Assessment of the main pathogens associated with clinical and subclinical endometritis in cows by culture and MALDI-TOF mass spectrometry identification.

Clinical endometritis (CE) and subclinical endometritis (SCE) are diseases that affect dairy cows during the puerperium, causing negative effects on the animals' milk production and fertility. The objective of this study was to assess the main bacteria related to cases of CE and SCE from uterine samples of dairy cows in Brazilian herds. Selective and differential media were used for isolation of aerobic and anaerobic bacteria and further MALDI-TOF mass spectrometry (MS) identification. A total of 279 lactating dairy cows with 28 to 33 d in milk from 6 commercial farms were evaluated. Initially, cows were classified in 3 groups: cytologic healthy cows (n = 161), cows with CE (n = 83), and cows with SCE (n = 35). Healthy animals presented 97 species, followed by the CE group with 53 identified species, and SCE cows presented only 21 bacterial species. We found a significantly higher isolation rate of Trueperella pyogenes in CE (26.5%) cows compared with healthy and SCE cows. Some anaerobic species were exclusively isolated from the CE group, even though they presented lower frequency. Interestingly, 18.1% of samples from CE cows and 40% of SCE cows were negative to bacterial isolation. Despite the use of culture-dependent methods instead of molecular methods, the present study enabled the identification of a complex community of 127 different species from 48 genera, composed of aerobic and anaerobic bacterial species among the 3 different animal groups. The method of sample collection, culture, and identification by MALDI-TOF MS were essential for the success of the analyses.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram-negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gramnegative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines

Abstract Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Resumo Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines.

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

PCDD, PCDF, dl-PCB and organochlorine pesticides monitoring in São Paulo City using passive air sampler as part of the Global Monitoring Plan.

The persistent organic pollutants (POPs), such as organochlorine pesticides and PCBs, are ordinarily monitored in the aquatic environment or in soil in the environmental quality monitoring programs in São Paulo, Brazil. One of the core matrices proposed in the POPs Global Monitoring Plan (GMP) from the Stockholm Convention list is the ambient air, which is not a usual matrix for POPs monitoring in the country. In this study POP levels were evaluated in the air samples from an urban site in São Paulo City over five years, starting in 2010 as a capacity building project for Latin America and the Caribbean region for POP monitoring in ambient air using passive samplers. Furthermore, after the end of the Project in 2012, the monitoring continued in the same sampling site as means to improving the analytical capacity building and contribute to the GMP data. The POPs monitored were 17 congeners of 2,3,7,8 chloro-substituted PCDDs and PCDFs, dioxin-like PCBs, indicator PCBs, organochlorine pesticides and toxaphene. The results show a slight decrease in PCDD/F, dl-PCBs and indicator PCBs levels along the five years. The organochlorine pesticide endosulfan was present at its highest concentration at the beginning of the monitoring period, but it was below detection level in the last year of the monitoring. Some other organochlorine pesticides were detected close to or below quantitation limits. The compounds identified were dieldrin, chlordane, α-HCH, γ-HCH, heptachlor, heptachlor epoxide, hexachlorobenzene and DDTs. Toxaphene congeners were not detected. These results have confirmed the efficacy of passive sampling for POP monitoring and the capacity building for POP analysis and monitoring was established. However more needs to be done, including expansion of sampling sites, new POPs and studies on sampling rates to be considered in calculating the concentration of POPs in ambient air using a passive sampler.

Microbiological quality assessment of sand and water from three selected beaches of South Coast, São Paulo State, Brazil.

This study aimed to assess the sanitary quality of water, and wet and dry sand from three beaches located in the South Coast region of São Paulo State, Brazil, selected taking into account the frequency of tourists and the water quality (good, fair and poor). Thirty-six water samples each of wet and dry sand and seawater were collected monthly over a period of one year and analyzed for fecal indicator bacteria (FIB: thermotolerant coliforms, Escherichia coli, and enterococci), presumptive Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and dermatophytes. The results revealed FIB concentrations more elevated in dry sand followed by wet sand and water. P. aeruginosa and presumptive S. aureus were detected with a similar frequency in water and sand samples, but maximum concentrations and geometric means were higher in dry sand. C. albicans was detected only in water samples whereas the dermatophyte Microsporum sp. was isolated exclusively from dry and wet sand samples. This evaluation showed also that the environment had a significant influence on P. aeruginosa but not on presumptive S. aureus concentrations. According to threshold values proposed in the literature for E. coli and enterococci dry sand densities, none of the beaches would be considered of sufficient quality for recreational activities.

