Bovine Mastitis Caused by Multidrug-Resistant Nocardia farcinica

Background: Mastitis caused by Nocardia is characterized by pyogranulomatous inflammation related to inadequate hygiene conditions and is difficult to treat. Prompted by the absence of documentation of Nocardia farcinica associated to bovine mastitis in the Northeast region of Brazil, this is the first report to describe bovine mastitis caused by multidrug-resistant N. farcinica. Case: Four milk samples (one from each teat) obtained from a 3-year-old Jersey cow raised on a property located in the metropolitan region of Recife, Pernambuco state, Brazil, were submitted to the Laboratory of Infectious-Contagious Diseases of the Veterinary Hospital at Universidade Federal Rural de Pernambuco. At the laboratory, samples were cultured in base agar enriched with 7% sheep blood (blood agar) in a microbiological incubator at 37°C under aerobic conditions for 72 h. After only 48 h, however, pure bacterial colony growth was observed in all samples. Macroscopic analysis revealed small colonies, with an irregular shape, dry aspect, and greyish in color. Gram-positive rods forming filaments and/or ramifications were observed using a Gram staining method. Nocardia spp. were identified according to morphotinctorial characteristics. Susceptibility testing using the disc-diffusion method in agar (antibiogram) was performed using the following antibiotics: penicillin (10 IU), tetracycline (30 µg), amoxicillin (10 µg), gentamicin (10 µg), cephalexin (30 µg), erythromycin (15 µg), cephalothin (30 µg) and ampicillin (30 µg). However, the organism exhibited resistance to all drugs; as such, a new milk sample was obtained at the same location the initial samples were collected. Samples (approximately 5 mL) were collected aseptically and separately from all four teats in sterile bottles, during which the presence of granular material was noted. Bacterial culture was performed as previously described and, after 48 h, colony growth with the same characteristics as the first isolation were observed, and with same morphotinctorial characteristics in the Gram stain. A resistance profile was observed for 14 of the antimicrobial drugs tested; sensitivity was verified only for ciprofloxacin and amoxicillin with clavulanic acid. One bacterial colony was selected and sent to the Center of Strategic Technology of Northeast (CETENE-PE) for species identification using a matrix-associated laser desorption ionization-time of flight (MALDI-TOF/MS) technique, which confirmed the species to be N. farcinica. Molecular characterization of 16s ribosomal DNA was performed using polymerase chain reaction with universal prokaryotic primers 516F-13R. Subsequently, the amplified product was subjected to sequencing, and the result was analyzed for quality using Phred base calling software; bases with a Phred value > 20 were kept. The sequence was evaluated using GenBank, in which the isolate exhibited 99% similarity to N. farcinica. Discussion: Clinical findings and animal history associated with microbiological culture and bacterial identification using the MALDI-TOF technique, as well as DNA sequencing, confirmed the case of clinical mastitis to be caused by N. farcinica. These bacteria are considered saprophytes, and their occurrence is associated with deficiencies in hygienic-sanitary management, such as not using pre- and post-dipping, which may favor mammary gland infection. Treatment of N. farcinica mastitis is effective only when properly performed, with agent identification and antibiotic sensitivity tests in vitro associated with the adoption of hygienic-sanitary measures. This is the first description of bovine mastitis caused by N. farcinica in the northeast of Brazil. Multidrug resistance should raise awareness of producers searching for laboratory aids in agent identification as well as antibiotic sensitivity tests, and to develop a proper therapeutic protocol based on results obtained in laboratory examinations

Metabolic and Hormonal Profile of overnourished sheep submited to Hyperketonemia and hyperlipidemia, and treated with recombinat bovine somatotropin

