The stable fly, Stomoxys calcitrans, has a great importance in medical and veterinary health due to its feeding and reproductive habits, which can disseminate some pathogenic agents among hosts even at long distances. This study aimed to describe, for the first time in Brazil, the bacterial microbiota in segments of the stable fly. Bacterial species were isolated from three different segments (cuticle, mouth parts and abdominal alimentary tract) of the stable fly. Twenty dairy farms were visited in four municipalities: Barra Mansa, Quatis, Resende and Rio Claro in the State of Rio de Janeiro in order to collect 20 flies in each site. Dissection of the flies and procedures of isolation and identification of bacterial species were performed. A total of 161 colonies of 33 distinct species were isolated, such as Escherichia coli, Staphylococcus aureus, and S. intermedius. Stomoxys calcitrans may harbor bacterial agents on their cuticle, mouth parts and abdominal alimentary tract and these bacteria may be pathogenic to their hosts.

Stomoxys calcitrans control is primarily based on the use of chemical products; however, new methods for control have been investigated. Biological control has been proposed as a promising alternative to traditional chemicals. The current study evaluated three methods in vitro to treat S. calcitrans eggs with the fungus Metarhizium anisopliae and assessed the virulence of this fungus to immature stages of this fly. In the first method of egg exposure, eggs were immersed in conidial suspensions and transferred to Petri plates with high humidity. A high mortality was observed in both treated and control groups. In the second method, the eggs were transferred to rearing medium right after immersion in conidial suspensions; the suspensions with highest conidial concentrations, 10(7) and 10(8) conidia mL(-1), caused 96.25% and 100% mortality, respectively. In the third method, the eggs were sprayed with conidial suspensions on the rearing medium. A high mortality was observed at 10(7) and 10(8) conidia mL(-1), 78.3% and 100%, respectively. The second method had the lowest mortality in the control group and was considered the most useful method for evaluating the effect of entomopathogenic fungi on S. calcitrans eggs. While eggs were very susceptible to M. anisopliae infection, larvae and pupae were not.

The present study verified the pathogenic potential of entomopathogenic nematodes (EPNs) of the genus Heterorhabditis (Heterorhabditis bacteriophora, isolate HP88 and Heterorhabditis baujardi isolate LPP7) to immature stages of Stomoxys calcitrans in the laboratory. All EPN concentrations of the H. bacteriophora HP88 strain caused mean larval mortality greater than 90% after four days. Higher concentrations of the H. baujardi LPP7 isolate (≥50 EPNs/larva) eliminated more than 70% of larvae after six days with the concentration 200 EPNs/larva reaching mortality levels of 93.3%. The larval mortality at all concentrations of EPNs (25, 50, 100, 150, and 200 EPNs/larva) for both strains was significant (p<0.05) when compared to the respective control groups. Concentrations of H. bacteriophora HP88 yielded an LC50 of 0.36 EPN/larva and LC90 of 29.1; while H. baujardi LPP7 yielded an LC50 of 39.85 and LC90 of 239.18. H. bacteriophora HP88 provided greater inhibition of the emergence of adults when compared to the response obtained with H. baujardi LPP7. EPNs did not cause considerable mortality when applied directly to pupae. The set of observed results suggests that the EPNs of the genus Heterorhabditis, isolates HP88 and LPP7, are a promising alternative in the control of the stable fly.Keywords: Stable fly, diptera, biological control, Heterorhabditis bacteriophora HP88, Heterorhabditis baujardi LPP7. ResumoO presente estudo verificou o potencial patogênico de nematoides (NEP) do gênero Heterorhabditis (H. bacteriophora -isolado HP88 e H. baujardi -isolado LPP7) para estágios imaturos de Stomoxys calcitrans em laboratório. Todas as concentrações de NEPs da cepa HP88 causaram mortalidade larval média maior que 90% após quatro dias. Concentrações mais elevadas da cepa LPP7 (≥50 NEPs/larva) eliminaram mais de 70% após seis dias, com a concentração 200 NEPs/larva atingindo níveis de 93,3%. A mortalidade larval em todas as concentrações de NEPs (25, 50, 100, 150, and 200 NEPs/larva) para ambas as cepas foi significativa (p<0,05), quando comparadas aos respectivos grupos controle. Concentrações de H. bacteriophora HP88 obtiveram LC50 de 0,36 NEP/larva e LC90 de 29,1; enquanto H. baujardi LPP7 obteve LC50 de 39,85 e LC90 de 239,18. O isolado H. bacteriophora HP88 propiciou maior inibição da emergência de adultos, quando comparado à resposta obtida com H. baujardi LPP7. NEPs não provocaram mortalidade considerável quando aplicados diretamente sobre pupas. O conjunto de resultados observados indica os nematoides entomopatogênicos do gênero Heterorhabditis (estirpes HP88 e LPP7) como uma alternativa promissora no controle da mosca dos estábulos.Palavras-chave: Mosca dos estábulos, diptera, controle biológico, Heterorhabditis bacteriophora HP88, Heterorhabditis baujardi LPP7.

