Effect of bacteriophage application on Campylobacter jejuni loads in commercial broiler flocks.

Campylobacteriosis is the most frequent food-borne human enteritis. The major source for infection with Campylobacter spp. is broiler meat. Risk assessments consider the reduction of Campylobacter in primary production to be most beneficial for human health. The aim of this study was to test the efficacy of a bacteriophage application under commercial conditions which had proved to be effective in previous noncommercial studies under controlled experimental conditions. A phage cocktail for Campylobacter reduction was tested on three commercial broiler farms each with a control and an experimental group. Colonization of Campylobacter was confirmed prior to phage application in fecal samples. Subsequently, a phage cocktail was applied via drinking water in the experimental group (log10 5.8 to 7.5 PFU/bird). One day after phage application, Campylobacter counts of one experimental group were reduced under the detection limit (<50 CFU/g, P=0.0140) in fecal samples. At slaughter, a significant reduction of >log10 3.2 CFU/g cecal content compared to the control was still detected (P=0.0011). No significant reduction was observed in the experimental groups of the other trials. However, a significant drop in cecal Campylobacter counts occurred in a phage-contaminated control. These results suggest that maximum reduction of Campylobacter at the slaughterhouse might be achieved by phage application 1 to 4 days prior to slaughter.

Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens.

BACKGROUND: Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. RESULTS: Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. CONCLUSION: The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

Antibody titers in turkeys increase after multiple booster vaccinations with an attenuated Salmonella live vaccine.

OBJECTIVE: Human Salmonellosis is one of the most frequently reported foodborne zoonoses in the European Union. The most common source of human infections is the consumption of poultry products. Besides management and hygiene practices vaccination of poultry livestock is seen as one way to reduce Salmonella infections in humans. Turkey flocks in Europe are frequently infected with Salmonella and until recently there was no live vaccine for turkeys available. The aim of the present study was to examine the development of humoral antibodies after repeated vaccination with a bivalent live Salmonella vaccine containing attenuated Salmonella Typhimurium and Salmonella Enteritidis strains. Furthermore the colonization of the caecum with the vaccine strains and their spread to liver and spleen as well as the course of their fecal excretion was observed. RESULTS: Antibody production was hardly detectable after the first vaccination but increased after booster vaccinations. Both the Salmonella Enteritidis and the Salmonella Typhimurium vaccine strain were reisolated from caecum contents and organ samples. After booster vaccinations the re-isolation rates were reduced. The shedding of the vaccine strains was most pronounced after the first vaccination.

Efficacy of a Salmonella live vaccine for turkeys in different age groups and antibody response of vaccinated and non-vaccinated turkeys.

OBJECTIVE: Human Salmonellosis continues to be one of the most important foodborne zoonoses worldwide, although a decrease in case numbers has been noted in recent years. It is a foodborne zoonotic infection most commonly associated with the consumption of raw egg products but also with meat consumption including the consumption of poultry products. Turkey flocks in Europe have been reported to be affected by Salmonella infection, too. The present study examines the efficacy of a newly licensed Salmonella life vaccine in reducing infections with the Salmonella serovars Typhimurium and Enteritidis in turkeys. Turkeys were vaccinated the first day of life and at the age of 6 and 16 weeks. Groups of birds which had received different numbers of vaccinations were then submitted to challenge trials with either SE or ST. RESULTS: In vaccinated birds Salmonella counts in liver and spleen and, less effectively, in caecum were reduced compared to unvaccinated birds. In several groups serum antibody-titers were statistically significantly higher in vaccinated turkeys than in non-vaccinated ones at day seven post infection, but only in one out of six groups at day 14 post infection.

Immune response to Salmonella infections in vaccinated and non-vaccinated turkeys.

Vaccination has been widely used to reduce the Salmonella burden in poultry and subsequently the transmission to humans. Concerning turkey, there is little knowledge on the immune response to colonization and invasion by Salmonella species or about efficacy of vaccination and involved immune mechanisms. In the present study, turkeys were vaccinated at the day of hatch and infected with Salmonella Typhimurium (ST) or Enteritidis (SE) field strains three weeks later. A control group was kept uninfected. After challenge infection, bacterial counts in the cecal content, liver and spleen were determined 7 and 14days post infection. They were often statistically significantly lower in vaccinated poults than in non-vaccinated ones. Production of iNOS, and the cytokines IL-8, IL-10 and IFN-γ were reduced in vaccinated birds. However, neither the influx of CD4+, CD8α+ and CD28+ cells into cecal mucosa after infection nor the antibody response were statistically significantly altered in vaccinated birds.

