Abstract. An epidemic of enterohemorrhagic colitis caused by Escherichia coli O157:H7 (EHEC-O157) occurred in a nursery school in a rural area of Japan in September 1996. The EHEC-O157 were isolated both from patients and houseflies collected at the school. The flies were suspected to be mechanical vectors of the pathogen. Feeding experiments of EHEC-O157 to houseflies showed that the ingested bacteria were harbored in the intestine of flies and continued to be excreted at least for 3 days after feeding. Scanning electron microscopy showed that a large number of EHEC-O157 adhered to the surface of the housefly mouthparts and actively proliferated in the minute spaces of the labellum. Food masses containing EHEC-O157 in the fly intestine were completely surrounded by a peritrophic membrane during digestion and discharged rapidly. The persistence of bacteria in the intestine and feces is mainly a result of proliferation in the mouthparts and accumulation in the crop. Our results strongly suggest that houseflies are not simple mechanical vectors of EHEC. The epidemiologic potential of houseflies to disseminate EHEC-O157 may be greater than initially suspected.The housefly (Musca domestica vicina) and flies in general are considered to be mechanical vectors of many kinds of pathogens such as bacteria, 1,2 protozoa, 3 viruses, 4 and helminth eggs. 5,6 In recent outbreaks of enterohemorrhagic colitis in nursery schools in Japan, the epidemiologic survey isolated enterohemorrhagic Escherichia coli O157:H7 (EHEC-O157) from both houseflies collected in the school and from patients. The DNA patterns and the type of verotoxin were identical in EHEC-O157 isolated from both flies and patients. 7,8 This result strongly indicated that houseflies in nursery schools disseminated EHEC-O157 to foods, drinks, plates, and utensils, although direct evidence of the transmission has not been clarified. The infective dose for EHEC-O157 is considered to be very low, similar to that of Shigella flexneri or S. dysenteriae. 9,10 There are many qualitative reports of microbial flora in flies, but there are no reports of the number of bacteria that are harbored in the intestine or crop or how long the potential for dissemination of bacteria persists. We investigated the vector potential of houseflies for EHEC-O157 by feeding the flies with bacteria and monitoring them for several days after feeding. We also used scanning electron microscopy to determine whether EHEC-O157 adhered to the surface of housefly mouthparts and transmission electron microscopy to determine whether bacteria proliferated and persisted in the alimentary canal of flies. MATERIALS AND METHODS Experimental feeding of EHEC-O157 to houseflies.Houseflies maintained in our insectary were used. Two strains of EHEC-O157 (verotoxin 1-or verotoxin 1Ϫ and 2Ϫproducing strains) were grown in trypticase soy broth (Becton Dickinson, Cockeysville, MD). The concentration of bacteria was approximately 10 9 colony-forming units (CFU)/ml. Adult (6-8-day old), female flies were allowed to fe...
We previously reported that enterohemorrhagic Escherichia coli O157: H7 (EHEC) proliferates in the mouthparts of the house fly Musca domestica vicina Macquart and are excreted for at least 3 d after ingestion. However, the role of the crop and excretory behavior of the house fly in contamination of human food with EHEC is not known. In the current experiments, EHEC persisted in the crop of house flies for at least 4 d. The number of EHEC in an excreted droplet was approximately 10(4) 1 h after bacterial feeding, > 1.8 x 10(5) 3 h after feeding, and then drastically decreased after 24 h. Excretion is one of the major mechanisms for decreasing number of EHEC in the crop and gut of the house fly. The frequency of excretion by females with developing eggs in their ovary was clearly higher (6.5 min per drop) than for males or females with mature eggs. Minute eosin-sign around a container filled with eosin-supplemented trypticase soy broth might be derived from frequent contact by house fly contaminated mouthparts. These results show that frequent excretion potentially enhances the dissemination of EHEC to foods, particularly during the first 24 h after ingestion of the bacteria.
