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).

Sensibilidade de bactérias causadoras de mastite bovinaaextratos de plantas nativas do cerrado / Sensitivity of bacteria causing bovine mastitis toextractsofnative plants of the cerrado / Sensibilidad de bacterias causantes de mastitis bovinaaextractos de plantas nativas en cerrado

A mastite bovina é a doença mais onerosa da produção leiteira e é caracterizadapelainflamação da glândula mamária. O tratamento da doença, sem o controle adequado, geramicrorganismos resistentes. Desta forma o uso de fitoterápicos tem se tornado uma fontedepesquisa como possível alternativa, como as plantas nativas do cerrado Dedaleiro (Lafoensiapacari), Jatobá (Hymenaea sp.) e Barbatimão (Stryphnodendron adstringens). No presentetrabalho, bactérias isoladas de vacas com mastite subclínica foram identificadas por testesbioquímicos. Posteriormente foram feitos testes de antibiograma e pesquisa por genesderesistência a antibióticos, por fim foi realizado o teste para avaliação da sensibilidadeaosextratos das plantas do cerrado. Foram identificados Staphylococcus aureus (S.aureus), Enterobacter sp. e Escherichia coli (E. coli). Entre os isolados de S. aureus, foramencontrados resistentes a meticilina (MARS), bem como resistentes à vancomicina (VARS). Foram encontrados isolados produtores de beta-lactamases de espectro extendido(ESBL)para Entreobacter sp. e E. coli. Os extratos, principalmente de Stryphnodendron adstringenseLafoensia pacari, são uma alternativa para a terapêutica antimicrobiana.(AU)

Bovine mastitis is the most costly disease of dairy production and is characterizedbyinflammation of the mammary gland. The treatment of the disease, without adequate control, generates resistant microorganisms. In this way, the use of herbal medicines has becomeasource of research as a possible alternative, such as the native plants of the cerrado Dedaleiro(Lafoensia pacari), Jatobá (Hymenaea sp.) and Barbatimão (Stryphnodendron adstringens). In the present work, bacteria isolated from cows with subclinical mastitis were identifiedthrough biochemical tests. Subsequently, antibiogram tests and research for antibioticresistance genes were carried out, finally the test was carried out to evaluate the sensitivitytoextracts of cerrado plants. Staphylococcus aureus (S.aureus), Enterobacter sp. andEscherichia coli (E. coli). Among the S. aureus isolates, methicillin resistant (MARS) as well as vancomycin resistant (VARS) were found. Extended spectrum beta-lactamases (ESBL)producing isolates were found for Entreobacter sp. and E. coli. The extracts, mainly from Stryphnodendron adstringens and Lafoensia pacari, are an alternative for antimicrobial therapy.(AU)

La mastitis bovina es la enfermedad más costosa de la producción lechera y se caracterizaporla inflamación de la glándula mamaria. El tratamiento de la enfermedad, sin uncontrol adecuado, genera microorganismos resistentes. De esta forma, el uso de fitoterápicos sehaconvertido en fuente de investigación como posible alternativa, como las plantas nativas del cerrado Dedaleiro (Lafoensia pacari), Jatobá (Hymenaea sp.) y Barbatimão (Stryphnodendronadstringens). En el presente trabajo se identificaron mediante pruebas bioquímicas bacteriasaisladas de vacas con mastitis subclínica. Posteriormente se realizaron pruebas deantibiograma e investigación de genes de resistencia a antibióticos, finalmente se realizólaprueba para evaluar la sensibilidad a extractos de plantas de cerrado. Staphylococcus aureus(S. aureus), Enterobacter sp. y Escherichia coli (E. coli). Entre los aislamientos de S. aureus, se encontraron resistentes a la meticilina (MARS) y resistentes a la vancomicina (VARS). Seencontraron aislamientos productores de betalactamasas de espectro extendido (ESBL) paraEntreobacter sp. y E. coli. Los extractos, principalmente de Stryphnodendron adstringensyLafoensia pacari, son una alternativa para la terapia antimicrobiana.(AU)

Termografia infravermelha aplicada ao diagnóstico de mastite subclínica / Infrared thermography applied in diagnosis of subclinical mastites

A mastite é uma doença inflamatória de alta incidência nos rebanhos leiteiros de todo o mundo, sendo responsável por grandes prejuízos econômicos na cadeia produtiva do leite. Existem diversos testes para a detecção da forma subclínica da doença, sendo os mais utilizados: a contagem de células somáticas (CCS) e o California Mastitis Test (CMT). No entanto, esses testes possuem algumas limitações práticas, como a interpretação subjetiva, o que pode resultar em diagnósticos falso-positivos. Diante desses aspectos, novas tecnologias vêm sendo estudadas para auxiliar o diagnóstico precoce da mastite subclínica, dentre as quais a termografia infravermelha (TIV), que é um método não invasivo de diagnóstico por imagem. Dessa forma, o objetivo desta revisão foi discutir sobre a técnica de termografia infravermelha e sua aplicabilidade no diagnóstico de mastite subclínica. A TIV tem sido empregada, com sucesso, em várias áreas da produção animal de precisão, inclusive no diagnóstico de mastite, sendo relatadas alterações térmicas na superfície do úbere antes do aparecimento dos sintomas clínicos e sua correlação com a CCS do leite. O uso dessa tecnologia se mostra promissor para a detecção do estado não saudável do úbere, mesmo na forma subclínica. Entretanto, mais estudos devem ser realizados para que se avalie a aplicabilidade da técnica em rebanhos comerciais.

