Investigations into Increased Incidence of Severe Gizzard Erosions and Ulcerations in U.S. Commercial Broilers.

During a series of pathology surveys in four production complexes of a U.S. broiler integrator, the technical services veterinarians of an animal health company noted a high incidence of severe gizzard erosions and ulcerations (GEU), prompting further clinical investigation and a battery trial. No growth-promoting antibiotics or ionophore coccidiostats were used during the period of these surveys. All used tribasic copper chloride (TBCC) at ≤120 ppm added copper in broiler rations. Clostridium perfringens was isolated from 83% and 67% of gizzard lesions cultured in two complexes, and cecal C. perfringens most probable number determinations were higher in severely affected than in mildly affected or unaffected birds. Histopathology revealed both acellular koilin fusion defects characteristic of copper toxicity, as well as inflammatory cell infiltrates. Intralesional bacilli suggestive of C. perfringens were noted in 78% of affected flocks examined. Species E Aviadenovirus was isolated from one bird in one complex, and that bird had a single intranuclear inclusion body; no other flocks had Adenoviruses isolated or detected on PCR, nor any inclusion bodies. Other viruses detected were thought to be incidental. A pilot study using feed with supplemental copper from TBCC or copper sulfate and challenge with one of the isolated C. perfringens strains reproduced the lesions. A battery study was conducted with an unchallenged negative control group fed a diet with 16 ppm added copper, a group fed the control diet and orally challenged with 108 organisms of a field strain of C. perfringens at 21 and 22 days, and a group treated with the same diet containing 250 ppm added copper from TBCC and orally challenged with C. perfringens. Birds were necropsied at 23 and 28 days. All challenged groups developed lesions, with those receiving both TBCC and C. perfringens having significantly higher gross and histopathological lesion scores than the unchallenged negative controls. Lesions were qualitatively similar to those in the field and contained suspected C. perfringens bacilli. Because the levels of TBCC used in the commercial birds and in the battery trial generally have been considered safe, and because C. perfringens is usually regarded as a pathogen of the lower GI tract, the possible association of these two agents with GEU is a novel observation and warrants further investigation.

Investigaciones sobre el aumento de la incidencia de erosiones y ulceraciones severas en la molleja en pollos de engorde comerciales en los Estados Unidos. Durante una serie de estudios de patología en cuatro complejos de producción de un integrador de pollos de engorde de los Estados Unidos, veterinarios de servicio técnico de una empresa de salud animal observaron una alta incidencia de erosiones y ulceraciones severas de la molleja (GEU), lo que motivó una mayor investigación clínica y un estudio en batería. Durante el período de estas encuestas no se utilizaron antibióticos promotores del crecimiento ni coccidiostáticos ionóforos. Todos utilizaron cloruro de cobre tribásico (TBCC) con un nivel de ≤120 ppm de cobre agregado en raciones para pollos de engorde. Se aisló Clostridium perfringens del 83% y el 67% de las lesiones de molleja cultivadas en dos complejos, y las determinaciones del número más probable de C. perfringens en los sacos ciegos fueron mayores en aves severamente afectadas que en aves levemente afectadas o no afectadas. La histopatología reveló defectos de fusión de la capa córnea acelular característicos de la toxicidad por cobre, así como infiltrados de células inflamatorias. Se observaron bacilos intralesionales sugestivos de C. perfringens en el 78% de las parvadas afectadas examinadas. La especie Aviadenovirus E se aisló de un ave en un complejo, y esa ave tenía un único cuerpo de inclusión intranuclear; en ninguna otra parvada se aislaron o detectaron adenovirus mediante PCR, ni se observaron cuerpos de inclusión. Se pensó que otros virus detectados fueron incidentales. Un estudio piloto que utilizó alimento con cobre suplementario de cloruro de cobre tribásico o sulfato de cobre y con desafío con una de las cepas aisladas de C. perfringens reprodujo las lesiones. Se realizó un estudio de batería con un grupo de control negativo no desafiado alimentado con una dieta con 16 ppm de cobre agregado, un grupo alimentado con la dieta de control y desafiado por vía oral con 108 organismos de una cepa de campo de C. perfringens a los 21 y 22 días, y un grupo tratado con la misma dieta que contenía 250 ppm de cobre agregado de cloruro de cobre tribásico y desafiados por vía oral con C. perfringens. A las aves se les realizó la necropsia a los 23 y 28 días. Todos los grupos desafiados desarrollaron lesiones, y aquellos que recibieron cloruro de cobre tribásico y C. perfringens tuvieron puntuaciones de lesiones macroscópicas e histopatológicas significativamente más altas que los controles negativos no desafiados. Las lesiones eran cualitativamente similares a las del campo y contenían bacilos sospechosos de C. perfringens. Debido a que los niveles de cloruro de cobre tribásico utilizados en las aves comerciales y en el ensayo en batería generalmente se han considerado seguros, y debido a que C. perfringens generalmente se considera un patógeno del tracto gastrointestinal inferior, la posible asociación de estos dos agentes con erosiones y ulceraciones severas de la molleja es una observación reciente y justifica una mayor investigación.

