Vectorial Competence of Larvae and Adults of Alphitobius diaperinus in the Transmission of Salmonella Enteritidis in Poultry

Abstract

Introduction:

The ingestion of food products originating from poultry infected with Salmonella spp. is one of the major causes of food poisoning in humans. The control of poultry salmonellosis is particularly difficult since birds are asymptomatic and numerous factors may expedite the maintenance of bacteria in poultry production facilities.

Objective:

The aim of the study was to determine the vectorial capacity of adults and larvae of Alphitobius diaperinus (Coleoptera: Tenebrionidae) in the experimental transmission of Salmonella Enteritidis phage type 4 to 1-day-old specific pathogen-free White Leghorn chicks.

Methods:

Adult insects and larvae were starved for 1 day, fed for 24 h or 7 days on sterile ration that had been treated with Salmonella Enteritidis phage type 4, and the levels of bacterial infection were determined. Infected adult insects and larvae were fed to groups of day-old chicks, after which bacteria were recovered from cecum, liver, and spleen samples over a 7-day period.

Results:

Infected larvae were more efficient than adult insects in transmitting Salmonella Enteritidis to chicks. Higher concentrations of bacteria could be reisolated from the cecum, liver, and spleen of chicks that had ingested infected larvae compared with those that had ingested infected adults.

Conclusions:

The control of A. diaperinus, and particularly of the larvae, represents a critical factor in the reduction of Salmonella spp. in poultry farms.

Introduction

T he lesser mealworm or litter beetle Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) is the most commonly found insect pest in poultry facilities in both Brazil (Chernaki-Leffer et al. 2002) and worldwide (De Las Casas et al. 1968). The first records of infestation of aviaries by this beetle date from 1950 (Gould and Moses 1951). Its introduction into animal production systems apparently occurred through the presence of contaminated feed (O'Connor 1987), but the pest spread rapidly by virtue of the agricultural practice of using spent litter as fertilizer (Le Torc'h 1979). A. diaperinus inhabits poultry droppings and litter, and is of particular importance as a vector of numerous pathogens, including viruses (Eidson et al. 1966, Snedeker et al. 1967, Goodwin and Waltman 1996, Watson et al. 2000), fungi (De Las Casas et al. 1970), protozoa (Goodwin and Watman 1996, Huber et al. 2007), and helminths (Karunamoorthy et al. 1994). Additionally, the beetle can act as a source of infection by Campylobacter spp. (Bates et al. 2004), Escherichia coli, Salmonella spp., and various other bacteria (McAllister et al. 1994, Chernaki-Leffer et al. 2002).

A. diaperinus completes its cycle from egg to adult in 42.5 days at a constant temperature of 28°C (Chernaki and Almeida 2001), and each female has the potential to produce more than 2000 eggs (Steelman 1996). Hence, in the absence of appropriate control measures, a new generation of insects would be available for each new lot of poults. The beetles often serve as an alternative food source for birds, resulting in the transmission of microorganisms, a reduction in the take-up of feed, and, consequently, a decline in weight gain (Axtell and Arends 1990).

The consumption of poultry and poultry-derived products that are infected with Salmonella constitutes a major source of human food poisoning. Hence, the carcasses of domestic fowl in which the presence of Salmonella has been detected should be destroyed. The lack of control of bacterial infection in a poultry production facility can result in significant economical losses. The presence of Salmonella Typhimurium in A. diaperinus adults and larvae has been reported (De Las Casas et al. 1968), and the bacteria could be isolated from the feces of beetles 28 days after a single 24 h exposure to a contaminated source (McAllister et al. 1994). The Salmonella serotypes Heidelberg, Worthington, Saint Paul, Typhimurium var. Copenhagen, Chester (Harein et al. 1970), and Indiana (Skov et al. 2004) have all been isolated from adult insects.

The objective of the present study was to determine whether A. diaperinus is able to harbor Salmonella Enteritidis and to transmit the bacteria to poults. For this purpose, adult beetles and larvae were artificially infected with the bacteria and analyzed through bacteriological examination. Infected adult insects and larvae were fed to groups of chicks, and Salmonella Enteritidis was subsequently reisolated from cecum, liver, and spleen samples.