Mastitis is an inflammatory disease of high incidence in dairy herds around the world, resulting in great economic losses in the milk production chain. There are several tests to detect the subclinical form of this disease, where the most applied are the somatic cell count (SCC) and the California Mastitis Test (CMT). However, due to their subjectivity, these tests can result in false-positive diagnoses. Therefore, new technologies have been studied for the early diagnosis of subclinical mastitis, including infrared thermography (IRT), which is a non-invasive diagnostic approach. Thus, this review aimed to discuss the infrared thermography technique and its applicability in the diagnosis of subclinical mastitis. The IRT has been successfully applied in numerous areas of precision animal production, including mastitis diagnosis, showing thermal alterations on the udder surface before the onset of clinical symptoms that are linked to milk SCC. Therefore, the use of this technology is a promising strategy to detect the unhealthy udder state, even in the subclinical form. However, further studies should be carried out to assess the applicability of this technique in commercial herds.

Economic and production aspects ofmilking management in dairy farming / Aspectos econômicos e produtivos do manejo de ordenha na pecuária leiteira

The Brazilian dairy sector is important for the world agricultural production; however, it presents problems in production efficiency and milk quality, since the occurrence of mastitis in the herds can reach 47%. The present study analyzed data from a rural property with 100 lactating cows in a semi-intensive breeding system, which did not adopt adequate hygiene management for milking processes. Corrective measures were proposed, such as daily strip cup test, biweekly California Mastitis Test, formation of a new milking line, and daily pre dipping and post dipping and cleaning of teat cups. Milk samples from 10 animals with positive clinical mastitis, 10 animals with positive subclinical mastitis, and from the cooling tank were collected at one day before and 90 days after the implementation of the proposed measures and sent to a laboratory for analysis of somatic cell counting (SCC) and total bacteria counting (TBC). An increase of 17% in milk production and decreases of 64% and 40% in clinical and subclinical mastitis, respectively, were found after 90 days, with a consequent decrease in SCC and TBC. The milking duration after this period denoted an adaptation of the employees to the new procedures. The production financial yield was calculated based on the implementation costs of the adopted adequations and increases in milk production, representing a profitability increase of 10.4%. The implemented adequations were efficient for the control of mastitis and increase in milk yield and quality, resulting in a positive economic return.(AU)

O setor lácteo brasileiro possui grande relevância na produção agrícola mundial e apesar disso, ainda são encontra-dos problemas na eficiência produtiva e na qualidade do leite, onde a prevalência de mastite nos rebanhos pode chegar a 47%. Neste estudo, foram analisados dados de uma propriedade rural com 100 vacas lactantes e sistema de criação semi-intensivo, que não adotava manejo higiênico adequado na ordenha. Foram propostas medidas de correção como execução diária do teste da caneca telada e teste quinzenal do CMT, formação de nova linha de ordenha, adoção diária do pré dipping e pós dipping e higienização das teteiras. Foram coletadas amostras de leite de 10 animais positivos para mastite clínica, 10positivos para mastite subclínica e do tanque de resfriamento um dia antes e 90 dias após a implementação das novas medidas e enviadas ao laboratório para análise da CCS e CBT. Após 90 dias, houve aumento na produção de leite em 17% e redução dos índices de mastite clínica e subclínica em 64% e 40%, respectivamente, com consequente diminuição dos valores de CCS e CBT. A duração da ordenha após esse período indicou adaptação dos colaboradores aos novos procedimentos instaurados. Por fim, calculou-se o rendimento financeiro da produção baseado nos custos de implantação das correções e no aumento da produção de leite, que reverteu-se em lucratividade 10,4% maior. Conclui-se que as adequações implementadas foram eficazes no con-trole de mastite e no aumento da produtividade dos animais e da qualidade do leite, resultando em retorno econômico positivo.(AU)

Lactoperoxidase potential in diagnosing subclinical mastitis in cows via image processing.

