MALDI-TOF bacterial subtyping for rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis.

AIMS: This study aimed to evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial subtyping for the rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis. METHODS AND RESULTS: A total of 229 S. aureus isolates were obtained from milk samples collected from cows with subclinical mastitis using microbiological culture. Staphylococcus aureus isolates were also submitted to PCR analysis targeting the mecA and mecC genes, which are indicative of methicillin resistance. Confirmation of the species was achieved through MALDI-TOF MS analysis. To analyze antimicrobial resistance patterns, the MALDI BioTyper Compass Explorer and ClinProTools Bruker software were employed, and dendrograms were generated using Bionumerics software. CONCLUSIONS: MALDI-TOF MS successfully identified S. aureus at the species level, but no methicillin resistance was observed. Moreover, spectral typing displayed limited similarity when compared to pulsed-field gel electrophoresis (PFGE).

First investigation of Staphylococcus argenteus in a Brazilian collections of S. aureus isolated from bovine mastitis.

BACKGROUND: Staphylococcus argenteus is a new specie positive coagulase staphylococci. We investigate the presence of S. argenteus in isolates previously classified as S. aureus, obtained from the milk of cows with mastitis in Brazil. RESULTS: Among 856 S. aureus tested in chocolate agar, tryptone soya agar and salt egg yolk agar, white or colorless colonies were observed in 185 (21.6%) isolates. Regarding the ctrOPQMN operon, 111 (60%) presented the complete cluster. Despite some missing genes in this cluster, the remaining strains (74) were confirmed as S. aureus using the nrps gene. CONCLUSIONS: As far as we know, this is the first review of S. aureus collection in Brazil and S. argenteus does not appear to be a significant problem in Brazilian herds.

Atypical Toxoplasma gondii genotype from a sheep and a pig on Fernando de Noronha Island, Brazil, showed different mouse virulence profiles.

Toxoplasma gondii is a zoonotic parasite which can infect almost all warm-blooded animals. Toxoplasma gondii isolates from Brazil have greater genetic diversity with a predominance of virulent and atypical genotypes, compared with the Northern Hemisphere. Considering that previous studies have demonstrated a high seroprevalence of T. gondii antibodies in animals from Fernando de Noronha Island, the aim of this study was to isolate, genetically characterize, and determine mouse virulence of isolates of T. gondii from livestock from this Brazilian island. Two T. gondii isolates were obtained by mouse bioassay from brain from one sheep and one pig. Genotyping was performed by PCR-RFLP using 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22- 8, c29-2, PK1, L358, and Apico) and an atypical genotype of T. gondii (ToxoDB #146) was identified for both isolates. Genotyping of four ROP loci indicated different alleles for ROP16 and mouse virulence analysis revealed different profiles (intermediate and low virulence). This is the first report of this genotype being described in a pig and a sheep.

Description of an atypical Toxoplasma gondii isolate from a case of congenital toxoplasmosis in northeastern Brazil.

We report a case of severe congenital toxoplasmosis that involved an atypical T. gondii genotype in a newborn baby from Alagoas state in Northeastern Brazil. A pregnant woman presented IgM and IgG anti-T. gondii antibodies, as detected by the chemiluminescence immunoassay on the second trimester of pregnancy. A mouse bioassay was performed using umbilical cord blood and one isolate was obtained. The isolate was designated TgCTBrAL1 and genetic characterization revealed genotype ToxoDB #162. Genotype results of the rhoptry genes, ROP5 and ROP18, could predict the high virulence of the isolate in mice, which was confirmed by an in vivo virulence assay. This is the first report of generating a T. gondii isolate from a newborn baby with congenital toxoplasmosis in Northeastern Brazil.

Antimicrobial potential of Copaiba Oil (Copaifera multijuga Hayne-Leguminosae) against bubaline mastitis multiresistant isolates.

Copaiba oil is a natural product used by Amazonian populations and recognized for its medicinal properties because it has significant antimicrobial activity for several pathogenic microorganisms. The present work aimed to evaluate and characterize the effect of natural oil produced by copaiba - Copaifera multijuga against multiresistant isolates of bubaline mastitis. The nitrocefin test was performed with isolates of Staphylococcus aureus from bubaline mastitis, which were 100% positive for beta-lactamase enzyme detection. Minimum Bactericidal Concentration (MBC) of 25% to 3.12% was obtained for Enterococcus faecalis and Escherichia coli and 50% and 25% for S. aureus, but Klebsiella pneumoniae and Bacillus subtilis were resistant. MBC with 12.5% and 6.25% oil were obtained for most multiresistant bubaline mastitis isolates from the states of Pernambuco, Ceará, Bahia and Alagoas. The results demonstrated the great potential of using copaiba natural oil in the treatment of buffalo mastitis.

Occurrence of ß-lactam-resistant Staphylococcus aureus in milk from primiparous dairy cows in the northeastern region of Brazil.