Investigating the uses of machine learning algorithms to inform risk factor analyses: The example of avian infectious bronchitis virus (IBV) in broiler chickens.

Infectious bronchitis virus (IBV) is a contagious coronavirus causing respiratory and urogenital disease in chickens and is responsible for significant economic losses for both the broiler and table egg layer industries. Despite IBV being regularly monitored using standard epidemiologic surveillance practices, knowledge and evidence of risk factors associated with IBV transmission remain limited. The study objective was to compare risk factor modeling outcomes between a traditional stepwise variable selection approach and a machine learning-based random forest Boruta algorithm using routinely collected IBV antibody titer data from broiler flocks. IBV antibody sampling events (n = 1111) from 166 broiler sites between 2016 and 2021 were accessed. Ninety-two geospatial-related and poultry-density variables were obtained using a geographic information system and data sets from publicly available sources. Seventeen and 27 candidate variables were screened to potentially have an association with elevated IBV antibody titers according to the manual selection and machine learning algorithm, respectively. Selected variables from both methods were further investigated by construction of multivariable generalized mixed logistic regression models. Six variables were shortlisted by both screening methods, which included year, distance to urban areas, main roads, landcover, density of layer sites and year, however, final models for both approaches only shared year as an important predictor. Despite limited significance of clinical outcomes, this work showcases the potential of a novel explorative modeling approach in combination with often unutilized resources such as publicly available geospatial data, surveillance health data and machine learning as potential supplementary tools to investigate risk factors related to infectious diseases.

Enumeration of Salmonella and Campylobacter spp. in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks.

A prospective cohort study was performed to evaluate the prevalences and loads of Salmonella and Campylobacter spp. in farm and processing plant samples collected from 55 commercial broiler chicken flocks. Environmental samples were collected from broiler houses within 48 h before slaughter, and carcass rinses were performed on birds from the same flocks at 4 different stages of processing. Salmonella was detected in farm samples of 50 (90.9%) flocks and in processing samples of 52 (94.5%) flocks. Campylobacter was detected in farm samples of 35 (63.6%) flocks and in processing samples of 48 (87.3%) flocks. There was a significant positive relationship between environmental farm samples and processing plant carcass rinses with respect to both Salmonella and Campylobacter prevalences and loads. Campylobacter loads were significantly higher than Salmonella loads, and the correlations between samples collected from the same flocks were higher for Campylobacter than they were for Salmonella. Boot socks were the most sensitive sample type for detection of Salmonella on the farm, whereas litter samples had the strongest association with Salmonella loads in pre- and postchill carcass rinses. Boot socks, drag swabs, and fecal samples all had similar sensitivities for detecting Campylobacter on the farm, and all were more strongly associated with Campylobacter loads in carcass rinses than were litter samples. Farm samples explained a greater proportion of the variability in carcass rinse prevalences and loads for Campylobacter than they did for Salmonella. Salmonella and Campylobacter prevalences and loads both decreased significantly as birds progressed through the processing plant.

Estimation of the prevalence of Salmonella species on the slatted area compared to the scratch area of broiler breeder chicken houses.

