Host Immune Response Modulation in Avian Coronavirus Infection: Tracheal Transcriptome Profiling In Vitro and In Vivo.

Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host-pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.

Determination of the virulence status of Clostridium perfringens strains using a chicken intestinal ligated loop model is important for understanding the pathogenesis of necrotic.

Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.

Colonization of the Gastrointestinal Tract of Chicks with Different Bacterial Microbiota Profiles.

This study aimed to investigate the consequences of early-life microbiota transplantation using different caecal content sources in broiler chicks. We hypothesized that chicks receiving at-hatch microbiota from organic hens would harbour a distinct microbiota from chicks receiving industry-raised broiler microbiota after six weeks of age. Three hundred Cobb broilers eggs were randomly assigned to one of four groups according to the caecal content received: organic laying hens (Organic); autoclaved caecal content of organic laying hens (Autoclaved); conventionally grown broilers (Conventional); and sterile saline (Control). caecal microbiota transplantation was given by gavage on day 1. Ten birds/group were euthanized on days 2, 7, 14, 28, and 42. The caecal tonsils and contents were collected for cytokines and microbiota analyses. The microbiota from chicks receiving live inocula resembled the donors' microbiota from day seven until day 42. The microbiota composition from the chickens who received the Organic inoculum remained markedly different. Starting on day 7, the Organic group had higher richness. Simpson and Shannon's indices were higher in the Conventional group on days 2 and 7. Chickens in the Conventional group presented higher production of IL-1ß and IL-6 in plasma on days 2 and 28, increased IL-6 expression in the caecal tonsils at days 7 and 42, and increased IL-12 expression on day 7. However, the Conventional group was infected with Eimeria spp., which likely caused inflammation. In conclusion, microbiota transplantation using different microbiota profiles persistently colonized newly hatched broiler chicks. Future studies evaluating the importance of microbiota composition during infections with common enteropathogens are necessary. This study also highlights the need for a strict screening protocol for pathogens in the donors' intestinal content.

Red osier dogwood extract versus Trimethoprim-sulfadiazine (Part 1). Effects on the growth performance, blood parameters, gut histomorphometry, and Salmonella excretion of broiler chickens orally challenged with Salmonella Enteritidis.

The poultry industry has not been spared from the prevalent incidence of diseases caused by invasive pathogens, especially Salmonella. Due to the pressing need to identify a suitable antibiotic alternative for use in poultry production, this study investigated the efficacy of red osier dogwood (ROD) extract on the growth, blood parameters, gut morphology, and Salmonella excretion in broiler chickens orally challenged with Salmonella Enteritidis (SE). A 4 × 2 factorial experiment was conducted based on 2 main factors, namely dietary treatments, and SE challenge. A total of 404, one-day-old male Ross broiler chicks were randomly assigned to 4 dietary treatments; 1) Negative control (NC), 2) NC + 0.075 ppm of Trimethoprim-sulfadiazine (TMP/SDZ)/kg of diet, 3) NC + 0.3% ROD extract, and 4) NC + 0.5% ROD extract. The absence of SE in the fecal samples obtained from chick delivery boxes was confirmed on d 0. On d 1, half of the birds were orally gavaged with 0.5 mL of phosphate-buffered saline each (noninfected group) and the remaining with 0.5 mL of 3.1 × 105 CFU/mL SE (infected group) in all treatment groups. Dietary treatments were randomly assigned to 8 replicate cages at 6 birds/cage. On 1-, 5-, 12-, and 18-day postinfection (DPI), cloacal fecal samples were collected on the 6 birds/cage to assess SE excretion. Average weight gain (AWG), average feed intake (AFI), feed conversion ratio (FCR), and mortality were determined weekly. On d 21, 10 chickens/treatment were euthanized to perform hematology, gut histomorphometry, serum immunoglobulins G and M (IgG and IgM), and superoxide dismutase measurements. Both ROD extract levels did not affect (P > 0.05) growth performance; however, the SE-infected birds showed increased (P < 0.05) AFI and FCR throughout the experimental period. Regardless of the SE-infection, both ROD extract levels improved (P < 0.05) duodenal villus height: crypt depth compared to other treatments. 0.5% ROD extract improved (P < 0.05) ileal villus width (VW) of noninfected birds and ileal crypt depth of infected birds, but it decreased (P < 0.05) the ileal VW of infected birds, compared to other treatments. The SE-infected birds showed lower (P < 0.05) lymphocytes (L) but increased (P < 0.05) heterophils (H), H:L, and monocytes (MON). Both ROD extract levels did not affect (P > 0.05) white blood cell differential, while dietary 0.3% ROD extract increased (P < 0.05) MON of the birds, regardless of infection model. Regardless of infection model, both TMP/SDZ and 0.5% ROD extract reduced the concentration of IgM in the serum, compared to the control and 0.3% ROD (P = 0.006). Conclusively, both ROD extract levels improved duodenal histomorphology and body defense against SE infection in broiler chickens; however, the 0.3% ROD extract was better.