Colonization pattern of C. jejuni isolates of human and avian origin and differences in the induction of immune responses in chicken.

Campylobacter jejuni (C. jejuni) is the most frequently reported bacterial food-borne pathogen. Poultry is regarded to be the main reservoir for human campylobacteriosis. By some authors C. jejuni is considered as a commensal of the chicken gut, but clinical signs may be observed indicating host-pathogen interaction. Little is known about C. jejuni strain dependent differences in stimulation of the immune response in chicken. Therefore we investigated the colonization pattern as well as humoral and cell-mediated immune parameters in three-week old specific pathogen-free (SPF) layer-type (LT) chicken after inoculation with different human and avian derived C. jejuni isolates between three and 21dpi. In a subsequent experiment we investigated earlier time points and additionally compared commercial broilers with SPF-LT chicken to identify possible differences after inoculation of selected C. jejuni strains from prior experiments. No clinical symptoms were observed during the experiments. The colonization pattern did not correlate with the strain origin or magnitude of circulating C. jejuni specific antibodies. Only minor changes were observed in caecal T cell populations after C. jejuni inoculation compared to non-inoculated controls. Interestingly the mRNA expression of IL-6 and IFN-gamma was down regulated at some time points after inoculation suggesting a possible immunomodulatory effect of some C. jejuni strains in the gut. Furthermore, broilers were colonized to a higher extend and the local immune cell response was different compared to the SPF -LT birds indicating an influence of genotype on C. jejuni colonization pattern. Overall, our study demonstrates that the outcome of C. jejuni infection in chicken is influenced by genotype and partially by the C. jejuni isolate, leading to differences in the early immune response and thereafter the control of colonization and infection.

Immune response of turkey poults exposed at 1 day of age to either attenuated or wild Salmonella strains.

Salmonellosis is a foodborne zoonosis that is most often acquired by consuming poultry products such as eggs and poultry meat. Amongst other measures the vaccination of food-producing poultry is thought to contribute to a reduction in human salmonellosis. In the European Union (EU) in 2014 the licence of a commercially available Salmonella vaccine for chickens and ducks was extended to turkeys. In the present study, we examined the course of infection with a virulent Salmonella enterica ssp. enterica serovar Enteritidis (SE) strain, a virulent S. enterica ssp. enterica serovar Typhimurium (ST) strain, and the respective live vaccine containing attenuated strains of both serovars in turkey poults. Besides collecting microbiological data and detecting invading Salmonella in the caecal mucosa via immunohistochemistry, we also assessed immune reactions in terms of antibody production, influx of CD4-, CD8α- and CD28-positive cells into the caecal mucosa and the expression of four different immune-related proteins. We found that the attenuated strains were able to invade the caecum, but to a lower degree and for a shorter duration of time compared to virulent strains. Infections with virulent Salmonellae also caused an increase in CD4-, CD8α- and CD28-positive cells in the caecal mucosa and an increased transcription of iNOS, IL-8-like chemokines, and IFN-γ. In poults treated with attenuated bacteria we could not detect any evidence of immune responses. In conclusion, the vaccine showed a lower degree of caecal invasion and induced weaker immune reactions compared to the virulent Salmonella strains in turkeys. The efficiency of the vaccine has to be verified in future studies.

Varying antibody responses of laying hens housed in an aviary system and in furnished cages.

Antibody titers after vaccination against Infectious Bronchitis Virus (IBV), Newcastle Disease Virus (NDV) and after natural infection with Campylobacter were analyzed in five trials with Lohmann Silver laying hens kept in two different housing systems. In these studies it could be demonstrated that antibodies against IBV and Campylobacter were in 4 out of 5 respectively in 2 out of 5 trials significantly higher in hens housed in an aviary system compared to those kept in furnished cages. The opposite trend was observed for antibodies against NDV which were on average significantly higher in cages. The mean mortality rate was slightly higher in hens kept in the aviary system compared to the cage system.

