Occurrence of Shiga-toxigenic Escherichia coli in Stomoxys calcitrans ( Diptera : Muscidae ) Ocorrência de Escherichia coli Shiga-Toxigênica em Stomoxys calcitrans ( Diptera : Muscidae )

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.


Introduction
Muscidae species such as the stable fly, Stomoxys calcitrans, stands out among the main insects that disperse infectious agents to farm animals.Besides using cattle feces and secretions for food and reproduction during its life phases, this fly takes blood meals from various animals.It can thus spread a number of infectious agents such as parasitic forms of helminths, protozoa, fungi and bacteria.Parasitism by this fly has specifically been associated with the outbreak of ailments among cattle (GRACZYK et al., 2001;BITTENCOURT;CASTRO, 2004).
Among the infectious agents carried by stable flies, enterobacteria have attracted attention because of their bio-reproductive characteristics.Therefore, although Escherichia coli is part of the indigenous microbiota of the intestinal tract of nearly all mammals, some strains, such as Shiga-toxigenic E. coli (STEC), can cause diseases in a great number of animals.STEC belongs to the Enterohemorrhagic group of E. coli ('EHEC') and have been described as being etiological agents of human illnesses, ranging from mild gastroenteritis to serious infections as hemorrhagic colitis and hemolytic uremic syndrome (KARMALI, 1989;GRIFFIN;TAUXE, 1991).
The natural reservoirs of STEC are domesticated and wild ruminants, mainly cattle, which disperse this agent through their feces (CAPRIOLI et al., 2005).Cattle do not usually present any clinical symptoms.Furthermore, products from these animals, such as milk and meat, are also potential sources of this agent, mainly when these products are eaten without proper heat treatment, as pasteurization or cooking (LIRA et al., 2004;HUSSEIN;SAKUMA, 2005).
Information about the occurrence and genetic-epidemiological aspects of these microorganisms in Brazil is still limited.Strains producing Shiga toxin, codified by the stx gene, have been isolated from apparently healthy cattle (CERQUEIRA et al. 1999;FARAH et al., 2007), with high prevalence of this strain and apparent predominance of non-O157 STEC (CERQUEIRA et al. 1999;IRINO et al. 2005;PIGATTO et al., 2008).
Flies of the Muscidae family play an important role in disseminating STEC and other strains of E. coli with high pathogenic potential (IWASA et al., 1999;AHMAD et al., 2007;TALLEY et al., 2009).
Therefore, the aim of this study was to evaluate the capacity of stable flies, collected on dairy farms in the state of Rio de Janeiro, Brazil, to carry STEC on/in three different structures: body surface, mouthparts and abdominal digestive tract.

Materials and Methods
In this study, we visited 10 dairy farms in the state of Rio de Janeiro, Brazil.On each farm, 20 specimens of Stomoxys calcitrans flying around the cattle were captured, placed in sterile glass tubes and kept under refrigeration until further use.
Briefly, for laboratory procedures, the captured flies were identified according to Furman and Catts (1982), and only the S. calcitrans specimens were killed by freezing at -10 °C.Each frozen fly was placed in a tube containing enriched brain-heart infusion (BHI) broth, agitated in this medium and transferred to another tube containing 70% alcohol for 2 minutes as described by Hillerton and Bramley (1985).Following the sterilization step of the outer surface, each fly was put on its back on 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).The anatomic segments were macerated in BHI broth and incubated in bacteriological chamber at 37 °C for 24 hours.
After the incubation period, the samples were transferred to Petri dishes containing MacConkey agar and Eosin Methyl Blue (EMB) agar culture media.Colonies were selected according to their morphophysiological characteristics and submitted to preliminary bacterial identification assays: Gram staining, for observation of morphological and staining characteristics; and hydrolysis test with 3% KOH, to confirm the Gram catalysis test results.For identification of bacterial species, the following tests were conducted: behavior in triple sugar iron agar; behavior in SIM agar; production of acids by glycosis; sugar fermentation; Voges Proskauer (VP) test; methyl red (MR) test; nitrate reduction; galatinase production; citrate and malonate degradation; and other differential tests according to the microorganism involved (KONEMAN et al., 2008).
After isolation and identification of E. coli, bacterial samples were submitted to detection of the stx1, stx2 and eae genes, which was performed by multiplex PCR reactions according to previous recommendations.Bacterial DNA extraction, amplification conditions and electrophoresis procedures were carried out according to the methodology proposed by China et al. (1996) with some modifications.The E. coli samples were seeded in 5 mL of Müeller Hinton broth (Merck) at 37 °C for 18 to 24 hours.After the incubation period, 300 µL of the culture was centrifuged for 30 seconds in a micro-centrifuge and the supernatant was discarded.The sediment was resuspended in 50 mL of sterile distilled water and boiled for 10 minutes, after that, the bacterial lysate was centrifuged for 30 seconds and the supernatant was used as DNA source.
Each PCR analysis included positive DNA controls E. coli E40705 (stx1 and eae positive) and E30121 (stx2 and eae positive) and a non-template control.Amplification reactions for the eae, stx1 and stx2 genes yielded DNA fragments of 570, 388 and 807 bp, respectively, which were analyzed after electrophoresis on 2.0% w/v agarose gels run for 3hs at 80V in 1X Tris-borate buffer (10X, Tris 1M, Boric Acid 1M, EDTA 0.01M, pH 8.0) and stained with ethidium bromide (0.5 mg/mL).A 100bp ladder (Invitrogen Brasil) was used as DNA molecular weight marker.Genetic profiles were visualized in an ultraviolet transilluminator and documented with the UVI Pro program (UVITEC, Cambridge, UK).