With the objective of detecting Salmonella species in the poultry house environment, sampling broiler breeder houses in the two-thirds slatted area and the one-third scratch area with pine shavings bedding can be difficult. The slatted area is where the females, consisting of approximately 90% of the population, eat, drink, and spend the majority of their time. The scratch area is where the males eat and drink, as well as where mating and mortality occur. Besides the nest boxes, the female feeders and water lines make the slatted areas more difficult to sample. However, it is important to take samples from the area with the greatest likelihood of isolating Salmonella. Therefore, boot swab samples were collected from 21 commercial broiler breeder chicken houses representing three different companies in north Georgia to estimate the prevalence of Salmonella on the slat vs. the scratch area in each chicken house. Every house sampled had a central scratch area covered with litter and two elevated slat areas constructed of wood. Four boot swab samples were collected on each side slat area by walking the entire length of the house for each swab. Eight samples were collected in the same manner from the scratch area. Results of 335 total samples were acquired, 167 of which were obtained from the slat area and 168 of which came from the scratch area, yielding 242 total Salmonella isolations. Salmonella was found in 117 of the slat samples, representing 70.1%, compared to 125 of the scratch area samples, representing 74.4%. Both the univariate (P = 0.311) and the multivariable (P = 0.303) analysis showed that sampling location was not associated with Salmonella prevalence.

Intra-cluster correlations for ceca Salmonella prevalence and enumeration from 40 experimental floor pen trials in broiler chickens using a seeder bird challenge model.

Floor pen trials are an efficient way to evaluate the effectiveness of potential Salmonella control interventions in broiler chickens. When treatments are allocated at the pen level, and outcomes are measured at the individual bird level, floor pen studies are considered to be cluster randomized trials. Estimating the sample size required to achieve a desired level of statistical power for a cluster randomized trial requires an estimate of the intra-cluster correlation (ICC) as an input. In this study, ICCs were estimated for the untreated challenged control group from 40 broiler chicken Salmonella pen trials performed using a seeder bird challenge model. The ICCs for ceca Salmonella prevalences ranged from 0.00 to 0.64, with a median of 0.17. The ICCs for ceca Salmonella log10(MPN/g + 1) ranged from 0.00 to 0.52, with a median of 0.14. These findings indicate that the effect of pen-level clustering is substantial in Salmonella floor pen trials, and it must be considered during both the study design and analysis. In a multivariable regression analysis, ICCs for ceca Salmonella prevalences were associated with the challenge status of sampled birds, age of birds at the time of challenge, and Salmonella serovar. ICCs were lower for studies in which a combination of direct (seeder) and indirect (horizontal) challenged birds were sampled, and for studies in which birds were challenged on the day of hatch or at one day of age. ICCs were higher for studies in which Salmonella Heidelberg was used as the challenge strain. These findings may be useful for investigators that are planning pen trials to evaluate Salmonella control interventions in broiler chickens. Choosing study design elements associated with a lower ICC may improve efficiency by leading to a larger effective sample size for the same number of experimental units.

A triple-sugar regulated Salmonella vaccine protects against Clostridium perfringens-induced necrotic enteritis in broiler chickens.

Gram-positive Clostridium perfringens type G, the causative agent of necrotic enteritis (NE), has gained more attention in the poultry industry due to governmental restrictions on the use of growth-promoting antibiotics in poultry feed. Our previous work has proved that regulated delayed lysis Salmonella vaccines delivering a plasmid encoding an operon fusion of the nontoxic C-terminal adhesive part of alpha toxin and a GST-NetB toxin fusion were able to elicit significant protective immunity in broilers against C. perfringens challenge. We recently improved our S. Typhimurium antigen delivery vaccine strain by integrating a rhamnose-regulated O-antigen synthesis gene enabling a triple-sugar regulation system to control virulence, antigen-synthesis and lysis in vivo traits. The strain also includes a ΔsifA mutation that was previously shown to increase the immunogenicity of and level of protective immunity induced by Salmonella vectored influenza and Eimeria antigens. The new antigen-delivery vaccine vector system confers on the vaccine strain a safe profile and improved protection against C. perfringens challenge. The strain with the triple-sugar regulation system delivering a regulated lysis plasmid pG8R220 encoding the PlcC and GST-NetB antigens protected chickens at a similar level observed in antibiotic-treated chickens. Feed conversion and growth performance were also similar to antibiotic-treated chickens. These studies made use of a severe C. perfringens challenge with lesion formation and mortality enhanced by pre-exposure to Eimeria maxima oocysts. The vaccine achieved effectiveness through three different immunization routes, oral, spray and in drinking water. The vaccine has a potential for application in commercial hatcher and broiler-rearing conditions.

