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Background: Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing., Results: The co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe 3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins., Conclusion: The results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens., (© 2024. The Author(s).)

In the current study, we investigated decreased hatchability and increased embryonic mortality in two farms of layer breeders (flocks A1 and B1) and a farm of broiler breeders (flocks C1 and C2) from Austria, which also presented discoloration of eggshells in 2% of the eggs. After conducting clinical evaluations and the approval that the feed operator was common for flocks A1 and B1, and C1 and C2, it was decided to investigate the feed. Our findings revealed that the feed contained levels of nicarbazin and narasin up to five and 14 times, respectively, above the maximum limits allowed by the European Union for nontarget species. On the other hand, there were no significant abnormalities in vitamin levels, which were also described as the etiology of the noticed abnormalities. Switching to a noncontaminated feed resulted in the clinical signs and production parameters returning to expected ranges. This report emphasizes the significance of considering feed contamination by nicarbazin and narasin as a potential cause of hatchery losses in nontarget species, even in the absence of other clinical signs.

In numerous countries, strict and targeted measures concerning Salmonella monitoring and control are implemented and high quality of surveillance is ensured by obligatory investigation of samples from the primary production level of animals according to EN/ISO standards. Here, 2 phenotypic characteristics of Salmonella exhibited on compulsory media are crucial, namely, motility demonstrated on modified semisolid Rappaport Vassiliadis agar (MSRV), and production of hydrogen sulfide (H 2 S) on xylose lysine deoxycholate agar (XLD). In the present study, we describe the detection of Salmonella Infantis variants found in broiler environmental samples with major alterations in their growth characteristics on MSRV, XLD, and brilliant green-phenol red-agar (BPLS). The variants proved to be non-motile on MSRV and displayed non-confirming colony appearances on the previously mentioned selective agars. The growth spectrum comprised pinhead sized yellow colonies with small black centers, but also pinpoint sized colorless colonies, both colony types of regular shape. Our work contributes to highlight the finding of S . Infantis variants which possess more than one phenotypic deviation from the "typical" growth characteristics and by this limit the detection power of the actual obligatory used media. IMPORTANCE Salmonellosis caused by non-typhoidal Salmonella serovars is the second most frequently reported zoonotic disease in humans in the EU. The transmission of these agents is mainly via contaminated food of animal origin. In this context, poultry products are the main source of infection. Therefore, continuous and standardized surveillance of the prevalence of such Salmonella serovars at the primary production level is essential. Our findings show the phenotypic heterogeneity of the serovar Infantis and provide growth characteristics of atypical variants. Such variants pass unnoticed official screening methods, resulting in incorrect identification and being underrepresented in epidemiological surveillance programs., Competing Interests: The authors declare no conflict of interest.

The cross-linking of structural proteins is critical for establishing the mechanical stability of the epithelial compartments of the skin and skin appendages. The introduction of isopeptide bonds between glutamine and lysine residues depends on catalysis by transglutaminases and represents the main protein cross-linking mechanism besides the formation of disulfide bonds. Here, we used a fluorescent labeling protocol to localize the activity of transglutaminases on thin sections of the integument and its appendages in mammals and birds. In human tissues, transglutaminase activity was detected in the granular layer of the epidermis, suprabasal layers of the gingival epithelium, the duct of sweat glands, hair follicles and the nail matrix. In the skin appendages of chickens, transglutaminase activity was present in the claw matrix, the feather follicle sheath, the feather sheath and in differentiating keratinocytes of feather barb ridges. During chicken embryogenesis, active transglutaminase was found in the cornifying epidermis, the periderm and the subperiderm. Transglutaminase activity was also detected in the filiform papillae on the tongue of mice and in conical papillae on the tongue of chickens. In summary, our study reveals that transglutaminase activities are widely distributed in integumentary structures and suggests that transglutamination contributes to the cornification of hard skin appendages such as nails and feathers.

Unlike in chickens, dynamics of the gut microbiome in turkeys is limitedly understood and no data were yet published in context of pathological changes following experimental infection. Thus, the impact of Histomonas meleagridis-associated inflammatory changes in the caecal microbiome, especially the Escherichia coli population and their caecal wall invasion in turkeys was investigated. Birds experimentally inoculated with attenuated and/or virulent H. meleagridis and non-inoculated negative controls were divided based on the severity of macroscopic caecal lesions. The high throughput amplicon sequencing of 16SrRNA showed that the species richness and diversity of microbial community significantly decreased in severely affected caeca. The relative abundances of operational taxonomic units belonging to Anaerotignum lactatifermentans, E. coli, and Faecalibacterium prausnitzii were higher and paralleled with a decreased abundances of those belonging to Alistipes putredinis, Streptococcus alactolyticus, Lactobacillus salivarius and Lactobacillus reuteri in birds with the highest lesion scores. Although the relative abundance of E. coli was higher, the absolute count was not affected by the severity of pathological lesions. Immunohistochemistry showed that E. coli was only present in the luminal content of caecum and did not penetrate even severely inflamed and necrotized caecal wall. Overall, it was demonstrated that the fundamental shift in caecal microbiota of turkeys infected with H. meleagridis was attributed to the pathology induced by the parasite, which only led to relative but not absolute changes in E. coli population. Furthermore, E. coli cells did not show tendency to penetrate the caecal tissue even when the intestinal mucosal barriers were severely compromised.

