Reduced rotavirus vaccine efficacy in protein malnourished human-faecal-microbiota-transplanted gnotobiotic pig model is in part attributed to the gut microbiota.

The low efficacy of human rotavirus (HRV) vaccines in low- and middle-income countries (LMIC) remains a major challenge for global health. Protein-calorie malnutrition (kwashiorkor) affects the gut microbiota and compromises immune development, leading to environmental enteropathy, vaccine failures, and increased susceptibility to enteric diseases in young children. Relationship between diet and reduced vaccine efficacy in developing countries is not well established; therefore, we investigated the interconnections between the host-microbiota-nutrition-HRV vaccine using HRV-vaccinated, human infant faecal microbiota (HIFM)-transplanted neonatal gnotobiotic pigs fed with a protein deficient or sufficient diet. The microbiota from faecal, intestinal (duodenum, ileum, jejunum, and colon), and systemic tissue (liver, spleen, and mesenteric lymph node [MLN]) samples was analysed before and after HRV challenge using MiSeq 16S rRNA sequencing. Overall, microbiota from deficient fed HIFM pigs displayed, compared to the sufficient group, significantly higher Shannon index, especially in the faeces and lower intestines; higher level of Proteus and Enterococcus, and lower level of Bifidobacterium, Clostridium, and Streptococcus in the three types of samples collected (P<0.05); and higher unique operational taxonomic units (OTUs), especially in the systemic tissues. Further, the multivariate analysis between microbiota and immunologic data showed that 38 OTUs at the genus level correlated (r2≤0.5 or ≥-0.5; P<0.05) with at least one host immune response parameter (regulatory [Tregs and transforming growth factor-ß], effectors [interferon (IFN)-γ+ CD4+ and CD8+ T cells, IFN-γ and interleukin (IL)-12], and inflammatory [tumour necrosis factor-α, IL-17 and IL-22]) and with opposite trends between diet groups. Differences described above were increased after HRV challenge. We demonstrated that a protein deficient diet affects the composition of the gut microbiota and those changes may further correlate with immune responses induced by HRV and perturbed by the deficient diet. Thus, our findings suggest that the reduced efficacy of HRV vaccine observed in Gn pig model is in part attributed to the altered microbiota composition.

Effect of antibiotic, probiotic, and human rotavirus infection on colonisation dynamics of defined commensal microbiota in a gnotobiotic pig model.

We developed a gnotobiotic (Gn) pig model colonised with defined commensal microbiota (DMF) to provide a simplified and controlled system to study the interactions between intestinal commensals, antibiotics (ciprofloxacin, CIP), probiotics (Escherichia coli Nissle 1917, EcN) and virulent human rotavirus (VirHRV). The DMF included seven gut commensal species of porcine origin that mimic the predominant species in the infant gut. Gn piglets were divided into four groups: DMF control (non-treated), DMF+CIP (CIP treated), DMF+CIP+EcN (CIP/EcN treated), DMF+EcN (EcN treated) and inoculated orally with 105 cfu of each DMF strain. The pig gut was successfully colonised by all DMF species and established a simplified bacterial community by post-bacteria colonisation day (PBCD) 14/post-VirHRV challenge day (PCD) 0. Overall, Bifidobacterium adolescentis was commonly observed in faeces in all groups and time points. At PCD0, after six days of CIP treatment (DMF+CIP), we observed significantly decreased aerobic and anaerobic bacteria counts especially in jejunum (P<0.001), where no DMF species were detected in jejunum by T-RFLP. Following HRV challenge, 100% of pigs in DMF+CIP group developed diarrhoea with higher diarrhoea scores and duration as compared to all other groups. However, only 33% of pigs treated with EcN plus CIP developed diarrhoea. EcN treatment also enhanced the bacterial diversity and all seven DMF species were detected with a higher proportion of Bifidobacterium longum in jejunum in the DMF+CIP+EcN group on PBCD14/PCD0. Our results suggest that EcN increased the proportion of B. longum especially in jejunum and mitigated adverse impacts of antibiotic use during acute-infectious diarrhoea. The DMF model with a simplified gut commensal community can further our knowledge of how commensals and probiotics promote intestinal homeostasis and contribute to host health.

