The identification of pathogens of viral (Rotavirus, Coronavirus), parasitic (Toxocara spp.) and bacterial (Escherichia coli, Salmonella spp., Rhodococcus equi) origin shed in feces, and the virulence profile of R. equi and E. coli isolates were investigated in 200 samples of sand obtained from 40 parks, located in central region of state of Sao Paulo, Brazil, using different diagnostic methods. From 200 samples analyzed, 23 (11.5%) strains of R. equi were isolated. None of the R. equi isolates showed a virulent (vapA gene) or intermediately virulent (vapB gene) profiles. Sixty-three (31.5%) strains of E. coli were identified. The following genes encoding virulence factors were identified in E. coli: eae, bfp, saa, iucD, papGI, sfa and hly. Phylogenetic classification showed that 63 E. coli isolates belonged to groups B1 (52.4%), A (25.4%) and B2 (22.2%). No E. coli serotype O157:H7 was identified. Eggs of Toxocara sp. were found in three parks and genetic material of bovine Coronavirus was identified in one sample of one park. No Salmonella spp. and Rotavirus isolates were identified in the samples of sand. The presence of R. equi, Toxocara sp, bovine Coronavirus and virulent E. coli isolates in the environment of parks indicates that the sanitary conditions of the sand should be improved in order to reduce the risks of fecal transmission of pathogens of zoonotic potential to humans in these places.

O objetivo deste trabalho foi detectar em 101 amostras de E. coli isoladas de bezerros com e sem diarreia, fatores de virulência associados aos patotipos de E. coli enterotoxigênica e enterohemorrágica. Os fatores de virulência de E. coli Stx1 (Shiga toxina), Stx2, Ehly (Enterohemolisina), o gene eae, LT-II (enterotoxina termo-lábil), STa (toxina termo-estável), e adesinas K99 e F41 foram detectados por PCR. Os sorogrupos foram determinados por métodos sorológicos e a produção de Stx foi observada através de ensaios biológicos em células Vero. A frequência de detecção do gene eae foi maior nos isolados de bezerros com diarreia (35/58, 60,3%) do que em bezerros saudáveis (8/43, 18,6%; P < 0.001). O gene da toxina Stx1 foi detectado em alta frequência em amostras diarreicas (24/58, 40,3%), bem como em amostras não diarréicas (19/43, 44,2%) e todas as amostras positivas para toxina Stx em PCR mostraram citotoxicidade em células Vero. Stx2 foi encontrada em dez amostras, Ehly em oito amostras, e LT-II em duas amostras. Vinte e seis amostras foram negativas para todos os ensaios de PCR, incluindo para as adesinas F41 e K99. Os sorogrupos O7, O23, O4, O8, O153 e O156 foram detectados com maior frequência. O trabalho mostra que amostras de E. coli isoladas de bovinos apresentam fatores de virulência semelhantes à isolados de casos de doenças em humanos e possivelmente é uma fonte para STEC potencialmente patogênicas para humanos.

The aim of this work was to test 101 strains of E. coli for virulence factors associated with enterotoxigenic and enterohemorrhagic pathotypes of E. coli isolated from diarrheic and non-diarrheic calves. The virulence factors of E. coli Stx1 (Shiga toxin), Stx2, Ehly (Enterohemolysin), the eae gene, LT-II (heat-labile enterotoxin), STa (heat-stable toxin), and adhesins K99 and F41 were detected by PCR. Serogroups were determined by serological methods and Stx production was observed by biological assays in Vero cells. The frequency of the eae gene was higher in isolates from diarrheic calves (35/58, 60.3%) than in non-diarrheic calves (8/43, 18.6%; P < 0.001). The gene for Stx1 occurred at high frequencies in the diarrheic strains (24/58, 41.3%) as well as in non-diarrheic (19/43, 44.2%) ones and all strains that were Stx positive by PCR showed cytotoxicity in Vero cells. Stx2 was found in ten strains, Ehly in eight strains, and LT-II in only two strains. Twenty-eight strains were negative for all of the PCR assays, including for F41 and K99 adhesins. The serogroups O7, O23, O4, O8, O153 and O156 were observed most frequently. Our results show that strains of E. coli isolated from cattle have similar virulence factors genes to strains isolated from cases of diseases in humans and may be a source of potentially pathogenic STEC for humans.