The present report describes an outbreak of Pullorum disease in a young layer parent stock in Austria. The flock, which comprised 14,220 Lohmann brown layer chickens, experienced high mortality from the first week of life, reaching a total of 1905 chickens in the fifth week, when the flock was depopulated. Clinical signs included uneven size of the chicks, pasty vents, apathy, and diminished water and feed intake, with some birds presenting central nervous system signs such as tremors and torticollis. The postmortem investigation of 43 birds, of ages 1 to 4 weeks, revealed retained yolk sacs filled with caseous exudate, purulent airsacculitis, hepatitis with whitish pinpoint coalescing necrotic foci, splenitis with splenomegaly, hemorrhagic-mucoid enteritis in the small intestine, fibrinous typhlitis, nephromegaly, and urate deposits in the ureters and cloaca. Inflammation and/or necrosis were identified in liver, spleen, kidney, small intestine, and heart by histopathology. However, no histopathologic lesions were observed in the brain. Salmonella enterica was isolated from heart, liver, spleen, and brain in pure culture. Group-specific serotyping determined the presence of group D, with S. enterica subspecies enterica serovar Gallinarum being confirmed based on the Kauffmann-White scheme. A duplex PCR further identified S. enterica subspecies enterica serovar Gallinarum biovar Pullorum as the responsible agent for the outbreak. Subsequently, the grandparent flocks, from which the affected flock originated, were tested and found to be negative for Salmonella Pullorum, with no other progenies from the same grandparents developing disease. Although the source of the pathogen could not be identified, such findings highlight the importance of "old" pathogens such as Salmonella Pullorum causing sudden high mortality in chicks, even in a highly controlled environment.

Bacterial infections in chicken eggs often cause mortality of embryos and clinical consequences in chicks but the pathological mechanism is unclear. We investigated the pathological changes and bacterial growth kinetics in dead and live embryos following infection with 2 Escherichia coli strains with a different clinical background and with 1 Salmonella Enteritidis strain. In 2 experiments, 12-day-old embryos were infected via the allantoic sac with 100 µl of 1 to 5 × 10 2 CFU/ml of one of the bacteria. In experiment 1, only dead embryos were sampled until 4 days postinfection (dpi), and surviving embryos were sampled at 5 dpi. In experiment 2, sampling was performed in dead and killed embryos sequentially at 1, 2, 3, and 4 dpi. The bacteria showed varying pathogenicity in embryos. The yolk sacs of dead embryos showed congestion, inflammation, damaged blood vessels, and abnormal endodermal epithelial cells. Such lesions were absent in the yolk sacs of negative control embryos and in those of embryos that survived infection. The livers and hearts of dead embryos showed congestion and lysed erythrocytes with no morphological changes in hepatocytes or myocardial cells. All bacteria multiplied rapidly in the yolks of infected embryos, although this did not predict survival. However, the livers of dead embryos contained significantly higher bacterial loads than the livers of the embryos that survived infection. The results provide evidence that lesions in the yolk sac, which have been neglected to date, coincide with embryonic mortality, underlining the importance of healthy yolk sacs for embryo survival.

The health status of broilers raised at lower stocking density and limited antimicrobial use (but routine anticoccidials) was assessed in order to identify prevalent causes of disease, mortality and reduced performance. "Dead-on-farm"(DOF) broilers from 145 commercial flocks were investigated at two different time points (TP1:7-14 and TP2:28-35 days of age); per sampling, 6-10 DOF broilers were selected for post-mortem investigation and gross pathomorphological changes were assessed from 2717 birds in total. Post-mortem findings were substantiated by bacteriological, virological and parasitological investigations. Furthermore, production data of all flocks were collected and used to perform comprehensive statistical analysis. Overall, colibacillosis was found most important with a significant negative impact on flock health, productivity and profitability through all ages of broiler production. At TP1, primary reasons for mortality comprised yolk sac infections, generally found together with fibrinous polyserositis due to E. coli . Furthermore, femoral lesions, which correlated with increased flock mortality, were associated with detection of E. coli . At TP2, ascites was detected frequently in DOF broilers, correlating with increased production losses in the fourth and fifth weeks of life. No aetiological link between the presence of ascites and the detection of the investigated pathogens was observed, instead a positive correlation was noticed with altitude above sea level of the farm, and with the sex of the birds. Disease conditions could not be linked with the housing system. Presence of infectious bronchitis virus, avian reovirus and fowl adenovirus did not correlate with macroscopic lesions or a specific disease. RESEARCH HIGHLIGHTS In young broilers lesions of visceral organs due to bacterial infections dominated. Colibacillosis impacts broiler health, productivity and profitability independent of the age of birds. Disorders of the locomotor system were frequently observed throughout production. Older broilers frequently showed pathologic changes due to metabolic disorders. Overall, a shift from infectious towards metabolic disease conditions was noticed.

In this investigation, data on antimicrobial resistance (AMR) profiles of 213 Gallibacterium anatis isolates were determined from 93 laying hens originating from 39 flocks. Each flock was sampled three times during its life time for the presence of G. anatis . The broth microdilution method was applied comprising 21 antimicrobial substances. Multidrug resistance was found in 96.2% of the G. anatis isolates. Most of the isolates were resistant to tetracycline (89.2%), tylosin (94.8%), enrofloxacin (58.2%), nalidixic acid (77.4%), and sulfamethoxazole (77.0%). Resistance against antimicrobial substances increased significantly with the age of birds. A total of 99 different AMR profiles were detected. On flock level, different AMR profiles were found in 71.8% of the flocks independent of the sampling time point. On bird level, identical AMR profiles were mostly found in isolates originating from the same organ of a single bird, but 22 such paired isolates differed in their AMR profile. Variations of AMR profiles were found within isolates from a single bird, but from different organs. Isolates from systemic organs were significantly more resistant to different antimicrobial substances compared to isolates from the reproductive tract. No influence could be found in regard to an increase of resistance and applied antibiotic treatment.