This report describes how image processing harnessed to multivariate analysis techniques can be used as a bio-analytical tool for mastitis screening in cows using milk samples collected from 48 animals (32 from Jersey, 7 from Gir, and 9 from Guzerat cow breeds), totalizing a dataset of 144 sequential images was collected and analyzed. In this context, this methodology was developed based on the lactoperoxidase activity to assess mastitis using recorded images of a cuvette during a simple experiment and subsequent image treatments with an R statistics platform. The color of the sample changed from white to brown upon its exposure to reagents, which is a consequence of lactoperoxidase enzymatic reaction. Data analysis was performed to extract the channels from the RGB (Red-Green-Blue) color system, where the resulting dataset was evaluated with Principal Component Analysis (PCA), Multiple Linear Regression (MLR), and Second-Order Regression (SO). Interesting results in terms of enzymatic activity correlation (R2 = 0.96 and R2 = 0.98 by MLR and SO, respectively) and of somatic cell count (R2 = 0.97 and R2 = 0.99 by MLR and SO, respectively), important mastitis indicators, were obtained using this simple method. Additionally, potential advantages can be accessed such as quality control of the dairy chain, easier bovine mastitis prognosis, lower cost, analytical frequency, and could serve as an evaluative parameter to verify the health of the mammary gland.

Evaluation of three Staphylococcus aureus proteins involved in the adhesion process as antigens for the detection of bovine intramammary infections.

Introduction. Fast and accurate diagnosis is one of the key strategies in the successful control of intramammary infections caused by Staphylococcus aureus. Immunoassays are one of the diagnostic tools that have been proposed for the detection of S. aureus infection because they offer an advantage in terms of cost and are fast and easy to use compared to other diagnostic tests.Gap statement. The main challenge of the immunoassays is to identify antigens or serological markers that allow accurate discrimination between infected and uninfected cows with S. aureus, since this bacterium can naturally colonize different areas of the animal body.Aim. To evaluate three S. aureus proteins (IsdA, ClfA, SdrD) involved in the adhesion process as antigens to detect indicator antibodies of bovine intramammary infections.Methodology. Ninety-six cows in lactation and not vaccinated against S. aureus were included. Forty-eight of these cows were infected with S. aureus, while the rest (n=48 cows) were uninfected. Blood and milk samples were collected from each animal to recover serum and whey. IgG titres against the three proteins individually and combined (Mix) were measured in each sample using an enzyme-linked immunosorbent assay (ELISA) test.Results. Significant differences in the IgG response against the proteins evaluated were observed, highlighting the antigenic potential of IsdA and demonstrating that some antigens can detect specific antibodies of infection better than others. According to receiver operating characteristic (ROC) curve analysis, the combined proteins showed the most remarkable capacity (sensitivity of 79 % and specificity of 77 %) to differentiate between infected and uninfected cows when blood samples were used. In addition, the combined proteins also showed the highest specificity (94 %) when using milk samples.Conclusion. Our findings provide information on the usefulness of three adhesion-associated S. aureus proteins in detecting serological markers of intramammary infections in bovines.

Training, development, and evaluation of on-farm culturing as a technical resource to reduce the use of antimicrobials in dairy herds in the Midwest Region of Santa Catarina State, Brazil / Capacitação, implementação e avaliação da cultura de leite na fazenda como recurso técnico voltado ao uso reduzido de antimicrobianos em rebanhos leiteiros no meio oeste catarinense

On-farm culturing is a technology booming in Brazil. It is based on the microbiological diagnosis of milk samples collected from clinical cases of bovine mastitis, on the farm where the animals are handled, quickly, simply, and at an affordable cost. With this resource, after 24 h of incubation, the isolated microorganisms are classified as gram positive or gram negative, helping to make decisions regarding the therapy of the animals. Considering the relevance of the Midwestern region of Santa Catarina State in the national dairy production scenario, the objectives of the study were: 1) to promote the technical training of the farmers in three dairy farms located in Treze Tílias-SC, 2) to assist in the implementation and evaluation of on-farm culturing as technological resource viewing the reduced use of antimicrobials in herds. In addition to the rapid acceptance by the farmers and their efficient training, with the use of the technology, there was a 45-50% reduction in the use of antimicrobials in dairy herds. This result demonstrated a significant effect on animal health, and determines an important cost reduction for farmers, as presented in detail in this manuscript.

A cultura de leite na fazenda (do inglês On farm culturing) é uma tecnologia em franca expansão no Brasil. Fundamenta-se no diagnóstico microbiológico de amostras de leite colhidas de casos clínicos de mastite bovina, na própria fazenda onde são manejados os animais, de forma rápida, simples e com custo acessível. Com este recurso, após 24 horas de incubação, os micro-organismos isolados são classificados em Gram-positivos ou Gram-negativos, auxiliando na tomada de decisões quanto à terapia dos animais. Considerando a relevância da região meio oeste catarinense no cenário nacional de produção leiteira, o objetivo do presente estudo foi promover a capacitação técnica dos produtores em três propriedades localizadas em Treze Tílias-SC, e auxiliar na implementação e avaliação da cultura de leite na fazenda como recurso tecnológico voltado ao uso reduzido de antimicrobianos nos rebanhos. Além da rápida aceitação pelos produtores e eficiente capacitação dos mesmos, verificou-se, com o emprego da tecnologia, redução de 45% a 50% do uso de antimicrobianos nos rebanhos. Este resultado, além de demonstrar significativo impacto em saúde animal, determinou importante redução de custos aos produtores, como apresentado detalhadamente no presente artigo.

