Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates.

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains.

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis.

AIM: To develop a TaqMan probe-based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. METHODS AND RESULTS: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay's specificity, detection limit, intra- and inter-assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100-fold more sensitive than the cPCR. No cross-reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10(11) and 2·75 × 10(8) target copies ml(-1) of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. CONCLUSION: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.

Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection.

Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher's exact test. The 635bp product amplified from 10 samples (10/67=14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI=0.00-0.451, p=0.0028), however other analyzed variables were not considered risk factors (p>0.05). Hematological abnormalities were not associated with infection (p>0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids.

Survey of owned feline and canine populations in apartments from a neighbourhood in Curitiba, Brazil.

Animal population estimates are essential for public health services to ensure the success of zoonoses control programmes. Canine and feline populations vary among different regions mainly because of local human income status and type of human residence. Accordingly, the present study estimated the pet population size living in apartments of a vertical neighbourhood in Curitiba, Brazil. We chose a neighbourhood with a predominance of apartment buildings. All apartment buildings were visited, and questionnaires were completed by doormen or residents. Data were obtained from 120 of 173 apartment buildings. Survey questions included the number of apartments, residents, dogs and cats. Two thousand nine hundred and sixty six apartments with a total of 7429 residents were surveyed. The number of dogs and cats was 569 and 86 respectively. Thus, the human:dog and human:cat ratios were 13.05:1 and 86.38:1. These ratios were higher than those observed in other neighbourhoods in Curitiba. The present study indicates that the number of pets from apartments may be different from houses, and different among distinct areas within the same city.

Exploratory study of Mycoplasma suis (Eperythrozoon suis) on four commercial pig farms in southern Brazil.

Mycoplasma suis (Eperythrozoon suis) was detected by PCR and Southern blot in 186 pigs (121 sows, 61 piglets and four boars) on four farms in southern Brazil. DNA was extracted from blood samples and a 16S rRNA gene fragment of M suis was amplified by PCR; Southern blot analysis was then performed on all the samples. Twenty-two of the sows (18.2 per cent) were positive by PCR, and 40 (33.1 per cent) were positive by Southern blot; only one piglet and one boar were positive. The packed cell volume and total plasma protein of the pigs and their PCR and Southern blot results were not significantly different on the four farms, but higher proportions of the pigs were positive by Southern blot than by PCR (P<0.05). The packed cell volume and total plasma protein concentrations of the M suis positive and negative sows were not significantly different.