Comparison of real-time PCR with conventional PCR and culture to assess the efficacy of a live attenuated Salmonella enterica serovar Typhimurium vaccine against Salmonella enterica serovar Enteritidis in commercial leghorn chicks vaccinated under field and laboratory conditions.

The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.

Research Note: First report on the detection of necrotic enteritis (NE) B-like toxin in biological samples from NE-afflicted chickens using capture enzyme-linked immunosorbent assay.

Necrotic enteritis (NE) is a devastating enteric disease caused by Clostridium perfringens type G. One of the pore-forming toxins, NE B-like (NetB) toxin, secreted by pathogenic C. perfringens type G, has been proposed to be the main virulent factor in NE pathogenesis. The present study aimed to detect the presence of NetB toxin in biological samples of NE-afflicted chickens using NetB-specific monoclonal-based enzyme-linked immunosorbent assay (ELISA). Biological samples, including serum, digesta, and fecal droppings, were obtained from three previous NE studies (designated as Trials 1 to 3). In Trials 1 and 2, broiler chicks were infected with Eimeria maxima strain 41A on day 1 and followed by the netB-positive C. perfringens on day 18. Serum samples were obtained at 20 d post-hatch (i.e., 2 d post C. perfringens infection). In addition, various samples, including serum, gut digesta, and fecal droppings, that had been collected 0, 6, 24, and 30 h post C. perfringens infection were obtained. In Trial 3, broiler chicks were indirectly infected with litter-contaminated E. maxima on d 14 and followed by netB-positive C. perfringens via drinking water on days 18, 19, and 20. Serum samples and fecal droppings were obtained 21 d post-hatch (i.e., 1 d post last C. perfringens infection). The results showed that NetB toxin was not detected in serum samples in Trials 1 and 3. No NetB toxin was detected in all samples obtained before C. perfringens infection in Trial 2. Low but detectable amounts of NetB toxin were found in the serum samples obtained 6 h post C. perfringens infection in Trial 2. While NetB toxin in digesta and fecal droppings was detected 6 h post C. perfringens infection, its level plateaued 24 and 30 h post C. perfringens infection. In Trial 3, NetB toxin was detected in fecal droppings from the NE group, and its concentration ranged from 2.9 to 3.1 ng/g of wet feces. In Trial 2, NE-specific lesions were not seen 0 and 6 h post C. perfringens infection but exhibited lesions were moderate to severe 24 h post infection, leading to a moderate association (r = +0.527) between NE lesions and NetB toxin in the gut digesta. This is the first study to use NetB-specific monoclonal-based capture ELISA to determine and report the presence of native NetB toxin in biological samples from NE-induced chickens.

Immunity and Protection Provided by Live Modified Vaccines Against Paratyphoid Salmonella in Poultry-An Applied Perspective.

Several serotypes of non-host-specific or paratyphoid Salmonella have been linked with contamination of poultry meat, and eggs, resulting in foodborne outbreaks in humans. Vaccination of poultry against paratyphoid Salmonella is a frequent strategy used to reduce the levels of infection and transmission, which ultimately can lead to lower rates of human infections. Live vaccines have been developed and used in poultry immediately after hatching as a result of their ability to colonize the gut, stimulate a mucosal immune response, induce a competitive inhibitory effect against homologous wild strains, and reduce colonization and excretion of Salmonella. Furthermore, vaccines can competitively exclude some heterologous strains of Salmonella from colonizing the gastrointestinal tract when young poultry are immunologically immature. In addition, various studies have suggested that booster vaccination with live vaccines a few weeks after initial vaccination is essential to increase the level of protection and achieve better cross-protective immunity. Vaccination of breeders, broilers, layers, and turkeys with modified live Salmonella vaccines is a common intervention that has become an important component in poultry companies' multistep prevention programs to meet increasingly demanding customer and regulatory food safety requirements. Both live and inactivated vaccines play a critical role in a comprehensive control program for chicken and turkey breeders and commercial layers. This review examines the response and protection conferred by live modified vaccines against non-host-specific Salmonella that can be considered for the design and implementation of vaccination strategies in poultry.

