The survival rate, p , of a measured inoculum of Staph. aureus in a standard volume of denbrinated blood, is a reliable quantitative measure of the bactericidal power of blood. The number of viable organisms in the inoculum and in the blood-bacterium mixture may be estimated with the necessary accuracy by counts of colonies developing from measured volumes of the fluids let fall on to the surface of solid media. Fildes' agar was the most suitable medium for this surface-viable count, and was selected on the basis of four criteria; of the media tested it yielded the highest counts, and the counts conformed most closely to a Poisson series; and on it the mean colony size was maximum, and the coefficient of variation of colony size was minimum. On this medium, the close conformity of the separate count values to a Poisson series enabled the standard error of the survival rate to be determined from a simplification of the general expression for the standard error of a ratio. The number of colonies growing from a sample of a blood-bacterium mixture may be reduced, not by killing of the individual cocci, but as a result of their aggregation either by agglutinins in the blood, or in the cytoplasm of leucocytes that are phagocytic but not bactericidal. It appears that these mechanisms are unlikely to operate in blood-bacterium mixture containing relatively few organisms; in such mixtures the survival rate is a reflexion of the killing power only. The immunological significance of p has not been investigated, but the range of values for healthy human adults differs significantly from that for sufferers from chronic staphylococcal infection. Moreover, by the technique employed differences may be detected between individual values of p that cannot reasonably be attributed to technical or sampling errors.

The Estimation of the Bactericidal Power of the Blood. With a Note by J. O. IRWIN.

Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni “supercolonizer mutant” with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.

IMPORTANCE Campylobacter jejuni is a common cause of human diarrheal disease worldwide and is listed by the World Health Organization as a high-priority pathogen. C. jejuni infection typically occurs through the ingestion of contaminated chicken meat, so many efforts are targeted at reducing C. jejuni levels at the source. We previously developed a vaccine that reduces C. jejuni levels in egg-laying chickens. In this study, we improved vaccine performance in meat birds by supplementing the vaccine with probiotics. In addition, we demonstrated that C. jejuni colonization levels in chickens are negatively correlated with the abundance of clostridia, another group of common gut microbes. We describe new methods for vaccine optimization that will assist in improving the C. jejuni vaccine and other vaccines under development.

Coadministration of the Campylobacter jejuni N-Glycan-Based Vaccine with Probiotics Improves Vaccine Performance in Broiler Chickens.

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features.

/pdf/promising-new-vaccine-candidates-against-campylobacter-in-2kyvmxs2r8.pdf

Promising new vaccine candidates against Campylobacter in broilers.

Immunogenicity and protective efficacy of mucosal delivery of recombinant hcp of Campylobacter jejuni Type VI secretion system (T6SS) in chickens.

Campylobacteriosis is the leading foodborne bacterial diarrheal illness in many countries, with up to 80% of human cases attributed to the avian reservoir. The only control strategies currently available are stringent on-farm biosecurity and carcass treatments. Heritable differences in the resistance of chicken lines to Campylobacter colonization have been reported and resistance-associated quantitative trait loci are emerging, although their impact on colonization appears modest. Recent studies indicated a protective role of the microbiota against colonization by Campylobacter in chickens. Furthermore, in murine models, differences in resistance to bacterial infections can be partially transferred between lines by transplantation of gut microbiota. In this study, we investigated whether heritable differences in colonization of inbred chicken lines by Campylobacter jejuni are associated with differences in cecal microbiota. We performed homologous and heterologous cecal microbiota transplants between line 61 (resistant) and line N (susceptible) by orally administering cecal contents collected from 3-week-old donors to day-of-hatch chicks. Recipient birds were challenged (day 21) with C. jejuni 11168H. In birds given homologous microbiota, the differential resistance of lines to C. jejuni colonization was reproduced. Contrary to our hypothesis, transfer of cecal microbiota from line 61 to line N significantly increased C. jejuni colonization. No significant difference in the overall composition of the cecal microbial communities of the two lines was identified, although line-specific differences for specific operational taxonomic units were identified. Our data suggest that while heritable differences in avian resistance to Campylobacter colonization exist, these are not explained by significant variation in the cecal microbiota. IMPORTANCECampylobacter is a leading cause of foodborne diarrheal disease worldwide. Poultry are a key source of human infections, but there are currently few effective measures against Campylobacter in poultry during production. One option to control Campylobacter may be to alter the composition of microbial communities in the avian intestines by introducing beneficial bacteria, which exclude the harmful ones. We previously described two inbred chicken lines which differ in resistance to intestinal colonization by Campylobacter. Here, we investigated the composition of the microbial communities in the gut of these lines and whether transferring gut bacteria between the resistant and susceptible lines alters their resistance to Campylobacter. No major differences in microbial populations were found, and resistance or susceptibility to colonization was not conferred by transferring gut bacteria between lines. The data suggest that gut microbiota did not play a role in resistance to Campylobacter colonization, at least in the lines used.

Role of Cecal Microbiota in the Differential Resistance of Inbred Chicken Lines to Colonization by Campylobacter jejuni.

