Maria Aparecida Vasconcelos Paiva Brito

Retail Survey of Brazilian Milk and Minas Frescal Cheese and a Contaminated Dairy Plant To Establish Prevalence, Relatedness, and Sources of Listeria monocytogenes Isolates

ABSTRACT A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.

Qualidade microbiológica do leite refrigerado nas fazendas

Avaliaram-se a qualidade microbiologica do leite obtido mecanicamente e refrigerado durante 48 horas, em 24 rebanhos, e a associacao entre a contaminacao microbiana e os procedimentos de higienizacao dos equipamentos de ordenha e armazenamento do leite. Os procedimentos de higiene foram avaliados in loco com auxilio de questionarios. Foram realizadas a contagem padrao em placas, a contagem de coliformes totais e a pesquisa de Staphylococcus aureus e Streptococcus agalactiae. No leite de 14 rebanhos, foram pesquisadas Salmonella spp. e Listeria monocytogenes. As medias geometricas da contagem padrao foram 103 UFC/ml foram verificadas em sete rebanhos. S. aureus e S. agalactiae foram isolados em 22 e 12 dos 24 rebanhos, respectivamente, e nao foram encontradas Salmonella spp. e L. monocytogenes. O uso de detergentes alcalino e acido, mais o de sanitizante foi associado (P 5×105 UFC/ml.

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.

Sensibilidade e especificidade do "California Mastitis Test" como recurso diagnóstico da mastite subclínica em relação à contagem de células somáticas

The California Mastitis Test (CMT) is a simple and widely used diagnostic tool for subclinical mastitis. It is used even in areas where laboratory facilities are available for diagnosis and monitoring program purposes. CMT usually score 1-5, where 1 indicates a completely negative reaction and 2-5 increasing degrees of inflammatory reaction of the udder. The reactions 2-5, 3-5 or 4-5 may be considered as indicative of subclinical mastitis, bever, may produce either false-positive or false-negative results. The aim of this study waecause they are related to an increase of the somatic cell count (SCC) in the milk. This variation, hows to assess the sensitivity and specificity of the CMT in comparison with SCC. A total of 3,012 quarter milk samples from 760 lactating cows were examined. CMT was evaluated at the moment of sampling at cow side and SCC in the laboratory, with a fluoro-opto-electronic method (Fossomatic 90). The average CCS (x 1,000 cells/ml) for CMT scores were: 1 (79.9), 2 (333.5), 3 (670.3), 4 (1,354.0) and 5 (4,455.6). Three options for CMT interpretation were evaluated in relation to a range of CCS, starting from 100,000 cells/ml: (a) 1 versus 2, 3, 4, and 5; (b) 1 and 2 versus 3, 4 and 5; and (c) 1, 2 and 3 versus 4 and 5. The sensitivities of CMT scoring to detect quarters with SCC >200,000/ml were 79%, 61% e 34% for options a, b and c, respectively. The sensitivities of CMT scores for SCC >500,000/ml for options a, b and c, were, respectively, 93%, 82% e 54%. The sensitivity of CMT scores to identify subclinical mastitis was satisfactory (above 80%) when options b and c were used. The interpretation of CMT score 3 as negative for subclinical mastitis was considered as adequate (sensitivity around 80%) only when CCS ranged between 1,200,000 and 1,400,000 cells/ml. The specificities of CMT scoring for CCS of 200,000 and 500,000 were, respectively, 90% and 80% (option a), 97% and 90% (option b) and 99% and 97% (option c).

Concentração mínima inibitória de dez antimicrobianos para amostras de Staphylococcus aureus isoladas de infecção intramamária bovina

The minimum inhibitory concentrations (MIC) for ampicillin, cephalothin, erythromycin, gentamicin, neomycin, norfloxacin, oxacillin, penicillin G, tetracycline and tylosin against 112 Staphylococcus aureus strains isolated from bovine intramammary infections were determined. The strains were originated from 33 dairy herds located in the Zona da Mata, Minas Gerais State. Twenty-four strains were isolated from clinical cases of mastitis, 66 from subclinical infections and 22 from chronic infections. The chronic infection strains were isolated from the same mammary quarters of nine cows of one herd over a period of 13 months. The MIC was performed on Mueller Hinton agar and concentrations, ranging from 0.015 to 128µgml-1, were evaluated for each antimicrobial agent. The American Type Culture Collection (ATCC) recommended quality control strains, S. aureus ATCC 29213, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, were included on each batch of test. All strains were susceptible to cephalothin, erythromycin, gentamicin, norfloxacin and oxacillin, 91% were susceptible to tetracycline (MIC50: 0.5µgml-1) and tylosin (MIC50: 2.0µgml-1), 65% to ampicillin (MIC50: 0.125µgml-1) and penicillin G (MIC50: 0.06µgml-1). All strains but one in the intermediate pattern, were susceptible to neomycin (MIC50: 0.5µgml-1). The resistance levels to ampicillin and penicillin were higher in strains isolated from clinical and subclinical (positive scores on CMT) cases (P<0.02). The resistance level to tylosin was also higher among the strains isolated from clinical infections (P<0.02). The strains with MIC³0.125µgml-1 to penicillin were positive for s-lactamase production.