Red osier dogwood and its use in animal nutrition: A review.

As the human population increases globally, the food animal industry has not been spared from the monumental demand for edible animal products, particularly meat. This has necessitated the simultaneous expansion of the productivity of the animal sector to meet the ever-growing human needs. Although antibiotics have been used in food animal production with commendable positive impacts on their growth performance, their sole contributive factor to the increasing incidence of antimicrobial resistance has ushered the strict restrictions placed on their use in the animal sector. This has handed a setback to both animals and farmers; thus, the intense push for a more sustainable antibiotic alternative for use in animal production. The use of plants with concentrated phytogenic compounds has gained much interest due to their beneficial bioactivities, including antioxidant and selective antimicrobial. While the reported beneficial activities of phytogenic additives on animals vary due to their varying total polyphenol concentrations (TPC), red osier dogwood (ROD) plant materials boast of high TPC with excellent antioxidant prowess and growth improvement capacities compared to some plant extracts commonly used in research. However, its adoption in research and commercial scale is still low. Thus, the present review aims to provide concise information on the dietary potential of ROD plant materials in animal feeding.

No change in avilamycin (Surmax® Premix) minimum inhibitory concentration for Clostridium perfringens isolates recovered from poultry up to 7 years post-approval in Canada.

BACKGROUND: Clostridium perfringens, the causative agent of necrotic enteritis (NE) in chickens, has an enormous economic impact on global broiler production. The non-medically important antibiotic avilamycin was approved in Canada in 2014 to prevent and control NE in broiler chickens. OBJECTIVES: To compare avilamycin susceptibility in C. perfringens isolates collected pre- and 7 years post-avilamycin approval in Canada and determine the avilamycin resistance mutation frequency rate in C. perfringens. METHODS: The MICs of avilamycin were determined for 89 strains of C. perfringens recovered from clinically relevant NE field cases pre-avilamycin approval between 2003 and 2013 (n = 50) and post-avilamycin approval between 2014 and 2021 (n = 39) across Canada. For determining the mutant prevention concentration (MPC) of avilamycin for C. perfringens strains, a strain with avilamycin MIC of 1 mg/L was randomly selected. RESULTS: MIC studies showed no difference in avilamycin susceptibility in pre-avilamycin and post-avilamycin isolates (MIC50/90: pre-avilamycin approval 2/2 mg/L and post-avilamycin approval 1/2 mg/L). The MPC was 8 × MIC (8 mg/L) for the selected strain. CONCLUSIONS: These findings suggest that the susceptibility of C. perfringens strains to avilamycin was not impacted by its continued use in the 7 years following its approval in Canada. Avilamycin, a non-medically important antibiotic, poses no threat to human health regarding cross-resistance or co-selection of other medically important antibiotics. These factors make avilamycin an appropriate choice for continued use in broiler chickens to prevent and control NE without increased antimicrobial resistance concerns.

Red osier dogwood extract vs. trimethoprim-sulfadiazine (Part 2). Pharmacodynamic effects on ileal and cecal microbiota of broiler chickens challenged orally with Salmonella Enteritidis.

