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.

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.

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.

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.

Emergence of livestock-associated Mammaliicoccus sciuri ST71 co-harbouring mecA and mecC genes in Brazil.

The discovery and tracking of antimicrobial resistance genes are essential for understanding the evolution of bacterial resistance and restraining its dispersion. Mammaliicoccus sciuri (formerly Staphylococcus sciuri) is the most probable evolutionary repository of the mecA gene, that later disseminated to S. aureus. In this study, we describe the first double mecA/mecC homologue-positive non-aureus staphylococci and mammaliicocci (NASM) from the American continent, also representing the first report of mecC-positive NASM in Brazil. Two clonally related methicillin-resistant M. sciuri strains co-carrying mecA and mecC genes were isolated from the teat skin swab and milk sample collected from an ewe's left udder half. Both M. sciuri strains belonged to the sequence type (ST) 71. Besides mecA and mecC genes, the M. sciuri strains carried broad resistomes for clinically important antimicrobial agents, including ß-lactams, tetracyclines, lincosamide, streptogramin, streptomycin, and aminoglycosides. Virulome analysis showed the presence of the clumping factor B (clfB), ATP-dependent protease ClpP (ClpP) and serine-aspartate repeat proteins (sdrC and sdrE) virulence-associated genes. Phylogenomic analysis revealed that these M. sciuri strains are part of a globally disseminated branch, associated with farm and companion animals and even with food. Our findings suggest that M. sciuri is likely to emerge as a pathogen of global interest, carrying a broad repertoire of antimicrobial resistance genes with a remarkable co-presence of mecA and mecC genes. Finally, we strongly encourage to monitor M. sciuri under the One Health umbrella since this bacterial species is spreading at the human-animal-environment interface.

Molecular detection of Burkholderia mallei in different geographic regions of Brazil.

Glanders is a contagious disease of equids caused by the Gram-negative bacterium Burkholderia mallei. In Brazil, the disease is considered to be reemerging and has been expanding, with records of equids with positive serology in most of the federative units. However, there are few reports describing the genotypic detection of the agent. This study demonstrated the detection of B. mallei by species-specific PCR directly from tissues or from bacterial cultures, followed by amplicon sequencing in equids (equines, mules, and asinines) with positive serology for glanders in all five geographic regions of Brazil. The molecular evidence of B. mallei infection in serologically positive equids in this study expands the possibility of strain isolation and the conduction of epidemiological characterizations based on molecular information. The microbiological detection of B. mallei in cultures from nasal and palate swabs, even in equids without clinical manifestations, raises the possibility of environmental elimination of the agent.

Serologic survey of brucellosis in captive neotropical wild carnivores in northeast Brazil.

Abstract. This study reports the detection of antibodies against Brucella abortus and B. canis in wild neotropical carnivores kept in captivity in three zoos in northeastern Brazil. A total of 42 serum samples were examined, 17 from coatis (Nasua nasua), eight from crab-eating raccoons (Procyon cancrivorus), three from crab-eating foxes (Cerdocyon thous), three from hoary foxes (Lycalopex vetulus), two from little spotted cats (Leopardus tigrinus), five from tayras (Eira barbara), two from greater grisons (Galictis vittata), and two from neotropical river otters (Lontra longicaudis). The Rose-Bengal test and complement fixation test (CFT) were performed to detect anti-Brucella spp. antibodies, whereas the agar gel immunodiffusion test (AGID) was employed to detect anti-B. canis antibodies. The overall seroprevalence varied by species and by test; in addition, CFT and AGID seemed better able to detect antibodies against B. abortus and B. canis, respectively. This is the first study on the presence of anti-Brucella spp. antibodies in captive carnivores from Brazil, as well as the first report of antibodies to Brucella spp. in coatis, crab-eating raccoons, hoary foxes, little spotted cats, tayras, and greater grisons.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

Protothecosis by Prototheca wickerhamii in goats.

This article reports a new case of protothecosis by Prototheca wickerhamii in goats. The animal presented severe respiratory difficulty and nodules, sometimes ulcerated, in the nasal vestibule, mucocutaneous junction of the nostrils and skin of the face. Prototheca wickerhamii was isolated from the lesions. The animal had no clinical or haematologiccl evidence of immunodepression. The agent was highly resistant to antimicrobial drugs. The goat was treated unsuccessfully with fluconazole and euthanised 10 months after the diagnosis of the disease. Histological lesions were necrotising pyogranulomatous dermatitis, rhinitis and osteomyelitis with myriads of walled sporangia characteristic of P. wickerhamii. It is suggested that in goats, protothecosis is characterised by a chronic, slowly progressive infection, which affects immunologically competent goats, causing multifocal, ulcerative, pyogranulomatous and necrotising lesions of the mucosa of the nasal vestibule, mucocutaneous junctions of the nostrils and skin of the face.

