Chapter 317 Other Infectious Causes of Infertility & Subfertility in Dogs & Cats PDF

CHAPTER 317 Other Infectious Causes of Infertility and Subfertility in Dogs and Cats Sophie Alexandra Grundy Client Information Sheets: Be Prepared: Reproductive Tract Sample Collection at Home Pre-Breeding Vaginal Cultures In...

CHAPTER 317 Other Infectious Causes of Infertility and Subfertility in Dogs and Cats Sophie Alexandra Grundy Client Information Sheets: Be Prepared: Reproductive Tract Sample Collection at Home Pre-Breeding Vaginal Cultures Introduction Infectious diseases can usually be identified with culture, polymerase chain reaction (PCR), or histology (see ch. 207). Obstacles to obtaining a definitive diagnosis of reproductive organ infectious disease in clinical practice include lack of sample, cost, and challenging interpretations. Thus, the true impact of infectious disease on canine and feline reproductive performance is difficult to establish. PCR techniques increase availability of cost effective multi-pathogen diagnostic screening, but sample collection, sample site selection, and laboratory quality control each have potential to impact positive and negative predictive values. Results may be confusing since reproductive pathogens may also be considered “normal flora” in healthy pets. Diagnostic panels that combine culture, PCR, and histopathology provide an opportunity to understand the true relationship between infectious disease and reproductive performance in cats and dogs. Client education regarding available testing options and sample requirements is an essential component of the diagnostic process as typically it is clients that have greater access to tissue types such as the placenta, or an aborted fetus. Sample Collection, Type and Site There are 4 main categories of infectious reproductive pathogens: bacterial, viral, protozoal, and fungal. Any of these can cause similar “storms” of reproductive failure within a breeding population. All cases of reproductive loss benefit from the collection of samples appropriate for culture, histology, and PCR.1 PCR, of great diagnostic value for identifying infectious disease, enables rapid identification of pathogens and is highly sensitive. As a result, it is important to consider sample collection protocols. Many reproductive pathogens are environmentally ubiquitous and normally found in oral, genital, urinary, or gastrointestinal flora. Interpretation must be made in the context of environment, sample type, location, and method of collection. All samples for PCR and culture should be collected in as sterile a manner as possible to avoid contamination. Plastic stemmed cotton tipped applicators, gloves, sterile instruments and a clean surface are advised when collecting samples. Samples should be refrigerated until submitted.2 Placental tissue can be extremely useful for understanding reproductive concerns in gravid bitches and queens. The placenta may be considered the immune guardian of the developing fetus. When sampling the placenta, a touch slide preparation should be made for prompt Brucella canis and Campylobacter jejuni screening. Two tissue samples should be collected: one for formalin fixation and one for PCR. If available, samples of fetal kidney, liver, spleen, and lung should also be collected in duplicate: one for formalin fixation and one for PCR. A sample of fetal liver or a swab of the abdomen is ideal for bacterial culture. EDTA whole blood and serum should be collected from the dam, in addition to a vaginal swab for PCR. PCR samples should be kept refrigerated. When evaluating the male for infectious reproductive pathogens, semen should be cultured and, ideally, the colony count compared to a urethral culture (see ch. 318). Samples of ejaculate may also be submitted for infectious disease screening by PCR. Various combinations of culture, PCR, and histology are available 4553 through private and university laboratories. These panel, or combination test packages offer a cost effective option for multi-pathogen screening. Pre-Breeding Vaginal Cultures Normal Flora It is common to be asked to perform a bacterial culture of the vaginal vault as a pre-breeding screen. There is an incorrect tendency to presume that a positive vaginal culture is abnormal and indicative of an infected and abnormal uterus.3-11 Some breeders incorrectly believe the normal vaginal canal to be sterile and require a negative pre-breeding culture prior to permitting live cover. However, the normal vaginal canal has aerobic and anaerobic bacterial populations and 60-100% of healthy bitches have positive vaginal cultures.3-10 Considering anatomical exposure to oral and gastrointestinal bacteria, the common vaginal isolates from bitches include Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pasteurella multocida and bacteria associated with the skin (Streptococcus spp. and Staphylococci spp.) (Table 317-1).3-10 Although bacterial cultures are typically limited to aerobes, anaerobes are also included among normal vaginal flora (Bacteroides