With the subsisting restrictions on the use of antibiotics in poultry production, the use of plant extracts has shown some promising antimicrobial capacity similar to antibiotics; however, such capacity is largely dependent on their total polyphenol concentration and profile. Given the emerging antimicrobial potential of red osier dogwood (ROD) extract, the study aimed to investigate the pharmacodynamic effect of ROD extract on the ileal and cecal microbiota of broiler chickens challenged orally with Salmonella Enteritidis (SE). A 21 d 4 × 2 factorial experiment was conducted based on 2 main factors, including diets and SE challenge. A total of 384 one-day-old mixed-sex Cobb-500 broiler chicks were randomly allotted to 4 dietary treatments; Negative control (NC), NC + 0.075 mg trimethoprim-sulfadiazine (TMP/SDZ)/kg of diet, and NC containing either 0.3 or 0.5% ROD extract. On d 1, half of the birds were orally challenged with 0.5 mL of phosphate-buffered saline (Noninfected group) and the remaining half with 0.5 mL of 3.1 × 105 CFU/mL SE (Infected group). Dietary treatments were randomly assigned to 8 replicate cages at 6 birds/cage. On d 21, 10 birds/treatment were euthanized and eviscerated to collect ileal and cecal digesta for gut microbiota analysis. The ileal and cecal microbiota was dominated by phyla Firmicutes, Proteobacteria, and Actinobacteriota. The SE infection decreased (P < 0.05) the relative abundance of Proteobacteria and Actinobacteriota in the ileum and ceca, respectively, however, it increased (P < 0.05) Proteobacteria in the ceca. Both 0.3 and 0.5% ROD extracts (P < 0.05) depressed the relative abundance of Actinobacteriota in the ileum but marginally improved (P < 0.05) it in the ceca compared to the TMP/SDZ treatment. Dietary TMP/SDZ increased (P < 0.05) genus Bifidobacterium at the ileal and cecal segments compared to other treatments. Dietary 0.3 and 0.5% marginally improved (P < 0.05) Bifidobacterium in the ceca and depressed (P < 0.05) Weissella and was comparably similar to TMP/SDZ in the ileum. Regardless of the dietary treatments and SE infection, alpha diversity differed (P < 0.05) between ileal and cecal microbiota. Beta diversity was distinct (P < 0.05) in both ileal and cecal digesta along the SE infection model. Conclusively, both ROD extract levels yielded a pharmacodynamic effect similar to antibiotics on ileal and cecal microbiota.

Pathogenesis and host responses in lungs and kidneys following Canadian 4/91 infectious bronchitis virus (IBV) infection in chickens.

The infectious bronchitis virus (IBV) 4/91 was one of the common IBV variants isolated in Eastern Canada between 2013 and 2017 from chicken flocks showing severe respiratory and production problems. We designed an in vivo experiment, using specific pathogen free (SPF) chickens, to study the pathogenesis of, and host response to, Canadian (CAN) 4/91 IBV infection. At one week of age, the chickens were infected with 4/91 IBV/Ck/Can/17-038913 isolate. Swab samples were collected at predetermined time points. Five birds from the infected and the control groups were euthanized at 3, 7- and 10-days post-infection (dpi) to collect lung and kidney tissues. The results indicate IBV replication in these tissues at all three time points with prominent histological lesions, significant immune cell recruitment and up regulation of proinflammatory mediators. Overall, our findings add to the understanding of the pathogenesis of 4/91 infection and the subsequent host responses in the lungs and kidneys following experimental infection.

Pathogenicity of the Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) on Female Reproductive Tract of Chickens.

