Effects of a novel, non-invasive pre-hatch application of probiotic for broilers on development of cecum microbiota and production performance.

BACKGROUND: Probiotics are used in the broiler industry to increase production performance. Most often a probiotic is applied by mixing it in the feed, but studies have shown that earlier application may be advantageous. Therefore, in ovo application where the probiotic is administrated into the egg before hatch has been investigated as an alternative application method. However, in ovo application may impact hatchability negatively and may not be feasible at all hatcheries. The purpose of this study was to investigate the effect of a novel non-invasive method for mass application before hatch. The probiotic (E. faecium 669) was applied as a single dose by spray on the unhatched eggs and production performance and development of the cecal microbiota until slaughter was compared with a control flock. Through 16S rRNA sequencing of cecal samples from 25 broilers at day 7, 21 and 37 we compared the microbiota composition and richness for each group. The study was repeated for additional recording of production performance and re-isolation of the probiotic E. faecium from the intestine. RESULTS: In both trials the probiotic E. faecium could be re-isolated from the yolk sac and intestine at hatch and at day 7. Broilers in the probiotic treated groups had a higher performance in terms of bodyweight at day 34 and European production efficiency factor. Finally, a significant reduction of first-week and overall mortality was observed in the probiotic group in the first trial. Based on 16S rRNA profiling, significant differences in alpha diversity were found exclusively at day 37. Estimation of beta diversities, however, identified significant differences in microbiota composition between the control and probiotic group at day 7, 21 and 37. CONCLUSION: The probiotic E. faecium strain successfully colonized broilers before/during hatch after a single spray application at day 18 of incubation. Positive effects of the probiotic were observed in multiple production parameters, including reduced mortality in trial 1, and microbiota analyses indicate significantly different microbiota compositions throughout the experimental phase. Taken together, the novel low-tech mass administration of E. faecium (669) may be considered a feasible strategy for improvements of production parameters in broiler production.

Complete association between CHCF1 genotype and enterotoxigenic Escherichia coli F4ab-associated post-weaning diarrhea in a pig challenge trial.

Enterotoxigenic E. coli (ETEC) susceptibility in pigs is highly influenced by their genotype. The aim of this study was to determine the association between CHCF1 genotype and ETEC F4ab susceptibility in experimentally infected pigs. We investigated ETEC diarrhea development in CHCF1 heterozygous susceptible (RS) (n = 12 pigs) compared to CHCF1 homozygous resistant (RR) (n = 12 pigs) for six days after ETEC F4ab challenge. Afterwards, we genotyped with MUC4 and MUC13 markers to relate performance in identifying ETEC F4ab diarrhea susceptible pigs. In the CHCF1 RS group, 12/12 pigs developed ETEC diarrhea compared with 0/12 pigs in the CHCF1 RR group. Weight gain was lower in CHCF1 RS pigs compared with RR pigs (mean ± SD: 208 ± 323 g and 987 ± 615 g, p = 0.0007). Further, the shedding of hemolytic E. coli was significantly higher in CHCF1 RS pigs from 2 to 6 days post inoculation and they shed the challenge strain for more days (mean ± SD: 3.5 ± 1.6 days versus 0.5 ± 0.5 days, p < 0.0001). Twelve pigs with ETEC diarrhea were misclassified as resistant with the MUC4 marker and four pigs without ETEC diarrhea were misclassified as susceptible with the MUC13 marker. We found complete association between CHCF1 genotype and ETEC diarrhea development in pigs from a herd with Danbred genetics. The CHCF1 marker was more likely to determine the true host susceptibility to ETEC F4ab than the other markers. The marker shows potential for improving reliability of PWD challenge models and potentially for use in breeding for ETEC F4ab/ac resistance.

Post-weaning diarrhea in pigs from a single Danish production herd was not associated with the pre-weaning fecal microbiota composition and diversity.

