In veterinary medicine, accurate and timely diagnosis of infectious diseases is crucial for effective treatment and management, especially in companion animals like dogs and cats. Molecular diagnostic assays, particularly Polymerase Chain Reaction (PCR), have revolutionized the diagnostic landscape, offering enhanced sensitivity and specificity for identifying various pathogens. This article delves into the application of molecular assays for diagnosing common infectious agents affecting dogs and cats, providing insights into their clinical utility and interpretation.
Blood-borne Pathogens: A Molecular Perspective
Blood-borne agents pose significant health threats to dogs and cats. Traditionally known as Hemobartonella felis, these feline hemoplasmas are now reclassified into Mycoplasma hemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus M. turicensis.” Among these, M. hemofelis is considered more pathogenic in cats, although all three species can induce fever and anemia. Canine hemoplasmas, formerly H. canis, are now M. hemocanis and “Candidatus M. hematoparvum,” generally causing disease in immunosuppressed dogs.
Diagnosis of these hemoplasma infections relies on detecting the organism on erythrocytes via blood film examination or through PCR assays. Blood film analysis can yield false negatives in over 50% of cases due to fluctuating organism numbers and difficulty in cytological detection, especially in chronic infections. PCR assays emerge as the preferred diagnostic tool due to their superior sensitivity, particularly when cytological examination is inconclusive. While both conventional and quantitative PCR assays offer comparable sensitivity and specificity, quantitative PCR can be valuable in research settings for monitoring DNA copy numbers during antimicrobial drug studies, although DNA copy numbers don’t directly correlate with disease presence in hemoplasma infections. It’s recommended to collect samples for PCR prior to antibiotic administration. Treatment with doxycycline or fluoroquinolones may be indicated upon PCR detection in symptomatic animals exhibiting fever or anemia. Furthermore, hemoplasma PCR screening is recommended for blood donor dogs and cats. Post-treatment PCR assays have limited clinical utility as pathogen elimination is often not achieved.
Ehrlichiosis, a significant concern in dogs within the United States, is caused by Ehrlichia canis, E. ewingii, E. chaffeensis, Anaplasma phagocytophilum, A. platys, and Neorickettsia risticii. These agents can induce fever and cytopenias. E. canis is the primary agent due to the distribution of its vector, Rhipicephalus sanguineous. Cats can also be infected by E. canis-like organisms and A. phagocytophilum. Serological cross-reactivity among these genera is variable, making single serological tests unreliable for comprehensive diagnosis. Moreover, some cats with E. canis-like infections may not seroconvert. Clinical illness can precede seroconversion in both dogs and cats. Cytology is often negative, and culture is impractical. PCR assays for Ehrlichia spp, Anaplasma spp, and N. risticii are therefore recommended for dogs and cats presenting with acute fever or cytopenias, prior to treatment. PCR assays can be organism-specific or designed as panels. Alternatively, broad-range primers followed by sequencing can identify infective species. While the predictive value of these PCR assays is still under investigation, treating clinically ill, PCR-positive animals is considered prudent. PCR can monitor treatment efficacy in canine ehrlichiosis, but reinfection is possible, necessitating ongoing tick control. Repeated PCR testing in cats has less established clinical utility. Blood donors should be screened for Ehrlichia/Anaplasma/Neorickettsia via PCR and excluded if positive, even post-treatment, due to blood sterilization challenges.
Rocky Mountain spotted fever, caused by Rickettsia rickettsii, and other spotted fever group Rickettsia organisms are relevant in the US. Serology against R. rickettsii is not specific for Rocky Mountain spotted fever due to cross-reactivity. Cats are susceptible to R. felis and may exhibit antibodies against R. rickettsii. Studies using PCR assays targeting citrate synthase and outer membrane protein B genes have detected R. felis DNA in flea extracts but not commonly in cat blood, suggesting exposure without necessarily active bacteremia. Antibody prevalence studies indicate exposure to R. felis and R. rickettsii in cats with fever, but blood PCR is often negative. Rickettsia spp DNA has been found in healthy dogs, but the species involved are unclear. The clinical predictive value of Rickettsia PCR assays in dogs and cats remains to be fully elucidated.
Bartonella spp infections, commonly B. vinsonii and B. henselae in dogs, and B. henselae and B. clarridgeiae in cats, can be diagnosed via blood culture, PCR, and serology. Cross-reactivity issues exist among Bartonella species, similar to Ehrlichia/Anaplasma/Neorickettsia. PCR assays targeting multiple Bartonella species, especially pre-treatment, can surpass serology in diagnostic utility. Culture combined with PCR might be necessary for detecting certain canine infections. Serology can indicate exposure, but bacteremia can occur in both seropositive and seronegative animals, limiting its diagnostic application. Routine Bartonella testing in healthy animals is not advised, reserved for suspected clinical bartonellosis cases. However, high prevalence in healthy animals means positive culture or PCR results do not definitively confirm clinical bartonellosis. For instance, while Bartonella DNA was more frequent in febrile cats compared to healthy controls, healthy cats were still commonly positive. Treatment generally doesn’t eradicate infection, thus repeat PCR assays post-treatment offer minimal clinical benefit.