RESUMO Este estudo avaliou o índice de patogenicidade, a produção de hemolisina e a determinação de sorogrupos de cepas deEscherichia coli isoladas de fígado de aves de postura comercial com um dia de idade. Para este estudo, foram analisados 32 lotes, dos quais 15 foram positivos para o isolamento de E. coli no fígado, totalizando vinte e quatro amostras. A patogenicidade dos isolados foi determinada por inoculação no saco aéreo de pintinhos e classificada como alta, intermediária, baixa ou não-patogênica. Os sorogrupos foram identificados utilizando um conjunto de antissoros anti-O (O1 a O180). A produção de hemolisina foi determinada por semeadura em ágar sangue de galinha (8%) e em placas de ágar sangue de carneiro (8%). Do total de amostras estudadas, 17 (70,83%) foram classificadas como não patogênica, 6 (25%) como de baixa patogenicidade e 1 (4,17%) de alta patogenicidade. Foram identificados 14 sorogrupos diferentes: O1, O2, O5, O8, O15, O18, O22, O36, O64, O70, O75, O115, O132, O141. Cinco cepas não tiveram o sorogrupo identificado. Com relação ao teste de produção de hemolisina, todas as cepas foram consideradas negativas, tanto para o teste realizado com ágar sangue de galinha quanto para o de carneiro. Os resultados obtidos neste estudo demonstram a importância de se identificar as cepas prevalentes deE. colinas diferentes regiões produtoras, podendo ser utilizados em estudos epidemiológicos.

ABSTRACT This work evaluated the index of pathogenicity, the production of hemolysin and determination of serogroups in Escherichia coli strains isolated from liver of commercial laying hens with one day of age. Thirtytwo lots were analyzed, of which 15 were positive for the isolation ofE. coli in the liver, for a total of 24 samples. The pathogenicity in one-day-old chicks was determined by inoculation in air sac and was classified as high, intermediate or low pathogenicity, or non-pathogenic. Serogroups were identified using a set of anti-O antisera (O1 to O180). The production of hemolysin was determined by plating on chicken blood agar (8%) and sheep blood agar (8%). Of the samples studied, 17 (70.83%) were classified as non-pathogenic, 6 (25%) as low pathogenicity and 1 (4.17%) as high pathogenicity. Fourteen different serogroups were identified: O1, O2, O5, O8, O15, O18, O22, O36, O64, O70, O75, O115, O132 and O141, while 5 samples were non-typable. Regarding the test for production of hemolysin, all strains were considered negative for both the test performed with chicken blood agar and that with sheep blood agar. The results of this study demonstrate the importance of identifying the prevalent strains of E. coli in different producing regions, as this information can be used in epidemiological studies.

Avaliou-se a ocorrência de fatores de virulência e do sorotipo O157:H7 em 120 linhagens de Escherichia coli, isoladas de 80 casos de mastite clínica bovina e 40 de mastite subclínica. Verificou-se alfa-hemolisina em oito (6,7%) linhagens, isoladas de cinco casos de mastite clínica e três de mastite subclínica e em nenhuma das estirpes detectou-se enteroemolisina. A presença de sideróforos foi encontrada em 11 (9,2%) linhagens, sete de mastite clínica e quatro de subclínica. Em duas (1,7%) estirpes isoladas de mastite subclínica, identificou-se enterotoxina STa. Observou-se efeito citopático em células vero compatível com a produção de verotoxina-VT em cinco (4,2%) linhagens, duas de mastite clínica e três subclínicas. Em uma (0,8%) linhagem isolada de mastite clínica, detectou-se efeito citopático compatível com o fator necrosante citotóxico. Nenhuma estirpe apresentou-se sorbitol-negativa no MacConkey-sorbitol, tampouco aglutinou com o sorotipo O157:H7. Os antimicrobianos mais efetivos foram polimixina B (97,5%) e norfloxacina (95,8%). Observou-se multi-resistência a dois ou mais antimicrobianos em 24 (20%) estirpes, principalmente com o uso de ampicilina e ceftiofur.

The occurrence of different virulence factors and O157:H7 serotype investigation in 120 Escherichia coli strains isolated from clinical (80 cases) and subclinical (40 cases) bovine mastitis was evaluated. Alpha-haemolysin was detected in 8 (6.7%) strains (5 clinical and 3 subclinical cases). None strain showed enterohaemolysin production. E. coli growth under iron restriction conditions (siderophores production) was observed in 11 (9.2%) strains (7 clinical and 4 subclinical cases). STa enterotoxin was detected in 2 (1.7%) strains from subclinical cases. Cytotoxic effect in vero cells compatible with verotoxin-VT production was observed in 5 (4.2%) strains (2 clinical and 3 subclinical cases). One strain (0.8%) isolated from clinical mastitis showed cytophatic effect in vero cells compatible with cytotoxic-necrotic-factor production. None strain showed non-sorbitol fermenting colonies using sorbitol MacConkey agar and agglutination with specific anti-sera for E. coli O157:H7 serotype investigation. Polymixin B (97.5%) and norfloxacin (95.8%) were the most effective drugs. Multiple-drug resistance for 2 or more antimicrobials was observed in 24 (20.0%) strains, mainly with use of ampicillin and ceftiofur.

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

This report describes the production of cytotoxic necrotizing factor (CNF) by an Escherichia coli strain isolated from clinical bovine mastitis with clinical signs of toxemia The animal had hemorrhages and necrosis of the mammary glands, and died within 24 hours after the onset of clinical signs. In addition to CNF identification, alpha-haemolysin and siderophores production were also characterized in this strain. This report reinforce the association of CNF and alpha-haemolysin production in E. coli virulence associated with clinical cases of severe bovine mastitis.