Campylobacter jejuni is the leading cause of foodborne bacterial gastroenteritis with contaminated poultry meat its main source. Control of C. jejuni is a priority for the poultry industry but no vaccines are available and their development hampered by poor understanding of the immunobiology of C. jejuni infection. Here we show the functional role of B lymphocytes in response to C. jejuni in the chicken through depletion of the B lymphocyte population (bursectomy) followed by challenge. B lymphocyte depletion has little effect on bacterial numbers in the ceca, the main site of colonisation, where C. jejuni persist to beyond commercial slaughter age, but reduces clearance from the small intestine. In longer-term experiments we show antibody leads to reduction in C. jeuni numbers in the ceca by nine weeks post infection. Whilst we did not examine any protective role to re-challenge, it illustrates the difficulty in producing a vaccine in a young, immunologically naive host. We believe this is first study of functional immunity to C. jejuni in chicken and shows antibody is ineffective in clearing C. jejuni from the ceca within the production lifetime of chickens, although is involved in clearance from the small intestine and longer-term clearance from the ceca.

/pdf/b-lymphocytes-play-a-limited-role-in-clearance-of-p3d6vjd9oh.pdf

B lymphocytes play a limited role in clearance of Campylobacter jejuni from the chicken intestinal tract

Over recent decades, Salmonella infection research has predominantly relied on murine infection models. However, in many cases the infection phenotypes of Salmonella pathovars in mice do not recapitulate human disease. For example, Salmonella Typhimurium ST313 is associated with enhanced invasive infection of immunocompromised people in Africa, but infection of mice and other animal models with ST313 have not consistently reproduced this invasive phenotype. The introduction of alternative infection models could help to improve the quality and reproducibility of pathogenesis research by facilitating larger-scale experiments. To investigate the virulence of S. Typhimurium ST313 in comparison with ST19, a combination of avian and insect disease models were used. We performed experimental infections in five lines of inbred and one line of outbred chickens, as well as in the alternative chick embryo and Galleria mellonella wax moth larvae models. This extensive set of experiments identified broadly similar patterns of disease caused by the African and global pathovariants of Salmonella Typhimurium in the chicken, the chicken embryo and insect models. A comprehensive analysis of all the chicken infection experiments revealed that the African ST313 isolate D23580 had a subtle phenotype of reduced levels of organ colonisation in inbred chickens, relative to ST19 strain 4/74. ST313 isolate D23580 also caused reduced mortality in chicken embryos and insect larvae, when compared with ST19 4/74. We conclude that these three infection models do not reproduce the characteristics of the systemic disease caused by S. Typhimurium ST313 in humans.

/pdf/the-use-of-chicken-and-insect-infection-models-to-assess-the-3ockn002o1.pdf

The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313.

BACKGROUND: Campylobacter jejuni , the most frequent cause of foodborne bacterial infection, is found on around 70% of retail chicken. As such there is a need for effective controls in chicken production. Microbial-based controls such as probiotics are attractive to the poultry industry, but of limited efficacy. Furthermore, as commercially-produced chickens have no maternal contact, their pioneer microbiome is likely to come from the hatchery environment. Early delivery of microbials that lead to a more &{prime]natural avian′ microbiome may, therefore, improve bird health and reduce susceptibility to C.jejuni colonisation. A faecal microbiota transplant (FMT) was used to transfer a mature cecal microbiome to newly-hatched broiler chicks and its effects on C.jejuni challenge assessed. We used both a seeder-bird infection model that mimics natural bird-to-bird infection alongside a direct-challenge model. We used a 16S rRNA gene sequencing-based approach to characterize the transplant material itself alongside changes to the chicken microbiome following FMT. RESULTS: FMT changes the composition of the chicken intestinal microbiome. We observed little change in species richness following FMT compared to untreated samples, but there is an increase in phylogenetic diversity within those species. The most significant difference in the ceca is an increase in Lactobacilli, although not a major component of the transplant material, suggesting the FMT results in a change in the intestinal milieu as much as a direct change to the microbiome. Upon direct challenge, FMT resulted in lower initial intestinal colonisation with C.jejuni . More significantly, in a seeder-bird challenge of infection transmission, FMT reduced transmission and intestinal colonisation until common UK retail age of slaughter. In a repeat experiment, transmission was completely blocked following FMT treatment. Delayed FMT administration at 7 days of-age had limited effect on colonisation and transmission. CONCLUSIONS: We show that transfer of a whole mature microbiome to newly-hatched chicks reduces transmission and colonisation of C.jejuni . This indicates that modification of the broiler chick microbiome can reduce intestinal colonisation of C.jejuni to levels projected to lead to lower the human infection rate. We believe these findings offer a way to identify key taxa or consortia that are effective in reducing C.jejuni colonisation and improving broiler gut health.

/pdf/campylobacter-jejuni-transmission-and-colonisation-in-23pyi4ny99.pdf

Rachel Gilroy

Papers

B lymphocytes play a limited role in clearance of Campylobacter jejuni from the chicken intestinal tract

The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313.

Campylobacter jejuni transmission and colonisation in broiler chickens is inhibited by Faecal Microbiota Transplantation