Evaluation of nanoparticle-encapsulated outer membrane proteins for the control of Campylobacter jejuni colonization in chickens.

Numerous vaccination strategies have been evaluated to develop effective vaccines against Campylobacter jejuni colonization in poultry but with limited success. The following experiments were conducted to investigate the effect of biodegradable and biocompatible poly (lactide-co-glycolide) nanoparticle (NP) encapsulated outer membrane proteins (OMP) of C. jejuni. Chickens were vaccinated with different routes [subcutaneous (s/c) or oral] and doses (25, 125, or 250 µg) of candidate nanoparticle vaccine with appropriate control groups. Serum and cloacal fecal samples were taken at regular intervals of time, and the birds were euthanized 7 d postchallenge with C. jejuni. The results were interpreted based on anti-OMP immunoglobulin response in chicken and intestinal colonization of C. jejuni. The C. jejuni colonization in cecal and cloacal contents at 7 d postchallenge was below the detection limit in the s/c vaccinated groups, but the other groups demonstrated varying degrees of colonization. The serum IgA was higher in the group vaccinated s/c with OMP only compared with the rest of the groups. The serum- and fecal-IgY titers were consistently higher in the s/c vaccinated groups (with or without NP) than the rest of the groups. Elevated levels of OMP specific serum antibodies correlated with below the limit of detection levels of Campylobacter colonization in broiler chickens receiving 125 µg of OMP alone and the OMP+NP vaccine s/c. In conclusion, the s/c route of vaccination with or without NP encapsulated OMP of C. jejuni may serve as a candidate vaccine for control of C. jejuni colonization in chickens.

An evaluation of the effect of sodium bisulfate as a feed additive on Salmonella enterica serotype Enteritidis in experimentally infected broilers.

The colonization of broiler chickens with Salmonella can pose serious health and economic risks for both consumers and the poultry industry. Because colonization with Salmonella can lead to subsequent contamination of chicken carcasses during processing, preemptive control measures should include the reduction of this pathogen in chickens before slaughter. In this study, we evaluated the effect of sodium bisulfate, a potential antimicrobial feed additive, on Salmonella colonization of experimentally infected broiler chickens. Two hundred and forty 1-d-old chickens were infected orally with Salmonella enterica serotype Enteritidis and divided into 4 groups (each comprised of 60 chickens). Three groups received different concentrations of sodium bisulfate integrated into their feed, while the feed of the fourth group (positive control) was not treated. At time points before the broilers' slaughter age, different organs/tissues (liver, spleen, cecum, and bone marrow) and feces were aseptically collected and tested for the occurrence and density of Salmonella Enteritidis. Our results show that at 3 d postinfection, high colonization with Salmonella Enteritidis was detected and affected all tested tissues and fecal samples. Although colonization decreased across time, Salmonella Enteritidis persisted in the cecum, feces, spleen, and bone marrow, but not in the liver, until slaughter age. Furthermore, the addition of sodium bisulfate to the feed did not significantly reduce Salmonella Enteritidis numbers in infected chickens or affect the shedding of the pathogen.

Use of bioluminescence imaging to monitor Campylobacter survival in chicken litter.

AIM: The aim of this study was to develop a novel approach for characterizing the growth and persistence of Campylobacter in different poultry-rearing environments. Specifically, we constructed bioluminescent Campylobacter strains and used them to monitor the survival of these pathogens in litter (bedding) material. METHODS AND RESULTS: We inserted shuttle plasmids carrying the luminescence genes (luxCDABE) into C. jejuni and C. coli to construct bioluminescent strains of these pathogens. The strains were spiked into microcosms containing samples of litter-washings and dry litter collected from different enclosures that housed broiler chickens. Our results show that C. jejuni and C. coli survived for at least 20 days in reused (old) litter while the growth of these pathogens was inhibited in clean (new) litter. Furthermore, our results suggest that the availability of nutrients and the condition of the litter (reused vs new) are important factors in the persistence of these pathogens. CONCLUSIONS: Reused litter can potentially predispose chickens to Campylobacter contamination and maintaining clean litter might reduce the incidences of colonization with these pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Bioluminescence provided a simple, sensitive, and rapid approach for analysing the growth dynamics of Campylobacter. Using this technology, we highlighted the potential role of litter material in maintaining these pathogens in the chicken environment.