Controle de qualidade analítica dos métodos utilizados para a detecção de protozoários patogênicos em amostras de água / Control of the analytic quality of the methods used for the detection of pathogenic protozoans in water samples

As espécies de protozoários patogênicos de veiculação hídrica Giardia spp. e Cryptosporidium spp. foram responsáveis por cerca 90% dos surtos de gastroenterite ocorridos nos últimos 25 anos, em vários países. Critérios de avaliação das etapas referentes à detecção destes agentes em amostras de água, tais como: precisão inicial, inoculação nas amostras em água bruta e, diagnóstico e localização de erros estão abordados neste trabalho. Estes critérios devem ser correntemente aplicados e constantemente avaliados pelos laboratórios, a fim de assegurar a confiabilidade dos resultados obtidos durante um monitoramento e, assim, fornecer subsídios para uma avaliação do risco de surtos.

The waterborne pathogenic protozoan species Giardia spp. and Cryptosporidium spp. have been responsible for the majority (90%) of the gastroenteritis outbreaks that have occurred in the last 25 years in various countries. This paper discusses evaluation criteria pertaining to the steps involved in the detection of these microorganisms in water samples, such as: initial precision, inoculation in environmental samples, diagnosis and error localization. These criteria should be often applied and evaluated by laboratories in order to assure the reliability of the results obtained in a program of monitoring, thus aiding in the evaluation of the risks of outbreaks.

Giardia and Cryptosporidium in source waters of São Paulo State, Brazil.

Giardia lamblia and Cryptosporidium parvum are two protozoan intestinal parasites responsible for many drinking-water-related disease outbreaks in recent years. They are very resistant to conventional water treatment processes, can persist for long times in the environment and are, therefore, of great concern for public health. This work aimed to evaluate the presence of Giardia and Cryptosporidium in water sources from São Paulo State, Brazil, as part of the "Evaluation of Inland Waters from São Paulo State" project from CETESB. Over a period of 19 months, 278 water samples from 28 sites located in 10 watersheds were analysed. The immunofluorescence assay was used after concentration of the samples by the calcium carbonate flocculation technique. Thermotolerant (faecal) coliforms, faecal streptococci and Clostridium perfringens were also determined in order to verify the existence of correlation between these bacterial indicators and the protozoa. Giardia and Cryptosporidium were detected in 27% and 2.5% of the samples, respectively, a lower figure compared with the results reported by other authors, especially for Cryptosporidium. A Spearman rank correlation test demonstrated a significant correlation between Giardia and faecal indicator concentrations. According to the American Regulation of Monitoring (ICR), treated water from 16 of these 28 collection sites should also be analysed to evaluate whether the treatment process could remove the parasites. Some technical deficiencies of these methods still limit the utilisation of the monitoring results for public health decisions, but the data here reported will help to improve the quality of drinking water in São Paulo State.

Evaluation of the genotoxic activity and acute toxicity of Euphorbia splendens latex, a molluscicide for the control of Schistosomiasis

The latex of euphorbia splendens var. hislopii has a molluscicidal action at low concentration (LD90 less than 1.5 ppm or 1.5 */ml) against the vector snails of schistosomiasis. In the present study, the latex in natura or after lyophilization was submitted to the Ames test and the chromotest to evaluate genotoxicity, to the Microtox System to determine acute toxicity, and to the Chinese hamster ovary cell assay (CHO) to measure cytotoxicity. The latex had no mutagenic activity in the presence or absence of S9 toward the TA98 and TA100 strains of Salmonella typhimurium (Ames test) at concentration up to 200 */plate (in natura) and of 200 *g/plate (lyophilized). The lyophilized latex had no genotoxic activity (Chromotest) and acute toxic effect on Photobacterium phosphoreum at concentrations up to 445 *g/ml, whereas the sample in natura had a toxic effect with an EC50 of 148,000 *l/l (or ppm). In the CHO/cytotoxicity assay, the lyophilized latex had no cytotoxicit effect in quantities up to 200 *g. The latex was found to have no acute toxicity or mutagenic at the concentrations of 10 to 12 *g/ml (or ppm) that are being proposed for molluscicidal use in the field