Padrão de infecção intramamária em rebanhos leiteiros: exame de todos os quartos mamários das vacas em lactação

Microbiological examination was carried out in 6,315 milk samples collected from all mammary quarters of 1,609 lactating cows from 48 herds, located in the regions of Zona da Mata and Campo das Vertentes, Minas Gerais State, Brazil. At sampling time, the udders were clinically examined and milk from all mammary quarters were evaluated by California Mastitis Test (CMT). A total of 3,919 agents were isolated, being 3,637 from quarters with single infection and 283 from mixed infection with two types of microorganisms. The mastitis agents found and the percentages of isolations were: Staphylococcus aureus, 19.2%, coagulase negative Staphylococcus sp. (SCN), 12.4%, Streptococcus agalactiae, 6.9%, esculin-positive Streptococcus sp. (ESCPOS), 4.0%, esculin-negative Streptococcus sp. (ESCNEG), 2.1%, Corynebacterium sp., 55.2%, yeast, 0.1% e Pseudomonas sp., 0.1%. Negative cultures were found in 2,463 (39%) samples and 216 samples were contaminated. Corynebacterium sp. was present in all 48 herds, with mammary quarters infection levels between 1.5% and 58.6%. S. aureus was isolated from 47 herds; in 37 of these there were up to 20% of infected mammary quarters. S. agalactiae was recovered from 29 herds (60%) and in 24 out of these the average of infected quarters was 2.7%. S. aureus, S. agalactiae, SCN, ESCPOS, ESCNEG and Corynebacterium sp. were isolated from mammary quarters with and without signs of inflammatory reaction (positive and negative scores on CMT, respectively). Comparing with minor pathogens (SCN and Corynebacterium sp.), the isolation frequency of major pathogens was significantly higher from quarters with positive scores on CMT (P<0.001). The results indicated that mammary quarters negative on CMT should also be considered when selecting samples for microbiological examination and that negative cows, infected with major pathogens, can be a source of infection for the others animals in the herd. The high prevalence of S. aureus, S. agalactiae and Corynebacterium sp. suggests that current control measures for contagious mastitis were not correctly practised in most of the herds.

Staphylococcus aureus of bovine origin: Genetic diversity, prevalence and the expression of adhesin-encoding genes

Staphylococcus aureus is a well-armed pathogen that is a leading cause of bovine mastitis. Attempts to define a set of bacterial proteins that are crucial for infection have failed. The identification of these proteins is important to define biomarkers that can be used for diagnostic purposes and to identify potential vaccine targets. In this study, seven genes that encode virulence factors were analyzed in 85 bacterial isolates that were derived from animals with bovine mastitis. The clfB, spa, sdrCDE and fnBP genes were detected in 91.8%, 85.9%, 85.9% and 63.5% of the isolates, respectively. At least one gene was present in all of the strains, while the most prevalent combination was clfB and sdrCDE (82.4%). The genetic diversity of the isolates was high and allowed for clustering into more than 40 groups, with each group containing bacteria collected from different locations. The gene expression of the four most prevalent adhesins was examined in nine genetically distinct strains. No common pattern of expression was observed for the genes, suggesting that the capacity of S. aureus to cause infection may rely on differential expression of the virulence factors in different isolates. Our results conclude that using only one antigen is unlikely to provide effective protection against bovine mastitis and suggest that a combination of at least three adhesins may be more suitable for developing preventive therapies. We also conclude that the characterization of isolates distributed worldwide is necessary to improve our understanding of pathogenesis in the natural populations of S. aureus.

Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing

Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

Variação da contagem de células somáticas em vacas leiteiras de acordo com patógenos da mastite

The influence of mastitis pathogens on variation of milk somatic cell count (SCC) was evaluated. Three thousand nine hundred eighty-seven milk samples were colected from 2,657 dairy cows in 24 herds located in the states of Minas Gerais and Rio de Janeiro. The milk samples were used to SCC and identification of mastitis pathogens. Descriptive statistics, T test for independent samples, and generalized linear model were used to data analysis. The generalized linear model identified the effects of herd, animal within herd, parity, year season, intramammary infection, and infection caused by Streptococcus agalactiae and Streptococcus spp. except S. agalactiae as significant on SCC variation. The effect of animal within herd was higher than the effect of herd. S. agalactiae was the pathogen responsible for higher SCC increasing and presented the average of 1,520,000 cells/mL. The specific effect on SCC variation was observed in the study.

Contagem de células somáticas e isolamento de agentes causadores de mastite em búfalas (Bubalus bubalis)

The research was accomplished in eight dairy water buffalo herds, randomically choosen in Regiao do Alto Sao Francisco, State of Minas Gerais, Brazil. Information was collected from March to November, 2003 during 270 days of observation. In order to determine the somatic cell count (SCC) in presence or absence of microbial isolation, 1,393 samples were collected from 285 lactating females and microbiological exams and SCC were done. Samples obtained from udders without evidence of clinical or subclinical inflammation showed infection for a great variety of microbial mastitis pathogens. The low SCC did not necessarily indicate the absence of intramammary infection, suggesting that SCC patterns used for bovine cannot be appropriate in order to control mastitis in buffalo herds.