Specific detection of avian pneumovirus (APV) US isolates by RT-PCR.

This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 x 10(-5) TCID50 (0.0323 microg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota.

Pathologic and bacteriologic findings in 27-week-old commercial laying hens experimentally infected with Salmonella enteritidis, phage type 4.

Two strains of 27-wk-old commercial laying chickens (strain A, brown-egg-laying type and strain B, white-egg-laying type) were inoculated either orally (PO) or intravenously (IV) with a field isolate of Salmonella enteritidis phage type 4. Chickens were sequentially necropsied at regular intervals throughout the 17-wk observation period. Gross and microscopic lesions were most evident between 1 and 14 days postinoculation (DPI). Gross lesions consisted of enlarged livers with white foci, enlarged and mottled white spleens, fibrinous exudate in the peritoneum, and atretic, misshapen ovarian follicles. Microscopic lesions included multifocal coagulative necrosis of hepatocytes and inflammation, fibrinous exudation in vascular sinuses of the spleen, and fibrinosuppurative inflammation of the peritoneum and ovarian follicles. The proportion of reproductive organ infections (ovary and oviduct) in the IV group, 83% (20/24, P = 0.007; 50% and 33% for strains A and strain B birds, respectively), was higher than that of the PO group, 46% (11/24; 29% and 17% for strains A and B, respectively), for the first 16 days of observation postinoculation. The proportion of fecal shedding for the IV group of birds was significantly (P = 0.009) lower, 29% (7/24; 33% and 25% respectively for strain A and strain B birds, respectively), than the PO group, 67% (16/24; 75% and 58% for strain A and strain B birds, respectively). Three (2.6%) of 234 egg pools were culture-positive for group D Salmonella from strain A chickens (1 of 119 pools from the IV group and 2 of 115 pools from the PO group of birds). Chickens infected with the field strain of S. enteritidis phage type 4 harbored the organism in tissues only for a brief time, most clearing within 8 DPI and nearly all within 16 DPI. Overall the percentage of culture-positive birds did not differ significantly (P > 0.05) between birds with and without lesions, but isolation of S. enteritidis tended to be more frequent when lesions were evident. This experiment also demonstrated that brown-egg-laying-type chickens were more susceptible than white-egg-laying-type chickens to S. enteritidis phage type 4 isolated from California based on gross and microscopic lesions and bacteriologic findings.

Pathogenic role of SEF14, SEF17, and SEF21 fimbriae in Salmonella enterica serovar enteritidis infection of chickens.

Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14(-) strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.

Multidrug-resistant Salmonella Typhimurium DT104 in poultry.

Salmonella Typhimurium isolates from feed ingredients or poultry sources isolated during 1995 to 1997 from different geographical locations within Minnesota were examined for the presence of Salmonella Typhimurium definitive type 104 (DT104). Antibiotic susceptibility studies indicated that 15 of 50 isolates of Salmonella Typhimurium had an antibiotic resistance pattern (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) that is usually observed with multidrug-resistant Salmonella Typhimurium DT104. Of the 15 isolates showing the antibiotic resistance pattern, 8 isolates were phage type 104, 3 isolates were typed as phage type 104 complex, and the remaining 4 isolates belonged to phage types 193, 81, and 126. DT104 was recovered from both feed ingredients and poultry samples. Of the seven feed ingredient-associated Salmonella Typhimurium isolates, four were DT104, whereas only 7 of 43 poultry-associated Salmonella Typhimurium isolates were DT104. A repetitive sequence-based polymerase chain reaction (rep-PCR) of 50 isolates of Salmonella Typhimurium representing 13 phage types identified seven distinct fingerprint profiles. No correlation between phage type and rep-PCR type was noticed. Eleven Salmonella Typhimurium isolates belonging to DT104 and its complex were grouped into two closely related rep-PCR types.