Gallibacterium is a genus of the family of Pasteurellaceae . It is well known as a commensal inhabitant of the respiratory and reproductive tract of healthy chickens. But in the last years, Gallibacterium anatis is increasingly reported in field cases with a decrease in laying performance due to infections of the reproductive tract. The aim of the present study was to investigate the implication of G. anatis infection in layer flocks facing a decrease in laying performance in Morocco. Birds were received from five different laying hen farms in two regions in Morocco showing a drop of egg production. Necropsy revealed 46.1 % (24/52) of sampled birds showed variable lesions in ovaries, salpinx, and trachea. In fact, 24 birds were affected by salpingitis, 18 by oophoritis, and 11 birds by atrophy of ovaries. Furthermore, tracheitis was observed in 24 birds. Bacteriological investigation was done from different organs, and G. anatis was found in ovaries ( n = 20), trachea ( n = 17), and cloaca ( n = 3). Identification was based on growth morphology, Gram staining, and biochemical properties. Additionally, polymerase chain reaction test using specific primers for the genus identification was carried out. All isolates showed bands of 925 bp specific for G. anatis expressing the virulent toxin GtxA. Antibiotic resistance testing was performed and revealed that isolates were sensitive to enrofloxacin, florfenicol, and gentamycin but resistant to ampicillin, erythromycin, oxytetracycline, and sulfamethoxazole-trimethoprim. The present study is the first report of G. anatis in Morocco, demonstrating the need for further epidemiologic investigations as well as in regard to antibiotic resistance development.

In recent years, the deleterious effects attributed to mycotoxins, in particular on the intestine, faced increased attention and it was shown that deoxynivalenol (DON) causes adverse effects on gut health. In this context, it has been repeatedly reported that DON can alter the intestinal morphology, disrupt the intestinal barrier and reduce nutrient absorption. The underlying mechanism of a compromised intestinal barrier caused by DON in chickens has yet to be illustrated. Although, DON is rapidly absorbed from the upper parts of the small intestine, the effects on the large intestine cannot be excluded. Additionally, a damaging effect of DON on the gut epithelium might decrease the resistance of the gut against infectious agents. Consequently, the objectives of the present studies were: (1) to investigate the impact of DON on the epithelial paracellular permeability by demonstrating the mucosal to serosal flux of 14 C-mannitol in the small and large intestine applying Ussing chambers and (2) to delineate the effects of DON on the colonization and translocation of Escherichia coli. Both parameters are well suited as potential indicators for gut barrier failure. For this, a total of 75 one-day-old Ross 308 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were randomly allocated to three different groups (n = 25 with 5 replicates/group) and were fed for 5 weeks with either contaminated diets (5 or 10 mg DON/kg feed) or basal diets (control). Body weight (BW) and BW gain of birds in the group fed with 10 mg/kg DON were significantly lower than in group with 5 mg/kg DON and the control group. Moreover, the mannitol flux in jejunum and cecum was significantly (P < 0.05) higher in DON-fed groups compared to control birds. Consistent with this, DON enhanced the translocation of E. coli with a higher number of bacteria encountered in the spleen and liver. Altogether, the actual results verified that DON can alter the intestinal paracellular permeability in broiler chickens and facilitates the translocation of enteric microorganisms such as E. coli to extra-intestinal organs. Considering that moderate levels of DON are present in feed, the consumption of DON-contaminated feed can induce an intestinal breakdown with negative consequences on broiler health.

Diets rich in various soluble nondigestible carbohydrates (sNDCs) were evaluated on different intestinal characteristics (histological, physico-chemical and microbiological) of chickens and compared with a maize-based diet as a control. A total of 160 Ross 308 male chickens were kept in deep litter pens (n = 40) and fed their appropriate diets from Day 1 to Day 35 of life. Four isocaloric and isonitrogenous diets, differing in their sNDC content, were composed; control (containing maize as the only cereal), maize-wheat-based (M + W) and maize-based supplemented with either 20 g/kg inulin (M + I) or 30 g/kg lactose (M + L). All of the diets tested decreased ileal crypt depth, ileal muscle layer thickness and increased caecal coliform counts relative to the control group. Villus-crypt ratio increased only in the M + L group. Ileal digesta of chickens fed the M + W diet had the highest ileal viscosity and the highest caecal butyrate, valerate and total short-chain fatty acid concentrations while the lowest pH was observed in caecal contents of chickens fed the M + I diet. The diet had no effect on ileal or caecal goblet cell and intraepithelial lymphocyte numbers. Lactobacillus counts in the caecal content remained unchanged. According to the present study, various sNDC sources may have beneficial gut health effects, however, some of the intestinal variables are dependent on the type of sNDCs., (© 2018 Blackwell Verlag GmbH.)

In laying and breeding chickens, pathomorphological signs of histomonosis often coincide with colibacillosis. Thus, we investigated the systemic spread of Escherichia coli in chickens affected with histomonosis and colibacillosis by characterizing their pheno- and genotypic profiles. For this, 29 birds from 11 affected flocks were necropsied and up to three E. coli isolates each from intestine, heart, and liver of the birds were isolated. A total of 251 isolates were characterized by serotyping, phylogenetic grouping, detection of virulence-associated genes (VAGs), and pulsed-field gel electrophoresis (PFGE). All birds showed egg peritonitis, and fibrinous typhlitis was additionally recorded in 18 birds. Presence of Histomonas meleagridis in ceca was confirmed by PCR and histopathology. Escherichia coli serotype O2:K1 was found to be the most prevalent (37.4%), whereas 31.1% of strains were not typeable. The majority of isolates collected from the intestine and extraintestinal organs belonged to phylogroups B2 (54.1%), D (21.5%), or A (19.5%). Isolates from these phylogroups harbored a higher number of VAGs. Macrorestriction analysis showed that 60.6% of total isolates from all organs tested were included in eight PFGE types. Isolation of E. coli with identical genomic profiles from the intestine and extraintestinal organs of the same or different birds in the same flock indicates for systemic dissemination of the bacteria, independent of E. coli genotype. Intestinal destruction due to H. meleagridis can be considered as the most plausible cause of bacterial dissemination, ultimately leading to colibacillosis.