Materials and Methods

Details of the project were submitted to and approved by the Ethics Committee on Animal Research of the University of São Paulo, São Paulo, SP, Brazil. All procedures were carried out in compliance with current Brazilian regulations relating to the use of experimental animals as contained in Guidelines for Ethical Conduct in the Care and Use of Animals—COBEA.

Insects

A. diaperinus adults, free from contamination with Salmonella spp., were collected at a commercial aviary in the municipal district of Louveira, SP, Brazil. Adults were maintained under abiotic conditions in plastic boxes (17 cm long × 17 cm wide × 14 cm high) containing sterile wood shavings and covered with a net lid. Insects were fed with a commercial diet consisting of corn and soybean meal (Cojac, Jacarei, SP, Brazil), without antibiotics, that had been moistened with distilled sterile water. Black corrugated paper was supplied for oviposition, and larvae of F1 and F2 generations were collected.

Salmonella Enteritidis: preparation of inoculum and reisolation from samples

Salmonella Enteritidis strain SA 109 phage type 4 (Nunes et al. 2003) was obtained from the culture collection of the Ornithopathology Laboratory, Faculty of Veterinary Medicine, University of São Paulo, Brazil. A stock solution of inoculum for the infection of insects was prepared by incubating the bacteria in Luria–Bertani broth for 4 h at 37°C with agitation (60 rpm). The number of colony-forming units (cfu)/mL was determined by inoculating 100 μL of the broth (diluted 10-fold with 0.1% peptone in water, pH 7.0) onto xylose lysine tergitol-4 (XLT₄) agar (Difco, Detroit, MI) and streaking with a Drigalsky's loop.

To recover Salmonella Enteritidis from chicks that had died during the experiment, samples of cecum, spleen, and liver were enriched by incubation in tetrathionate broth (Difco) for 36 h at 37°C, and the resulting cultures streaked onto XLT₄ agar and incubated for 48 h at 37°C.

The biochemical identification of isolated bacteria was carried out using the Lac EMIC 9T system (Probac do Brasil Produtos Bacteriológicos, São Paulo, SP, Brazil), and positive isolates were submitted for serological identification.

Experimental infection of A. diaperinus with Salmonella Enteritidis

Sets of A. diaperinus adults (n = 120) and larvae of approximately 1 cm (n = 120) were each separated into 6 groups of 20 individuals and maintained without food for 24 h before the start of the experiment. After this time, five test groups of adults and five of larvae each received 5 g of ration that had previously been sterilized (humid heat at 121°C for 15 min and subsequently dried at 37°C) and treated once with 5 mL of Salmonella Enteritidis phage type 4 containing 1 × 10⁹ cfu/mL. The control groups of adults and larvae each received 5 g of ration that had been moistened with 5 mL of Luria–Bertani broth. Insects were maintained in plastic containers (5.5 cm diameter × 6.0 cm high) covered with a sterile screen at a temperature of 27 ± 1°C with a scotophase of 24 h.

Salmonella Enteritidis was reisolated from five insects from each of the five treatment groups 24 h after receiving contaminated ration, and from the remaining 15 insects (3 replicates; n = 5) from each group on day 7. In each case, the insects were killed by cooling at − 80°C for 10 min and then submitted to an external decontamination process as described by De Las Casas et al. (1968) with modifications. Briefly, insects were washed sequentially with 2% sodium hypochlorite, 50% ethanol, and twice with 0.1% peptone in water (pH 7.0). Insects were then macerated in 0.1% peptone in water (pH 7.0; five insects/mL), the samples diluted 10 times in the same solution, and aliquots (100 μL) of the suspension inoculated onto XLT₄ agar. After incubation for 48 h at 37°C, the number of cfu/mL was determined.