Training, development, and evaluation of on-farm culturing as a technical resource to reduce the use of antimicrobials in dairy herds in the Midwest Region of Santa Catarina State, Brazil / Capacitação, implementação e avaliação da cultura de leite na fazenda como recurso técnico voltado ao uso reduzido de antimicrobianos em rebanhos leiteiros no meio oeste catarinense

On-farm culturing is a technology booming in Brazil. It is based on the microbiological diagnosis of milk samples collected from clinical cases of bovine mastitis, on the farm where the animals are handled, quickly, simply, and at an affordable cost. With this resource, after 24 h of incubation, the isolated microorganisms are classified as gram positive or gram negative, helping to make decisions regarding the therapy of the animals. Considering the relevance of the Midwestern region of Santa Catarina State in the national dairy production scenario, the objectives of the study were: 1) to promote the technical training of the farmers in three dairy farms located in Treze Tílias-SC, 2) to assist in the implementation and evaluation of on-farm culturing as technological resource viewing the reduced use of antimicrobials in herds. In addition to the rapid acceptance by the farmers and their efficient training, with the use of the technology, there was a 45-50% reduction in the use of antimicrobials in dairy herds. This result demonstrated a significant effect on animal health, and determines an important cost reduction for farmers, as presented in detail in this manuscript.(AU)

A cultura de leite na fazenda (do inglês On farm culturing) é uma tecnologia em franca expansão no Brasil. Fundamenta-se no diagnóstico microbiológico de amostras de leite colhidas de casos clínicos de mastite bovina, na própria fazenda onde são manejados os animais, de forma rápida, simples e com custo acessível. Com este recurso, após 24 horas de incubação, os micro-organismos isolados são classificados em Gram-positivos ou Gram-negativos, auxiliando na tomada de decisões quanto à terapia dos animais. Considerando a relevância da região meio oeste catarinense no cenário nacional de produção leiteira, o objetivo do presente estudo foi promover a capacitação técnica dos produtores em três propriedades localizadas em Treze Tílias-SC, e auxiliar na implementação e avaliação da cultura de leite na fazenda como recurso tecnológico voltado ao uso reduzido de antimicrobianos nos rebanhos. Além da rápida aceitação pelos produtores e eficiente capacitação dos mesmos, verificou-se, com o emprego da tecnologia, redução de 45% a 50% do uso de antimicrobianos nos rebanhos. Este resultado, além de demonstrar significativo impacto em saúde animal, determinou importante redução de custos aos produtores, como apresentado detalhadamente no presente artigo.(AU)

Intramammary treatment using allogeneic pure platelet-rich plasma in cows with subclinical mastitis caused by Gram-positive bacteria.

The aims of the study were (1) to compare the cure risk of intramammary treatment of pure platelet rich plasma (P-PRP) or cefquinome sulfate (CS) in cows with subclinical mastitis (SCM) caused by Gram-positive bacteria, evaluated via somatic cell count (SCC) and the microbiological analysis of milk; (2) to compare the inflammatory/anti-inflammatory response of mammary gland to both treatments through the analyses of interleukins (IL), interferon gamma (IFN-γ), and tumour necrosis factor alpha (TNF-α) in milk. A non-inferiority randomized clinical trial was conducted. The null hypothesis was that cure risk in the experimental group (P-PRP) was inferior to the reference group (CS). A total of 103 cows were selected according to SCC and presence of Gram-positive bacteria, 49 cows were treated with CS and 54 cows were treated with P-PRP after determination of its cellular and molecular quality control. Cure was assessed by milk analyses at day 21 and 22 after treatment. Cows that remained with SCM were retreated at day 26, and cure assessed at day 47 and 48. Overall, bacteriological cure was observed in 16 cows (30%) of the P-PRP group, and 35 cows (71%) in CS group. Staphylococcus aureus cure risk was higher in CS group, but inconclusive for Streptococcus spp. The mean SCC increased in relation to time only in the P-PRP group. A direct relation between time and treatment for IL-1, IL-2, and IL-6 was observed, while no differences were observed for IL-4. Furthermore, IL-1 and IL-2 increased in cows treated twice in both groups. IL-8, IFN-γ, and TNF-α showed a significant interaction between time and treatment. IFN-γ concentration was lower in the P-PRP group compared to the CS on days 0 and 22. Leukocyte counts were lower in P-PRP when compared to whole blood. TGF-ß1 and PF4 concentrations were higher in platelet lysates in comparison to P-PRGS and plasma. Moreover, PDGF-BB concentration was significantly higher in platelet lysates in comparison to plasma. Results obtained in this study demonstrate that SCM treated with PRP showed a lower rate of bacteriologic cure when compared to animals treated with CS.

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis.

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.

