of Adult Dogs
Aerobic and anaerobic microflora were identified and quantitated in 82 vaginal and 78 uterine samples obtained from mature bitches during different stages of the estrous cycle. The mean + SD of total bacterial counts/100 mg of vaginal contents of the 82 bitches was log 5.0 + 1.5 ranging from log 2.4 to log 8.8. The count during the estrous stage (log 7.8 + 0.7) was markedly higher than that at anestrus (log 4.4+1.0), pregnancy (log 5.9+1.3), and postpartum (log 5.1+1.5) stages. The common organisms isolated from the vaginas were Bacteroidaceae, streptococci, Pasteurella spp, and mycoplasmas. Organisms were isolated from 48 (68%) of 78 uterine samples. The range of total counts/100 mg of uterine contents was from log 1.6 to log 8.3. Staphylococci and mycoplasmas were frequently isolated from the uterine contents. Although many uterine microfloras were similar to vaginal microfloras, some uterine culture had a single isolate identified. There were no pathologic findings in most of the uteri. Seemingly, vaginal bacteria frequently flow into the uterus, yet they rarely cause uterine infection.—E. Baba, H. Hata, T. Fibkata, et al in Am J Vet Res, 44 (Apr 1983): 606.
Long-term study of aerobic bacteria of the genital tract in breeding bitches
The aerobic bacterial flora of the genital tract of bitches was characterized in an 18-month study. The 59 bitches represented 4 breeds and were from 3 kennels. Collection of vaginal swab specimens for bacterial culturing was performed every month, except during estrus when specimens were collected every week (n=826). The capsule of the swab containing transport medium was broken before specimen collection to moisten the tip, which helped to reduce the number of negative culture results. All bitches whelped at least once during the study and, thus, had known reproductive functions. Pregnancy rates, litter sizes, and pup mortality were within normal limits. Pasteurella multocida, 9-hemolytic streptococci group G1 and Escherichia coli were the most common bacteria isolated. Although these species generally were isolated from mixed cultures, pure cultures were obtained from 18% of the specimens. There was a tendency for the various breeds to differ in their vaginal bacterial flora. Pasteurella multocida was isolated significantly more often during proestrus, estrus, metestrus, and pregnancy than during anestrus and the postpartum period, and hemolytic streptococci were isolated significantly more often during proestrus than dunng estrus, pregnancy, or the postpartum period. Staphylococcus intermedius was almost exclusively found after parturition. Culture results were negative for only 5.2% of specimens. On the basis of our findings, bacterial culturing of vaginal swab specimens from bitches without signs of genital disease is of little value.—Lena Bjurstrom and Catharina Linde-Forsberg in Am J Vet Res 53 (May 1992).