Chickens are recognized as an imperative source of thermophilic Campylobacter spp., carrying this microorganism in high numbers in their intestinal tract. For a long time, Campylobacter jejuni has been considered as a commensal microorganism which colonizes its primary host rather than infecting it, in the absence of any obvious clinical signs. However, recent studies question this and argue for a deeper understanding of the host-bacteria interaction. Following oral uptake, it was demonstrated that C. jejuni interacts intimately with the gut epithelium and influences cellular functions of the host, with consequences on nutrient absorption. The immune reaction of the host which was revealed in some studies confirmed the infectious nature of C. jejuni. In agreement with this, an increased expression of pro-inflammatory cytokine genes was noticed. The ability to induce intestinal damage and to modulate the barrier function of the intestinal epithelia has further consequences on gut integrity, as it facilitates the paracellular passage of C. jejuni into the underlying tissues and it supports the translocation of luminal bacteria such as Escherichia coli to internal organs. This is associated with an alteration of the gut microbiota as infected birds have a significantly lower abundance of E. coli in different parts of the intestine. Some studies found that the gut microbiota influences the infection and translocation of C. jejuni in chickens in various ways. The effects of C. jejuni on the intestinal function of chickens already indicate a possible interference with bird performance and welfare, which was confirmed in some experimental studies. Furthermore, it could be demonstrated that a Campylobacter infection has an influence on the movement pattern of broiler flocks, supporting experimental studies. The intense interaction of C. jejuni with the chicken supports its role as an infectious agent instead of simply colonizing the gut. Most of the findings about the impact of Campylobacter on chickens are derived from studies using different Campylobacter isolates, a specific type of bird and varying experimental design. However, experimental studies demonstrate an influence of the aforementioned parameters on the outcome of a certain trial, arguing for improved standardization. This review summarizes the actual knowledge of the host-pathogen interaction of C. jejuni in chickens, emphasizing that there are still major gaps despite recently gained knowledge. Resolving the cascade from oral uptake to dissemination in the organism is crucial to fully elucidating the interaction of C. jejuni with the chicken host and to assess the clinical and economic implications with possible consequences on preventive interventions.

The pheno- and genotypic relatedness among Escherichia coli isolates from broilers with and without macroscopic lesions of the femoral head were investigated. In total, 219 isolates obtained from the bone marrow were characterized by serotyping, antimicrobial resistance (AMR) profiles, phylogenetic grouping, detection of virulence-associated genes (VAGs) and pulsed-field gel electrophoresis (PFGE). Serotyping revealed that 48.4% of the isolates were assigned to one of the three serotypes (O78:K80: 21.0%, O2:K1: 18.7%, O1:K1: 8.7%). Substantial phenotypic variation was also noticed in AMR testing as most of the birds harboured E. coli isolates with different AMR profiles, which is of high clinical relevance. The majority of isolates could be classified into phylogenetic groups D (54.3%) and B2 (25.6%), followed by A (11.4%) and B1 (8.7%). Virulotyping showed that the highest number of isolates contained genes iucD (86.8%) and iss (84.9%), whereas papC (16.0%) and astA (12.3%) were present in least number of isolates. PFGE resulted in 58 different profiles from 200 typeable isolates. No correlation was found between specific serotypes, AMR profiles, phylogenetic groups, PFGE types or VAG profiles of E. coli and the occurrence of bacterial chondronecrosis with osteomyelitis, contradicting the hypothesis of a specific bacterial pheno- or genotype being involved in the disease.

In the present study 66 layer flocks housed in different alternative husbandry systems were monitored from placement of birds on the farm until slaughter to evaluate a possible influence on the occurrence of selected non-infectious as well as infectious diseases. Postmortems were performed and the occurrence of extraintestinal Escherichia coli and Gallibacterium anatis was investigated. No specific postmortem findings were seen in pullets. Non-infectious diseases were mainly found in layers. Most prominent postmortem findings in layers were reproductive tract lesions and the presence of intestinal helminths. From each flock E coli and from approximately 65 per centof the flocks G anatis were isolated. No significant differences were seen in regard to the housing system, but the prevalence of G anatis increased with the age of birds. The majority of reproductive tract lesions could be associated with E coli alone or G anatis co-infections. The prevalence of Ascaridia galli and Heterakis gallinarum was not influenced by the housing system, but significantly increased with age. Cestodes were present in six flocks. Histomonosis was detected twice. Dermanyssus gallinae was found in 5 pullet and 20 layer flocks. Additional investigations were performed on demand. Again, reproductive tract lesions were the most prominent postmortem findings. In one flock each histomonosis or erysipelas was diagnosed, respectively. Severe affection by D gallinae was found once. Necrotic enteritis was seen in two layer flocks., Competing Interests: Competing interests: None declared., (© British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

A longitudinal survey was conducted to investigate the presence of Brachyspira species in layer flocks. A total of 66 layer flocks kept in alternative husbandry systems were sampled at three time points: end of rearing, at peak of lay and at end of lay. Content from caecal samples of freshly killed birds was cultured at each sampling time point and processed for further investigations. Gross pathological lesions in caeca were recorded during post mortem investigation. Spirochaetes were isolated from 50 flocks: three flocks were positive at all three sampling points, 25 flocks at two and 22 flocks at one sampling point, respectively. The presence of Brachyspira spp. could not be related to specific gross pathological caecal lesions or antibiotic treatments. The number of positive flocks increased with the age of birds. Furthermore, organic flocks were more often positive than flocks from barn systems. In total 80 spirochaetal cultures were obtained. B. intermedia (43.8%) was the most common species, followed by B. pulli (13.8%) and B. pilosicoli (12.5%). Brachyspira murdochii and B. innocens were found in 5.0% and 2.5%, respectively, whereas 11.3% of Brachyspira isolates could not be identified to species level. In 11.3% of the samples mixed infections were detected. Finally, the longitudinal survey revealed for the first time a possible shift in the Brachyspira species in a substantial number (32%) of layer flocks during their lifetime.

