Genetic relationship between Escherichia coli strains isolated from dairy mastitis and from the stable fly Stomoxys calcitrans

Pesquisa Veterinária Brasileira

Regua-Mangia, Adriana H.³  Souza, Miliane M.S.¹  Bittencourt, Avelino J.¹  Castro, Bruno G.² 

[1] Universidade Federal Rural do Rio de Janeiro, Seropédica, Brazil;Universidade Federal de Mato Grosso, Sinop, Brazil;Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

关键词:  Random amplification of polymorphic DNA;    RAPD;    milk;    stable fly;    clonal species;   

DOI  :  10.1590/S0100-736X2016000600004

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

:The stable fly Stomoxys calcitrans (Linnaeus, 1758) has been described as a potential spreader of infectious agents to cattle herds. Among the agents transmitted by this fly, Escherichia coli has attracted attention due to its potential to cause gastrointestinal disorders as well as environmental mastitis in dairy cows. Therefore, the aim of this study was to isolate and to assess the genetic diversity and the clonal relatedness among E. coli isolates from the milk of dairy mastitis and from stable flies anatomical sites by the Random Amplification of Polymorphic DNA (RAPD-PCR) technique. The molecular typing revealed a high degree of genetic polymorphism suggesting that these microorganisms have a non-clonal origin. Identical electrophoretic profiles were observed between E. coli isolates from different flies, different mammary quarters of the same cow and from cows on a single farm. These results reveal the circulation of the same bacterial lineages and suggest the role of the stable fly in bacterial dispersion. Considering the high pathogenic potential of this bacterial species, our findings alert to a more effective health surveillance.Index Terms: Random amplification of polymorphic DNA; RAPD; milk; stable fly; clonal speciesResumo:A mosca dos estábulos Stomoxys calcitrans é descrita como um importante dispersor de agentes infecciosos aos bovinos. Dentre os agentes veiculados por esta mosca a bactéria Escherichia coli ganha relevância devido ao seu potencial em desenvolver alterações gastroentéricas, bem como mastite bovina ambiental. Desta forma, objetiva-se com este estudo isolar e acessar a diversidade genética e relação de clonalidade entre isolados de E. coli provenientes de casos de mastite e de moscas dos estábulos utilizando a técnica da Amplificação Randômica do DNA Polimórfico (RAPD). A tipagem molecular revelou elevado polimorfismo genético sugerindo que esses microrganismos têm origem não clonal. Perfis eletroforéticos idênticos entre si foram observados entre amostras isoladas de diferentes moscas, quartos mamários de uma mesma vaca, bem como de diferentes vacas dentro de uma mesma propriedade. Esses resultados revelam a circulação de uma mesma linhagem bacteriana e sugerem o papel da Stomoxys calcitrans na dispersão bacteriana. Considerando o elevado potencial patogênico dessa espécie bacteriana, nossos achados alertam para uma vigilância sanitária mais efetiva.Termos de Indexação: Amplificação randômica do DNA polimórfico; RAPD; leite; mosca dos estábulos; clonesIntroductionDespite the reduction in the number of cases of bovine mastitis of contagious origin in the world, several authors have reported that cases of environmental origin have been increasing, mainly caused by Escherichia coli (Fairbrother et al. 2015). The proportion of E. coli isolated as an etiological agent from the milk of cows suffering from mastitis varies according to the place of study. According to Nevala et al. (2004), in Finland fewer than 20% of mastitis cases are caused by E. coli, while Shpigel et al. (1998) and Shpigel et al. (2008) reported 60% in Israel, as well as the E. coli is the leading cause of acute mastitis in dairy animals in that country. In Brazil the cases of mastitis caused by E. coli have a prevalence ranging from 4.0% to 15%, possibly due to the stock-raising system used in the country, where the animals generally are confined to stables only at the time of milking, thus reducing the possibility of contaminating the mammary quarters by feces (Ferreira et al. 2007).Hyper-acute and acute udder infections by E. coli occur in the first weeks of clinical and sub-clinical form. They are difficult to cure therapeutically, and cases of systemic involvement can lead to death by toxemia (Hertl et al. 2010). The main source of contamination is by direct contact of the recently milked mammary quarters with materials contaminated by the agent, such as feces, unhygienic equipment, dirty hands of stable workers and possibly some insects, as muscid flies (Pyörälä 2002, Ryman et al. 2013).Some muscid flies have been studied for their potential to transmit agents causing mastitis. Among these, the flies Hydrotea irritans, Haematobia irritans and Musca domestica are most often studied in Europe and USA and have been implicated on carriage of bacterial mastitis strains in experimental cases (Chirico et al. 1997, Braverman et al. 1999, Anderson et al. 2012).The stable fly has also been studied among the Muscidae family causing injury to Brazilian cattle herds. Some authors have found similarity in the isolation of agents from cases of bovine mastitis and in Stomoxys calcitrans (Castro et al. 2001, Moraes et al. 2004). However, these authors did not confirm the real potential of stable flies to transmit agents of bovine mastitis; they only reported the possibility of this occurrence.This study was designed to isolate and to assess the genetic diversity among E. coli isolates from mastitic milk of dairy cattle and stable fly from dairy farms in Rio de Janeiro state.Materials and MethodsFor this study 10 dairy farms in the municipalities of Barra Mansa and Resende located at Rio de Janeiro state, Brazil, were visited. These two municipalities were selected because these locations are the main dairy region of the state of Rio de Janeiro (IBGE 2009). Also, previous studies detected a high prevalence of the stable fly and in these municipalities it was identified common bacterial species from the microbiota of macerated flies and mastitic milk, but the genetic relationship was not evaluated (Castro et al. 2001, Moraes et al. 2004).At the visited farms, the cows ready for milking were submitted to the California Mastitis Test (CMT). After detected the positive quarters, milk samples were taken from these after washing the teats with neutral soap and water and then drying and disinfecting them with iodized alcohol. The milk samples were placed in sterile tubes and taken under refrigeration to the laboratory for bacterial isolation.Twenty flies were also collected at each farm, using an insect sweep net, giving preference to those that were feeding or flying at most one half meter from the animals (Bramley et al. 1985, Puri-Giri et al. 2015). Each fly was placed in a sterile test tube and taken under refrigeration to the laboratory.All the dissection procedures of the flies were performed under a fume hood near a Bunsen burner. The flies were identified according to Bowman (2014), and only the S. calcitrans specimens were killed by freezing at -10oC for five minutes. Then, each frozen fly was placed in a test tube containing enriched brain-heart infusion (BHI) Broth, agitated in this tube and transferred to another test tube containing 70% alcohol for 10 minutes for sterilization of the outer surface, as described by Hillerton & Bramley (1985) and Castro et al. (2013).Then each fly was placed on its back in a sterile Petri dish and a stereoscopic microscope was used to aid removal of the mouth apparatus and abdominal digestive tract, as described by Castro et al. (2007). Each segment was then macerated in BHI Broth and incubated in a bacteriological chamber at 37 ºC for at least 24 hours.After the incubation period, the samples were transferred to Petri dishes containing the MacCon

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