Artículo regular­Inmunidad y protección que brindan las vacunas vivas modificadas contra salmonelas paratíficas en la avicultura­Una perspectiva aplicada. Varios serotipos de Salmonella paratífica no específica de huésped se han relacionado con la contaminación de la carne de pollo y huevos lo que ha provocado brotes de origen alimentario en los seres humanos. La vacunación de aves comerciales contra Salmonella paratífica es una estrategia que se utiliza con frecuencia para reducir los niveles de infección y transmisión, que en última instancia puede conducir a tasas más bajas de infecciones en humanos. Se han desarrollado y utilizado vacunas vivas en aves comerciales inmediatamente después de la eclosión como resultado de su capacidad para colonizar el intestino, estimular una respuesta inmunitaria de la mucosa, inducir un efecto inhibidor competitivo contra cepas silvestres homólogas y reducir la colonización y excreción de Salmonella. Además, las vacunas pueden excluir competitivamente algunas cepas heterólogas de Salmonella de colonizar el tracto gastrointestinal cuando las aves jóvenes son inmunológicamente inmaduras. Además, varios estudios han sugerido que la vacunación de refuerzo con vacunas vivas unas semanas después de la vacunación inicial es esencial para aumentar el nivel de protección y lograr una mejor inmunidad de protección cruzada. La vacunación de reproductoras, pollos de engorde, aves de postura y pavos con vacunas vivas modificadas contra Salmonella es una intervención común que se ha convertido en un componente importante en los programas de prevención de múltiples pasos de las empresas avícolas para cumplir con los requisitos de los clientes y regulatorios de seguridad alimentaria. Tanto las vacunas vivas como las inactivadas desempeñan un papel fundamental en un programa de control integral para productores de pollos, pavos y aves ponedoras comerciales. Esta revisión examina la respuesta y la protección conferidas por las vacunas vivas modificadas contra Salmonella no específica del huésped que pueden ser consideradas para el diseño e implementación de estrategias de vacunación en la avicultura.

Bacteriophage therapy for control of necrotic enteritis of broiler chickens experimentally infected with Clostridium perfringens.

Several lytic bacteriophages effective at destroying a genetically diverse population of Clostridium perfringens were isolated from the environment, extensively characterized, and used to formulate a multivalent bacteriophage cocktail designated -401." Two in vivo studies were conducted to determine the cocktail's efficacy in controlling necrotic enteritis (NE) caused by C. perfringens. The first study investigated the efficacy of INT-401 and a bacteriophage-derived, toxoid-type vaccine in controlling NE in C. perfringens-challenged broiler chickens. The study was designed as a proof-of-concept battery cage study with birds reared until 28 days old. Compared with the mortality observed with the C. perfringens-challenged but untreated chickens, oral administration of INT-401 significantly (P < 0.05) reduced the mortality of the C. perfringens-challenged birds by 92%. Overall, INT-401 was more effective than the toxoid vaccine in controlling active C. perfringens infection. The second study was conducted to investigate the effectiveness of the cocktail when administered via oral gavage, feed, or drinking water. The study was conducted in floor pens, with birds reared to 42 days old. INT-401 administered by all three methods significantly (P < 0.05) reduced mortality. Weight gain and feed conversion ratios were significantly better in the C, perfringens-challenged chickens treated with INT-401 than in the C. perfringens-challenged, phage-untreated control birds. The data indicate that delivering INT-401 to broiler chickens via their drinking water or feed may be an effective means for controlling NE caused by C. perfringens and may improve weight gain and feed conversion ratios in birds with clinical or subclinical NE.

Effect of a radiant energy-treated lysozyme antimicrobial blend on the control of clostridial necrotic enteritis in broiler chickens.