Introduction: The association between the porcine pre-weaning gut microbiota composition and diversity, and subsequent post-weaning diarrhea (PWD) susceptibility is currently being studied. In this longitudinal study, we examined the association between pre-weaning fecal microbiome composition and diversity, and PWD development in a Danish sow herd. Methods: Forty-five pigs were followed from birth until 7 days after weaning (post-natal day (PND) 33). At PND 33, the pigs were categorized as PWD cases or healthy controls based on fecal consistency. We compared their fecal microbiomes at PND 8, late lactation (PND 27) and 7 days post weaning (PND 33) using 16S rRNA V3 region high-throughput sequencing. At PND 27 and 33, we also weighed the pigs, assessed fecal shedding of hemolytic Escherichia coli by culture and characterized hemolytic isolates by ETEC virulence factors with PCR and by whole genome sequencing. Results: A total of 25 out of 45 pigs developed PWD and one Enterotoxigenic E. coli strain with F18:LT:EAST1 virotype was isolated from most pigs. At PND 33, we found differences in beta diversity between PWD and healthy pigs (R2 = 0.027, p = 0.009) and that body weight was associated with both alpha and beta diversity. Pre-weaning fecal microbiome diversity did not differ between PWD and healthy pigs and we found no significant, differentially abundant bacteria between them. Conclusion: In the production herd under study, pre-weaning fecal microbiome diversity and composition were not useful indicators of PWD susceptibility.

Prevalence of Potential Pathogenic and Antimicrobial Resistant Escherichia coli in Danish Broilers.

Avian pathogenic Escherichia coli (APEC) are important bacteria in broiler production in terms of economy, welfare, and use of antibiotics. During a previous outbreak of APEC in the Nordic countries, it was suggested that the pathogenic clones of E. coli causing the outbreak originated from grandparent stock and were transmitted to the offspring, causing increased first week mortality. This study investigated whether the pathogenic potential of E. coli at the parent and broiler level differs in relation to pathogenic potential described by the level of virulence-associated genes and pattern of antimicrobial resistance. The hypothesis was that, due to higher biosecurity at the parent level, the E. coli population will show a lower level of antimicrobial resistance and carry fewer virulence-associated genes, as a result of fewer E. coli infections observed. From four parent flocks and eight broiler flocks, 715 E. coli were isolated from cloacal swabs of newly hatched chickens (Ross 308). The isolated E. coli were characterized by eight virulence-associated genes and phenotypic resistance against six antimicrobials. It was found that the prevalence of virulence-associated genes and phenotypic antimicrobial resistance varied significantly between flocks, and the virulence-associated genes papC and irp2 and resistance against ampicillin were significantly more prevalent in breeder flocks compared to broiler flocks.

Pilot study on CHCF1 genotype in a pig challenge model for enterotoxigenic Escherichia coli F4ab/ac associated post-weaning diarrhea.

Host genotype is important for enterotoxigenic E. coli (ETEC) susceptibility. We conducted two trials to evaluate the effect of CHCF1 genotype on incidence of ETEC diarrhea. In trial 1 (n = 15 pigs), pigs were inoculated with 108 CFU or 1010 CFU doses of an ETEC F4ac strain. In trial 2 (n = 33 pigs), pigs were inoculated with ETEC F4ab or F4ac. Across trials, all inoculated pigs that developed ETEC diarrhea were CHCF1 heterozygous susceptible (6/6). No inoculated CHCF1 homozygous resistant pigs developed ETEC diarrhea (0/26). Susceptibility towards ETEC F4ac/ab infection might correspond with CHCF1 genotype.

Research Note: The effect of a probiotic E. faecium 669 mitigating Salmonella Enteritidis colonization of broiler chickens by improved gut integrity.

In this study, we investigate the effect of the probiotic E. faecium 669 strain on the gut integrity of broilers and the effect on intestinal colonization with Salmonella Enteritidis. In the in vivo experiment, 120-day-old broilers (Ross 308) were divided into 4 equally sized groups. Group A received the probiotic as a single dose by spray at d 18 of incubation and group B received the probiotic in the drinking water daily throughout the experiment. Group C was untreated control. Group D received the antibiotic Apramycin sulfate in the drinking water. Broilers in all four groups were challenged with S. Enteritidis by oral gavage at d 8 of life. From d 9 to 12, a cloacal swab was collected from all broilers for culturing on Salmonella selective media to determine the shedding. At d 12, birds were euthanized and S. Enteritidis in ceca were enumerated and intestinal samples for histology and host gene expression were collected. The group receiving the probiotic in the drinking water shed significantly less S. Enteritidis compared to the untreated control group at all times. The group receiving a single probiotic application before hatch showed a reduced shedding of Salmonella at d 9 and 10. S. Enteritidis was not detected in the ceca of the antimicrobial treated broilers. Histology of jejuni samples and host gene expression showed that intestinal integrity was enhanced by adding probiotic to the drinking water. Overall, the study shows that pre-hatch and daily application of the probiotic strain E. faecium 669 reduces the colonization of broilers with S. Enteritidis and daily application enhances gut integrity. Application of the probiotic E. faecium strain can be recommended as a method to reduce the colonization of broilers with S. Enteritidis and enhance their gut integrity.

