Use of conventional and real-time polymerase chain reaction to determine the epidemiology of hemoplasma infections in anemic and nonanemic cats.

BACKGROUND: The goals of this study were to develop and apply conventional (c) and real-time TaqMan polymerase chain reaction (PCR) assays for Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haematoparvum' (Mhp), and 'Candidatus Mycoplasma haemominutum' (Mhm) to blood samples of cats to determine the epidemiology of these infections in cats. HYPOTHESIS: Cats are infected with >2 hemoplasma species, and organism load correlates with disease induced by these organisms. ANIMALS: Blood samples from 263 anemic and nonanemic cats were used. METHODS: A retrospective study was conducted. RESULTS: Forty-seven (18%) samples were positive. Three samples (1%) yielded 170 base pair cPCR products, 1 of which was positive for Mhf using real-time PCR. Forty-four samples (17%) yielded 193 base pair cPCR products, 40 of which were positive for Mhm using real-time PCR. Organism loads ranged from 375 X 10(6)/mL to 6.9 x 10(6)/mL of blood. Sequencing of cPCR products from samples testing negative using real-time PCR identified 2 Mhp-like sequences, 1 Mhm-like sequence, and 1 sequence resembling 'Candidatus Mycoplasma turicensis'. Cats infected with Mhm were less likely to be anemic than uninfected cats. Older age, outdoor exposure, feline immunodeficiency virus (FIV) seropositivity, cutaneous squamous cell carcinoma (SCC), and stomatitis were associated with Mhm infection. Cats from the Sacramento Valley were more often infected with Mhm than cats from the San Francisco bay area. CONCLUSIONS AND CLINICAL IMPORTANCE: Cats may be infected with 4 hemoplasma species. The association between Mhm infection, FIV, and SCC may reflect outdoor roaming status of infected cats. The clustered distribution of infection suggests an arthropod vector in transmission.

Detection of mixed infections with "Candidatus Mycoplasma haemominutum" and Mycoplasma haemofelis using real-time TaqMan polymerase chain reaction.

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

The geographic distribution of the putative agent of epizootic bovine abortion in the tick vector, Ornithodoros coriaceus.

Epizootic bovine abortion (EBA), also known as "foothill abortion", is a vector borne disease of beef cattle that graze in the mountainous regions of California, southern Oregon and western Nevada transmitted by the argasid tick Ornithodoros coriaceus. Recently, the putative agent of EBA was identified as a novel Deltaproteobacter in the order Myxococcales. In this study, a TaqMan real-time PCR (TM-PCR) protocol specific to the putative EBA agent was developed. The new real-time TM-PCR assay functioned sensitively and specifically to detect pathogen DNA in field-collected O. coriaceus ticks. The assay had an analytical sensitivity of a single plasmid copy and, when evaluated with a collection of tick-borne pathogens, yielded a positive PCR-result only for the agent of EBA. Use of the TM-PCR represents an effective tool for rapid and highly sensitive assessment of environmental risk and spatial and statistical analysis to highlight areas where there may be increased risk for EBA in susceptible cattle.

A retrospective study of cutaneous equine sarcoidosis and its potential infectious aetiological agents.

Nine horses from ages 5 to 21 years were diagnosed with cutaneous equine sarcoidosis (ES) over an 18-year period. In addition to skin, the lungs were frequently involved, with other organ systems affected less commonly. A predisposition for thoroughbreds and geldings was noted. Cutaneous lesions and signs included crusts, scales, alopecia and pruritus. These were found at various sites, particularly the legs/thighs/elbows, thorax, neck, face and ventral abdomen. Three horses were euthanized shortly after hospitalization; others survived as long as 12 years. Histopathologic stains, immunohistochemistry and polymerase chain reaction assays on paraffin-embedded cutaneous specimens from eight horses for Mycobacterium spp., Coccidioides immitis, Cryptococcus neoformans, Corynebacterium pseudotuberculosis, and Borrelia burgdorferi were all negative. The aetiology of ES is unlikely microbial and continues to be a diagnosis of exclusion. ES, when limited to the skin, is associated with a good prognosis, with either partial or complete response to glucocorticoid therapy in all the surviving horses.

Use of imidacloprid-permethrin to prevent transmission of Anaplasma phagocytophilum from naturally infected Ixodes scapularis ticks to dogs.

One group of eight beagles was treated with a combination of imidacloprid and permethrin 7 days before exposure to Ixodes scapularis ticks that were naturally infected with Anaplasma phagocytophilum. A second group of eight beagles was not treated and was also exposed to infected ticks. Seven of eight non-treated dogs--but none of the treated dogs--developed specific antibodies to A. phagocytophilum. Results of this study indicate that a combination of imidacloprid and permethrin can prevent transmission of A. phagocytophilum to dogs if administered before exposure to infected ticks.

A comparison of routine culture with polymerase chain reaction technology for the detection of Mycoplasma species in feline nasal samples.

Nasal flush samples were collected from 20 cats and submitted for Mycoplasma culture and polymerase chain reaction (PCR). Nasal biopsy samples were also obtained from each cat and simultaneously evaluated for Mycoplasma by standard culture and PCR. Concordance of the test results was determined through calculation of the kappa statistic. In 6 cats, nasal flush samples were culture positive for Mycoplasma. PCR was positive in each culture-positive cat and also positive in 1 flush sample that was culture negative. DNA sequencing of the PCR product from the culture negative flush sample identified the organism as Mycoplasma arginini. All other flush samples that were culture negative were also PCR negative (kappa = 0.89). Nasal biopsy samples from 7 cats were culture positive for Mycoplasma, and all were PCR positive. Biopsy samples that were culture negative for Mycoplasma were also PCR negative (kappa = 1.0). Results of culture and PCR for both nasal flush and biopsy were concordant in 19 of 20 cats, and PCR was able to identify an unusual Mycoplasma species that did not grow in culture. In most cats, organisms could be detected in either nasal flush or biopsy samples. In this study, PCR provided rapid and sensitive detection of Mycoplasma species in nasal samples from cats and detected 1 organism that did not grow in culture.

Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays.

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.