The distribution of prothoracicotropic hormone in the pupal brain of Manduca sexta has been determined by an in vitro assay for prothoracic gland activation. Prothoracicotropic activity was observed in both the brain and retrocerebral complex, but predominantly in the dorsolateral regions of the protocerebrum. Of the two groups of neurosecretory cells present in this area of the brain, only the two lateral type III neurosecretory cells exhibited significant prothoracicotopic hormone activity. Further analysis revealed that the neurohormone was localized in only one of the two type III cells, suggesting that a single neurosecretory cell in each hemisphere is the source of the hormone at the stage examined (day 0). Prothoracicotropic hormone activity was detected in both the corpora allata and the corpora cardiaca, but the corpora allata contained 2 to 9 times the activity of the corpora cardiaca, depending on developmental stage. The significantly higher level of activity in the corpora allata suggests that they may be the neurohemal organs through which the prothoracicotropic hormone of Manduca is released.The role of the brain in the neuroendocrine control of insect molting was demonstated about 4 decades ago in a series of classical experiments utilizing ligation, decapitation, and implantation techniques (1). Since that time, various other approaches, both indirect (2-5) and direct (6), have confirmed the tenet that the brain produces a neurohormone that stimulates the prothoracic glands to synthesize the prohormone ecdysone (7), whose hydroxylation product ecdysterone then elicits the molt. Although the prothoracicotropic hormone (PTTH) is the primary effector of insect postembryonic development (8), neither the source of PTTH within the brain nor the site of its release into the hemolymph has been directly demonstrated.It is presumed that PTTH is the product of specific cerebral neurosecretory cells (NSC) (9). Studies attempting to localize PTTH by the implantation of different groups of NSC or injection of homogenates of portions of the brain have yielded various results among the several insect species investigated. For example, in the reduviid bug Rhodnius prolixus, and the aphid Megoura viciae, PTTH activity appears to be located in the medial neurosecretory cells (M-NSC) of the protocerebrum (10, 11), whereas in the sphingid Manduca sexta the lateral neurosecretory cells (L-NSC) of the pupal brain may be the source of the neurohormone (5). By contrast, in the saturniid Hyalophora cecropia, both M-NSC and L-NSC are thought to produce PTTH (12).Definitive identification of the NSC that produce PTTH has not been made, in part due to the nature of the bioassays used to assess hormone activity (6, 9). However, with the recent development of a specific and sensitive in vitro assay for PTTH (6), the localization of this neurohormone in specific cerebralThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in ac...
An in vitro assay for the prothoracicotropic hormone has been developed that utilizes an ecdysone radioimmunoassay to quantify the increase in the rate of ecdysone synthesis elicited by the neurohormonal activation of the prothoracic glands. The rapidity, reproducibility, and accuracy of the assay were maximized by using one member of a gland pair as the control and the other as the test gland. This was possible because the basal rates of ecdysone synthesis by the members of a gland pair were equivalent. Activation was demonstrated to be dose dependent'and specific, with prothoracicotropic hormone activity present only in homogenates of brain. The in vitro activation of the prothoracic glands was verified with the Manduca bioassay for the prothoracicotropic hormone in which the morphological responses to the hormone were correlated with increased in vivo ecdysone titers. These results provide unequivocal evidence that the activation of the prothoracic glands by the prothoracicotropic hormone is direct and suggest that activation represents an increase in a basal rate of ecdysone synthesis. The endocrine function of the insect brain in the process of metamorphosis was first demonstrated in 1922 (1), but nearly 2 decades passed before it was demonstrated that this function involved the control of molting (2, 3). The initial observations on the role of a cerebral neurohormone in the molting process have since been confirmed by various implantation, extirpation, and ligation experiments (4-6). On the basis of these in vvo studies, it has been assumed that this brain hormone, now termed prothoracicotropic hormone (PTTH), acts directly on the prothoracic glands (PGs) to stimulate the synthesis of the steroid prohormone ecdysone (7). Hydroxylation of ecdysone at C-20 to form ecdysterone (20-hydroxyecdysone) then occurs in tissues peripheral to the PGs, and it is ecdysterone that initiates the molting process (7).Although PTTH was the first hormone to be discovered in insects, of the three major hormones involved in metamorphosis it alone remains to be chemically characterized. A necessary prerequisite to the chemical characterization of this putative peptide hormone is the unequivocal demonstration that PTTH directly activates the PGs. Recent attempts have not been successful due in part to the assay methods used to measure PTTH activity (8-10). Current PTTH assays are biological in nature and indirect by definition, in that they measure secondary morphological responses presumably elicited by a PTTH-stimulated increase in ecdysterone titer (10-12). However, with the. availability of a radioimmunoassay for ecdysteroids (13) and culture techniques for maintaining prothoracic glands in vitro (14), it is now possible to demonstrate the direct activation of the PGs by PTTH.This study details the development of an assay for PTTH that utilizes these two methods and proves that PTTH acts directly on the PGs to stimulate ecdysone synthesis.MATERIALS AND METHODS Animals. The tobacco hornworm (Manduca sexta) was used for...
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