Innervation of the southern green stink bug [Nezara viridula (L.) (Hemiptera: Pentatomidae)] stylet bundle.

The southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae), continues to threaten high-value cash crops, including cotton. Earlier reports confirmed ingestion and transmission of disease-causing pathogens of cotton, including elucidation of the dimensions for the food and salivary canals of the southern green stink bug stylet bundle. During this earlier work, innervation of the stylet bundle was observed. Here, we present the first imagery and descriptions of the innervations (i.e., dendrites) within the southern green stink bug stylets. Two types of dendrites innervate each mandibular stylet, and the number of dendrites differed depending on location. Within the head, six dendrites (3 within a thick-walled and 3 within a thin-walled dendrite sheath) are present in each mandibular stylet; only 3 dendrites within a thin-walled sheath are present at the most distal labial segment. Transmission electron microscopy (TEM) suggests innervation of the maxillary stylets, and the presence of stained tissue within the dendritic canal of the maxillary stylets was observed via light microscopy, thereby supporting the TEM analyses. These new observations regarding types and spatial differences in numbers of dendrites within the mandibular stylets - and the new revelation of innervation within maxillary stylets - improve the current knowledge base regarding internal stylet morphology and feeding mechanics.

Controlling the Colonization of Clostridium perfringens in Broiler Chickens by an Electron-Beam-Killed Vaccine.

Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.

Assessment of microbiological correlates and immunostimulatory potential of electron beam inactivated metabolically active yet non culturable (MAyNC) Salmonella Typhimurium.

This study investigates the microbiological and immunological basis underlying the efficacy of electron beam-inactivated immune modulators. The underlying hypothesis is that exposure to eBeam-based ionization reactions inactivate microorganisms without modifying their antigenic properties and thereby creating immune modulators. The immunological correlates of protection induced by such eBeam based Salmonella Typhimurium (EBST) immune modulators in dendritic cell (DC) (in vitro) and mice (in vivo) models were assessed. The EBST stimulated innate pro inflammatory response (TNFα) and maturation (MHC-II, CD40, CD80 and CD86) of DC. Immuno-stimulatory potential of EBST was on par with both a commercial Salmonella vaccine, and live Salmonella cells. The EBST cells did not multiply under permissive in vitro and in vivo conditions. However, EBST cells remained metabolically active. EBST immunized mice developed Salmonella-specific CD4+ T-cells that produced the Th1 cytokine IFNγ at a level similar to that induced by the live attenuated vaccine (AroA- ST) formulation. The EBST retained stable immunogenic properties for several months at room temperature, 4°C, and -20°C as well as after lyophilization. Therefore, such eBeam-based immune modulators have potential as vaccine candidates since they offer the safety of a "killed" vaccine, while retaining the immunogenicity of an "attenuated" vaccine. The ability to store eBeam based immune modulators at room temperature without loss of potency is also noteworthy.

Disinfectant and Antimicrobial Susceptibility Profiles of Campylobacter coli Isolated in 1998 to 1999 and 2015 from Swine and Commercial Pork Chops.

Susceptibility profiles were determined for 111 Campylobacter coli strains obtained in 1998 to 1999 and 2015 from market age pigs and pork chops against 22 disinfectants and 9 antimicrobials. Resistance to tetracycline (TET) was observed in 44.4% of 1998 to 1999 strains, and the antibiotic resistance profile was TET. But strains obtained in 2015 from swine and retail pork chops had 75% TET resistance and the antibiotic resistance profile was TET, followed by azithromycin-erythromycin-TET-telithromycin-clindamycin. Antimicrobial resistance increased in 2015 strains. All strains were resistant to triclosan, and 84.1% and 95.8% of strains in 1998 to 1999 and 2015, respectively, were chlorhexidine resistant. All strains were susceptible to benzalkonium chloride. There was a shift toward higher susceptibility to chlorhexidine, triclosan, P-128, OdoBan, CPB, and CPC in 2015 swine and pork chop strains compared with 1998 to 1999 strains. The disinfectants Tek-Trol and providone-iodine, tris(hydroxylmethyl)nitromethane (THN) and formaldehyde demonstrated the highest susceptibilities. Didecyldimethylammonium chloride (C10AC) appeared to be about equally effective as benzyldimethyltetradecylammonium chloride (C14BAC) for inhibiting C. coli, and both were more effective than C8AC and C12BAC, but C16BAC was not efficient at inhibiting C. coli. The BACs, C12BAC and C14BAC, were the most effective ingredients in DC&R. Also, C12BAC and C14BAC, or these two in synergy with C10AC were responsible for inhibition of C. coli at high P-128 MICs. No cross-resistance was observed between antibiotics and disinfectants. The continued use of THN and formaldehyde in DC&R should be evaluated since these components are not effective, and their inclusion adds unwanted chemicals in the environment. PRACTICAL APPLICATION: Campylobacter species cause diarrheal disease throughout the world. Disinfectants are often used on the farm, in veterinary medicine, by the food processing industry, in restaurants, and in consumer's homes. Limited information is available in the literature showing how disinfectants or disinfectant components may affect the many different foodborne pathogens, and, specifically, Campylobacter coli studied here. The knowledge generated in this study concerning the interactions of a broad array of disinfectants against C. coli may well affect the types of disinfectants and disinfectant formulations allowable for use by medical personnel, producers, food processors, restaurants, and consumers.

