Stable Flies ( Stomoxys calcitrans L.) from Confined Beef Cattle Do Not Carry Shiga-Toxigenic Escherichia coli (STEC) in the Digestive Tract

Introduction

Beef and dairy cattle are asymptomatic reservoirs of the important human foodborne pathogen Shiga-toxigenic Escherichia coli (STEC) (Karmali et al., 2010). Stable flies are blood-feeding insects that cause the U.S. cattle industry about $2.2 billion in economic losses annually (Taylor et al., 2012). Stable fly larval survival and development depend on an active bacterial community in animal manure (Romero et al., 2006) and adult stable flies may acquire STEC from the cattle environment by contact and during feeding from the contaminated cattle skin. However, very little is known about their potential role in the ecology of STEC. Recently, a study from Brazil reported six STEC isolates from stable flies from dairy farms (de Castro et al., 2013); however, they did not provide any information on STEC serotypes and prevalence of STEC-positive stable flies. In the present study, we assessed the prevalence of eight serotypes of STEC in stable flies collected from a commercial cattle feedlot using culturing and molecular approaches.

Materials and Methods

Stable flies (n = 50) were collected on a weekly basis from June 9 to August 25 from a commercial feedlot in central Nebraska using a sweep net and processed using two approaches for isolation of STEC. In the parallel study, the fecal samples from the same feedlot were collected on a weekly basis and processed for detection of STEC (Dewsbury et al., 2015).

Direct plating involved surface sterilization (Zurek et al., 2000) to eliminate cross-contamination during collection followed by homogenization of individual flies in 1.0 mL phosphate-buffered saline (pH 7.2) and dilution plating of the homogenate on sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) and on modified Posse agar (mP) as described by Dewsbury et al. (2015). Colony-forming units (CFU) were counted on each medium and the means were expressed as mean ± standard error of the mean throughout the study. Non-sorbitol fermenting (colorless) colonies from CT-SMAC were screened by the latex agglutination test (Oxoid, Basingstoke, England) for detection of E. coli O157:H7.

Enrichment: The fly homogenate (700 μL) was added to 10 mL of EC broth and incubated at 40°C for 6 h at 50 rpm and were subjected to immunomagnetic separation (IMS) following the manufacturer's instructions (Dynal Biotech, New Hyde Park, NY). Enriched samples were pooled into group A (O103+O104+O26) and group B (O145+O45+O121+O111) and dilution plated on mP.

For non-O157 serogroups, 6 colonies from direct plating and 12 (2 × 6) colonies from enrichment/IMS were tested per fly for STEC-8 as described by Dewsbury et al. (2015) using multiplex polymerase chain reaction (PCR) with STEC-7 (Bai et al. 2012) and O104 (Paddock et al. 2013) specific primers. Individual isolates from positive samples were further confirmed for serotype by single PCR and also screened for virulence genes (stx1, stx2, eae, ehxA) using 4-plex PCR (Bai et al., 2012).

Correlation between prevalence of enteric-positive flies and bacterial concentration on mP was assessed by the multiple regression analysis (p < 0.05). One-way analysis of variance was performed to compare the enteric concentrations over the 12-week period on each of mP and CT-SMAC (p < 0.05) in Origin 7 (OriginLab, Northampton, MA).

Results and Discussion

Of 180 stable flies, 67 (37.2%) and 55 (30.5%) were positive for bacteria on mP and CT-SMAC, respectively. The concentration of enterics ranged from 1.0 × 10¹ to 3.2 × 10⁷ (mean: 3.6 ± 1.05 × 10⁶) CFU/fly on mP and 1.0 × 10¹ to 6.0 × 10⁵ (mean: 1.2 ± 1.08 × 10⁴) CFU/fly on CT-SMAC (Fig. 1). Interestingly, the weekly prevalence of positive flies peaked (86.6%) in week 12 and corresponded to the highest enteric concentration (mean: 1.7 ± 0.32 × 10⁷ CFU/fly). A moderate correlation was found between the prevalence of enteric positive flies and the bacterial concentration (R ² = 0.509). There was no significant difference in enteric concentrations on CT-SMAC over the 12-week period (p = 0.856); however, a significant difference (p = 0.001) was observed in enteric concentrations on mP between week 12 and all other weeks with exception of week 4 and week 10 (Fig. 1).

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FIG. 1.

Prevalence and concentration of enterics associated with stable flies on a beef cattle farm. The 12-week sampling period (June 9–August 25) and a total of 15 stable flies were processed each week. Log colony-forming unit (CFU) per fly is presented as mean ± standard error of the mean. Detection limit for enterics was 10 CFU/fly on both modified Posse agar (mP) and MacConkey agar with cefixime and tellurite (CT-SMAC). Numbers above the gray bars represent total number of flies with detectable colony count on mP following direct plating technique/total number of flies processed each week by enrichment and immunomagnetic separation. The numbers above the white bars represent number of flies with detectable colony count on CT-SMAC. Black down arrows above the bars indicate flies positive for Escherichia coli O26 (week 8) and O45 (week 11). Different letters above gray bars indicate significant differences (p < 0.05) among CFU counts over 12 weeks.

Our previous studies reported 44.3% (411/982) stable flies from the pastured and confined cattle environment carried enterics with a mean concentration of 6.4 × 10⁴ CFU/fly. We also found 12.9% (120/928) of stable flies carried fecal coliforms with a mean concentration of 8.7 × 10³ CFU/fly (Mramba et al., 2006). In contrast to stable flies, the majority (95.4%) of house flies collected from a beef cattle feedlot carried fecal coliforms with a mean concentration 2.1 × 10⁵ CFU/fly (Alam and Zurek, 2004).

All sorbitol-negative colonies on CT-SMAC tested negative for the O157 antigen. Of 180 stable flies, only 2 flies were positive for the serotypes of interest: O45 (1 isolate) and O26 (2 isolates), all from the enrichment/IMS approach and neither of them carried the virulence genes tested. In a parallel study, cattle feces were collected from the same feedlot during the same collection period and screened for the presence of STEC following a similar approach. They showed that 0.8–41.4% of fecal samples in summer were positive for one or more of the STEC serotypes O157, O103, O26, and O145 (Dewsbury et al., 2015). Overall, our current data indicate that adult stable flies do not carry STEC-8 despite the presence of STEC in cattle feces on the same farm. These data are in agreement with the previous reports of Rochon et al. (2004, 2005) where they suggested based on laboratory bioassays that stable fly larvae and pupae retain and accumulate E. coli in the gut; however, most teneral adults were bacteria free. Their bioassays also showed that survival and retention of E. coli throughout various life stages of stable flies were significantly lower compared to that of house flies (Rochon et al., 2004, 2005).

Adult blood-feeding flies secrete salivary compounds such as complex mixtures of serine proteases, endonucleases, antithrombins, and antimicrobial peptides to evade host defense response (Wang et al., 2009) and may help in digestion of microbes during their passage through the crop and intestinal tract of the fly. Based on our study, we conclude that stable flies do not play a major role as a biological vector of STEC.