O objetivo deste trabalho consistiu em esclarecer os mecanismos bioquímicos e hormonais da ação da somatotropina recombinante bovina (r-bST), recomendada no tratamento de toxemia da prenhez (TP). Para tal, foram realizados dois experimentos nos quais induziram-se quadros de hipercetonemia ou hiperlipidemia em 39 ovelhas com elevada condição corporal tratadas ou não previamente com 160 mg de um produto comercial de r-bST, pela via subcutânea. A hipercetonemia foi provocada pela infusão IV de 5 mM/kg P.V. de beta hidroxibutirato (BHB) no decorrer de duas horas e a hiperlipidemia com injeção IV de solução 20% de óleo de peixe, perfazendo 0,54g de triacilglicerol/kg P.V. no decorrer de quatro dias. Essas quantidades infundidas lograram provocar os quadros esperados. No primeiro experimento, o tratamento com r-bST promoveu aumento significativo da gliconeogênese por meio da conversão de BHB em glicose, maiores teores de insulina, com consequente aumento da resistência insulínica, incremento na concentração de IGF1 que levou a mobilização de nitrogênio ureico para os depósitos e maior atividade de GGT. No segundo experimento, o rbST gerou destacado aumento da gliconeogênese, pela metabolização dos AGNEs e de BHB em glicose, com maior destaque aos AGNEs, elevação dos teores de insulina e do percentual de resistência insulínica, e maior atividade da AST e GGT e bilirrubinal direta e total. Assim, conclui-se que a atuação farmacológica do r-bST se baseia na vigorosa capacidade do aumento da gliconeogênese, diminuindo concomitantemente os nefastos catabólitos (AGNEs e BHB) no processo da TP. Foi realizado ainda um experimento subsidiário para comparar a capacidade diagnóstica da detecção de BHB, em sangue total, entre o kit diagnóstico enzimático (Randox®), padrão ouro, e tiras reativas comerciais (Precision Xceed®) lidas em dosímetro portátil. Foram obtidas 247 amostras de sangue das ovelhas nas quais tinham sido infundida solução de BHB, no experimento um. Duas faixas de acetonemia foram consideradas: de 0,7 a 1,5 mM caracterizada como cetose inaparente e acima deste valor como cetose clínica. A correlação entre os métodos foi alta (r = 0,98 ; P < 0,001). Na cetose inaparente as tiras reativas apresentaram sensibilidade de 0,97 e especificidade de 0,89 e na cetose aparente sensibilidade de 0,99 e especificidade de 0,75, respectivamente. As tiras reativas podem ser empregadas eficientemente no diagnóstico precoce de casos de TP, com custo baixo e rápida leitura ao pé dos animais.The aim of this study was to clarify the biochemical and hormonal mechanisms of action of recombinant bovine somatotropin (r-bST), recommended in the treatment of pregnancy toxemia (PT). Two experiments were conducted inducing hyperlipidemia or hyperketonemia in 39 high body condition sheep pretreated or no with 160 mg of a commercial product of rbST subcutaneously. The hyperketonemia was caused by IV infusion of 5 mM / kg BW of beta-hydroxybutyrate (BHB) during two hours and hyperlipidemia with IV injection of a 20% fish oil solution corresponding to 0.54 g of triglyceride / kg BW in four days course. These infusions successfully caused the expected effects. In the first experiment, treatment with rbST caused a significant increase of gluconeogenesis by converting BHB into glucose, higher levels of insulin, with consequent increased insulin resistance, increase in the concentration of IGF1 leading to mobilization of urea nitrogen for deposits and increased activity of GGT. In the second experiment, the r-bST greatly increased gluconeogenesis generated by metabolism of non esterified fatty acids (NEFA), BHB and glucose, with emphasis on NEFA, elevated levels of insulin and the percentage of insulin resistance, and increased activity of AST and GGT and bilirrubin direct and total. In conclusion, the pharmacological action of r-bST is based on vigorous increase capacity on gluconeogenesis, concomitantly reducing the adverse catabolites (NEFA and BHB) in the PT process. An additional experiment was performed to compare the diagnostic capability of detecting BHB in whole blood, between the enzymatic diagnostic kit (Randox®), considered gold standard, and commercial test strips (Precision Xceed®) read in hand-held meter. 247 sheep blood samples were obtained in the first experiment in which BHB solution had been infused. Two ketonemic tracks were considered: 0.7 to 1.5 mM characterized as unapparent ketosis and above this value as a clinical ketosis. The correlation between the methods was high (r = 0.98, P <0.001). In test strips method, ketosis unapparent showed sensitivity of 0.97 and specificity of 0.89 and in ketosis apparent sensitivity of 0.99 and specificity of 0.75, respectively. The test strips can be used efficiently in the early diagnosis of cases of TP, with fast reading and low cost

Comparative study of hormonal and metabolic profile of pregnant ewes carrying one, multiple or no pregnant fed high density energetic diet