Use of Foaming Disinfectants and Cleaners to Reduce Aerobic Bacteria and Salmonella on Poultry Transport Coops.

Transport coops are infrequently washed and have been demonstrated to cross-contaminate broiler carcasses. We hypothesized that peracetic acid or a chlorinated cleaner, commonly used within poultry processing plants, can also be used to disinfect transport coops when applied via a compressed air foam system (CAFS). A mixture of fresh layer manure and concentrated Salmonella Typhimurium (ST) was evenly applied to the floors of four pre-cleaned transport coops and allowed to dry for thirty minutes. Treatments consisted of a (1) water rinse only, (2) product application with a water rinse, (3) product application followed by power washing and (4) power washing followed by application of product. Each foaming treatment was applied with a compressed air foam system and allowed 10 min of contact time. Samples were aseptically collected from the transport coops prior to and following treatment using a sterile 2 × 2-inch stainless steel template and a gauze swab pre-enriched with buffered peptone water. The chlorinated cleaner significantly (p < 0.05) reduced aerobic bacteria and ST by 3.18 to 4.84 logs across application methods. The peroxyacetic acid (PAA) disinfectant significantly (p < 0.05) reduced aerobic bacteria and ST by 3.99 to 5.17 logs across application methods. These data indicate that a compressed air foam system may be used in combination with a commercially available cleaner or disinfectant to reduce aerobic bacteria and ST on the surfaces of commercial poultry transport coops.

Interactions of organic acids with Campylobacter coli from swine.

Campylobacter coli is a bacterial species that is a major cause of diarrheal disease worldwide, and Campylobacter spp. are among the top 5 foodborne pathogens in the United States. During food production organic acids (OAs) are often used to remove bacteria from animal carcasses. The interactions of six OAs with 111 C. coli strains obtained from swine and retail pork chops were studied by determining the molar minimum inhibitory concentrations (MICMs) of the C. coli strains, and the pH at the MICMs. The Henderson-Hasselbalch equation was used to calculate the concentrations of the undissociated and dissociated OAs at the MICMs of the C. coli strains. The results for the 111 different C. coli strains obtained from different locations were treated as a single group for each OA since many of the C. coli strains behaved similarly to each different OA. Inhibition of C. coli was not dependent on pH or on the undissociated OA species, but C. coli inhibition correlated with the dissociated OA species. Therefore, if the concentration of the dissociated OAs decreases from optimum, one may then expect that C. coli bacteria would escape disinfection. The concentration of the dissociated OA should be carefully controlled in a carcass wash. We suggest maintaining a concentration of the dissociated acetic, butyric, citric, formic, lactic and propionic acids at 29, 23, 11, 35, 22 and 25 mM, respectively, when using a carcass wash with these OAs to remove C. coli bacteria. However, due to C. coli utilization of acetate, formate, lactate and propionate, these four OAs may not be the best choice to use for a carcass wash to remove C. coli contamination. Of the six OAs, citric acid was the most efficient at inhibiting C. coli.

Electron-Beam-Inactivated Vaccine Against Salmonella Enteritidis Colonization in Molting Hens.