Campylobacter infections in four poultry species in respect of frequency, onset of infection and seasonality.

Over a seven-year period (2001-2007) flocks of four poultry species, 52 broiler flocks, 46 Pekin duck flocks, 22 Muscovy duck flocks, 20 turkey flocks, which were kept on the same farm, were continuously investigated for Campylobacter (C). Altogether 76.1% of the broiler flocks, 59.6% of the Pekin duck flocks, 68.2% of the Muscovy duck flocks and 90.0% turkey flocks were Campylobacter positive. The prevalence during the course of the fattening period increased steadily. There was no specific point of time for the onset of infection. More detailed examination over a one-year period showed the highest isolation rates of C. coli from July to September and a higher isolation rate of the same agent with increasing age, in all species except Muscovy ducks. Moreover, C. coli was isolated more often from the lungs of broilers and Muscovy ducks than from the other two bird species. Flocks of all species housed during the summer months featured a higher prevalence of Campylobacter colonisation than those housed in winter. This was statistically significant for broilers. Another approach for evaluating the seasonality of Campylobacter colonisation was to compare the age of the respective poultry species when the onset occurred in summer and in winter. All poultry species were younger when infection was introduced into a flock in summer. This was statistically significant for broilers and for Pekin ducks.

Effects of carry-over of fluoroquinolones on the susceptibility of commensal Escherichia coli in the intestinal microbiota of poultry.

Due to the frequent use of antibacterials in veterinary medicine as well as in human medicine the occurrence of antibacterial resistance rises worldwide. But independent of the usage of antimicrobials the microbiota from animals as well as from humans already harbour a diversity of resistance genes. As a consequence of manufacturing animal production the treatment of livestock in case of illness is carried out via feed or drinking water. This automatically implies several risks. It has been demonstrated that an antibiotic treatment of livestock via feed or drinking water cause an accumulation of antibiotics and their metabolites in the direct environment of animals. This can lead to a carry-over or rather a resumption of the antimicrobials and their metabolites. Thus, the aim of this study was to determine the influence of carry-over of enrofloxacin as a representative of the fluoroquinolones on the development of bacterial resistance of commensal E. coli in the intestinal microbiota of poultry. Therefore four different treatment groups were provided and the minimal inhibitory concentrations (MICs) of commensal E. coli were measured: One group acted as untreated control, another one was therapeutically treated with the recommended dosage. The third and fourth group were exposed to different "carry-over dosages" for three weeks, 3% of the recommended dosage were applied to the third and 10% to the fourth group. To determine the influence of a therapeutic treatment on a prestressed microbiota, both groups were treated with the recommended dosage for five days. The present study demonstrates that every kind of exposure of the commensal microbiota of poultry with enrofloxacin leads to an amplification and selection of resistant E. coli, which persist in the commensal microbiota. A long-term exposure of gut microbiota, which already harbour non-wild type E. coli, with high levels of carry-over of fluoroquinolones may lead to a development of high-level clinically resistant E. coli in the commensal microbiota. It has to be investigated to which extent antimicrobial leftovers occur in animal production.

Colonisation of a phage susceptible Campylobacter jejuni population in two phage positive broiler flocks.

The pathogens Campylobacter jejuni and Campylobacter coli are commensals in the poultry intestine and campylobacteriosis is one of the most frequent foodborne diseases in developed and developing countries. Phages were identified to be effective in reducing intestinal Campylobacter load and this was evaluated, in the first field trials which were recently carried out. The aim of this study was to further investigate Campylobacter population dynamics during phage application on a commercial broiler farm. This study determines the superiority in colonisation of a Campylobacter type found in a field trial that was susceptible to phages in in vitro tests. The colonisation factors, i.e. motility and gamma glutamyl transferase activity, were increased in this type. The clustering in phylogenetic comparisons of MALDI-TOF spectra did not match the ST, biochemical phenotype and phage susceptibility. Occurrence of Campylobacter jejuni strains and phage susceptibility types with different colonisation potential seem to play a very important role in the success of phage therapy in commercial broiler houses. Thus, mechanisms of both, phage susceptibility and Campylobacter colonisation should be further investigated and considered when composing phage cocktails.

Microplate-test for the rapid determination of bacteriophage-susceptibility of Campylobacter isolates-development and validation.