Recently we demonstrated that co-infection with Avibacterium paragallinarum and Gallibacterium anatis leads to increased severity of clinical signs of infectious coryza in birds. The present study examined the interaction of these two pathogens in chickens by evaluation of histologic lesions in sinus infraorbitalis and nasal turbinates, applying a defined scoring scheme ranging from 0 to 3. Furthermore, for the first time, an in situ hybridization (ISH) technique was applied to detect A. paragallinarum in tissues. The samples were received from vaccinated and nonvaccinated birds that were infected with A. paragallinarum and/or G. anatis. Vaccinated birds were mostly devoid of any histopathologic lesions except a few birds with lesion score 1 at 7 and 14 days postinfection (dpi). Likewise, nonvaccinated birds infected with G. anatis only did not present microscopic changes in the sinus infraorbitalis, except in a single bird at 7 dpi. Interestingly, median lesion scores caused by G. anatis infection were significantly higher in the nasal turbinates of infected birds than in negative control at 7 and 14 dpi. The most prominent histologic changes were recorded from sinus infraorbitalis and nasal turbinates of nonvaccinated birds that were infected either with A. paragallinarum only or together with G. anatis. ISH demonstrated positive signals for A. paragallinarum in exudates present in the lumen or attached to the epithelial layer of investigated tissues. Such signals were mainly detected in tissues from birds with the highest histopathologic lesion scores.

Despite the importance of gut microbiota for broiler performance and health little is known about the composition of this ecosystem, its development and response towards bacterial infections. Therefore, the current study was conducted to address the composition and structure of the microbial community in broiler chickens in a longitudinal study from day 1 to day 28 of age in the gut content and on the mucosa. Additionally, the consequences of a Campylobacter (C.) jejuni infection on the microbial community were assessed. The composition of the gut microbiota was analyzed with 16S rRNA gene targeted Illumina MiSeq sequencing. Sequencing of 130 samples yielded 51,825,306 quality-controlled sequences, which clustered into 8285 operational taxonomic units (OTUs; 0.03 distance level) representing 24 phyla. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria , and Tenericutes were the main components of the gut microbiota, with Proteobacteria and Firmicutes being the most abundant phyla (between 95.0 and 99.7% of all sequences) at all gut sites. Microbial communities changed in an age-dependent manner. Whereas, young birds had more Proteobacteria, Firmicutes , and Tenericutes dominated in older birds (>14 days old). In addition, 28 day old birds had more diverse bacterial communities than young birds. Furthermore, numerous significant differences in microbial profiles between the mucosa and luminal content of the small and large intestine were detected, with some species being strongly associated with the mucosa whereas others remained within the luminal content of the gut. Following oral infection of 14 day old broiler chickens with 1 × 10 8 CFU of C. jejuni NCTC 12744, it was found that C. jejuni heavily colonized throughout the small and large intestine. Moreover, C. jejuni colonization was associated with an alteration of the gut microbiota with infected birds having a significantly lower abundance of Escherichia (E.) coli at different gut sites. On the contrary, the level of Clostridium spp. was higher in infected birds compared with birds from the negative controls. In conclusion, the obtained results demonstrate how the bacterial microbiome composition changed within the early life of broiler chickens in the gut lumen and on the mucosal surface. Furthermore, our findings confirmed that the Campylobacter carrier state in chicken is characterized by multiple changes in the intestinal ecology within the host.

Background: Various information about the genetic diversity of Escherichia coli isolates from chickens are available but a detailed epidemiological investigation based upon isolates obtained from interrelated pullet and layer flocks is still missing. Therefore, in the course of a longitudinal epidemiological study on pullets and layers, 144 E. coli isolates from chickens with or without pathological lesions of the reproductive tract were serotyped and genotyped with pulsed-field gel electrophoresis (PFGE). These isolates were collected during rearing, peak and at the end of production. The actual study is the first of its kind so as to elucidate genetic relatedness among extraintestinal E. coli isolated from chickens with varying pathological conditions in interrelated layer farms/flocks at different stages of rearing., Results: Serotyping revealed that 63.19 % of the isolates could not be assigned to any of the three serotypes tested whereas 30.55 % of the isolates belonged to serotype O1:K1, 4.86 % to O2:K1 and 1.38 % to O78:K80. After macrorestriction digest with XbaI, 91.66 % of the isolates were typeable resulting in 96 distinct PFGE profiles. Among them, five PFGE types included isolates collected from diseased chickens as well as from birds without pathological lesions. This finding shows that pathogenicity of E. coli in layers seems to be largely influenced by concurrent susceptibility factors. Furthermore, in six out of eight cases where two isolates were collected from each of eight birds, different PFGE types were found in the same or different organs of the same bird. The existence of predominant or persistent E. coli genotypes was only observed in two cases., Conclusions: It is concluded that extraintestinal E. coli genotypes and serotypes in pullets and layers are heterogenous and also do not maintain a single clonality within the same bird. The facts that E. coli strains did not show any definite clonal population structure based on geographical region, age of the host and pathological lesions should have relevance in further epidemiological studies and control strategies.