spp., Peptostreptococcus spp., Clostridium spp.).9 Whilst there are slightly different relative frequencies for each isolate, the “normal organisms” are consistent across studies.3-10 The types of bacteria isolated per healthy bitch are relatively stable during the reproductive cycle, but count and number of isolates tend to increase during proestrus and estrus.4,5 During proestrus and estrus, it has been shown that the uterine bacterial population reflects the vaginal bacterial population, presumably due to relaxation of the cervix and hormonal changes that decrease uterine immune responses.4 During diestrus and anestrus, uterine bacterial populations typically clear, making it rare to isolate bacteria during this time.4,8 TABLE 317-1 Frequency of Vaginal Bacterial Isolates in Healthy Bitches across Selected Studies, 1978-2012 BACTERIAL PERCENT POSTIVE BACTERIAL ISOLATES (HEALTHY BITCHES) PER STUDY ORGANISM LING, ODSBALDISTON, HIRSCH, OLSON, BABA, BJURSTROM, GROPETTI, 197810 197211 19773 19786 19839 19928 20125 Arcanobacterium pyogenes 2.9 Bacillus spp. 3.7 Bacteroidaceae spp. 10 55 Corynebacterium spp. 35 6 2.5 40.7 Enterococci 10 44.1 Enterococcus faecalis 4 23.5 Escherichia coli 25 50 22 18.5 23 84.7 2.9 Haemophilus 10 Klebsiella pneumoniae 2.9 Moraxella spp. 10 Mycoplasma spp. 30 43 59.3 Pasteurella spp. 5 9.9 34 67.8 8.8 Pasteurella multocida 98.3 Peptococcaceae 27 (Clostridium spp.) Proteus spp. 5* 6* 4.9 9 25.4* 2.9* Pseudomonas aeruginosa 5 10 2 Staphylococci spp. 70 10 13 6.2 20 2.9 4554  coagulase-positive Staphylococci spp. 5 6 6.2 4 22.0 coagulase-negative Streptococcus spp. 10 52 2.9 Beta-hemolytic 35 9 14.8 89.8 20.6 Streptococci 20 14 13.6 55.9 26.4 “viridans” *Proteus mirabilis. Bacteria in shaded rows indicate that they are more frequently isolated across all studies. Vaginal Cultures During Proestrus and Estrus Specific concerns are often raised with respect to positive cultures of E. coli or Streptococcus spp. as they are commonly associated with pyometra, mastitis, and neonatal septicemia. However, a large majority of vaginal cultures from healthy bitches are positive for E. coli.4,6,8,12 One study associated a positive proestrus Streptococcus spp. vaginal culture with a decreased risk of uterine infection during diestrus.5 With years of literature support, there is no evidence to support routine culture or antibiotic treatment of an asymptomatic bitch with a positive pre-breeding vaginal culture. Future studies quantifying microbial levels of the normal vaginal microbiome may be used to define pathogenic vaginal bacterial populations. Since vaginal cytology is frequently evaluated during proestrus of bitches (see ch. 119 and 312) and often reveals large numbers of bacteria or phagocytized bacteria, caregivers may request treatment of these bacteria. However, no correlations have been made between vaginal bacteria (phagocytized or not), vaginal neutrophils, and fertility in an asymptomatic bitch.5 Bacteria in the vaginal canal are normal and cytologically observed bacteria are not valid reasons for antimicrobial treatment during proestrus. Caregivers should be informed that most bitches undergoing pre-breeding vaginal cultures are in proestrus or estrus, times when results are expected to be positive. While there may be pressure from owners of the bitch or stud dog to treat a bitch with a positive vaginal culture with antibiotics prior to breeding, one should also note the increasing evidence that indiscriminate use of antimicrobials in breeding bitches is correlated with induction of multi- resistant bacteria which can alter vaginal flora and promote growth of pathogenic bacterial populations. This places kennels and neonates at greater risk and does not improve reproductive performance.13-15 When should a bitch with a positive pre-breeding culture be treated? As a general rule, treatment during proestrus should be considered for a bitch that is exhibiting a clearly abnormal vaginal discharge and with significant growth of either a single pure normal bacterial species, or atypical bacterial isolate (see provided Client Information Sheet online). Vaginal Discharge and Culture Bacterial cultures of vaginal discharge generally differ from normal flora only with respect to the number of bacteria isolated.3 In symptomatic dogs or cats, treatment is advised for a single bacterial isolate present in high numbers, typically a 3+ to 4+ on a four-point scale.3,16 Antimicrobial choice should be made with consideration to sensitivity and minimum inhibitory concentration (MIC). Mycoplasma and Ureaplasma Positive Mycoplasma vaginal cultures are common in bitches and not of clinical significance with respect to fertility. In stud dogs, however, positive Mycoplasma culture may be associated with infertility if the quantitated culture count of the semen is 2 log10 times greater than that of the urethra.22* Many studies evaluating canine vaginal cultures do not comment on Mycoplasma. Mycoplasma spp. do not grow easily under standard aerobic culture conditions.4-6,17 When specifically evaluated, positive