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 species.

This study aimed to verify the occurrence of Shiga toxin-producing Escherichia coli (STEC) strains in three distinct anatomic parts of the stable fly Stomoxys calcitrans by multiplex polymerase chain reaction (PCR Multiplex). According to the results obtained, E. coli was identified in 19.5% of the stable flies. Shiga toxin genes were detected in 13% of the E. coli isolated, most frequently from the surface, followed by abdominal digestive tract and mouth apparatus of insects, respectively. This is the first study to detect presence of STEC in Stomoxys calcitrans in Brazil; it has also revealed the potential role of stable flies as carriers of pathogenic bacterial agents.Keywords: Stomoxys calcitrans, Shiga toxin-producing Escherichia coli, Multiplex-PCR. ResumoEste estudo teve por objetivo avaliar a ocorrência de Escherichia coli Shiga-Toxigênica (STEC) em três diferentes partes anatômicas da mosca dos estábulos pela Reação de Polimerase em Cadeia Multiplex (PCR Multiplex). De acordo com os resultados obtidos, foi identificada E. coli em 19,5% das moscas dos estábulos colhidas. Foram detectados genes de produção de Shiga toxina em 13,63% das Escherichia coli isoladas, sendo mais frequente a superfície externa, seguido pelo trato digestivo abdominal e pelo aparelho bucal, respectivamente. Este foi o primeiro estudo no Brasil que detectou a presença de STEC em Stomoxys calcitrans e revelou o potencial papel da mosca dos estábulos em carrear um agente bacteriano patogênico.

The microbiota present in Stomoxys calcitrans larvae may assist their survival in contaminated environments through production of inhibitory substances. Bacteriological identification methods, the polymerase chain reaction (PCR) and scanning electron microscopy (SEM) were used to detect a bacterium naturally present in mucus and macerated S. calcitrans larvae. The antifungal activity was determined based on the results from disk diffusion tests on an artificial solid medium. The bacterium was identified as Stenotrophomonas maltophilia and presented antifungal activity against Beauveria bassiana sensu lato isolates CG 138, CG 228 and ESALQ 986. This result suggests that the larval microbiota is a factor that can compromise the use of B. bassiana s.l. fungus for biological control of S. calcitrans larvae.Keywords: Stenotrophomonas maltophilia, Stomoxys calcitrans, antifungal activity. ResumoA microbiota presente em larvas de Stomoxys calcitrans pode auxiliar na sua sobrevivência em ambientes contaminados, devido à produção de substâncias inibidoras. Métodos bacteriológicos de identificação, reação em cadeia da polimerase (PCR) e microscopia eletrônica de varredura (MEV) foram utilizados para detectar uma bactéria naturalmente presente no muco e macerado de larvas de S. calcitrans. A atividade antifúngica foi baseada nos resultados obtidos no teste de difusão em meio sólido artificial. A bactéria foi identificada como Stenotrophomonas maltophilia e apresentou atividade antifúngica contra os isolados CG 138, CG 228 e ESALQ 986 de Beauveria bassiana sensu lato. Estes resultados sugerem que a microbiota larval é um fator que pode comprometer o uso de B. bassiana s.l. no controle biológico de larvas de S. calcitrans.

III Isolamento de espécies enterobacterianas em Stomoxys calcitrans Isolation of enterobacterial species in Stomoxys calcitrans RESUMO

RESUMOPor ser um díptero hematófago, a mosca Stomoxys calcitrans (Linnaeus, 1758)