Infectious bronchitis virus (IBV) infection causes significant economic losses to various sectors of the poultry industry worldwide. Over the past few years, the incidence of false layer syndrome in Eastern Canadian layer flocks has been associated with the increased prevalence of the IBV Delmarva (DMV)/1639 strain. In this study, 1-day-old specific-pathogen-free (SPF) hens were infected with the Canadian DMV/1639 strain and observed until 16 weeks of age in order to determine if the IBV DMV/1639 strain is causing false layer syndrome. Early after infection, the virus showed a wide tissue distribution with characteristic gross and histopathological lesions in the respiratory tract and kidney. Around 60-70% of the infected hens demonstrated continuous cloacal viral shedding until the end of the experiment (at 16 weeks) which was associated with high IBV genome loads detected in the cecal tonsils. The experiment confirmed the field observations that the Canadian DMV/1639 strain is highly pathogenic to the female reproductive tract causing marked cystic lesions in the oviduct. Moreover, significant histopathological damage was observed in the ovary. Our study provides a detailed description of the pathological consequences of the IBV DMV/1639 strain circulating in an important poultry production sector.

Molecular Characterization of 4/91 Infectious Bronchitis Virus Leading to Studies of Pathogenesis and Host Responses in Laying Hens.

Infectious bronchitis virus (IBV) initially establishes the infection in the respiratory tract and then spreads to other tissues depending on its virulence. During 2011-2018, the 4/91 IBV strain was isolated from poultry flocks affected by decreased egg production and quality in Eastern Canada. One of the Canadian 4/91 IBV isolates, IBV/Ck/Can/17-038913, was propagated in embryonated chicken eggs and molecularly characterized using whole genome sequencing. An in vivo study in laying hens was conducted to observe if IBV/Ck/Can/17-038913 isolate affects the egg production and quality. Hens were infected with IBV/Ck/Can/17-038913 isolate during the peak of egg lay, using a standard dose and routes maintaining uninfected controls. Oropharyngeal and cloacal swabs were collected at predetermined time points for the quantification of IBV genome loads. At 6 and 10 days post-infection, hens were euthanized to observe the lesions in various organs and collect blood and tissue samples for the quantification of antibody response and IBV genome loads, respectively. Egg production was not impacted during the first 10 days following infection. No gross lesions were observed in the tissues of the infected birds. The IBV genome was quantified in swabs, trachea, lung, proventriculus, cecal tonsils, kidney, and reproductive tissues. The serum antibody response against IBV was quantified in infected hens. In addition, histological changes, and recruitment of immune cells, such as macrophages and T cell subsets in kidney tissues, were measured. Overall, data show that IBV/Ck/Can/17-038913 isolate is not associated with egg production issues in laying hens infected at the peak of lay, while it demonstrates various tissue tropism, including kidney, where histopathological lesions and immune cell recruitments were evident.

Persistence of a Clostridium perfringens Strain in a Broiler Chicken Farm over a Three-Year Period.

Clostridium perfringens, a commensal of the intestinal tract of many animal species, has been associated with necrotic enteritis (NE), an economically significant poultry disease. Clostridium perfringens is known to survive in the environment for extended periods of time through the formation of spores. These spores have the potential to be transmitted to subsequent flocks. Persistence of a single C. perfringens strain in a broiler chicken farm environment has, however, been poorly documented. The aim of this study was to compare multiple isolates of C. perfringens collected over time in a single farm with recurrent episodes of NE. Isolates were recovered from the intestines of chickens affected with NE (2014 and 2016 outbreaks) and from healthy chickens (2017), as well as from environmental samples (2016). PCR characterization of those isolates showed that all sampling groups contained netB-positive isolates except for the environmental samples. Moreover, results showed that all environmental isolates were positive for the cna adhesin whereas other groups had lower numbers of cna-positive isolates. Biofilm formation assays showed that most of the isolates were able to form biofilm. Pulsed-field gel electrophoresis analysis showed that one clone was present in every sampling group, with the exception of the 2014 outbreak. However, one clone found in the latter group was highly similar, having 94% similarity with the persistent C. perfringens clone. This study describes for the first time the persistence of a C. perfringens strain on a broiler chicken house over a 3-yr period.

Impacts of antibiotic reduction strategies on zootechnical performances, health control, and Eimeria spp. excretion compared with conventional antibiotic programs in commercial broiler chicken flocks.