Mycoplasma and Ureaplasma vaginal culture rates in the healthy bitch are reported to be as high as 88% and 50%, respectively; Ureasplasma, when present, is typically in association with Mycoplasma.18-20 In one study, positive Ureaplasma vaginal culture rates were higher in bitches with a vaginal discharge (75%), but there was no significant relationship between Mycoplasma, Ureaplasma, and fertility.19 Seventy-two percent to 84% of semen samples from healthy 4555 stud dogs culture positive for Mycoplasma.19 Whilst implicated in some cases of canine orchitis, Mycoplasma is not isolated more frequently in infertile dogs.17,19,21 Ureaplasma are generally not present in the prepuce or semen of fertile males, but are found in around 70% of infertile males, typically isolated in association with Mycoplasma.19 For both bitch and stud, Mycoplasma canis is the most frequent isolate. In males, Mycoplasma cynos is also noted.19 Females often have multiple species identified.18-20 Concurrent semen evaluation is advised. Campylobacter Jejuni Campylobacter jejuni has been reported in association with abortion in the bitch.23-25 In all cases, the classic feature is late term abortion or intrauterine fetal death about one week prior to the anticipated whelping date. Bitches may also have a hemorrhagic vaginal discharge.23-25 Entire litters may be aborted dead, but partial live litter delivery is also reported. As the fetuses are not yet at term, they may be born live, but not viable at less than 61 (females) or 62 (males) days gestation with day 0 defined by the luteinizing hormone surge.26 An important differential for this clinical presentation is Brucella canis (see ch. 213), the major cause of bacterial abortion in the dog. Personal protective equipment should be used when evaluating these cases due to the zoonotic potential of both organisms. Diagnosis is based on either positive bacterial culture, or positive PCR, of the placenta, and fetal liver, or lung.23-25 Successful treatment is rare. Leptospirosis Leptospiral organisms induce marked uterine inflammation and abortion is the most common reproductive sign of leptospiral infection in the bitch.27,28 There is return to fertility after successful treatment. In stud dogs, leptospirosis-induced vasculitis may cause breakdown of the blood testes barrier and any stud dog diagnosed with leptospirosis should have a semen evaluation performed initially at 65 days post recovery to evaluate reproductive potential (see ch. 217). Other Bacteria Salmonella and Listeria are both reported as a cause of reproductive tract disease in the bitch and queen. Salmonella is an uncommon cause of reproductive loss and most often associated with systemic disease.29 Listeria has been reported as a cause of abortion in the literature but is considered an uncommon pathogen.30 Bartonella spp. have been associated with reproductive losses under experimental conditions but this is challenging to prove in clinical cases due to the difficulties isolating the organism (see ch. 215 and 216).31 Viral Pathogens Viral pathogens are an important consideration when evaluating reproductive losses in the bitch and queen as they may cross the placenta, infecting embryo or fetus, in addition to debilitating the dam.32 Many viral infections can be confirmed by PCR on tissue samples in combination with histology. For an in-depth discussion of the major viral pathogens, the reader is referred to the individual discussions in the infectious disease section (see ch. 222-225 and 229). Although herpesvirus (see ch. 227 and 228) is reviewed, it is worth highlighting that the reproductive impact of canine herpesvirus (CHV) is extremely difficult to define as a result of its latency, poor immunogenicity, short-lived antibody production, and transient viral excretion.33 Reproductive hormone fluctuations appear to alter viral reactivation, but it is not clear how this impacts the course of clinical disease.33,34 Indeed, given that there is no difference in antibody titers between healthy bitches and those with reproductive abnormalities, in addition to zero recovery rates from vaginal PCR in kennels with endemic disease, reproductive loss due to CHV is extremely difficult to document unless associated with neonatal loss.33-35 Vaccination when available is advised 10 days after mating and again 6 weeks later as it is thought that higher antibody titers in diestrus may protect against reproductive failure.33,36 Successful medical management of neonatal CHV using acyclovir is described (see ch. 162).37 Protozoal Pathogens 4556 Toxoplasma may be associated with reproductive loss in the queen mainly due to systemic effects of disease in the dam.12 Transplacental transmission of Neospora is known to occur in the bitch but the impact of this organism on reproductive performance is unclear.12 A single case report of Leishmania-associated abortion is reported in the literature.38 The reader is referred to ch. 221 for an in-depth discussion of protozoal disease. 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