Whole genome sequence comparison of avian pathogenic Escherichia coli from acute and chronic salpingitis of egg laying hens.

BACKGROUND: Infection in the oviduct (salpingitis) is the most common bacterial infection in egg laying hens and is mainly caused by Escherichia coli. The disease is responsible for decreased animal welfare, considerable economic loss as well as a risk of horizontal and vertical transmission of pathogenic E. coli. The outcome of salpingitis may be either acute or chronic. It has not yet been clarified whether the pathological manifestation is a result of the characteristics of the E. coli or whether the manifestation is associated with host factors such as host immunity. RESULTS: From the core- and accessory genome analysis and comparison of 62 E. coli no genetic markers were found to be associated to either acute or chronic infection. Twenty of the 62 genomes harboured at least one antimicrobial resistance gene with resistance against sulfonamides being the most common. The increased serum survival and iron chelating genes iss and iroN were highly prevalent in genomes from both acute and chronic salpingitis. CONCLUSION: Our analysis revealed that no genetic markers could differentiate the E. coli isolated from acute versus chronic salpingitis in egg laying hens. The difference in pathological outcome may be related to other factors such as immunological status, genetics and health of the host. These data indicate that salpingitis is another manifestation of colibacillosis.

Correlation between footpad lesions and systemic bacterial infections in broiler breeders.

Footpad lesions are an important factor in evaluation of animal welfare in broilers regulated by law; however, no legal requirements have been set for the parent birds. Nevertheless, the present study confirms that foot health in broiler breeders declines significantly with increasing age, thus potentially impairing the animal welfare due to pain and discomfort from footpad dermatitis. Furthermore, this is the first report demonstrating a correlation between the presence of footpad lesions and systemic bacterial infections with Gram-positive cocci in broiler breeder birds.

Longitudinal Study on Causes of Mortality in Danish Broiler Breeders.

Broiler production is highly dependent on good health in the parent flocks. The so-called normal mortality in these flocks remains to be addressed to further reduce mortality of the breeders and to improve the quality of broilers. The aim of the present study, therefore, was to investigate the etiology of this breeder mortality to map out possible critical periods during production in relation to possible risks of importance to the offspring. Dead birds from four flocks were subjected to postmortem and bacteriologic examination from onset of lay until slaughter (20-60 weeks). Causes of mortality were divided into noninfectious and infectious etiology. The infectious group could be subdivided into suppurative salpingitis/peritonitis caused by Escherichia coli and other infections (e.g., sepsis, endocarditis, and arthritis) mainly caused by Gram-positive cocci. Data analysis showed that 41% of the birds died from noninfectious causes, while 55% died from infectious causes, and 4% had no known cause of death. The prevalence of noninfectious mortality was highest in the youngest birds and lowest in the oldest birds. In contrast, the infectious mortality was lowest in the young birds and highest at the end of production. Within each age group, the prevalence of salpingitis/ peritonitis was 26% in young birds (20-29 weeks) and progressed throughout production to 41% in the oldest birds (≥50 weeks of age). Mortality due to other infections was low at onset of production (12%), peaking at 40-49 weeks of age (25%). Consequently, 40-49 weeks of age is identified as a critical period with regard to causes of mortality, possible vertical transmission of E. coli to the offspring, and increased risk of Gram-positive coccal infections.