Increasing efforts have been made in recent years to reduce antimicrobial use in animal production. The objective of this prospective study was to evaluate, in commercial broiler chicken farms, 2 antibiotic reduction strategies that eliminated the use of antibiotics important for human medicine, in comparison with the conventional use of antibiotics. On 7 broiler chicken farms, a house was allocated to the antibiotic reduction treatments for 6 consecutive flocks, whereas a similar house on the same premises was assigned to the conventional use of antibiotics (CONV) for 6 consecutive flocks. The antibiotic reduction strategies consisted of continuous in-feed use of ionophores (TX1) and continuous in-feed use of ionophores with butyric acid (TX2). In the 84 flocks, zootechnical performance was recorded, lesion scoring at 21 and 28 D of age was performed, and fecal samples were recovered during grow out for Eimeria spp. oocysts counts. There was no statistical difference between TX1, TX2, and CONV for weights at slaughter, feed conversion ratios, average daily gains, age at slaughter, total mortalities, and condemnations. The probability of identifying oocysts in the fecal samples significantly increased with the age of the flock, but there was no significant treatment effect between 7 and 16 D of age. At 19 D of age, the probability of a sample containing oocysts was higher in TX1 than in CONV, but TX2 was not statistically different from TX1 and CONV. Predicted oocysts per gram in CONV flocks were significantly lower between 22 and 34 D of age than in TX1 and TX2 flocks, whereas there were no significant differences between TX1 and TX2 for all ages. Lesion scoring of the gastrointestinal system showed no differences for coccidiosis scores between TX1, TX2, and CONV. No lesions of necrotic enteritis were observed. In conclusion, it was possible to adequately control intestinal diseases and maintain zootechnical performances by relying exclusively on ionophores, when compared with broiler chicken flocks using standard shuttle programs with antibiotic growth promoters.

Diagnostic Accuracy of Ultrasonography to Detect False Layers in a Commercial Laying Flock Infected by an Infectious Bronchitis Virus Delmarva Genotype Causing Cystic Oviducts.

Infection of the oviduct by an infectious bronchitis virus (IBV) in laying hens has been associated with the false layer syndrome. Because the diagnostic procedure for the detection of cystic oviducts by postmortem examinations in IBV-positive replacement pullet flocks could involve the unnecessary sacrifice of numerous healthy pullets without reproductive tract anomalies, the development of a noninvasive and nonlethal diagnostic procedure would be desirable. The first objective of the study was to evaluate the diagnostic accuracy of a transcutaneous ultrasonography method to predict the presence of cystic oviducts compared to postmortem examinations in a commercial pullet flock positive for an IBV genotype Delmarva (DMV) variant. The second objective was to evaluate the performance of the same ultrasonography method to later detect false layers in the same flock in sexually mature hens by identifying the presence of an egg in the oviduct due to the presence of atretic oviducts undetectable by ultrasonography and the absence of cystic oviducts at that age. In replacement pullets, the sensitivity (Se) and specificity (Sp) of the ultrasonography (index test) compared to the postmortem examination (reference standard test) were 73% and 91%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 67% and 93%. The ultrasonography technique showed a positive likelihood ratio (LR+) of 7.82 and a negative likelihood ratio (LR-) of 0.30. In sexually mature hens, the Se, Sp, PPV, and NPV of the ultrasonography compared to the laying status were 98%. The LR+ was 49.00 and the LR- was 0.02 when compared to the laying status. In conclusion, the ultrasonography could replace postmortem examinations to detect cystic oviducts in commercial flocks of replacement pullets previously infected with an IBV-DMV 1639 variant. Although the test accuracy of ultrasonography was excellent for the hens at production peak to identify laying and nonlaying hens based on the presence of an egg in the reproductive tract, its practicality was limited due to atretic oviducts being not detectable.