A survey on the prevalence of Salmonella (S) species was carried out on 39 layer farms in Kosovo between April and September 2012. In total 367 samples, comprising feces, dust, eggs, and internal organs from dead birds, were investigated using bacteriological culture methods. Additionally, data on the location of the farm, the total number of birds on the farm, age of birds, and laying performance were collected. Salmonella were isolated from 38 samples obtained from 19 (49%) farms. The most common serovar identified was Salmonella enteritidis, found on 18 farms. The most common S. enteritidis phage type was PT29 followed by PT6, PT7, PT21, PT13a, PT8, PT14b, and PT4. One S. enteritidis isolate was not typable. Six farms had more than one phage type. Furthermore, serovar S. Bovismorbificans also was found in samples from 3 farms. Flock size or production stage was not associated with the probability of isolating Salmonella. The only flock factor found to be significantly associated was percent hen/day production: It was 2.8 times more likely to isolate Salmonella from flocks with production above 80% hen/day production compared to flocks producing at a lower level. Analysis of antimicrobial resistance patterns of 30 isolates revealed that all isolates were sensitive to gentamicin, ampicillin, sulphamethoxazole trimethoprim, and oxytetracycline, and 29 (97%) were sensitive to ciprofloxacin. All isolates showed intermediate resistance or were resistant to minocycline and cloxacillin. Twenty-six isolates (86%) had intermediate resistance to amoxicillin and 27 isolates (90%) were fully resistant to streptomycin. The present survey revealed a high prevalence of Salmonella enteritidis in layer flocks in Kosovo, indicating that table eggs have to be suspected as an important source of human salmone-llosis., (© 2016 Poultry Science Association Inc.)

The progression of Gallibacterium anatis infection in immunosuppressed versus immunocompetent chickens was investigated. Before experimental infection, birds were treated with corticosterone for general immunosuppression, or 5-fluorouracil, cyclosporine-A, cyclophosphamide for depletion of specific leukocyte populations. Necropsy and sampling were performed at 0, 3, 7, 10 and 28 days post infection. The used drugs did not cause selected depletion of B cells, T cells, heterophils and monocytes/macrophages, as determined by quantification of leukocytes in blood and lymphoid organs using different technologies. Bacterial re-isolation and counts of colony forming units (CFU) showed that G. anatis colonization pattern in various organs, and the numbers of bacteria in trachea were not affected by immunosuppression. However, the treatments acutely increased CFU counts derived from the spleen, which demonstrates that chemically induced immunosuppression intensifies systemic multiplication of G. anatis in chickens., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Asymptomatic carriage of Campylobacter jejuni is highly prevalent in chicken flocks. Thus, we investigated whether chronic Campylobacter carriage affects chicken intestinal functions despite the absence of clinical symptoms. An experiment was carried out in which commercial chickens were orally infected with C. jejuni (1 × 10(8) CFU/bird) at 14 days of life. Changes in ion transport and barrier function were assessed by short-circuit current (I(sc)) and transepithelial ion conductance (G(t)) in Ussing chambers. G(t) increased in cecum and colon of Campylobacter-infected chicken 7 d post-infection (DPI), whereas G t initially decreased in the jejunum at 7 DPI and increased thereafter at 14 DPI. The net charge transfer across the epithelium was reduced or tended to be reduced in all segments, as evidenced by a decreased I sc. Furthermore, the infection induced intestinal histomorphological changes, most prominently including a decrease in villus height, crypt depth and villus surface area in the jejunum at 7 DPI. Furthermore, body mass gain was decreased by Campylobacter carriage. This study demonstrates, for the first time, changes in the intestinal barrier function in Campylobacter-infected chickens and these changes were associated with a decrease in growth performance in otherwise healthy-appearing birds., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Aims: To evaluate the impact of diet composition on colonization dynamics of Camp. jejuni and on related physiological parameters in the chicken intestine., Methods and Results: A total of 54 1-day-old Ross 308 broiler chicks were randomly divided into three isocaloric and isonitrogenous dietary groups: maize-based (MB), wheat-based (WB) diet and wheat-based diet with NSP-degrading enzyme supplementation (WBES). Chickens were orally infected with 10(8)  CFU Camp. jejuni on day 14, and samples (n = 6) were collected on 7, 14 and 21 days postinfection (DPI), respectively. Colony forming units of Camp. jejuni of caecum and jejunum, short-chain fatty acid (SCFA) concentrations, pH values of the caecum, jejunal histomorphology and viscosity of jejunal chymus were measured. In case of WBES diet, lower Camp. jejuni colonization 14 DPI, higher jejunal viscosity, higher total SCFA concentrations in the caecum and enhanced jejunal histomorphology were observed compared to those measured in chickens fed MB diet., Conclusions: The WBES diet altered Camp. jejuni colonization dynamics in the chicken intestine which resulted by higher SCFA concentrations in the caecum and by the change of gut morphology., Significance and Impact of the Study: Our study proves that diet composition can modify Camp. jejuni colonization depending on sampling time point postinfection., (© 2014 The Society for Applied Microbiology.)