Electron-beam (eBeam) irradiation technology has a variety of applications in modern society. The underlying hypothesis was that eBeam-inactivated Salmonella enterica serovar Enteritidis (SE) cells can serve as a vaccine to control SE colonization and shedding in poultry birds. An eBeam dose of 2.5 kGy (kilograys) was used to inactivate a high-titer (10(8) colony-forming units [CFU]) preparation of SE cells. Microscopic studies revealed that the irradiation did not damage the bacterial cell membranes. The vaccine efficacy was evaluated by administering the eBeam-killed SE cells intramuscularly (1 x 10(6) CFU/bird) into 50-wk-old single comb white leghorn hens. On day 14 postvaccination, the hens were challenged orally with live SE cells (1 x 10(9) CFU) and SE colonization of liver, spleen, ceca, and ovaries determined on day 23. Blood samples were collected on days 0, 14, and 23 postvaccination and the sera were analyzed to quantify SE-specific IgG titers. The vaccinated chickens exhibited significantly (P < 0.0001) higher SE-specific IgG antibody responses and reduced SE ceca colonization (1.46 ± 0.39 logi10 CFU/g) compared to nonvaccinated birds (5.32 ± 0.32 log10 CFU/g). They also exhibited significantly lower SE colonization of the ovaries (1/30), spleen (3/30), liver (4/30), and ceca (7/30) compared to nonvaccinated birds. These results provide empirical evidence that eBeam-based SE vaccines are immunogenic and are capable of protecting chickens against SE colonization. The advantages of eBeam-based vaccine technology are that it is nonthermal, avoids the use of formalin, and can be used to generate inactivated vaccines rapidly to address strain-specific infections in farms or flocks.

The acquisition and internalization of Salmonella by the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

In poultry broiler production facilities, it is important to understand the sources and contribution of reservoir populations of pathogens. The lesser mealworm beetle, Alphitobius diaperinus (Panzer), is a common pest in poultry litter that is reported to carry pathogens affecting both human and animal health. This study investigates whether the carriage of a bacterial pathogen occurs by the harboring of bacteria internally by these insects. Beetles were exposed to a marker bacterium, Salmonella enterica serovar Typhimurium-green fluorescent protein (ST-GFP), at concentrations up to 10(7) colony-forming units (cfu)/mL for 0.5 to 12 h, and then subsequently surface disinfected and dissected. The head, gastrointestinal tract and hemolymph were cultured for the presence of ST-GFP. This study definitively demonstrates the internal carriage of Salmonella by this insect and found that the beetles rapidly acquired bacteria from external sources and harbored the bacteria within their alimentary canal after exposure for 30 min at 10(4) cfu/mL and within the hemolymph after exposure for 2 h at 10(6) cfu/mL. Beetles internalized an average of 9.5 × 10(1) and 3.2 × 10(3) after a 2-h exposure to 2 × 10(4) and 2 × 10(6) cfu/mL, respectively. The lesser mealworm is a serious pest within the poultry brooder and laying industry and because of their mobility, voracious feeding habits, and prey potential may represent an active source facilitating the dissemination of Salmonella.

In vitro cultivation of a newly recognized Babesia sp. in dogs in North Carolina.

A novel large Babesia sp. from an infected dog was cultivated in vitro by microaerophilous stationary phase culture methodology. A primary culture initiated in enriched RPMI-1640 medium supplemented with 40% canine serum and incubated in a 2% oxygen environment supported parasite growth in vitro. Subsequent subcultures into enriched HL-1 medium with 20% fetal bovine serum also supported parasite propagation. Cultures were successfully introduced to 5% carbon dioxide in air atmosphere at passage 4. To date, the parasites have been continuously cultured through 35 passages, although the parasitemias are low, ranging from 0.2 to 0.3%. Parasites cultured in RPMI with canine serum were cryopreserved and successfully recovered from liquid nitrogen storage. The small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived parasites, differing in a single base position from the previously reported sequence for this Babesia sp. The ultrastructure of the parasite was consistent with that of other large Babesia spp., except that the spherical body contained numerous round particles unlike the inclusions previously described in Babesia spp.

Qualitative and quantitative comparison of gut bacterial colonization in enterally and parenterally fed neonatal pigs.

Total parenteral nutrition (TPN) has been associated with mucosal atrophy, impaired gut barrier function, and translocation of luminal bacteria with resultant sepsis in preterm human infants. Currently, we examined the effects of enteral (ENT) or TPN treatments on translocation events in neonatal pigs and on colonization and composition of microbiota in the neonatal gut. Newborn, colostrum-deprived pigs (<24 hours old) were fitted with intravenous catheters and were fed either ENT (n = 13) or TPN (n = 13) for 7 days. After 7 days of treatment, pigs were euthanized and samples were collected for bacterial culture from the blood, intestinal tract and organs. ENT pigs had increased numbers of bacterial genera isolated, higher concentrations of bacteria (CFU/g), and increased colonization of all segments of the intestinal tract compared to the TPN pigs. Translocation of bacteria from the intestinal tract to tissues or blood was similar (8 of 13) for both groups. The ENT group had 1/13 positive for Clostridium difficile toxin A whereas the TPN group had 5/13. We concluded that ENT favored increased bacterial concentrations comprised of more speciation in the gastrointestinal tract compared to TPN, and that TPN-treated piglets were at higher risk of colonization by toxin-expressing strains of C. difficile.