Precisión diagnóstica de la ultrasonografía para detectar gallinas falsas ponedoras en una parvada comercial infectada por un virus de la bronquitis infecciosa genotipo Delmarva que causa oviductos quísticos. La infección del oviducto por el virus de bronquitis infecciosa (IBV) en gallinas de postura se ha asociado con el síndrome de la falsa ponedora. Debido a que el procedimiento de diagnóstico para la detección de oviductos quísticos mediante exámenes post mortem en parvadas de pollitas de reemplazo positivas para bronquitis infecciosa podría involucrar el sacrificio innecesario de numerosas pollitas sanas sin anomalías del tracto reproductivo, por lo tanto es deseable el desarrollo de un procedimiento de diagnóstico no invasivo y no letal. El primer objetivo del estudio fue evaluar la precisión diagnóstica de un método de ultrasonografía transcutánea para predecir la presencia de oviductos quísticos en comparación con los exámenes post mortem en un lote comercial de pollitas que resultó positivo para una variante del genotipo Delmarva (DMV) del virus de la bronquitos infecciosa. El segundo objetivo fue evaluar el desempeño del mismo método de ultrasonografía para detectar posteriormente gallinas falsas en la misma parvada en las gallinas sexualmente maduras mediante la identificación de la presencia de un huevo en el oviducto debido a la presencia de oviductos atrésicos indetectables por ultrasonografía y la ausencia de oviductos quísticos a esa edad. En las pollitas de reemplazo, la sensibilidad (Se) y la especificidad (Sp) de la ultrasonografía (prueba de índice) en comparación con el examen post mortem (prueba estándar de referencia) fueron de 73% y 91%, respectivamente. El valor predictivo positivo (VPP) y el valor predictivo negativo (VPN) fueron 67% y 93%. La técnica de ultrasonografía mostró una razón de probabilidad positiva (LR+) de 7.82 y una razón de probabilidad negativa (LR­) de 0.30. En las gallinas sexualmente maduras, la Se, Sp, PPV y NPV de la ultrasonografía en comparación con el estado de postura fueron del 98%. El LR + fue 49.00 y el LR­fue 0.02 en comparación con el estado de la postura. En conclusión, la ultrasonografía podría reemplazar los exámenes post mortem para detectar oviductos quísticos en parvadas comerciales de pollitas de reemplazo previamente infectadas con una variante DMV-1639 del virus de la bronquitis infecciosa. Aunque la precisión de la prueba de la ecografía fue excelente para las gallinas en el pico de producción para identificar gallinas ponedoras y no ponedoras en función de la presencia de un huevo en el tracto reproductivo, su funcionalidad fue limitada debido a que los oviductos atrésicos no fueron detectables.

Antimicrobial Resistance and Virulence Gene Profiles Among Escherichia coli Isolates from Retail Chicken Carcasses in Vietnam.

This study evaluated virulence and resistance profiles of Escherichia coli in chicken carcasses from three retail systems in Vietnam. Fresh chicken carcasses from traditional markets and fresh and frozen chicken carcasses from supermarkets were sampled in Vietnam. E. coli isolates from carcass rinses were characterized for extraintestinal pathogenic E. coli (ExPEC) virulence factors (iucD, cnf, papC, tsh, KpsMT II, afa, and sfa) and for phenotypical antimicrobial resistance by Sensititre ARIS® as well as genotypically by polymerase chain reaction. An elevated proportion (30% to 70%) of samples resistant to antimicrobials critically important for human medicine was observed in routine isolates, with no significant differences between the three retail systems. Multidrug-resistant (MDR) ExPEC isolates of phylogroup B1 and, of greater concern, of phylogroup F were detected. Extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing E. coli possessing blaCTX-M or blaCMY-2 resistance genes, respectively, were found. The presence of ExPEC with a high level of antimicrobial resistance (more than 50% of isolates) and MDR (91% of isolates) and detection of ESBL-producing E. coli underline the potential health threat for humans associated with mishandled chicken carcasses or consumption of undercooked chicken meat in Vietnam.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence.

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

The use of a live non-attenuated coccidiosis vaccine modifies Eimeria spp. excretion in commercial antibiotic-free broiler chicken flocks compared to conventional shuttle anticoccidial programs.