Estudio longitudinal sobre las causas de mortalidad en reproductores pesados daneses. La producción de pollo de engorde depende en gran medida de la buena salud de las parvadas de reproductores. La llamada mortalidad normal en estas parvadas aún debe analizarse para reducir aún más la mortalidad de los reproductores y mejorar la calidad del pollo de engorde. El objetivo del presente estudio, por lo tanto, fue investigar la etiología de esta mortalidad en los reproductores para determinar posibles períodos críticos durante la producción con relación con los posibles riesgos que sean de importancia para la progenie. Las aves muertas de cuatro parvadas se sometieron a un examen post mortem y bacteriológico desde el inicio de la postura hasta la edad de sacrificio (20­60 semanas). Las causas de mortalidad se dividieron en etiología no infecciosa e infecciosa. El grupo de causas de origen infeccioso se subdividió en salpingitis/peritonitis supurativa (SP) causada por Escherichia coli y otras infecciones (por ejemplo, sepsis, endocarditis y artritis) causadas principalmente por cocos Gram positivos. El análisis de los datos mostró que el 41% de las aves murieron por causas no infecciosas, mientras que el 55% murió por causas infecciosas y el 4% por causas desconocidas. La prevalencia de mortalidad no infecciosa fue más alta en las aves más jóvenes y más baja en las aves con mayor edad. En contraste, la mortalidad infecciosa fue más baja en las aves jóvenes y más alta al final de la producción. Dentro de cada grupo de edad, la prevalencia de salpingitis/peritonitis supurativa fue del 26% en aves jóvenes (20­29 semanas) y aumentó a lo largo de la producción hasta el 41% en las aves con más edad (≥50 semanas de edad). La mortalidad debida a otras infecciones fue baja al inicio de la producción (12%), alcanzando un máximo de 40 a 49 semanas de edad (25%). En consecuencia, la edad entre 40 a 49 semanas se identifica como un período crítico con respecto a las causas de mortalidad, con la posible transmisión vertical de E. coli a la progenie y con el aumento del riesgo de infecciones por cocos Gram positivos.

Characterization of Escherichia coli causing cellulitis in broilers.

The aim of the study was to investigate cellulitis caused by Escherichia coli which has been responsible for economic and welfare problems in Danish broiler production between 2014 and 2016. The study included 13 flocks with unusually high condemnation rates due to cellulitis during a period of approximately one year. From six flocks, 126 condemned carcasses were collected at a Danish slaughterhouse. Further 272 broilers dead on their own were collected on nine broiler farms from flocks with increased mortality and cellulitis (2 farms included both birds from the rearing period and broilers subsequently condemned). All broilers were subjected to post mortem investigation including bacteriology and 247 E. coli isolates were obtained in pure culture from typical lesions of cellulitis. Two-hundred-thirty six E. coli isolates were investigated by pulsed field gel electrophoresis for clonality and 21 selected strains representing major clones were subsequently multi locus sequence typed allowing comparison to sequence types (ST) in the databases. One dominating PFGE type (A) was found to cause cellulitis on all 13 flocks (67% of all isolates). The clone belonged to ST117, which is well described as a pathogen in poultry, and was the primary agent responsible for cellulitis. Whole genome sequencing of eight E. coli isolates confirmed the close genetic relationship between isolates from the outbreaks and showed the presence of genes predicted to encode for the autotransporter proteins aatA, pic and upaG, reported to be of importance for adhesion of E. coli to eukaryotic cells.

Staphylococcus agnetis, a potential pathogen in broiler breeders.

In this study, four broiler parent flocks have been followed from the onset of the production period (week 20) until slaughter (week 60). Every week, approximately ten dead broiler breeders, randomly selected among birds dead on their own, were collected and subjected to a full post mortem analysis including bacteriological examination. In total 997 breeders were investigated and for the first time Staphylococcus agnetis was isolated in pure culture from cases of endocarditis and septicemia from 16 broiler breeders. In addition, the cloacal flora from newly hatched chickens originating from the same four flocks were characterized and S. agnetis was found in pure culture of several newly hatched chickens (n=12) and only in one case in combination with another species. Clonality of the isolates was examined by pulsed-field-gel-electrophoresis which showed indistinguishable patterns in isolates from both broiler breeders and broilers. Three isolates were whole genome sequenced to obtain knowledge on virulence genes. The isolates harbored a number of genes encoding different fibrinogen binding proteins and toxins which might be important for virulence. The present findings demonstrate that S. agnetis may be associated with mortality in broiler breeders. No disease was associated with the broilers which were found positive for S. agnetis in the cloaca.

Longitudinal study of transmission of Escherichia coli from broiler breeders to broilers.

Escherichia coli is of major importance in industrial broiler production as the main cause of salpingitis and peritonitis in broiler breeders. Furthermore E. coli is the most common cause of first week mortality in broiler chickens. The aim of the present study was to investigate the transmission of E. coli, isolated from broiler breeders with salpingitis, to the progeny and the possibility of subsequent first week mortality. Four parent flocks were followed during the whole production period (20-60 weeks) by post mortem and bacteriological examination of randomly selected dead birds. Newly hatched chickens from each flock were swabbed in the cloaca on four occasions (parent age 30, 40, 50, 60 weeks) and E. coli was isolated. Causes of first week mortality were determined pathologically and bacteriologically. E. coli isolates from parents, newly hatched chickens and first week mortality were selected for Pulsed-Field-Gel-Electrophoresis (PFGE) and Multi-Locus-Sequence-Typing (MLST) to determine their clonal relationships. E. coli was the main cause of both salpingitis in parents and first week mortality in broilers, and E. coli dominated the bacterial flora of the cloaca of newly hatched chickens. PFGE of E. coli showed identical band patterns in isolates from the three different sources indicating a transmission of E. coli from parent birds to chickens. In conclusion, E. coli isolated from salpingitis in broiler parents were found to be transmitted to broilers in which some sequence types contributed to the first week mortality.