The gastrointestinal tract represents the first barrier against pathogens. However, the interaction of Campylobacter with intestinal epithelial cells and its effects on the intestinal function of chickens are poorly studied. Therefore, the goal of the present study was to characterize the effects of C. jejuni oral infection on the mRNA expression of nutrient transporters in the intestine. Newly hatched specific pathogen-free (SPF) chickens were orally infected with C. jejuni (NCTC 12744; 1 × 10(8)CFU/bird) at 14 days of age. Quantitative RT-PCR analyses at 14 days-post infection (dpi) revealed that the relative gene expression of the sodium/glucose cotransporter (SGLT-1) and the peptide transporter (PepT-1) was down-regulated (P<0.05) in all investigated segments (duodenum, jejunum and cecum) of Campylobacter-infected birds, while the facilitated glucose transporter (GLUT-2) was down-regulated (P<0.05) in jejunal and cecal tissues only. Furthermore, down-regulation (P<0.05) of the cationic amino acid transporter (CAT-2) and the excitatory amino acid transporter (EAAT-3) was seen in the jejunum, and down-regulation (P<0.05) of the l-type amino acid transporter (y(+)LAT-2) was noticed in the duodenum of infected birds. The decreased expression of intestinal nutrient transporters coincided with a decrease (P<0.05) in body weight and body weight gain during a 2-week post infection period. For the first time, it can be concluded that nutrient transporter expression is compromised in the small and large intestine of Campylobacter-infected birds with negative consequences on growth performance. Furthermore, the down-regulation of mRNA expression of glucose and amino acid transporters may result in accumulation of nutrients in the intestinal lumen, which may favor C. jejuni replication and colonization., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Escherichia coli (E. coli) infections are very widespread in poultry. However, little is known about the interaction between the intestinal epithelium and E. coli in chickens. Therefore, the effects of avian non-pathogenic and avian pathogenic Escherichia coli (APEC) on the intestinal function of broiler chickens were investigated by measuring the electrogenic ion transport across the isolated jejunal mucosa. In addition, the intestinal epithelial responses to cholera toxin, histamine and carbamoylcholine (carbachol) were evaluated following an E. coli exposure. Jejunal tissues from 5-week-old broilers were exposed to 6×10(8) CFU/mL of either avian non-pathogenic E. coli IMT11322 (Ont:H16) or avian pathogenic E. coli IMT4529 (O24:H4) in Ussing chambers and electrophysiological variables were monitored for 1 h. After incubation with E. coli for 1 h, either cholera toxin (1 mg/L), histamine (100 μM) or carbachol (100 μM) were added to the incubation medium. Both strains of avian E. coli (non-pathogenic and pathogenic) reduced epithelial ion conductance (Gt) and short-circuit current (Isc). The decrease in ion conductance after exposure to avian pathogenic E. coli was, at least, partly reversed by the histamine or carbachol treatment. Serosal histamine application produced no significant changes in the Isc in any tissues. Only the uninfected control tissues responded significantly to carbachol with an increase of Isc, while the response to carbachol was blunted to non-significant values in infected tissues. Together, these data may explain why chickens rarely respond to intestinal infections with overt secretory diarrhea. Instead, the immediate response to intestinal E. coli infections appears to be a tightening of the epithelial barrier.

Pathogenesis of Gallibacterium anatis was investigated in specific pathogen free cockerels. Birds aged 35 weeks were infected intranasally with G. anatis whereas negative controls were left uninfected. Following infection, necropsy, bacteriological and histopathological investigations were performed in birds killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.). Additionally, semen samples were collected twice a week until 5 weeks post infection for quality assessment. No clinical signs and gross pathological lesions were seen throughout the experiment. Bacteriological investigation revealed that G. anatis colonized the upper respiratory tract at 3 d.p.i. and could be isolated from testis and epididymis at 7 d.p.i. onwards. Bacterial persistence was found in the respiratory tract, gut and testis until the termination of the study at 38 d.p.i. Furthermore, G. anatis was isolated from semen arguing for the possibility of vertical transmission. Histopathological examination showed infiltration of mononuclear cells in epididymal tissue, indicating an inflammation. Density, total motility, progressive motility and membrane integrity of sperms were significantly decreased in infected birds as compared with control chickens. Along with these findings, an increase in spermatozoa with morphological defects was observed at different time points. In conclusion, the present study provides novel data on the impact of a G. anatis infection in cockerels in a natural infection model, thus helping to elucidate bacterial distribution, pathological lesions as well as influences on semen quality.

Pathogenicity of Gallibacterium anatis was studied in specific pathogen free layers in a controlled environment, applying the intranasal route for experimental infection. At 30 weeks, 37 hens were infected with 0.4 ml of 1.53 × 10(8) colony-forming units/ml suspension of G. anatis strain 07990 whereas equal numbers of hens were left uninfected for control. Following experimental infection, clinical signs and the number and weight of the eggs were recorded daily until 5 weeks post infection. Three birds from each group were killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.) for necropsy and sampling for bacteriological and histopathological examinations. Additionally, necropsy examination was performed on all remaining birds at 38 d.p.i. G. anatis infection was found to have an immediate and severe effect on egg production, showing early and persistent colonization in respiratory and reproductive organs as well as in the gut of infected layers. In birds killed at various time points, G. anatis infection caused focal necrosis in the liver (1/37), folliculitis (2/37), pericarditis (3/37), haemorrhagic follicles (2/37), ruptured follicles (20/37), yolk in the body cavity (2/37) and egg peritonitis (1/37). The inflammation of the ovaries could be further confirmed by histopathological examination. Recovery of G. anatis from yolk at 10 d.p.i. indicates the potential of vertical transmission. Altogether, lesions reflect typical findings of G. anatis infection reported in natural cases. Thus, for the first time, lesions and the consecutive disease caused by G. anatis infection have been reproduced experimentally in a natural infection model.