Prevalence of mastitis and characterization of milking management of dairy cows in northwestern Sao Paulo State / Prevalência de mastite e caracterização do manejo de ordenha de bovinos leiteiros da região de Fernandópolis, São Paulo

The lack of application of good practices in milking management can lead to the occurrence of one of the main diseases affecting the Brazilian dairy herd - mastitis. This study was developed to examine the prevalence of clinical and subclinical mastitis as well as to evaluate the contribution of milking management to this prevalence, thereby providing bases for a better control of mastitis in dairy herds at Fernandopolis region, Sao Paulo State, Brazil The study involved nine farms with 223 lactating cows. All cows were evaluated by the strip-cup test and the California Mastitis Test (CMT). In addition, the employees' conduct during milking; the physical structure of the farm; animal handling; and dry cow treatment were observed. Results were tabulated and evaluated for the development of a flowchart of good practices. Subclinical mastitis was detected in 30.5% of the farms; 66.7% perform mechanical milking and 89% have a cooling tank, but 77.7% do not sanitize the equipment after milking. Pre-and post-dipping were not performed by 89%; 66.6% do not adopt a milking line; and none of the farms realized mastitis detection tests. In conclusion, subclinical mastitis is present in the evaluated herds. By observing the management adopted on each farm, the main critical points were found to be related to failures in handling and in milking and equipment hygiene. The implementation of a flowchart of good milking practices is extremely important for the producer, since the main method to avert and control infection is prevention.(AU)

A falta da aplicação de boas práticas no manejo de ordenha pode resultar na ocorrência de uma das principais doenças que acomete o rebanho leiteiro no Brasil, a mastite. O objetivo deste trabalho foi avaliar a prevalência de mastite clínica e subclínica bem como avaliar a contribuição do manejo da ordenha nessa prevalência para que, dessa forma, se tenham subsídios que proporcionem um melhor controle da mastite em rebanhos leiteiros da região de Fernandópolis, São Paulo. Participaram da pesquisa 9 propriedades rurais, com 223 vacas em lactação. Todas as vacas em lactação foram avaliadas por meio da realização do teste da caneca de fundo escuro e CMT (“California Mastitis Test”). Além disso foi realizada observação da conduta do funcionário durante a ordenha, estrutura física da propriedade, manejo com os animais e tratamento das vacas secas. Os resultados obtidos foram tabulados, avaliados para elaboração de um fluxograma de ações de boas práticas. A prevalência de mastite subclínica detectada foi de 30,5%, sendo que, 66,7% possuíam ordenha mecânica e 89% tanque de expansão, mas 77,7% não efetuavam higiene dos equipamentos após ordenha. O pré e o pós-dipping não eram realizados por 89%, sendo que, 66,6% não faziam linha de ordenha e ninguém realizava testes de detecção de mastite. Conclui-se que a mastite subclínica está presente nos rebanhos avaliados. Pode-se observar o manejo adotado em cada propriedade e elencar os principais pontos críticos sendo eles relacionados principalmente a falhas de manejo e higiene de ordenha e equipamentos. A implantação do fluxograma de boas práticas de ordenha é de extrema importância para o produtor, uma vez que, o principal método para evitar e controlar a infecção é a prevenção.(AU)

Evaluation of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical and subclinical mastitis.

This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32-0.81; Staphylococcus aureus) to 0.90 (0.80-0.99 Streptococcus uberis), Sp varied from 0.94 (0.92-0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26-0.67; Staph. aureus) to 0.84 (0.78-0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15-0.85; E. coli) to 0.94 (0.87-1.00; Staph. aureus), Sp varied from 0.95 (0.93-0.97; Strep. uberis) to 0.99 (0.98-1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00-0.40; Escherichia coli) to 0.88 (0.80-0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00-0.26; Serratia spp.) to 0.94 (0.85-1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92-0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00-0.24; Serratia spp.) to 0.85 (0.75-0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10-0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90-0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98-1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00-0.72; E. coli) to 0.72 (0.60-0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.

Use of the rumination profile through collar sensors for mastitis diagnosis in dairy cows

Background: Mastitis is an inflammatory disease of the mammary gland, mostly associated with bacterial infections. Itis responsible for great economic losses due to decreased milk yield, discarded milk, milk composition alterations andtreatment costs, besides it impairs the animal health and welfare. The rumination time is an important behavioral markerand its assessment can be used as an early diagnosis tool, which can improve cure rate. Therefore, the aim of the presentstudy was to evaluate the sensitivity of behavior monitoring system collars in the diagnosis of mastitis and the averagerumination time (RT) of Holstein cows during the healthy period and affected by the disease.Materials, Methods & Results: The study was conducted on a commercial property located in the municipality of RioGrande, Rio Grande do Sul, Brazil. The RT data from 39 multiparous Holstein cows with an average milk yield of 38.4 L/day was collected. RT monitoring was performed using C-Tech1 collars combined with CowMed® software, which assessbehavior data from the animals and emits warning signals when it finds abnormalities in any parameter. In order to verifywhether the animals were determined correlated with diseases, the sensitivity of the data was evaluated, when the systemhad given the alert to animals considered ill, they underwent to a further clinical evaluation performed by a veterinarian toconfirm the diagnosis. From the diagnosis, the cows were divided into subclinical mastitis (SM) and clinical mastitis (CM)groups. SM was detected by the California Mastitis Test (CMT) and cows that were graded 1 (++), 2 (++) or 3 (+++) withoutthe presence of any other clinical sign were assigned to the SM group. CM was assessed by observation of abnormalitiesin milk such as changes in color and consistency, as well as the presence of lumps, clots or blood; and clinical examinationof the udder was performed for detection of hot, hard, swollen or painful quarters...(AU)