A cage study was conducted to demonstrate the effect of Entegard REV, a lysozyme-based antimicrobial blend, on the performance of broiler chickens and necrotic enteritis (NE) disease reduction of birds that were challenged with Eimeria maxima and Clostridium perfringens. In the experiment, challenge by the infectious agents without medication resulted in impaired feed consumption, weight gain, and feed conversions and caused high incidence of gross NE lesions and NE mortality rate. Entegard REV included in feed at 200 g/metric ton (MT) was very effective in reducing negative health effects in the birds after NE challenge, and its ability to control the disease was not statistically different from a commonly used antibiotic growth promotant, bacitracin methylene disalicilate, at 55 g/MT.

Efficacy of Trace Mineral-Amino Acid Complexes in a Necrotic Enteritis Challenge Model.

Necrotic enteritis (NE) is a common and costly disease of poultry caused by virulent toxigenic strains of Clostridium perfringens. Although the importance of trace minerals for intestinal integrity and health is well documented, there is little information on their role in ameliorating the effects of NE. The two studies reported here examined the effects of replacing a portion of the dietary zinc (Zn), copper (Cu), and manganese (Mn) supplied as sulfates in the control diets with metal-amino acid-complexed minerals in a NE-challenge model consisting of coccidiosis and Clostridium perfringens. In a 28-day battery study, the treatments were the following: (1) no additional Zn or Mn, unchallenged (negative control); (2) no added Zn or Mn, challenged (positive control); (3) added ZnSO4 and MnSO4 at 100 ppm each, challenged; (4) additional ZnSO4 at 60 ppm, Availa-Zn at 40 ppm (Low), and MnSO4 at 100 ppm, challenged; (5) added ZnSO4 at 60 ppm, Availa-Zn at 60 ppm (high), and MnSO4 at 100 ppm, challenged; and (6) added ZnSO4 at 60 ppm, Availa-Zn at 40 ppm, MnSO4 at 60 ppm, and Availa-Mn at 40 ppm, challenged. None of the treatments ameliorated gross lesion scores, but all reduced NE-associated mortality compared with the positive control. At 28 days, the group supplemented with Availa-Zn at 40 ppm (low) had a lower body weight than challenged groups supplemented with Zn and the negative control. In a floor pen study, the five treatment groups were the following: (1) Zn, Mn, and Cu from sulfate sources at 100, 100, and 20 ppm respectively; (2) Zn, Mn, and Cu from sulfate sources at 40, 100, and 20 ppm, respectively, plus Zn from Availa-Zn at 60 ppm; (3) Zn and Mn from sulfate sources at 40 and 100 ppm, respectively, plus Zn from Availa-Zn at 60 ppm and Cu from Availa-Cu at 10 ppm; (4) Zn, Mn, and Cu from sulfate sources at 60, 60, and 20 ppm, respectively, plus Zn and Mn from Availa-Zn/Mn at 40 and 40 ppm, respectively; and (5) bacitracin methylene disalicylate at 55 g/metric ton with Zn, Mn, and Cu from sulfate sources at 100, 100, and 20 ppm, respectively (Zoetis, Inc., Kalamazoo, MI). None of the treatments reduced lesion scores. The Availa-Zn and Availa-Zn/Mn had lower mortality than the sulfate-supplemented feed, whereas Availa-Zn/Cu and bacitracin methylene disalicylate were intermediate and did not differ from the other groups. Considering both trials together, and by using NE mortality as the discriminating factor, we found that adding Zn and Mn exceeding National Research Council requirements reduced NE-associated mortality, and in the floor pen study, complexed Zn and complexed Zn plus Mn appeared to be superior to sulfates.

Efficacy of Butyric and Valeric Acid Esters in a Necrotic Enteritis Challenge Model.