Paralelamente ao aumento da criação intensiva de ovinos há o aumento da ocorrência de doenças metabólicas, dentre elas a Toxemia da Prenhez. Embora existam estudos envolvendo esta enfermidade, ainda pouco se sabe sobre a etiologia da mesma. O presente estudo tem por objetivo analisar o perfil metabólico e hormonal de ovelhas vazias, gestando um, dois e três fetos, alimentadas com dieta de alta densidade energética durante toda a gestação. 42 ovelhas foram divididas em três grupos experimentais, G3 (prenhes de 3 fetos), G2 (prenhes de 2 fetos), G1 (prenhes de 1 feto) e G0 (não gestantes) As avaliações iniciaram ao 60º dia pós-cobertura e a cada 14 dias até o primeiro mês pós-parto. Pretende-se encontrar, dentre as variáveis analisadas, colesterol, triglicérides, uréia, creatinina, albumina, proteínas totais, globulina, glicose, AGLs, BHB, AST, GGT, glucagon, insulina, cortisol, T3 e T4 e pesquisa de corpos cetônicos qual a indicadora mais precoce de distúrbio metabólico durante a gestação.Foi encontrada diminuição das concentrações de glicose, de uréia, creatinina, proteína total, globulina e albumina nos momentos referentes ao terço médio de gestação (M2 e M3) e aumento das concentrações de AGLs, T3 e T4 séricos no terço final de gestação (M6) nos grupos de ovelhas gestantes G3, G2 e G1 feto em relação ao grupo de ovelhas não gestantes. As ovelhas que apresentaram comprometimento com sua prole ao final da gestação e que apresentaram sinais clássicos da TP apresentaram aumento dos índices protéicos no M3, aumento dos teores de AGLs, BHB, creatinina e T4 no M6. AGLs e BHB mostraram-se melhores e mais precoces indicadores que a glicose, no que se refere a alteração do metabolismo energético de ovelhas prenhes no terço final de gestação. As variáveis protéicas mostraram-se indicadores importantes no perfil comparativo entre ovelhas gestantes e não gestantes e quanto a alteração do metabolismo que se instala em fêmeas que apresentaram comprometimento. Nestas, foi constatado diminuição do metabolismo refletidas pelas baixas concentrações dos hormônios T3 e T4. As demais variáveis hormonais não apresentaram diferenças notáveis no estudo comparativo entre os perfis. O presente estudo permitiu a comparação do perfil metabólico e perfil hormonal entre ovelhas prenhes de um, dois, três fetos e ovelhas vazias. Permitiu a identificação de algumas alterações destes perfis entre ovelhas que não apresentaram comprometimento com as que apresentaram comprometimento ao final da gestação, no entanto, numero maior de animais comprometidos são necessários na pesquisa das variáveis preditoras do distúrbio metabólico e nutricional relacionado à TP.Pregnancy Toxemia (PT) is a metabolic disease that commonly affects pregnant ewes and does during late gestation. The intensive management conditions provided to ewes herds in the last decades, cause impairment on the occurrence of this disease. The mainly cause that starts the metabolic disturbance involved in PT is not already established. The aim of this study is to evaluate the metabolic and hormonal profile of pregnant ewes with one, two, three and non gestating ewes fed a high density energetic diet during the period of gestation. 42 Santa Ines sheep breeding were divided in four experimental groups, G3 (pregnant of 3 lambs); G2 (pregnant of 2 lambs); G1 (pregnant of 1 lamb); G0 (non gestating). The study started on 60º day of pregnancy and repeated after fourteen days until the first month after parity. We collected and analyzed blood serum and plasma samples and urine samples. We determined the concentrations of cholesterol, triglycerides, urea, creatinin, albumin, globulin, total protein, glucose, nonsterified fatty acids (NEFA), &beta; hidroxibutyrate (BHB), AST, GGT, glucagon, insulin, cortisol, T3, T4 and BHB urines. The aim is to find, which one of these will be the earliest marked sign to predict the occurrence of PT. According to the results of this experiment, we conclude that plasma NEFA and plasma BHB, showed be the mainly energetic indicators related to energetic status in late gestating ewes and non gestating ones. The protein variables showed be very useful in prediction protein status of breed and prediction any metabolism change in affected ewes. The hormonal analyses didnt show much usefulness in the prediction of metabolic or hormonal change between the four groups or in relation of affected gestating ewes.The present study allowed the comparison of metabolic and hormonal profile of pregnant ewes of three, two, one and non gestating ewes. Allowed the identification of some changes of these profiles between them and related to affected ones, however a bigger number of animals should be necessarily in the search on predicted variables in the disturbance related to PT