Administration of infected A. diaperinus to chicks

One-day-old specific pathogen-free White Leghorn chicks, kindly donated by Laboratório BioVet, Vargem Grande Paulista, SP, Brazil, were housed in batteries (each 1 m²) and supplied with water and rations ad libitum. Birds were divided into four groups (n = 40), and, within each group, the treatment was replicated four times (n = 10 per replicate). Control group 1 was fed normal adult beetles, control group 2 received normal larvae, treatment group 3 was fed adult beetles that had received Salmonella-contaminated ration for 7 days, and treatment group 4 was fed on larvae that had received Salmonella-contaminated ration for 24 h. Larvae and adult beetles (five insects per chick) were placed in separate containers and offered as food on the first day of the experiment. Chicks ingested the larvae spontaneously, but they had difficulty with and showed lack of interest in consuming adult beetles, so these were administered directly via the oropharynx using forceps. The number of cfu/mL administered was determined after maceration of five insects in duplicate.

After 7 days, chicks were euthanized with CO₂ and the liver, spleen, and cecum obtained aseptically. Samples were placed into tared sterile plastic bags, reweighed, and macerated in an amount of 0.1% peptone in water (pH 7.0) equivalent to 90% of the weight of the organs. Macerated samples were inoculated onto 100 μL of XLT₄ agar, streaked with a Drigalsky's loop, and incubated for 48 h at 37°C. The number of cfu/mL was determined and log (cfu/g) was calculated. The presence of Salmonella Enteritidis was determined in the liver, spleen, and cecum of each of the birds that had died during the course of the experiment, after enrichment of the samples in tetrathionate broth and inoculation onto XLT₄ agar.

Statistical analysis

Statistical analyses were carried out with the aid of Minitab^® Statistical Software, version 15 (Minitab, State College, PA; 2005). Values of log (cfu/mL) of Salmonella Enteritidis obtained after reisolation of bacteria from control and infected insects (larvae and adults), and values of log (cfu/g) from liver, spleen, and cecum of control and treated chicks were submitted to comparative analysis using the Mann–Whitney test. Differences between median values were considered significant for values of p < 0.05.

Results

Experimental infection of A. diaperinus with Salmonella Enteritidis

All groups of insects tested positive for the presence of Salmonella Enteritidis after exposure to contaminated ration for a period of 24 h. A range of 3.5 to 5.0 log (cfu/mL) of Salmonella Enteritidis was recovered from A. diaperinus adults (n = 5), while bacteria in the range 4.0 to 6.6 log (cfu/mL) were recovered from larvae (Table 1). After 7 days feeding on contaminated ration, 13 out of the 15 replicates of adult insects examined were positive with a range of 4.0 to 7.3 log (cfu/mL) of Salmonella Enteritidis. Regarding the larvae, only five of the replicates tested were positive for the bacteria, and the log (cfu/mL) values ranged from 5.0 to 7.0 (Table 2). The potential of A. diaperinus adults and larvae for the transmission of Salmonella Enteritidis was not significantly different (p = 0.08) after feeding for 24 h on contaminated ration, whereas after feeding for 7 days on the same ration, adult insects showed a significantly (p < 0.01) higher capability than larvae did. No Salmonella spp. could be isolated from any of the insects in the control groups (Tables 1 and 2).

Table 1.

Levels of Infection in Groups of Alphitobius diaperinus Adults and Larvae After Feeding for 24 h on 5 g of Ration Containing 5 mL of Salmonella Enteritidis (1 × 10⁹ cfu/mL)

	Salmonella Enteritidis recovered [log (cfu/mL)]
Experimental groups (n = 5 insects per group)	Adult insects	Larvae
Group 1	5.0	5.0
Group 2	3.5	5.0
Group 3	3.7	5.9
Group 4	4.0	4.0
Group 5	4.5	6.6
Median (Q1–Q3)	4.0 (3.6–4.7)a	5.0 (4.5–6.3)a
Infected group (%)	100	100
Control groupb	0	0

There were no differences between the groups (p > 0.05).

Control insects were fed on 5 g of ration moistened with 5 mL of LB broth.

Q1, first quartile; Q3, third quartile; LB, Luria–Bertani.

Table 2.