Two separate bird trials were performed to establish a reliable route of infection for Gallibacterium anatis in chickens, comparing intranasal (i.n.) and intravenous (i.v.) applications. Additionally, three mutually divergent isolates from three geographical locations, as shown by MALDI-TOF-MS and partial rpoB gene sequence analysis, were compared. In the first trial, birds were infected with one of the selected isolates by the i.v. or i.n. route. Subsequently, birds were killed 3, 12 and 24 h post infection following i.v. infection while at 3, 7 and 10 days post infection (dpi) in the case of i.n. infection along with birds of the control group. As a result, i.n. infection showed prominent and consistent bacterial tissue distribution in different organs persisting until 10 dpi, which was a striking contrast to the i.v. infection route. Likewise, histopathology revealed mild to severe tracheal lesions following i.n. infection. The second trial was set up to confirm both the achieved results and the robustness of i.n. infection but with an extended observation period, until 28 dpi In agreement with the preceding trial, identical results for bacteriological and histopathological examinations were obtained with persistency of bacteria until 28 dpi Comparing the three different isolates from Mexico, China and Austria, the Mexican isolate showed a somewhat higher pathogenicity than the other strains. Consequently, pathogenesis of G. anatis strains was studied in chickens elucidating i.n. infection as the most reliable route characterized by a long-lasting bacteraemia, targeting the respiratory and reproductive tract.

Salmonella infection of chickens that leads to potential human foodborne salmonellosis continues to be a major concern. Chickens serve as carriers but, in contrast to humans, rarely show any clinical signs including diarrhea. The present investigations aimed to elucidate whether the absence of diarrhea during acute Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) infection may be linked to specific changes in the electrophysiological properties of the chicken gut. Immediately after slaughter, intestinal pieces of the mid-jejunum and cecum of either commercial broiler or specific pathogen-free (SPF) chickens were mounted in Ussing chambers in 2 separate experimental series. Living Salmonella Enteritidis (3 × 10(9)) or Salmonella Enteritidis endotoxin (20 mg/L), or both, were added to the mucosal side for 1 h. In both experimental series, the Salmonella infection decreased the trans-epithelial ion conductance G(t) (P < 0.05). In the jejunum of SPF chickens, there was also a marked decrease in net charge transfer across the epithelium, evidenced by decreased short-circuit current (I(sc), P < 0.05). Interestingly, the mucosal application of Salmonella endotoxin to the epithelial preparations from jejunum and cecum of SPF chicken had an effect similar to living bacteria. However, the endotoxin had no additional effect on the intestinal function in the presence of bacteria. The decreasing effect of Salmonella and or its endotoxin on G(t) could be partly reversed by serosal addition of histamine. To our knowledge, this is the first study to address the functional response of native intestinal epithelium of chicken to an in vitro Salmonella infection. For the first time, it can be reported that intestinal ion permeability of chicken decreases acutely by the presence of Salmonella. This type of response could counteract ion and fluid secretion and may thus, at least in part, explain why chickens do not develop overt diarrhea after Salmonella infection.

Salmonella (S.) Infantis is the most common serovar in broilers and broiler meat in the European Union. In the field, fast-growing broilers are reported to be more affected than slow-growing and layer birds. The present study investigated the infection dynamics and immunological response of four chicken lines in the course of a S. Infantis infection. Two commercial chicken lines, Ross 308 and Hubbard ISA-JA-757, and two experimental chicken lines, specific pathogen free (SPF) layers and broilers, were infected at 2 days of age. Investigations focused on faecal shedding, bacterial colonization, humoral and cellular immune response. Ross and SPF broilers proved mainly as high shedders followed by Hubbard. SPF layers showed the least shedding. This is in agreement with the caecal colonization; SPF layers harboured significantly less bacteria. Systemic spread of S. Infantis to liver and spleen was highest in Ross broilers compared to the other lines. Spread of infection to in-contact birds, was noticed 5 days post infection in every line. Antibody response occurred in every chicken line from 21 days of age onwards. In contrast to the other chicken lines, significant differences in T cell subsets and monocytes/macrophages were found between infected and negative Hubbard birds at 7 days of age. Uninfected SPF birds had significantly higher immune cell counts than uninfected commercial birds, a fact important for future experimental settings. The results illustrate that the infection dynamics of S. Infantis is influenced by the chicken line resulting in a higher risk of transmission to humans from fast-growing broilers. RESEARCH HIGHLIGHTS Infection dynamics of Salmonella Infantis differs between chicken lines. Layers showed less faecal shedding and caecal colonization compared to broilers. Fast-growing broilers proved more susceptible than slow-growing broilers. [ABSTRACT FROM AUTHOR]

Five different Artemisia annua-derived materials (i.e. dry leaves, pure artemisinin, and hexane, dichloromethane or methanol extracts of leaves) were screened for their in vitro activities against six clonal cultures of Histomonas meleagridis. Except for the methanol extract, all tested materials displayed in vitro activity against all tested protozoal clones. Neither the dry plant material, extracts nor artemisinin showed any antibacterial activity against the xenic bacteria accompanying the six H. meleagridis clones at concentration levels identical to the antihistomonal setting. The dichloromethane extract of dry leaves (Ext-DCM) (minimal lethal concentration=1.0 mg/ml) and artemisinin (half-maximal inhibitory concentration=1.295 mg/ml) had the most promising antihistomonal properties and were therefore subsequently tested in a standardized experimental infection model in both turkeys and chickens infected with clonal H. meleagridis. There were no differences between treatment groups, where all infected turkeys showed severe clinical histomonosis and demonstrated severe typhlohepatitis typical for histomonosis. Consistent with the infection model used, the infected chickens did not show any adverse clinical signs but contracted severe lesions in their caeca 7 and 10 days post infection (d.p.i.), liver lesions were absent to mild after 7 d.p.i. and progressed to severe lesions at 10 d.p.i.; thus no differences between treatment groups were observed. In conclusion, neither artemisinin nor Ext-DCM was able to prevent experimental histomonosis in turkeys and chickens at the given concentrations, which is contrary to the antihistomonal effect noticed in vitro even though the same clonal culture was used. The results of this study therefore clearly demonstrate the importance of defined in vivo experimentation in order to assess and verify in vitro results. [ABSTRACT FROM AUTHOR]