The objectives were to compare the effects of an antibiotic-free (ABF) program in commercial broiler chicken flocks using a live non-attenuated coccidiosis vaccine on fecal Eimeria spp. excretion and growth performances with those of conventionally raised commercial broiler chicken flocks. Fecal samples were collected every 3 d from 7 d of age to slaughter in 44 flocks of 7 participating farms for oocyst counts by the McMaster method. A live non-attenuated anticoccidial vaccine was administered by spray cabinet at the hatchery in ABF flocks only. Shuttle programs in conventional flocks consisted of in-feed chemical anticoccidials from 0 to 20 d of age, followed by polyether ionophores until slaughter. In-feed antibiotic growth promoters were included from the starter to finisher diets in conventional flocks only. Age of the flock at the oocyst excretion peak (AGE_PEAK) and the number of oocysts at that excretion peak (OPG_PEAK) were recorded. There was a significant difference of 2.7 d (P = 0.0001) for the AGE_PEAK, from 26.4 d in the conventional treatment to 23.7 d in the ABF program. There was no significant difference for the OPG_PEAK between the 2 treatments (P = 0.626). There was a significant decrease of 2.28 g in the average daily gain (P = 0.004) and increase of 0.08 for the feed conversion ratio (P < 0.0001) in the ABF program compared to the conventional program. There were no significant differences for body weights at slaughter (P = 0.0563), livability (P = 0.2694), and condemnations (P = 0.6775). From this study, it can be concluded that an ABF program using a live non-attenuated vaccine will show an earlier oocyst excretion peak compared to a shuttle program, but no significant effect was observed on the total number of oocysts at that excretion peak between the 2 programs.

Esophagitis and Pharyngitis Associated with Avian Infectious Laryngotracheitis in Backyard Chickens: Two Cases.

Infectious laryngotracheitis (ILT) is a contagious viral respiratory disease of great economic importance for the global poultry industry caused by Gallid herpesvirus 1 (GaHV-1). Lesions of the upper digestive tract caused by this virus have not been reported before. Two small flocks of backyard chickens experienced an outbreak of ILT, one in 2006 and the other in 2014. These birds had typical ILT lesions, characterized by a necrohemorrhagic laryngitis and tracheitis but were also affected by a severe erosive and necrotic esophagitis and pharyngitis. On microscopic examination of the esophagus and pharynx, numerous individual epithelial cells were degenerated or necrotic. Syncytial cells were present in the mucosa or sloughed in the overlying inflammatory crust, and some of these cells contained an amphophilic intranuclear viral inclusion. GaHV-1 was detected in tissues, from respiratory and digestive tracts, either by PCR, immunohistochemistry, or both diagnostic assays. This case stresses the importance for veterinarians, owners, and technicians to pay attention to different or atypical clinical manifestations of ILT given its highly contagious nature.

Recurring Necrotic Enteritis Outbreaks in Commercial Broiler Chicken Flocks Strongly Influence Toxin Gene Carriage and Species Richness in the Resident Clostridium perfringens Population.

Extensive use of antibiotic growth promoters (AGPs) in food animals has been questioned due to the globally increasing problem of antibiotic resistance. For the poultry industry, digestive health management following AGP withdrawal in Europe has been a challenge, especially the control of necrotic enteritis. Much research work has focused on gut health in commercial broiler chicken husbandry. Understanding the behavior of Clostridium perfringens in its ecological niche, the poultry barn, is key to a sustainable and cost-effective production in the absence of AGPs. Using polymerase chain reaction and pulsed-field gel electrophoresis, we evaluated how the C. perfringens population evolved in drug-free commercial broiler chicken farms, either healthy or affected with recurring clinical necrotic enteritis outbreaks, over a 14-month period. We show that a high genotypic richness was associated with an increased risk of clinical necrotic enteritis. Also, necrotic enteritis-affected farms had a significant reduction of C. perfringens genotypic richness over time, an increase in the proportion of C. perfringens strains harboring the cpb2 gene, the netB gene, or both. Thus, necrotic enteritis occurrence is correlated with the presence of an initial highly diverse C. perfringens population, increasing the opportunity for the selective sweep of particularly virulent genotypes. Disease outbreaks also appear to largely influence the evolution of this bacterial species in poultry farms over time.