Levels of Infection in Replicate Groups of Alphitobius diaperinus Adults and Larvae After Feeding for 7 Days on 5 g of Ration Contaminated in the First Day with 5 mL of Salmonella Enteritidis (1 × 10⁹ cfu/mL)

	Salmonella Enteritidis recovered [log (cfu/mL)]
Experimental groups (n = 5 insects per replicate)	Adult insects	Larvae
Group 1
Replicate 1	5.3	7.0
Replicate 2	5.0	0
Replicate 3	5.7	6.0
Group 2
Replicate 1	4.0	0
Replicate 2	0	0
Replicate 3	7.3	0
Group 3
Replicate 1	6.5	5.0
Replicate 2	7.0	5.5
Replicate 3	5.0	Not determined
Group 4
Replicate 1	5.9	0
Replicate 2	7.3	0
Replicate 3	6.0	0
Group 5
Replicate 1	4.5	0
Replicate 2	6.0	5.5
Replicate 3	0	0
Median (Q1–Q3)	5.8 (4.9–6.6)a	0 (0–5.5)a
Infected group (%)	86.7	33.3
Control groupb	0	0

There were significant differences between the groups (p < 0.05).

Control insects were fed on 5 g of ration moistened with 5 mL of LB broth.

Q1, first quartile; Q3, third quartile; LB, Luria–Bertani.

Administration of infected A. diaperinus to chicks

The mean levels [log (cfu/mL)] of Salmonella Enteritidis present in the adults and larvae of A. diaperinus fed to chicks were 5.3 ± 2.259 and 5.0 ± 0.995, respectively.

During the experiment, seven chicks died—specifically, five from the test groups and two from the control groups. Salmonella Enteritidis could be isolated from the birds in the test groups, but not from those of the control groups.

Insects were efficient in the transmission of Salmonella Enteritidis to chicks, since 88.7% and 100% of birds tested positive for the bacteria after ingestion of adult insects and larvae, respectively (Table 3). Of the chicks that had been fed on infected adult insects, 22.4% were positive for the presence of Salmonella Enteritidis in the cecum and the median recovery of Salmonella spp. was zero log (cfu/mL) (Tables 3 and Table 4). These results were significantly different (p = 0.01) from the equivalent values [i.e., 55.26% and 1.097 log (cfu/mL), respectively] recorded for birds that had been fed on infected larvae. With respect to the recovery of bacteria from liver and spleen, there were significant differences between adult insects and larvae (p = 0.0004) with median values of 1.644 log (cfu/mL) (87.35%) and 2.603 log (cfu/mL) (97.37%), respectively. Higher levels of Salmonella Enteritidis could be isolated from systemic sites than from the cecum irrespective of the source of infection (adult insects or larvae). A further interesting aspect was that, independent of the organ analyzed, chicks fed on infected larvae presented higher concentrations of bacteria than those fed on adult beetles.

Table 3.

Capability of Salmonella Enteritidis–Infected Alphitobius diaperinus Adults and Larvae to Transmit Infection to 1-Day-Old Specific Pathogen-Free Chicks

	Treatment group 3 fed on adult insects a		Treatment group 4 fed on larvae a
Replicate experiments (n = 10 birds per replicate)	Cecum	Liver/spleen	Cecum	Liver/spleen
Replicate 1	44.4b (9)	88.9b,c (9)	55.6b (9)	100b (9)
Replicate 2	25 (8)	75 (8)	70 (10)	90c (10)
Replicate 3	10 (10)	100 (10)	55.6 (9)	100 (9)
Replicate 4	10 (10)	80 (10)	40 (10)	100 (10)
Organs infected (%)	22.4	86.0	55.3	97.5
Birds infected (%)	88.7c		100c

Samples were obtained 7 days after the experimental birds were fed with five infected insects (per chick) in the first day.

Percentile of positive (number of chicks tested).

Bird presenting liver/spleen negative but cecum positive.

Table 4.