The objective of the current study was to isolate and identify Staphylococcus (S.) aureus strains resistant to beta-lactam antibiotics from primiparous cows' milk. A total of 432 milk samples were collected from all primiparous dairy cows in early lactation that originated from 9 dairy properties. All samples were cultured in Mannitol salt agar enriched with egg yolk emulsion. Determination of genotypic resistance of S. aureus was achieved by polymerase chain reaction (PCR) for amplification of the blaZ, mecA, and mecC genes. Phenotypic resistance of S. aureus strains was evaluated by minimal inhibitory concentration (MIC) technique using broth microdilutions of penicillin G and oxacillin. From all the mammary quarters examined, S. aureus strains were detected in 27 out of 432 (6.25%) milk samples (CI95%, 4.33-8.84). From all dairy properties visited, only two out of 9 were found to have S. aureus. Hence, it was possible to evaluate genotypic and phenotypic resistance in 27 samples from two dairy farms. The isolates of S. aureus had a frequency of (20/27) 74.07% to blaZ gene (CI95%, 57.5-90.6), whereas mecA and mecC genes were not observed. According to MIC results, penicillin G had a 74.07% (20/27) resistance rate (CI95%, 57.5-90.6) and oxacillin had a 14.81% (4/27) resistance rate (CI95%, 1.4-28.2). Thus, the circulation of S. aureus strains resistant to beta-lactams has been confirmed in primiparous dairy cows in the northeastern region of Brazil, indicating the need for new management strategies involving the use of beta-lactam drugs to treat mastitis, discouraging and/or limiting their use. Also, it is important to highlight the need for further studies on epidemiology and traceability of the pathogen.

Low frequency of antibodies anti-Neospora caninum in rodents in Fernando de Noronha Island, Brazil.

This study aimed to evaluate the frequency of Neospora caninum antibodies in rodents from the Island of Fernando de Noronha, Brazil, and to contribute to epidemiological data on neosporosis in this region. Blood samples were collected from 154 black-rats (Rattus rattus) and 12 rock-cavies (Kerodon rupestris). The search for anti-N. caninum antibodies was made using the NAT technique, with a cut-off of 1:20. The frequency of positive mice was 5.19% (8/154 - 1:20 titer) and all rock-cavies were negative. The low frequency of antibodies against N. caninum in rodents found in the Island shows the low contamination of the environment where these animals live.

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

IDENTIFICATION OF BACTERIA IN BLOOD CULTURES FROM CLINICALLY ILL CAPTIVE ANTILLEAN MANATEES (TRICHECHUS MANATUS MANATUS).

Between September 2001 and March 2013, 62 bacterial cultures (37 aerobic and 25 anaerobic) were performed on 37 blood samples from 23 Antillean manatees ( Trichechus manatus manatus) that were kept in captivity at the Brazilian National Center for Research and Conservation of Aquatic Mammals (CMA) in Pernambuco (CMA-PE) and Alagoas (CMA-AL), Brazil. All of the animals sampled exhibited clinical signs at the time of sampling including abscesses (n = 8), debilitation and anorexia (n = 22), and profound lethargy-moribundity (n = 7). The 4 animals with profound lethargy-moribundity died shortly after sampling of unknown causes. Bacteria were isolated from 15/37 (40.5%) and aerobic blood cultures from 13/23 animals (56.5%). None of the anaerobic cultures were positive. Aeromonas caviae , Aeromonas hydrophila , Aeromonas sp., Escherichia coli , Leclercia adecarboxylata , Pantoea agglomerans , Pseudomonas aeruginosa , Pseudomonas stutzeri , Pseudomonas sp., Sphingomonas paucimobilis , coagulase-negative Staphylococcus, and Staphylococcus epidermidis were each found in only one animal; Staphylococcus spp. was found in two; and Vibrio fluvialis in four. Thirteen samples had only one bacteria isolated, one sample had two bacteria, and one sample had three bacteria isolated. Regarding sex, age group, and origin among the manatees examined, 54.5% (6/11) of the females, 58.3% (7/12) of the males, 40% (2/5) of the calves, 66.7% (8/12) of the juveniles, 50% (3/6) of the adults, 55.5% (10/18) at CMA-PE, and 60% (3/5) at CMA-AL were found to be positive for bacterial growth during at least one sampling time. All Antillean manatees were clinically ill. Regarding clinical signs, bacteria were found in 50% (11/22) of blood samples of the animals showing debilitation and anorexia, 1 of 8 (12.5%) of blood samples of the animals showing abscesses, and 3 of 7 (42.9%) of blood samples of the animals showing profound lethargy-moribundity.

Osteosarcoma in a free-living yellow armadillo (Euphractus sexcinctus).

An adult male free-living yellow armadillo (Euphractus sexcinctus) was found by hunters and referred for clinical evaluation because of a tumour-like lesion on the carapace. The animal was lethargic and weak with severe dehydration, enophthalmos and cachexia, and was euthanized because of its very poor clinical condition. Necropsy revealed a whitish, exophytic, irregular and moist mass (7 × 6.5 × 1.5 cm) in the caudal third of the carapace. On cut section, the mass was hard, compact, irregular and whitish. Histopathology revealed a densely cellular, expansive, poorly delimited neoplasm composed of malignant mesenchymal cells arranged in islands and cords, interspersed by numerous areas of irregularly mineralized osteoid matrix. Neoplastic cells were intensely immunolabelled for vimentin. The diagnosis of a high-grade osteosarcoma of osteoblastic subtype was based on the clinical, gross, histopathological and immunohistochemical findings. This first report of an osteosarcoma in an armadillo expands the list of armadillo diseases and will assist the management of these animals by veterinarians.