Cytauxzoon felis is readily identifiable in clinically affected cats through cytological examination of blood smears or splenic aspirates. PCR can detect organism DNA in cytologically negative cats, though nonpathogenic strains in healthy cats reduce the positive predictive value (PPV) of PCR. Given the high pathogenicity of some strains, treatment of clinically ill, PCR-positive cats is recommended. The clinical benefit of post-treatment PCR remains undefined.
Babesia canis and B. gibsoni infect dogs in the United States, with no feline-specific species in this region. Serology is available but doesn’t confirm Babesia-induced anemia. Commercially available PCR assays for both species are valuable for high-risk breeds like greyhounds and pit bull terriers or dogs exposed to pit bulls with hemolytic anemia. While the predictive values of Babesia PCR assays are not fully established, treatment for clinically ill, PCR-positive dogs is warranted. The clinical utility of repeat PCR assays post-treatment is unknown.
Feline immunodeficiency virus (FIV) diagnosis commonly employs serum ELISA to detect antibodies. Most assays show comparable results. Persistent infection is indicated by antibody presence in unvaccinated cats. Clinical signs can precede seroconversion, and some infected cats may remain seronegative, resulting in false negatives. Virus isolation or RT-PCR on blood can detect infection in antibody-negative cats, but false negatives are common due to low viral loads in blood, limiting accuracy, especially in differentiating vaccinated from naturally infected cats.
Feline leukemia virus (FeLV) infection typically results in antigenemia, reducing the necessity for molecular assays in routine practice. However, sensitive RT-PCR assays are utilized to characterize infection stages, though not widely available commercially.
Feline infectious peritonitis (FIP) and feline enteric coronavirus (FECV) RNA can be detected in blood and feces. Positive PCR results alone do not confirm FIP development. RT-PCR for M gene mRNA yielded variable results; one study showed FECV mRNA in blood of 13 of 26 normal cats, indicating low PPV for FIP diagnosis.
Gastrointestinal Pathogens: Molecular Insights
Key bacterial agents associated with gastrointestinal disease in dogs and cats include Salmonella spp, Campylobacter spp, Clostridium spp, and Helicobacter spp. Culture with antimicrobial susceptibility testing is preferred over PCR for Salmonella and Campylobacter. In canine Clostridium infections (C. perfringens and C. difficile), combining PCR and enterotoxin assays on feces is optimal, although positive results can occur in healthy dogs, reducing PPV. Helicobacter spp suspicion arises from cytological or histopathological detection of gastric spiral organisms and urease-positive biopsies. PCR can detect Helicobacter DNA in tissues, but its presence in healthy animals lowers the PPV. Gastrointestinal bacterial infections are often not permanently eliminated by treatment, limiting the benefit of repeat PCR assays.
Common enteric protozoans include Cryptosporidium spp, Giardia spp, Isospora spp, and Tritrichomonas fetus. Cryptosporidium felis and C. canis oocysts are small and often missed by fecal flotation in cats. Acid-fast staining is insensitive, and human-titered antigen assays are inaccurate for canine/feline feces. Immunofluorescent antibody (IFA) staining, commercially available for human C. parvum and G. lamblia, appears effective for detecting Cryptosporidium spp and Giardia spp in dog and cat feces. Combined with fecal flotation, IFA is useful for initial screening in small bowel diarrhea cases. PCR assays are more sensitive than IFA for C. felis in cats, but canine sensitivity data is limited. PCR’s high sensitivity may detect subclinical carriers, lowering PPV. Cryptosporidium felis and C. canis are less zoonotic, making positive PCR results significant mainly in diarrheic animals. Cryptosporidium PCR is indicated in IFA-negative cases of unexplained small bowel diarrhea or for genotyping. Repeat PCR post-treatment has minimal clinical benefit.
Giardia spp diagnosis is readily achieved in small bowel diarrhea cases using fecal flotation and wet mount examination. Fecal antigen assays and Giardia IFA are also accurate. Fecal PCR assays are less reliable due to PCR inhibitors and should be reserved for genotyping. Giardia infection is often not permanently eliminated, and reinfection is common. Testing healthy animals or repeat PCR post-diarrhea resolution is not clinically beneficial.
Tritrichomonas foetus (formerly Toxoplasma fetus) can cause large bowel diarrhea in young animals, though rare. Cytology often reveals infection, and culture is feasible. PCR is more sensitive and faster than culture. Subclinical carriers are common, reducing PPV. Treatment and reinfection dynamics limit the clinical benefit of testing healthy animals or repeat PCR after diarrhea resolution.