Levels of Salmonella Enteritidis in Cecum and Liver/Spleen of 1-Day-Old Specific Pathogen-Free Chicks Transmitted by Infected Alphitobius diaperinus Adults and Larvae

	Salmonella Enteritidis (log cfu/mL) recovered from cecum					Salmonella Enteritidis (log cfu/mL) recovered from liver/spleen
	Interquartile range					Interquartile range
Insect stage fed to chicks a	Minimum	Q1	Median	Q3	Maximum	Minimum	Q1	Median	Q3	Maximum
Adult	0.000	0.000	0.000	0.000	3.852	0.000	0.897	1.644	2.543	3.395
Larvae	0.000	0.000	1.097	2.511	3.689b	0.000	1.816	2.603	3.073	6.895c

Experimental groups (n = 40 birds per group initially).

There were significant differences between the cecum of the groups (p < 0.05).

There were significant differences between the liver/spleen of the groups (p < 0.05).

Q1, first quartile; Q3, third quartile.

Discussion

Information concerning the participation of vectors in the transmission of pathogens is essential for the development of strategies for the control of viruses or bacteria (Axtell and Arends 1990). In the present study, it was demonstrated that 100% of the groups of A. diaperinus adults and larvae were infected with Salmonella Enteritidis after exposure to contaminated ration for 24 h. However, after consumption of contaminated feed over a period of 7 days, 86.7% of adults but only 33.3% of larvae were infected. Crippen et al. (2009) demonstrated that the exposure to Salmonella Typhimurium (2 × 10⁶ cfu/mL) for only 2 h was sufficient to infect 100% of A. diaperinus, and that the bacteria were present throughout the alimentary canal. As insects possess a complex and efficient defense system, it is feasible that Salmonella Enteritidis might have been eliminated from the larvae over time. However, it is also possible that the insect immune system had not been fully activated in the first 24 h of infection.

Lipopolysaccharides present in the external membrane of bacteria can trigger defense mechanisms in insects either by covalent association to the hemocyte surface (Charalambidis et al. 1996) or by cascade activation of the prophenoloxidase system (Söderhall and Cerenius 1998), a process that involves a number of enzymes that are present in the hemolymph, plasma, or circulating hemocytes. Lectins also link to lipopolysaccharides and participate in clearing bacteria from the hemolymph, and this suggests that the binding of lectins to bacterial surfaces may be a signal for recognition and subsequent response of hemocytes (Gillespie et al. 1997). Takahashi et al. (1986) reported that the largest production of lectin in larvae of Sarcophaga peregrina occurred within 24 h after challenge, suggesting that the cfu of Salmonella/larvae was higher after 24 h than after 7 days. Bulet et al. (1991) detected a strong antibacterial activity initiated by the injection of heat-killed bacteria into larvae of Zophobas atratus. These authors identified peptide A, which acts on Gram-negative bacteria, as the principal component responsible for the observed activity.

It is possible that in the present study the activation of hemocytes by bacterial lipopolysaccharides induced the synthesis of antibacterial peptides in the larvae together with other defense mechanisms that were responsible for the lower levels of infection observed after 7 days. Our results corroborate those of McAllister et al. (1994), who were able to isolate Salmonella Typhimurium from the feces of adult A. diaperinus for up to 28 days after the ingestion of contaminated ration for a 24 h period. According to De Las Casas et al. (1972), factors that may influence the concentration of bacteria that can be recovered from an insect include the age and sex of the insect, and the quantity of food present in the alimentary channel. Another relevant point is the change in developmental stage that may be associated with a reduction in the level of infection of an insect (McAllister et al. 1994). In Sarcophaga peregrina, for example, a lectin was described that was activated at the larval stage in response to chemical challenge (Takahashi et al. 1986).

In the experimental infection of chicks by administration of infected A. diaperinus, adult insects and larvae presented similar potential for infecting the chicks, that is, the bacterial concentration was 5.3 ± 2.259 and 5.0 ± 0.995 log (cfu/mL), respectively, and both were efficient in transmitting Salmonella Enteritidis. Higher concentrations of bacteria could be reisolated from systemic sites compared with the cecum, irrespective of whether the chick had ingested adult insects or larvae. This result shows the high competence of Salmonella Enteritidis strain SA 109 phage type 4 to cause systemic infection.