Prevalence of mastitis and characterization of milking management of dairy cows in northwestern Sao Paulo State / Prevalência de mastite e caracterização do manejo de ordenha de bovinos leiteiros da região de Fernandópolis, São Paulo

The lack of application of good practices in milking management can lead to the occurrence of one of the main diseases affecting the Brazilian dairy herd - mastitis. This study was developed to examine the prevalence of clinical and subclinical mastitis as well as to evaluate the contribution of milking management to this prevalence, thereby providing bases for a better control of mastitis in dairy herds at Fernandopolis region, Sao Paulo State, Brazil The study involved nine farms with 223 lactating cows. All cows were evaluated by the strip-cup test and the California Mastitis Test (CMT). In addition, the employees' conduct during milking; the physical structure of the farm; animal handling; and dry cow treatment were observed. Results were tabulated and evaluated for the development of a flowchart of good practices. Subclinical mastitis was detected in 30.5% of the farms; 66.7% perform mechanical milking and 89% have a cooling tank, but 77.7% do not sanitize the equipment after milking. Pre-and post-dipping were not performed by 89%; 66.6% do not adopt a milking line; and none of the farms realized mastitis detection tests. In conclusion, subclinical mastitis is present in the evaluated herds. By observing the management adopted on each farm, the main critical points were found to be related to failures in handling and in milking and equipment hygiene. The implementation of a flowchart of good milking practices is extremely important for the producer, since the main method to avert and control infection is prevention.

A falta da aplicação de boas práticas no manejo de ordenha pode resultar na ocorrência de uma das principais doenças que acomete o rebanho leiteiro no Brasil, a mastite. O objetivo deste trabalho foi avaliar a prevalência de mastite clínica e subclínica bem como avaliar a contribuição do manejo da ordenha nessa prevalência para que, dessa forma, se tenham subsídios que proporcionem um melhor controle da mastite em rebanhos leiteiros da região de Fernandópolis, São Paulo. Participaram da pesquisa 9 propriedades rurais, com 223 vacas em lactação. Todas as vacas em lactação foram avaliadas por meio da realização do teste da caneca de fundo escuro e CMT (“California Mastitis Test”). Além disso foi realizada observação da conduta do funcionário durante a ordenha, estrutura física da propriedade, manejo com os animais e tratamento das vacas secas. Os resultados obtidos foram tabulados, avaliados para elaboração de um fluxograma de ações de boas práticas. A prevalência de mastite subclínica detectada foi de 30,5%, sendo que, 66,7% possuíam ordenha mecânica e 89% tanque de expansão, mas 77,7% não efetuavam higiene dos equipamentos após ordenha. O pré e o pós-dipping não eram realizados por 89%, sendo que, 66,6% não faziam linha de ordenha e ninguém realizava testes de detecção de mastite. Conclui-se que a mastite subclínica está presente nos rebanhos avaliados. Pode-se observar o manejo adotado em cada propriedade e elencar os principais pontos críticos sendo eles relacionados principalmente a falhas de manejo e higiene de ordenha e equipamentos. A implantação do fluxograma de boas práticas de ordenha é de extrema importância para o produtor, uma vez que, o principal método para evitar e controlar a infecção é a prevenção.

Use of the rumination profile through collar sensors for mastitis diagnosis in dairy cows

Background: Mastitis is an inflammatory disease of the mammary gland, mostly associated with bacterial infections. Itis responsible for great economic losses due to decreased milk yield, discarded milk, milk composition alterations andtreatment costs, besides it impairs the animal health and welfare. The rumination time is an important behavioral markerand its assessment can be used as an early diagnosis tool, which can improve cure rate. Therefore, the aim of the presentstudy was to evaluate the sensitivity of behavior monitoring system collars in the diagnosis of mastitis and the averagerumination time (RT) of Holstein cows during the healthy period and affected by the disease.Materials, Methods & Results: The study was conducted on a commercial property located in the municipality of RioGrande, Rio Grande do Sul, Brazil. The RT data from 39 multiparous Holstein cows with an average milk yield of 38.4 L/day was collected. RT monitoring was performed using C-Tech1 collars combined with CowMed® software, which assessbehavior data from the animals and emits warning signals when it finds abnormalities in any parameter. In order to verifywhether the animals were determined correlated with diseases, the sensitivity of the data was evaluated, when the systemhad given the alert to animals considered ill, they underwent to a further clinical evaluation performed by a veterinarian toconfirm the diagnosis. From the diagnosis, the cows were divided into subclinical mastitis (SM) and clinical mastitis (CM)groups. SM was detected by the California Mastitis Test (CMT) and cows that were graded 1 (++), 2 (++) or 3 (+++) withoutthe presence of any other clinical sign were assigned to the SM group. CM was assessed by observation of abnormalitiesin milk such as changes in color and consistency, as well as the presence of lumps, clots or blood; and clinical examinationof the udder was performed for detection of hot, hard, swollen or painful quarters...