Viral agents in feline gastrointestinal disease include feline coronaviruses, feline panleukopenia virus, FeLV, and FIV. Canine gastrointestinal viruses include canine coronavirus, canine distemper virus, and canine parvoviruses. FeLV and FIV assays are blood-based, not fecal. Fecal coronaviruses can be detected by electron microscopy, virus isolation, or molecular assays. RT-PCR can detect FECV/FIPV RNA in cat feces and canine coronavirus in dog feces, but positive results merely indicate coronavirus presence, not necessarily FIP or diarrhea causation. Enteric coronaviruses are rarely problematic in well-managed young animals and less often cause adult diarrhea. Routine coronavirus RT-PCR on feces is generally not warranted. Feline panleukopenia virus and canine parvoviruses are detectable in feces via electron microscopy, virus isolation, antigen assays, and molecular assays. Parvovirus fecal antigen assays are widely available, user-friendly, and detect current strains, making routine parvovirus PCR assays unnecessary. Other viruses like astroviruses and reoviruses are less frequently associated with disease, and the need for specific assays is unclear.
Respiratory Pathogens: Molecular Diagnostics in the Airway
For bacterial respiratory pathogens in dogs and cats, PCR assays are available for Bordetella bronchiseptica, Mycoplasma spp, Streptococcus zooepidemicus, and Chlamydophila felis. While B. bronchiseptica is a primary canine pathogen, it’s common in healthy cats, reducing the PPV of culture and PCR in felines. Culture is preferred for B. bronchiseptica due to antimicrobial susceptibility testing capability. Post-treatment culture or PCR offers limited benefit as eradication is uncommon. Chlamydophila felis is a differential diagnosis for feline conjunctivitis and rhinitis, less so for lower airway disease. Culture is challenging, making PCR useful. However, not all PCR-positive cats are clinically ill, lowering PPV. PCR can confirm cattery clearance post-treatment. Mycoplasma spp are commensals of mucous membranes in dogs and cats. Mycoplasma felis is linked to feline conjunctivitis and possibly rhinitis. Pathogenicity varies among Mycoplasma species, and nonpathogenic species can contribute to disease in the presence of other conditions. Culture is difficult for Mycoplasma, and susceptibility testing is limited. Mycoplasma PCR assays have moderate clinical utility. Genus-specific primers are recommended due to varying pathogenicity. However, as commensals, PPV is likely low. Post-treatment PCR offers minimal benefit.
Feline respiratory viruses commonly involve feline calicivirus (FCV) and feline herpesvirus 1 (FHV-1), prevalent in crowded environments. Canine respiratory viruses include adenovirus 2, parainfluenza, influenza, respiratory coronavirus, canine distemper virus, and canine herpesvirus. FCV exhibits high strain diversity and is associated with rhinitis, stomatitis, conjunctivitis, and less frequently polyarthritis, kitten lower airway disease, and systemic disease. Virus isolation is slow and not universally available. Serology has poor PPV due to widespread exposure and vaccination. RT-PCR offers rapid FCV RNA detection but also amplifies vaccine strains. FCV RNA detection in both healthy carriers and ill cats reduces PPV. FCV RNA presence did not correlate with stomatitis in one study. RT-PCR can also yield false negatives, impacting NPV. Treatment doesn’t eliminate FCV, negating post-treatment testing.
FHV-1 is linked to feline rhinitis, stomatitis, conjunctivitis, keratitis, and facial dermatitis. Serology has poor PPV due to exposure and vaccination. FHV-1 detection methods include direct fluorescent staining, virus isolation, and PCR. FHV-1 DNA detection in conjunctival cells of healthy cats lowers PCR PPV. Current PCR assays also detect vaccine strains, further reducing PPV. FHV-1 DNA presence did not correlate with stomatitis in one study. Quantitative PCR may correlate with disease presence but failed to correlate with conjunctivitis in another study. PCR NPV is questionable, potentially due to immune clearance of FHV-1 DNA. Tissue biopsies are more sensitive than conjunctival swabs. FHV-1 DNA detection in aqueous humor is of unclear significance for uveitis. Treatment doesn’t eliminate FHV-1, making post-treatment testing unhelpful.
Canine respiratory viruses typically cause acute, self-resolving disease. Diagnostic test results often return after resolution, and molecular assay results can be negative by symptom onset. Lack of specific viral treatments reduces the clinical utility of definitive diagnosis in individual dogs. However, in outbreaks, differentiating bacterial from viral etiology aids treatment (bacteria) or prevention (viral) strategies.
Toxoplasma gondii (dogs and cats) and Neospora caninum (dogs) can cause interstitial pneumonia progressing to alveolar disease. Serology has low PPV due to high seroprevalence. Cytology of airway washings may reveal tachyzoites, but inflammation can obscure them. PCR can amplify microbial DNA and differentiate T. gondii from N. caninum.
Diagnosis of fungal respiratory infections like Aspergillus spp, Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum relies on serology and organism demonstration assays. Molecular assays have been evaluated in research, but predictive value data is limited, making other methods the primary diagnostic tools currently.
Conclusion
Molecular diagnostic assays, particularly PCR, are powerful tools in veterinary medicine for the diagnosis of infectious diseases in dogs and cats. While offering superior sensitivity and speed, their interpretation requires careful consideration of pre-test probability, clinical context, and potential limitations such as PPV and NPV. Integrating molecular diagnostics judiciously with clinical findings and other diagnostic modalities optimizes patient care and disease management in veterinary practice.