The capability of Salmonella spp. to cause disease depends on a range of parameters associated with bacterial virulence, the immune system of the host, environmental factors, including pH, detergents, and digestive enzymes, and competition with the existent microbiota (Slauch et al. 1997). The invasion of epithelial cells by Salmonella spp. involves the participation of type III secretion system proteins, effector proteins, and adenosine diphosphate (Humphries et al. 2001, Kimbrough and Miller 2002, Knodler et al. 2002). Aabo et al. (2002) compared the capacities of different Salmonella serotypes to invade epithelial cells and reported that Enteritidis, Typhimurium, and Berta were the most efficient while Hadar, Virchow, Infantis, and Tennessee presented much lower capabilities.

In the present study, the concentration of Salmonella Enteritidis recovered from the cecum, liver, or spleen of experimental chicks was larger in birds that had been fed infected larvae than in those who had received infected adult insects. Therefore, it is evident that a larger number of viable bacteria from the larvae had managed to reach the intestine compared with the bacteria from adult insects. Although, the form of administration could have influenced the result, since ingestion of larvae might have allowed the liberation of Salmonella Enteritidis in the crop, the environment of which could lead to the activation of genes for the synthesis of acid-stress proteins permitting adaptation of the bacteria to the extreme pH conditions of the proventricle and gizzard. However, there are reports that the growth of bacteria in a moderately acid atmosphere induces the synthesis of proteins that protect microorganism from extreme conditions of pH (Bearson et al. 1997, 1998, Audia et al. 2001). Such results suggest that inducement of acid tolerance would prepare the bacterial cell to support the inhospitable enteric microenvironment (Foster 1995, 1999, Audia et al. 2001).

Considering that the bacterial inoculum (5.3 ± 2.259) from adult insects presented great variability, chicks fed on adults may have ingested a greater concentration of bacteria than chicks fed on larvae. It is possible that Salmonella Enteritidis was released and inactivated in the crop and proventriculus, and hence the low-observed bacterial concentration in the cecum, liver, and spleen. This hypothesis is supported by the study of Ford (1974), who determined the pH of the gastrointestinal tract in chickens and found pH values of 5.1 in the crop, 2.1 in the proventriculus and gizzard, and 6.8 to 7.4 in the duodenum, jejunum, and ileum. However, it is also possible that the lower bacterial concentrations found in cecum, liver, and spleen resulted from the smaller bacterial inoculum presented by adult insects compared with the larvae.

The epidemiology of Salmonella spp. is complex and involves various environmental factors. Persistence of Salmonella Enteritidis in the environment is an important issue since it has been demonstrated that this bacteria can be isolated from litter, dry feces, feed, centipedes, and beetles over a period of 26 months after birds had vacated the chicken shed, and for up to 8 months from the adjacent soil (Davies and Breslin 2003). Moreover, A. diaperinus is one of the vectors of Salmonella spp. (McAllister et al. 1994, Davies and Wray 1995, Liebana et al. 2003, Skov et al. 2004, Kinde et al. 2005, Hazeleger et al. 2008), and the transmission of Salmonella Indiana over two consecutive hatches of chicks was associated with the presence of bacteria in litter beetles remaining in the empty shed after disinfection (Skov et al. 2004).

The results of the present study demonstrate that both adults and larvae of A. diaperinus are efficient in the transmission of Salmonella spp. in chickens. Chicks fed enthusiastically on the larvae of the beetle, and it is possible that this form of ingestion specifically enabled large numbers of bacteria to survive the acid stress of the gastrointestinal tract and to multiply in the intestine. Clearly, the control of this insect, particularly of the larvae, represents a critical factor in the reduction of Salmonella spp. in poultry farms.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Aabo

, Christensen

, Chadfield

, Carstensen

et al. Quantitative comparison of intestinal invasion of zoonotic serotypes of Salmonella enterica in poultry. Avian Pathol, 2002; 31:41–47.

Audia

, Webb

, Foster

. Breaking through the acid barrier: an orchestrated response to proton stress by enteric bacterium. Int J Med Microbiol, 2001; 291:97–106.

Axtell

, Arends

. Ecology and management of arthropod pests of poultry. Annu Rev Entomol, 1990; 35:101–126.