Outer membrane proteins for serologic detection of Ornithobacterium rhinotracheale infection in turkeys.

Ornithobacterium rhinotracheale (ORT) is a bacterium responsible for a respiratory disease in turkeys and chickens and has been identified as one of the emerging respiratory bacterial pathogens. The clinical signs and lesions caused by ORT are very similar to those caused by other respiratory infectious agents; therefore, an accurate diagnostic test is necessary to identify the infection. In this study, we have investigated the use of outer membrane proteins of ORT in an indirect enzyme-linked immunosorbent assay (ELISA) to detect the exposure to ORT infection. Outer membrane proteins of ORT were extracted and used as an antigen in ELISA to detect infection in turkeys exposed to different serotypes of ORT. The ELISA results were compared with the conventional serum plate agglutination test. The agglutination test detected specific antibodies for ORT in 65% of experimentally infected turkeys during the first 2 wk of infection. The ELISA detected up to 100% of infected birds for 8 wk postinfection. The results suggest that ELISA is able to detect the exposure to ORT in later stages of the infection and this assay can be used in serologic surveillance of ORT infection for poultry in the field.

A rapid strip immunoblot assay for the specific detection of Salmonella enteritidis infection in chickens.

A rapid strip immunoblot assay (RSIA) was developed using recombinant SEF14 antigen. The rSEF14-RSIA was very specific in detecting antibodies to Salmonella enteritidis in chickens. When serum samples obtained from groups of chickens (N = 5) inoculated with six different Salmonella serovars were tested in rSEF14-RSIA, only serum samples obtained from S. enteritidis inoculated birds reacted with rSEF14 antigen except for a group of chickens that had been inoculated with S. dublin. To assess the sensitivity of the rSEF14-RSIA, groups of chickens were inoculated with either 10(4) cfu or 10(10) cfu of S. enteritidis and the serum and egg yolk were analyzed for SEF14 antibodies. By 1 week after infection 66-78% of chickens were found positive for SEF14 antibodies in the serum and the number of positive birds increased subsequently to 89-100%. The S. enteritidis specific antibodies appeared as early as 6 days after infection in the egg yolk of infected chickens. The antibodies to SEF14 in both the serum and egg yolk persisted for at least 7 weeks after infection in a significant proportion of chickens. Our results suggest that rSEF14-RSIA can be a practical and efficient screening test for identifying S. enteritidis infected chickens.

SERE, a widely dispersed bacterial repetitive DNA element.

The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis.

Application of recombinant fimbrial protein for the specific detection of Salmonella enteritidis infection in poultry.

A number of disease outbreaks of Salmonella enterica serotype enteritidis (SE) in humans have been traced to the consumption of SE-contaminated egg and egg products. A rapid, specific, and inexpensive method of detecting SE infection in poultry is necessary to reduce human outbreaks. We evaluated rSEF14 fimbrial antigen of SE for specific detection of SE-infected birds in latex agglutination test and enzyme-linked immunosorbent assay. rSEF14 antigen was highly specific in identifying birds infected with SE. The sera from birds infected with closely related serogroup-D Salmonella and other avian pathogens did not react with rSEF14 antigen. The rSEF14 antigen identified antibodies in serum of 88% of birds during the first 2 weeks of infection, and 100% of the birds subsequently. The SE-specific antibodies were detected in egg yolk as early as 6 days post-infection in rSEF14-enzyme-linked immunosorbent assay. Our results suggest that rSEF14-based assays could be used as screening tests for detection of SE antibodies and would overcome the cross reactions observed with existing serological tests.