Seroprevalence and associated factors of infectious bovine rhinotracheitis and bovine viral diarrhea in dairy cows in the Caparaó region, Espírito Santo, Brazil / Soroprevalência e fatores associados à rinotraqueíte infecciosa bovina e diarreia viral bovina em vacas leiteiras na região do Caparaó, Espírito Santo, Brasil

This study evaluated the seroprevalence and associated factors of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD), and to analyze the possible relationship between IBR, BVD, and the occurrence of mastitis. For this purpose, 854 crossbred dairy cows were evaluated from 69 properties allocated in the 12 municipalities that make up the Caparaó region, Espírito Santo (ES), Brazil. The seroprevalence of IBR and BVD was determined using the indirect ELISA test. Associations between variables were estimated using the prevalence ratio (PR) with a 95% confidence interval. The chi-square test was used to verify the significance of the associations (P < 0.05). The average prevalence of IBR and BVD was 48.59% and 26.46%, respectively. Animals seroreactive for IBR were more likely to develop subclinical mastitis (P < 0.01; PR: 1.27), and cows that were seroreactive for BVD were more likely to develop clinical mastitis (P < 0.01; PR: 2.24). Mechanical milking was considered a factor associated with IBR (P < 0.01; PR: 1.36) and BVD (P < 0.01; PR: 1.25). Reproductive management by natural breeding was considered a factor associated with IBR (P < 0.01; PR: 1.22). Animals seroreactive for BVD were more likely to develop reproductive problems (P < 0.01; PR: 1.34). It was concluded that the agents that cause IBR and BVD are widely disseminated in dairy cattle herds in the municipalities of the Caparaó region, ES, Brazil. The presence of IBR and BVD increased the chances of cows developing subclinical mastitis and clinical mastitis, respectively, and the cows that were mechanically milked were more likely to be seroreactive for IBR and BVD.(AU)

O objetivo do presente estudo foi avaliar a soroprevalência e os fatores de risco associados à Rinotraqueíte Infecciosa Bovina (IBR) e Diarreia Viral Bovina (BVD), e analisar a possível relação entre IBR, BVD e à ocorrência de mastite. Para tanto, foram avaliadas 854 vacas leiteiras mestiças de 69 propriedades localizadas nos 12 municípios que compõem a região do Caparaó, Espírito Santo (ES), Brasil. A soroprevalência de IBR e BVD foram determinadas pelo teste ELISA indireto. As associações entre variáveis ​​foram estimadas pela razão de prevalência (PR) com intervalo de confiança de 95%. O teste do qui-quadrado foi utilizado para verificar a significância das associações (P < 0,05). A prevalência média de IBR e BVD foi de 48% e 26%, respectivamente. Os animais sororreagentes para IBR foram mais propensos a desenvolver mastite subclínica (P < 0,01; PR: 1,27), e as vacas sororreagentes para BVD foram mais propensas a desenvolver mastite clínica (P < 0,01; PR: 2,24). À ordenha mecânica foi considerada um fator associado a IBR (P < 0,01; PR: 1,36) e BVD (P < 0,01; PR: 1,25). O manejo reprodutivo por monta natural foi considerado um fator associado IBR (P < 0,01; PR: 1,22). Os animais sororreagentes para BVD foram mais propensos a desenvolverem problemas reprodutivos (P < 0,01; PR: 1,34). Concluiu-se que os agentes causadores de IBR e BVD estão amplamente disseminados em rebanhos leiteiros nos municípios da região de Caparaó, ES, Brasil. A presença de IBR e BVD aumentaram as chances das vacas desenvolverem mastite subclínica e mastite clínica, respectivamente, e as vacas que foram ordenhadas mecanicamente apresentaram maior probabilidade de serem sororreagente para IBR e BVD.(AU)

In vitro efficacy of iodine in the pre and post dipping against coagulase negative Staphylococcus isolated in milk of cows with subclinical mastitis / Eficácia in vitro do iodo no pré e pós dipping frente a Staphylococcus coagulase negativa isolados em leite de vacas com mastite subclínica