Bates

, Hiett

, Stern

. Relationship of Campylobacter isolated from poultry and from darkling beetles in New Zealand. Avian Dis, 2004; 48:138–147.

Bearson

, Bearson

, Foster

. Acid stress responses in enterobacteria. FEMS Microbiol, 1997; 147:173–180.

Bearson

, Wilson

, Foster

. The low pH-inducible, PhoPQ-dependent acid tolerance response protects Salmonella Typhimurium against inorganic acid stress. J Bacteriol, 1998; 180:2409–2417.

Bulet

, Cociancich

, Dimarcq

J-L

, Lambert

et al. Insect immunity. Isolation from a Coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family. J Biol Chem, 1991; 266:24520–24525.

Charalambidis

, Foukas

, Marmaras

. Covalent association of lipopolysaccharide at the hemocyte surface of insects as an initial step for its internalization, protein-tyrosine phosphorylation requirement. Eur J Biochem, 1996; 236:200–236.

Chernaki

, Almeida

. Exigências térmicas, período de desenvolvimento e sobrevivência de imaturos de Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) Neotrop Entomol, 2001; 30:365–368.

10.

Chernaki-Leffer

, Biesdorf

, Almeida

, Leffer

EVB

et al. Isolamento de enterobactérias em Alphitobius diaperinus e na cama de aviários no Oeste do Estado do Paraná, Brasil. Revista Brasileira de Ciência Avícola/Braz J Poult Sci, 2002; 4:243–247.

11.

Crippen

, Sheffield

, Esquivel

, Droleskey

, Esquivel

. The aquisition and internalization of Salmonella by the lesser mealworm, Alphitobius diaprinus (Coleóptera: Tenebrionidae) Vector Borne Zoonot Dis, 2009; 9:65–72.

12.

Davies

, Breslin

. Persistence of Salmonella Enteritidisphage type 4 in the environment and arthropod on an empty free-range chicken farm. Environ Microbiol, 2003; 5:79–84.

13.

Davies

, Wray

. Contribution of the lesser mealworm beetle (Alphitobius diaperinus) to carriage of Salmonella Enteritidis in poultry. Vet Poult, 1995; 137:407–408.

14.

De Las Casas

, Harein

, Mirocha

. Detection of the mycotoxin F-2 in the confused flour beetle and the lesser mealworm. J Econ Entomol, 1970; 63:412–415.

15.

De Las Casas

, Harein

, Pomeroy

. Bacterium and fungi within the lesser mealworm collected from poultry brooder houses. Environ Entomol, 1972; 1:27–30.

16.

De Las Casas

, Pomeroy

, Harein

. Infection and quantitative recovery of Salmonella Typhimurium and Escherichia coli from within the lesser mealworm, Alphitobius diaperinus (Panzer) Poult Sci, 1968; 47:1871–1875.

17.

Eidson

, Schmittle

, Goode

, Lal

. Induction of leukosis tumors with the beetle Alphitobius diaperinus. Am J Vet Res, 1966; 27:1053–1057.

18.

Ford

. The effect of the microflora on gastrointestinal pH in the chick. Brit Poult Sci, 1974; 15:131–140.

19.

Foster

. Low pH adaptation and the acid tolerance response of Salmonella Typhimurium. Crit Rev Microbiol, 1995; 21:215–237.

20.

Foster

. When protons attack: microbial strategies of acid adaptation. Curr Opin Microbiol, 1999; 2:170–174.

21.

Gillespie

, Michael

, Kanost

, Trenczek

. Biological mediators of insect immunity. Annu Rev Entomol, 1997; 42:611–643.

22.

Goodwin

, Waltman

. Transmission of Eimeria, viruses, and bacteria to chicks: darkling beetles (Alphitobius diaperinus) as vectors of pathogens. J Appl Poult Cattle, 1996; 5:51–55.

23.

Gould

, Moses

. Lesser mealworm infestation in the broiler house. J Econ Entomol, 1951; 44:265–265.

24.