Enterococcus and Streptococcus spp. associated with chronic and self-medicated urinary tract infections in Vietnam.

BACKGROUND: Urinary tract infections (UTI) are one of the most common infections among women worldwide. E. coli often causes more than 75% of acute uncomplicated UTI, however, little is known about how recurrent UTIs and indiscriminate use of antimicrobials affect the aetiology of UTIs. This study aimed to establish the aetiology of UTI in a population of recurrent and self-medicated patients referred from pharmacies to a hospital in Hanoi, Vietnam and to describe genotypes and antimicrobial susceptibility of the associated bacterial pathogens. The aetiology of bacterial pathogens associated with UTI (defined as ≥ 104 CFU/ml urine) was established by phenotypic and molecular methods. Enterococcus faecalis isolates were typed by Multi Locus Sequence Typing (MLST), Pulsed-Field Gel Electrophoresis (PFGE) and antimicrobial susceptibility testing. METHODS: Urine samples from 276 patients suffering symptoms of urinary tract infection were collected and cultured on Flexicult agar® allowing for detection of the most common urine pathogens. Patients were interviewed about underlying diseases, duration of symptoms, earlier episodes of UTI, number of episodes diagnosed by doctors and treatment in relation to UTI. All tentative E. faecalis and E. faecium isolates were identified to species level by PCR, 16S rRNA and partial sequencing of the groEL gene. E. faecalis isolates were further characterized by Multi Locus Sequence Typing and antimicrobial susceptibility testing. RESULTS: Mean age of 49 patients was 48 yrs (range was 11-86 yrs) and included 94% women. On average, patients reported to have suffered from UTI for 348 days (range 3 days-10 years, and experienced 2.7 UTIs during the previous year). Cephalosporins were reported the second drug of choice in treatment of UTI at the hospital. E. faecalis (55.1%), E. coli (12.2%) and Streptococcus gallolyticus subsp. pasteurianus (8.2%) were main bacterial pathogens. MIC testing of E. faecalis showed susceptibility to ampicillin, penicillin and vancomycin, but high-level resistance against gentamicin (48.1%). MLST revealed 12 Sequence Types (ST) of which ST 16 made up 44.5% and showed closely related PFGE types. CONCLUSION: The different aetiology of UTI compared with reports elsewhere, where E. coli dominates, may be a result of chronic and recurrent UTIs together with indiscriminate use of antimicrobials. The similar genotypes shown by epidemiologically unrelated ST 16 isolates in Vietnam and elsewhere, suggest that E. faecalis ST 16 might represent a globally distributed clone. Treatment of UTI with cephalosporins may select for E. faecalis as it is intrinsic resistant and further studies are needed to establish the source(s) and role of E. faecalis ST 16 in acute UTI.

Enterococcus faecalis clones in poultry and in humans with urinary tract infections, Vietnam.

Enterococcus spp. as pathogens have increased, but the sources of infection often remain unclear. To investigate whether poultry might be a reservoir for E. faecalis-associated urinary tract infections (UTIs) in humans, we characterized E. faecalis isolates from patients in Vietnam with UTIs during January 2008-January 2010 and poultry living in close contact with them by multilocus sequence typing (MLST), pulsed-field gel electrophoresis, analysis of antimicrobial drug susceptibility patterns, and sequencing of virulence genes. In 7 (23%) of 31 UTI cases, we detected identical MLST, indistinguishable or closely related pulsed-field gel electrophoresis patterns, and similar antimicrobial drug susceptibility patterns. Isolates from urine and poultry showed identical virulence gene profiles, except for 1 variation, and individual genes showed identical sequences. The homology of isolates from urine and poultry further indicates the zoonotic potential and global spread of E. faecalis sequence type 16, which recently was reported in humans with endocarditis and in pigs in Denmark.