The efficiency of the disinfectants used in the milking management is fundamental to the success in the dairy activity, being a critical point to the control of mastitis. The objective was to evaluate the in vitro efficacy of iodine used in pre and post-dipping against coagulase negative Staphylococcus (CNS). Thus, 53 CNS isolates were studied for the action of the 1.0% disinfectant and their serial dilutions of 0.5%, 0.375% and 0.25%, in addition to two commercial presentations of iodine in concentrations of 0.5% and 0.25%. The rate of CNS inhibition achieved by iodine at 0.375%, 0.5% and 1.0% for 60 seconds, was 60.4%. In 30 seconds, iodine at 0.5% and 1.0% showed a microbial inhibition rate of 52.8% and 56.6%, respectively. The other protocols tested were less efficient. It is concluded that the greatest in vitro disinfectant activity for CNS was demonstrated by iodine at 0.375%, 0.5% and 1.0%, for 60 seconds. Microbial susceptibility tests should be carried out periodically, as well as health education practices and corrective training on the property, in order to ensure udder health and mastitis control.(AU)

A eficiência dos desinfetantes empregados no manejo de ordenha é fundamental no sucesso na atividade leiteira, sendo um ponto crítico ao controle da mastite. Objetivou-se avaliar a eficácia in vitro do iodo utilizado no pré e pós-dipping frente à Staphylococcus coagulase negativa (SCN). Foram estudados 53 isolados de SCN quanto à ação do desinfetante a 1,0% e suas diluições seriadas de 0,5%, 0,375% e 0,25%, além de duas apresentações comerciais nas concentrações de 0,5% e 0,25%. A taxa de inibição de SCN alcançada pelo iodo a 0,375%, 0,5% e 1,0% durante 60 segundos, foi de 60,4%. Em 30 segundos, o iodo a 0,5% e 1,0% apresentaram taxa de inibição microbiana de 52,8% e 56,6%, respectivamente. Os demais protocolos testados foram menos eficientes. Conclui-se que a maior atividade desinfetante in vitro para SCN foi demonstrada pelo iodo a 0,375%, 0,5% e 1,0%, durante 60 segundos. Testes de susceptibilidade microbiana devem ser realizados periodicamente, assim como práticas de educação em saúde e treinamentos corretivos na propriedade, visando garantir a saúde do úbere e o controle da mastite.(AU)

Genetic traceability of Staphylococcus aureus strains isolated from primiparous dairy cows mastitis, humans and environment in the Northeast region of Brazil / Rastreabilidade genética de cepas de Staphylococcus aureus isoladas de mastite de vacas primíparas, ordenhadores e ambiente de ordenha na região Nordeste do Brasil

This research aimed to investigate the genotypic relatedness of 18 Staphylococcus aureus strains isolated from intramammary infections in primiparous cows and extramammary sites on five dairy herds by rep-PCR using RW3A primers, and by PFGE using the endonuclease SmaI. The isolates were also evaluated in vitro for the susceptibility against beta-lactam antimicrobials drugs (penicillin and oxacillin), considering that beta-lactams are frequently used for treating staphylococcal intrammamary infections. The rep-PCR typing was highly discriminatory (D value= 0.9804) and a total of 15 patterns were detected. The PFGE method was also highly discriminatory (D value= 0.9667) and a total of 13 patterns were observed. A total of 15 out of 18 (83%) isolates were resistant to penicillin and one out of 18 (6%) to oxacillin. In conclusion, these findings confirmed the occurrence of a high genetic diversity of S. aureus strains at the herds and the presence of clonally-related strains only at the same herd, emphasizing a variety of genotypic profiles among the isolates.(AU)

Objetivou-se com este estudo investigar a correlação genética de 18 cepas de Staphylococcus aureus isoladas de infecções intramamárias em vacas primíparas e de locais extramamários em cinco propriedades leiteiras através das técnicas de PCR por sequências palindrômicas extragênicas repetitivas (rep-PCR), usando iniciadores RW3A, e de eletroforese em gel de campo pulsado (PFGE), usando a endonuclease SmaI. Os isolados também foram avaliados in vitro quanto à suscetibilidade aos antimicrobianos beta-lactâmicos (penicilina e oxacilina). A tipagem por rep-PCR foi altamente discriminatória (valor D = 0,9804) e um total de 15 padrões foram detectados. Os isolados de S. aureus foram agrupados em três grupos diferentes (A a C), com 80% de similaridade. A técnica de PFGE também foi altamente discriminatória (valor D = 0,9667) e um total de 13 padrões foi observado. A análise do dendrograma com um coeficiente de similaridade de 80% gerou dois grupos diferentes (A e B). Além disso, cepas clonais isoladas do leite foram identificadas na mesma propriedade pelos dois métodos de tipificação e, apesar da presença de cepas dominantes, nossos resultados sugerem uma alta diversidade genética dentre as cepas de S. aureus analisadas. Um total de 15, dos 18 (83%) isolados, eram resistentes à penicilina e um dos 18 (6%) à oxacilina. Assim, esses achados confirmam a ocorrência de uma alta diversidade genética de cepas de S. aureus nas propriedades e a presença de cepas clonalmente relacionadas apenas na mesma propriedade, enfatizando uma variedade de perfis genotípicos entre os isolados.(AU)