A simple susceptibility test using 800 isolates of one Campylobacter strain with different degrees of susceptibility and four bacteriophages of the British phage typing scheme was developed and examined for its suitability. The test presented is economically cheaper and less time consuming than the conventional agar overlay plate assay and therefore enables the monitoring of changes in the susceptibility pattern during phage therapy under practical field conditions. The main objective of this study was to compare the simplified test with the conventional agar overlay plate assay. The conventional test describes for a population of Campylobacter: i. the rate of resistant isolates (0 plaques) and ii. the degree of susceptibility, also called relative efficiency of plating (EOP), for the remaining isolates. The simplified test divides the isolates into four susceptibility ranks, which are easily distinguishable to the naked eye. Ten Campylobacter isolates out of each rank were subjected to the conventional method for validation of the simplified test. Each resistance rank contained isolates showing certain degrees of susceptibility, reflecting decreasing susceptibility by an increase of the rank. Thus, the simplified test correlated well with the conventional method. Nevertheless, it can be suggested for a clear cut to summarise the first thee ranks as "high susceptible" and to mark out the fourth rank as reduced susceptible. Further test improvements will enable the monitoring of the degree of susceptibility and potentially also of resistance during phage therapy in the field. To ensure a long-lasting successful use of phage therapy, further studies on both the loss of susceptibility and the development of resistance of Campylobacter against phages combined with their impact on phage therapy will be necessary.

Impact of a single phage and a phage cocktail application in broilers on reduction of Campylobacter jejuni and development of resistance.

Campylobacteriosis is currently the most frequent foodborne zoonosis in many countries. One main source is poultry. The aim of this study was to enhance the knowledge about the potential of bacteriophages in reducing colonization of broilers with Campylobacter , as there are only a few in vivo studies published. Commercial broilers were inoculated with 104 CFU/bird of a Campylobacter jejuni field strain. Groups of 88 birds each were subsequently treated with a single phage or a four-phage cocktail (107 PFU/bird in CaCO3 buffered SM-Buffer). Control birds received the solvent only. Afterwards, subgroups of eleven birds each were examined for their loads with phages and Campylobacter on day 1, 3, 7, 14, 21, 28, 35 and 42 after phage application. The susceptibility of the Campylobacter population to phage infection was determined using ten isolates per bird. In total 4180 re-isolates were examined. The study demonstrated that the deployed phages persisted over the whole investigation period. The Campylobacter load was permanently reduced by the phage-cocktail as well as by the single phage. The reduction was significant between one and four weeks after treatment and reached a maximum of log10 2.8 CFU/g cecal contents. Phage resistance rates of initially up to 43% in the single phage treated group and 24% in the cocktail treated group later stabilized at low levels. The occurrence of phage resistance influenced but did not override the Campylobacter reducing effect. Regarding the reduction potential, the cocktail treatment had only a small advantage over the singe phage treatment directly after phage administration. However, the cocktail moderated and delayed the emergence of phage resistance.

Carry-over of thermophilic Campylobacter spp. between sequential and adjacent poultry flocks.

Nineteen flocks of four poultry species were monitored at a veterinary field station to investigate the distribution and spread of Campylobacter genotypes between sequential and adjacent flocks. Caecal and liver samples were obtained at frequent intervals from birds of all flocks and examined for Campylobacter. Amplified fragment length polymorphism (AFLP) analysis was performed to genotype Campylobacter isolates. Of the 1643 caecal and liver samples investigated, 452 (27.5%) caecal samples and 11 (0.7%) liver samples contained Campylobacter. Of the caecal isolates 76.3% were identified as Campylobacter jejuni and 23.7% were identified as Campylobacter coli. Poultry flocks were largely colonized by more than one AFLP type and an intense exchange of Campylobacter genotypes between different poultry flocks occurred. These findings indicate that multiple genotypes can constitute the Campylobacter population within single poultry flocks, hinting to different sources of exposure and/or genetic drifts within the Campylobacter population. Nevertheless, in most flocks single Campylobacter genotypes predominated. Some strains superseded others resulting in colonization by successive Campylobacter genotypes during the observation period. In conclusion, the data demonstrate that the large genetic diversity of Campylobacter must be considered in epidemiological evaluations and microbial risk assessments of Campylobacter in poultry.