Results and Discussion
Isolation and identification of colonies showed a total of 159 bacterial agents in the three parts of the 200 flies collected.The majority (44, 27.7%) was identified as E. coli.This result coincides with those of earlier studies on stable flies, which revealed a greater prevalence of E. coli compared to the remaining enterobacteria isolated (MORAES et al., 2004;CASTRO et al., 2007).
In the present study, 52.3% of the E. coli samples identified were isolated from the body surface, 31.8% from the mouthparts and 15.9% from the abdominal digestive tract of the flies analyzed.According to the results, E. coli strains were isolated in 19.5% of the stable flies.This is possibly explained by the usual occurrence of this microorganism in environments where feces are deposited and decomposing food is located near feed troughs, where all the developmental phases of the fly are present.
The highest prevalence of isolates observed on the body surface shows the great capacity of flies to carry bacteria on their body or secretions, as reported by Nazni et al. (2005).Moreover, identification of E. coli in both the mouth apparatus and abdominal digestive tract confirms the potential of this fly to act as a vector of E. coli in the environment, especially considering its ability to fly long distances (BAILEY et al., 1973).Castro, B.G. et al. Rev. Bras. Parasitol. Vet.According to Rochon et al. (2004Rochon et al. ( , 2005)), E. coli seems to play an important role in the development of the non-parasitic phases of the stable fly.The authors reported that in experiments with laboratory-reared stable flies, a greater number of adult flies emerged from entomological cages containing media enriched with E. coli than from cages with no enriched media.The authors also reported that the newly emerged flies hold living E. coli bacteria in their digestive tube, so that E. coli is part of the microbiota of the immature forms of the stable fly, favoring their development.This observation highlights the role of flies in spreading E. coli to the environment.
After the initial laboratory process of isolation and identification, all the E. coli isolates were submitted to multiplex chain polymerase reaction (Multiplex-PCR) to detect the stx1, stx2 and eae genes.Of the 44 E. coli samples isolated from the stable flies collected, 6 contained genes characteristic of the STEC pathotype.
Of all the STEC isolated from S. calcitrans, one sample carried only the stx1gene; four carried both the stx1 and stx2 genes; and only one was positive for the three genes investigated: stx1, stx2 and eae.
Among the six E. coli strains carrying virulence genes, three came from the body surface, two from the intestinal content and one from the mouth apparatus of the stable flies.Considering that from all these three anatomical parts, particularly from the body surface, viable bacterial cells were isolated; it is assumed that these stable flies can spread STEC from all three parts evaluated.Keen et al. (2006) previously reported presence of STEC in some muscid flies, including S. calcitrans.However, the present findings are the first showing the capacity of this fly to disseminate STEC in Brazil.
According to the results, most of the samples investigated carried the stx2 gene, a fact that indicates the need for increased epidemiological vigilance and public health surveillance because of its high pathogenic potential for humans, as also described by Aktories and Just (2000).
Stable flies can contaminate both human and animal food with this agent, because this fly species utilizes organic compounds for oviposition and stalls and feeding troughs as resting places, where it usually defecates.
Another noteworthy observation in this study was the presence of STEC in the mouth apparatus of the flies collected.Such findings reveal the potential risk to spread this agent while flies take blood meals, allowing the contamination of animals and humans with bacteria at the time of inserting the proboscide or when regurgitating during feeding.
The stable fly normally uses substrates with high potential to shelter bacterial agents with pathogenic capacity (CASTRO et al., 2008).Therefore, further studies on this fly are important to shed light on the impact of parasitism on livestock and public health.
These results show the importance of the stable fly as a carrier of microorganisms with recognized high pathogenic potential.Greater attention should thus be focused on its control in order to prevent E. coli transmission, which can cause diarrheal disease and severe clinical syndromes characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency, with high risk of human death, especially for those who work or live near stables.