Restrictions on the use of antibiotics in poultry production have increased interest in nonantibiotic alternatives to control necrotic enteritis (NE). Volatile fatty acids, and in particular butyric acid preparations, have shown potential as aids in controlling NE. Valeric acid compounds may be a new additional alternative. This series of three trials compared the effects of tributyrin, monovalerin, which is an organic acid mixture, and bacitracin in a NE challenge model consisting of challenge with coccidiosis followed by Clostridium perfringens. Trial 1 was a pen trial comparing tributyrin at 0.5 kg/metric ton continuously in the feed, a proprietary organic acid blend at 1 kg per 1000 L as a metaphylactic treatment in the water, and bacitracin in the feed at 55 g/metric ton. Tributyrin and the organic acid mixture were at least as effective as bacitracin in controlling the growth- and efficiency-suppressing effects of the NE challenge, and the organic acid mixture reduced NE lesion scores. None of the treatments reduced mortality. Trial 2 was a battery study comparing monovalerin at 1.5 kg/metric ton and bacitracin in the feed. Both interventions provided significant control of both clinical and subclinical NE, with bacitracin being slightly superior to monovalerin. Trial 3 was a pen trial comparing monovalerin at 1 kg or 1.5 kg/metric ton continuously, or 0.5 kg/metric ton from 0 to 14 days and 0.25 kg/metric ton from 14 to 42 days (variable dose), to tributyrin at the same variable-dose schedule. The higher dose of monovalerin appeared to suppress feed intake and weight gain prechallenge but also produced the lowest NE mortality and the lowest total mortality of the challenged groups. All of the treatments except the variable-dose monovalerin treatment demonstrated reductions in NE lesion scores compared with the positive challenge control group; however, they did not control mortality and had fewer effects on the performance effects of subclinical NE. Results of these studies indicate that the organic acid products monovalerin and tributyrin may be useful adjuncts to reduce NE in antibiotic-free broiler production.

Salmonella Enteritidis reduction in layer ceca with a Bacillus probiotic.

BACKGROUND AND AIM: Salmonella Enteritidis (SE) is a significant foodborne pathogen that can often be traced to poultry and poultry products. This study aims to evaluate the ability of three commonly used non-antimicrobial feed additives in reducing the amount of SE in the ceca of laying type pullets. MATERIALS AND METHODS: On day 0, 60 Hy-Line Brown pullets aged 9 weeks were allocated to individual cages in 15 replicate blocks of four pens. Pullets were administered a mash feed provided ad libitum without supplementation (control) or with dietary supplementation of 454 g/ton yeast cell wall (YCW), or 454 g/ton Bacillus spp. probiotic, or 1133 g/ton yeast culture (YC). On day 3 of the trial, all birds were orally administered 3×107 CFU of a nalidixic acid-resistant SE. On day 10, 7 days after inoculation, all birds were humanely euthanized, and the ceca were aseptically removed for analysis. RESULTS: There was no significant difference in the prevalence of SE among treatments. The mean quantity of SE detected in the ceca expressed in log10 most probable number/g was 2.52 in the control, 2.49 in the YCW treatment, 1.73 in the probiotic treatment, and 1.66 in the YC treatment. The reduction between control and probiotic and control and YC was significant (p=0.021). CONCLUSION: This study demonstrates the ability of the novel probiotic and the YC to reduce the load of SE in layer ceca.

A rapid screen of broth enrichments for Salmonella enterica serovars enteritidis, Hadar, Heidelberg, and Typhimurium by Using an allelotyping multiplex PCR that targets O- and H-antigen alleles.

Salmonella continues to cause significant foodborne outbreaks, best illustrated with recent outbreaks associated with peanut butter, raw tomatoes, and serrano peppers. To ascertain the likely source of the outbreak, bacterial typing is essential to this process. While PCR has become an important detection tool for pathogens in foods, PCR can also identify strain differences by targeting gene(s) or sequences exhibiting polymorphisms or variability in its distribution within the bacterial population. Over 2,500 Salmonella enterica serovars identified based on antigenic differences in lipopolysaccharide and flagellin have been identified to date. We developed an allelotyping PCR scheme that identifies the O and H alleles associated with S. enterica serovars Enteritidis, Hadar, Heidelberg, Typhimurium, and others, with the same antigen alleles but in different O- and H-allele combinations (e.g., S. enterica Kentucky), and validated it as a screen to identify samples contaminated with these Salmonella serovars. We correctly identified poultry samples containing S. enterica serovars Enteritidis, Kentucky, and Typhimurium from our multiplex screen of primary enrichments of environmental drag swabs. PCR agreed well (kappa values = 0.81 to 1.0) with conventional serotyping methods used to type salmonellae isolated from primary enrichment. Coupled with Salmonella-specific PCR, such as invA, this allelotyping PCR could prove useful in the identification of Salmonella and specific S. enterica serovars present in foods or the environment and could decrease the time and cost associated with conventional serotyping methods.