Staphylococcus aureus mastitis in a white-eared opossum (Didelphis albiventris) in the Atlantic Forest of northeast Brazil.

This is the first brief communication of clinical mastitis caused by Staphylococcus aureus occurring in a white-eared opossum (Didelphis albiventris) captured in a remnant of the Atlantic Forest in northeast Brazil. The bacterium was cultured from a milk sample by using sheep blood and Levine agars. Phenotyping of S aureus was performed with coagulase biochemical tests, DNase agar, mannitol agar, and anaerobic glucose and mannitol fermentation.

Detection of Toxoplasma gondii DNA in heart tissue from common marmoset (Callithrix jacchus) monitored for yellow fever and rabies in Pernambuco state, Northeastern of Brazil.

There is limited available information concerning the prevalence of Toxoplasma gondii in noncaptive monkeys. Also, New World monkeys (NWM) are highly susceptible to toxoplasmosis, which is a conservation concern. This study aimed to investigate apicomplexan parasites in common marmosets (Callithrix jacchus) collected for yellow fever and rabies surveillance program in Northeastern region of Brazil. Heart fragments of 39 free-ranging common marmosets were analyzed for the presence of the 18S rDNA gene of apicomplexan parasites by nested PCR. Positive samples were sequenced. T. gondii DNA was detected in 17.9% (7/39) of the analyzed animals. This study is the first report on T. gondii in Callithrix jacchus in Brazil. These findings should be an alert for wildlife conservation institutions, as high susceptibility and mortality were reported for captive NWM.

First record of Burkholderia mallei Turkey 10 strain originating from glanderous horses from Brazil.

Burkholderia (B.) mallei is the causative agent of glanders in Equidae. This study describes the first record of the Turkey 10 strain of B. mallei in glanderous horses in Northeastern of Brazil. This description should contribute to the future actions of diagnosis, control, and eradication of this disease in Brazil.

Looking for alternative treatments for bovine and caprine mastitis: Evaluation of the potential of Calliandra surinamensis leaf pinnulae lectin (CasuL), both alone and in combination with antibiotics.

This work aimed to evaluate the effects of CasuL on growth and viability of 15 mastitis isolates from cows and goats, to determine the synergistic potential between CasuL and antibiotics, and to investigate the effects on bacterial ultrastructure and antibiofilm activity. The lectin inhibited the growth of Staphylococcus isolates from either bovine (Ssp6PD and Sa) or caprine (Ssp5D and Ssp01) mastitis. The minimal inhibitory concentrations were ranged from 3.75 to 15 µg/ml. Synergistic effect was observed for CasuL-tetracycline against Sa and Ssp6PD and CasuL-ampicillin against Ssp01. No structural damage was observed under the scanning electron microscope in CasuL treatments. Flow cytometry analysis using thiazol orange and propidium iodide demonstrated that CasuL was unable to reduce the cell viability of the isolates tested. At sub-inhibitory concentrations, CasuL reduced biofilm formation by the isolates Sa and Ssp5D. However, CasuL-tetracycline and CasuL-ampicillin combinations inhibited biofilm formation by Ssp6PD and Ssp01, respectively. In conclusion, CasuL is a bacteriostatic and antibiofilm agent against some mastitis isolates and displayed a synergistic potential when used in combination with either ampicillin (against one isolate) or tetracycline (against two isolates). The results stimulate the evaluation of CasuL for the treatment of mastitis, particularly when used in conjunction with antibiotics.

Molecular and serological investigation of infectious diseases in captive and free-range crab-eating fox (Cerdocyon thous - Linnaeus, 1776) from northeastern Brazil.

The aim of this study was to detect DNA and antibodies anti-Leishmania spp., Neospora caninum and Toxoplasma gondii in captive and free-range crab-eating fox (Cerdocyon thous) from northeastern Brazil. Twenty-five crab-eating foxes from different states of northeastern Brazil were sampled by this study. Blood samples were collected by cephalic or jugular vein punctures. The whole blood was submitted to PCR, and the sera samples to the serological analysis as follows: MAT for T. gondii, NAT for N. caninum, and ELISA for L. chagasi. The frequence of antibodies anti-T. gondii was 50% and 29.41% for free-range and captive wild canids, respectively. The frequence of antibodies anti-N. caninum observed by this study was 62.50% and 23.52% for free-range and captive wild canids, respectively. The frequence of antibodies anti-L. chagasi was 4.0% for captive wild canids. Co-infections cases were identified as follows: one captive wild canid seropositive for T. gondii and L. chagasi and two free-range animals seropositive for T. gondii and N. caninum. All PCR assays performed were negative for the pathogens analyzed. This study describes the presence of antibodies anti-T. gondii, N. caninum e L. chagasi in wild canids from northeastern Brazil and highlights the necessity of further studies on infectious diseases in free-range and captive wild canids.