Transmission electron microscopy.

Transmission electron microscopy has long been an important analytical tool in the field of microbiology. This unit describes preparation techniques for examining particulate samples as well as samples presenting more complex ultrastructural considerations that require analysis in thin sections. Negative staining is a useful technique for routine examination of particulate samples in suspension ranging from bacteria to purified macromolecules. In order to investigate the relationships between microbes and the environments with which they interface, fixed samples can be prepared for imaging in sections of 60- to 90-nm thickness. Due to the many steps in sample preparation for ultrastructural analysis of thin-sectioned samples, the major steps in the process are divided into fixation and initial processing of samples for thin sectioning, the embedment of samples into a plastic resin for sectioning, ultramicrotomy, and staining of samples. Procedures for immunolocalization of antigens in negatively stained and thin-sectioned preparations are also considered.

In vitro cultivation of a zoonotic Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts.

A Babesia sp. found in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, is the same organism that caused human babesiosis in Missouri and Kentucky, on the basis of morphology and identical small-subunit rRNA (SSU rRNA) gene sequences. Continuous cultures of the rabbit parasite were established from infected blood samples collected from two cottontail rabbits livetrapped on Nantucket Island. HL-1 medium or minimal essential medium alpha medium supplemented with 20% human serum best supported in vitro propagation of the parasite in human or cottontail erythrocytes, respectively. Parasite growth was not sustained in domestic-rabbit erythrocytes or in medium supplemented with domestic-rabbit serum. The cultured parasites were morphologically indistinguishable from the Kentucky human isolate. Transmission electron microscopy revealed similar fine structures of the parasite regardless of the host erythrocyte utilized in the cultures. Two continuous lines of the zoonotic Babesia sp. were established and confirmed to share identical SSU rRNA gene sequences with each other and with the Missouri and Kentucky human Babesia isolates.

News and notes: An atypical Campylobacter coli exhibiting unusual morphology.

Campylobacter coli cells are characterized by a comma, or spiral shape, and a single polar flagellum. Here we report stable spontaneous changes in morphology to an aflagellated straight rod. The ability to colonize the chick cecum was lost. The atypical cells were compared with the original C. coli 67 culture from which they were derived, with PCR, Riboprinting, and PFGE.

Further characterization of Campylobacter isolated from U.S. dairy cows.

The objective of the present study was to compare polymerase chain reaction (PCR) identification with ribotype results and to use pulsed field electrophoresis (PFGE) to correlate genotypic patterns with antibiotic resistance of Campylobacter isolated from lactating dairy cows in the United States. Thirty isolates were studied. Twenty-seven of the isolates were identified by PCR as Campylobacter jejuni and three were identified as Campylobacter coli. Genotypic patterns of 15 isolates were determined by PFGE, and although isolates originated from geographically separated regions of the United States, some were genotypically identical. In contrast to their genetic similarity, antibiotic sensitivity patterns differed within some genotypes. Under the conditions of our study, we concluded that ribotyping is not as discriminatory as PCR for speciation, and that a phenotypic trait such as antimicrobial resistance cannot always be predicted within the same genotype.

Campylobacter prevalence in lactating dairy cows in the United States.

The objective of the present study was to determine the prevalence of intestinal Campylobacter in lactating dairy cows from various regions of the United States. Participating commercial dairy farms were chosen at random and were part of a national survey to determine E. coli O157:H7 and Salmonella prevalence in dairy cows. Farms had no previous history of Campylobacter problems. Fecal samples were collected rectally from 720 cows on farms in the northeast (four farms), in the desert southwest (three farms), and in the Pacific west (two farms). A minimum of 60 fecal samples per visit were collected from each farm. Thirty isolates were analyzed using the RiboPrinter Microbial Characterization System to obtain ribosomal RNA patterns. Twenty isolates were tentatively identified as Campylobacter jejuni, two as Campylobacter coli, three as Campylobacter spp., and five as unknown. Individual single-visit farm prevalence ranged from 0 to 10%. The disk diffusion method, employing 11 antibiotics, was used to test the antibiotic sensitivities of 27 of the isolates. Eight isolates were resistant to two or more antibiotics, 13 isolates were resistant to one antibiotic, and 6 were totally susceptible. Under the conditions of this study, the authors conclude that Campylobacter prevalence in lactating dairy cows in the United States is low, there is no difference in prevalence on the basis of geographical location, the predominant species is C. jejuni, and that the majority of these isolates are sensitive to antibiotics.