Feed Supplementation with Red Seaweeds, Chondrus crispus and Sarcodiotheca gaudichaudii, Reduce Salmonella Enteritidis in Laying Hens.

Salmonella Enteritidis is vertically transmitted to eggs from laying hens through infected ovaries and oviducts. S. Enteritidis can also penetrate the eggshell from contaminated feces. Reducing S. Enteritidis in laying hens is vital to provide safer eggs and minimize the spread of salmonellosis to humans. Antibiotics have been widely used to control bacterial diseases in broilers and laying hens. However, there is a major concern that the use of antibiotics leads to the development of antibiotic resistance and adverse effects on microbiota of the treated birds. Thus, there is an interest in developing alternatives to antibiotics, such as dietary prebiotics. In the present study, feed supplemented with the red seaweeds: Chondrus crispus (CC) or Sarcodiotheca gaudichaudii (SG), was offered to laying hens late in production to control S. Enteritidis. Diets contained one of the following; 2% or 4% Chondrus crispus (CC2, and CC4, respectively) or Sarcodiotheca gaudichaudii (SG2 and SG4, respectively). Chlortetracycline was used in the positive control diet. During week-4, 48 birds were orally challenged with 2 × 109 CFU/mL of S. Enteritidis. Eggs and fecal samples were collected 1, 3, 5, and 7 days' post inoculation. Birds were euthanized and organs (ceca, ovary, liver, and spleen) were sampled and analyzed for the presence of S. Enteritidis, 7 days' post inoculation. Results showed that seaweed reduced the negative effect on body weight and egg production in S. Enteritidis-challenged laying hens. Analysis of fecal samples showed that the antibiotic (CTC) reduced S. Enteritidis in the intestinal tract and fecal samples, 3 days' post inoculation. Fecal samples from Chlortetracycline and CC4 supplemented birds tested negative for S. Enteritidis on days 5 and 7 post inoculation (lowest detection limit = 10-1). S. Enteritidis colonization in the ceca was also significantly reduced in birds fed CC (4%) and Chlortetracycline. Blood serum profiles revealed that there were no significant differences in serum aspartate aminotransferase (AST) and sodium. However, the level of serum immunoglobulin (IgA) was higher in the CC4 treatment. The relative abundance of Lactobacillus acidophilus was significantly higher in CC4 while, the abundance of the pathogenic bacteria, Clostridium perfringens and Salmonella Enteritidis were reduced compared to control. Results indicate that feed supplemented with 4% CC is effective in providing protection against Salmonella Enteritidis colonization in laying hens.

Tolerance of Clostridium perfringens biofilms to disinfectants commonly used in the food industry.

Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Recently, it was shown to form mono-species biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. Biofilms have been associated with tolerance to antibiotics, disinfectants, and physical and environmental stresses. Very little is known about the tolerance of C. perfringens biofilm toward disinfectants. In the present study, susceptibilities of C. perfringens biofilms to five types of commonly used disinfectants on farms and in food processing environments were analysed. In this paper, we show that C. perfringens mono-species biofilms can protect the bacterial cells from the action of potassium monopersulfate, quaternary ammonium chloride, hydrogen peroxide and glutaraldehyde solutions. However, sodium hypochlorite solution was shown to be effective on C. perfringens biofilms. Our investigation of dual-species biofilms of C. perfringens with the addition of Staphylococcus aureus or Escherichia coli demonstrated that overall, the mono-species biofilm of C. perfringens was more tolerant to all disinfectants than the dual-species biofilms. For the anaerobic grown biofilms, the mono-species biofilm of C. perfringens was more tolerant to sodium hypochlorite and quaternary ammonium chloride than the dual-species biofilms of C. perfringens with S. aureus or E. coli. This study demonstrates that C. perfringens biofilm is an effective protection mechanism to disinfectants commonly used on farms and in food processing environments.