Harein

, De Las Casas

, Pomeroy

, Cork

. Salmonella spp. and serotypes of Escherichia coli isolated from the lesser mealworm collected in poultry brooder houses. J Econ Entomol, 1970; 63:80–82.

25.

Hazeleger

, Bolder

, Beumer

, Jacobs-Reitsma

. Darling beetles (Alphitobius diaperinus) and their larvae as potential vectors for the transfer of Campylobacter jejuni and Salmonella enterica serovar Paratyphi B variant Java between successive broiler flocks. Appl Environ Microbiol, 2008; 74:6887–6891.

26.

Huber

, Gouilloud

, Zenner

. A preliminary study of natural and experimental infection of the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) with Histomonas meleagridis (Protozoa: Sarcomastigophora) Avian Pathol, 2007; 36:279–282.

27.

Humphries

, Townsend

, Kingsley

, Nicholson

et al. Roll of fimbriae as antigens and intestinal colonization factors of Salmonella serovars. FEMS Microbiol Lett, 2001; 201:121–125.

28.

Karunamoorthy

, Chellappa

, Anandan

. The life history of Subulura brumpti in the beetle Alphitobius diaperinus. Indian Vet J, 1994; 71:12–15.

29.

Kimbrough

, Miller

. Assembly of the type III secretion needle complex of Salmonella Typhimurium. Microbes Infect, 2002; 4:75–82.

30.

Kinde

, Castellan

, Kerr

, Campbell

et al. Longitudinal monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar Enteritidis and other Salmonellae. Avian Dis, 2005; 49:189–194.

31.

Knodler

, Celli

, Hardt

W-D

, Vallance

et al. Salmonella effectors within the single pathogenicity island are differentially expressed and translocated by separate type III secretion systems. Mol Microbiol, 2002; 43:1089–1103.

32.

Le Torc'h

. The new pest of rearing buildings (Alphitobius diaperinus in pig sties, Brittany) Phytoma, 1979; 308:31–33.

33.

Liebana

, Garcia-Migura

, Clouting

, Clifton-Hadley

et al. Molecular fingerprinting evidence of the contribution of wildlife vectors in the maintenance of Salmonella Enteritidis infection in layer farms. J Appl Microbiol, 2003; 94:1024–1029.

34.

McAllister

, Steelman

, Skeeles

. Reservoir competence of the lesser mealworm (Coleoptera: Tenebrionidae) for Salmonella Typhimurium (Eubacteriales: Enterobacteriaceae) J Med Entomol, 1994; 31:369–372.

35.

Minitab, Inc. Minitab^® Statistical Software, Release 15 for Windows. State College: PA, 2005.

36.

Nunes

, Helmuth

, Schroeter

, Mead

et al. Phage typing of Salmonella Enteritidis from different sources in Brazil. J Food Prot, 2003; 66:324–327.

37.

O'Connor

. Alphitobius diaperinus (Panzer) (Col. Tenebrionidae) damaging polystyrene insulation in Irish piggery. Entomol Mon Mag, 1987; 123:1472–1475.

38.

Skov

, Spencer

, Hald

, Petersen

et al. The role of litter beetles as potential reservoirs for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks. Avian Dis, 2004; 48:9–18.

39.

Slauch

, Taylor

, Maloy

. Survival in the cruel world: how Vibrio cholerae and Salmonella respond to an unwilling host. Genes Dev, 1997; 11:1761–1774.

40.

Snedeker

, Wills

, Moulthrop

. Some studies on the infectious bursal agent. Avian Dis, 1967; 11:519–528.

41.

Söderhall

, Cerenius

. Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol, 1998; 10:23–28.

42.

Steelman

. Darkling beetles are costly pests. Poult Dig, 1996; 55:22–23.

43.

Takahashi

, Komano

, Natori

. Expression of the lectin gene in Sarcophaga peregrine during normal development and under conditions where the defence mechanism is activated. J Insect Physiol, 1986; 32:771–779.

44.

Watson

, Guy

, Stringham

. Limited transmission of turkey coronavirus in young turkeys by adult Alphitobius diaperinus (Coleoptera: Tenebrionidae) J Med Entomol, 2000; 37:480–483.