Failure of viable nonculturable Campylobacter jejuni to colonize the cecum of newly hatched leghorn chicks.

Campylobacter jejuni cells entered the viable but nonculturable (VBNC) state upon suspension in sterile water. Cell viability was determined with tetrazolium violet. VBNC cells suspended in water for 7, 10, or 14 days were given, by gavage, to day-of-hatch leghorn chickens. The ceca of control and challenged birds were examined for the presence of campylobacteria by conventional microbiological methods at 1 wk and 2 wk after challenge inoculation and by polymerase chain reaction methods at 1 wk after challenge. We did not find culturable Campylobacter cells in the ceca. Neither was Campylobacter DNA found in cecal samples. Therefore, VBNC cells did not revert to the culturable colonizing form, nor did VBNC cells persist within the cecal environment.

The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons.

The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome contains 13 putative fimbrial operons termed agf (csg), fim, pef, lpf, bcf, saf, stb, stc, std, stf, sth, sti and stj. Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf, fim and pef. We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be detected by flow cytometry and immunoelectron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study the expression of fimbrial antigens in S. Typhimurium by flow cytometry, we constructed strains carrying deletions of agfAB, pefBACDI, lpfABCDE, bcfABCDEFG, stbABCD, stcABC, stdAB, stfACDEFG, sthABCDE or stiABCDE. Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 h after infection of bovine ligated ileal loops with S. Typhimurium. FimA was the only fimbrial antigen expressed by S. Typhimurium after static growth in Luria-Bertani (LB) broth. Injection of static LB broth cultures of S. Typhimurium into bovine ligated ileal loops resulted in the expression of BcfA, FimA, LpfA, PefA, StbA, StcA, StdA, StfA and StiA. These data show that in vivo growth conditions drastically alter the repertoire of fimbrial antigens expressed in S. Typhimurium.

In vitro inhibition of Salmonella enterica serovars choleraesuis and typhimurium, Escherichia coli F-18, and Escherichia coli O157:H7 by a porcine continuous-flow competitive exclusion culture.

A competitive exclusion (CE) culture of porcine cecal bacteria was developed as a continuous-flow culture in chemostats, was designated RPCF, and was used as a model to determine its usefulness against in vitro colonization by Salmonella enterica serovars Typhimurium and Choleraesuis, Escherichia coli strain F-18, and E. coli serotype O157:H7 (933). Chemostats with or without RPCF were inoculated with 10(6) colony-forming units (CFU)/ml of Typhimurium, Choleraesuis, F-18, or O157:H7. Chemostats were sampled for salmonellae and E. coli at 15 min, 7 h, and every 24 h thereafter. In control chemostats without RPCF, Typhimurium, Choleraesuis, F-18, and O157:H7 rapidly established colonization and had concentrations of 10(6) CFU/ml for 96-120 h post-inoculation. In the chemostats that contained RPCF, reductions (P < 0.05) of Choleraesuis, F-18, and O157:H7 were observed at 24 h post-inoculation. Typhimurium was decreased (P < 0.05) at 48 h post-inoculation, and by 120 h post-inoculation, all chemostats were negative for the four challenge microorganisms. These results demonstrate that RPCF cultures were able to inhibit the growth of Typhimurium, Choleraesuis, and E. coli strains F-18 and O157:H7 in vitro and suggest the potential for the use of CE in swine to prevent disease induced by these microorganisms.

Campylobacter coli pulsed field gel electrophoresis genotypic diversity among sows and piglets in a farrowing barn.

Genotypes of Campylobacter coli isolates from feces of three sows and rectal swabs of 17 piglets were examined by pulsed field gel electrophoresis (PFGE). All of the animals originated from a single farrowing barn of a farrow-to-finish swine operation. Five Campylobacter colonies were picked from a single agar plate for each sample after broth enrichment and growth on Campy-Cefex agar. Genotypes were examined by PFGE after genomic DNA digestion with SmaI and SacII restriction endonucleases. Twenty SmaI genotypes and 12 SacII genotypes were detected among 99 Campylobacter coli isolates. There was no pattern of shared genotypes between sows and their respective piglets, nor between littermates. Results indicate that a high number of Campylobacter genotypes may coexist in related pigs from a single housing facility.