Human contacts and potential pathways of disease introduction on Georgia poultry farms.

As highly pathogenic avian influenza H5N1 virus continues to circulate in the world, poultry farm biosecurity and timely reporting of morbidity and mortality among commercial poultry farms in the United States are major concerns. To assess the vulnerability of poultry farms to the introduction and spread of a highly infectious pathogen, such as the currently circulating H5N1 influenza virus, a survey was administered to growers in two counties in Georgia representing areas of low and high poultry densities. Survey questions regarding horizontal contacts and management were sent to commercial broiler and breeder-layer chicken producers. Responses were used to estimate and compare contact rates and patterns between the two regions. The distribution of high-risk visitors (i.e., those going inside the poultry houses) to poultry farms did not vary significantly between growers in counties with high and low poultry densities or between breeder-layer and broiler growers. Compared with broiler producers in the county with high poultry density, broiler growers in the county with low poultry density were more likely to hire non-family employees to help with poultry management (62% vs. 17%; P = 0.001) and assist other growers with their poultry (31% vs. 6%; P = 0.025). Use of contracted litter services was significantly higher (P = 0.019) among broiler growers in the poultry-dense county (40%) compared with the low-density county (6%). Compared with broiler growers, breeder-layer producers also were significantly more likely to hire non-family employees to help on the farm (53% vs. 17%; P = 0.008). Poultry growers in the highly poultry-dense county were more likely to have a public road or field receiving poultry litter within a quarter mile of their poultry houses, compared with those in the lower density county. Data obtained in this study support the observations of published poultry disease outbreak investigations and highlight the differences in farm vulnerability to disease introduction within areas of different poultry densities and management practices.

Maternally-Derived Antibodies Protect against Challenge with Highly Pathogenic Avian Influenza Virus of the H7N3 Subtype.

Vaccination of hens against influenza leads to the transfer of protective maternally-derived antibodies (MDA) to hatchlings. However, little is known about the transfer of H7N3 vaccine-induced MDA. Here, we evaluated transfer, duration, and protective effect of MDA in chickens against H7N3 HPAIV. To generate chickens with MDA (MDA (+)), 15-week-old White Leghorn hens were vaccinated and boosted twice with an inactivated H7N3 low pathogenic avian influenza virus vaccine, adjuvanted with Montanide ISA 71 VG. One week after the final boost, eggs were hatched. Eggs from non-vaccinated hens were hatched for chickens without MDA (MDA (-)). Both MDA (+) and MDA (-) hatchlings were monitored weekly for antibody levels. Anti-HA MDA were detected by hemagglutination inhibition assay mostly until day 7 post-hatch. However, anti-nucleoprotein MDA were still detected three weeks post-hatch. Three weeks post-hatch, chickens were challenged with 106 EID50/bird of Mexican-origin H7N3 HPAIV. Interestingly, while 0% of the MDA (-) chickens survived the challenge, 95% of the MDA (+) chickens survived. Furthermore, virus shedding was significantly reduced by day 5 post-challenge in the MDA (+) group. In conclusion, MDA confers partial protection against mortality upon challenge with H7N3 HPAIV, as far as three weeks post-hatch, even in the absence of detectable anti-HA antibodies, and reduce virus shedding after challenge.

Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.

BACKGROUND: Classical Salmonella serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different Salmonella enterica serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. RESULTS: By analyzing the nucleotide sequences of the genes for O-antigen biosynthesis including wba operon and the central variable regions of the H1 and H2 flagellin genes in Salmonella, designated PCR primers for four multiplex PCR reactions were used to detect and differentiate Salmonella serogroups A/D1, B, C1, C2, or E1; H1 antigen types i, g, m, r or z10; and H2 antigen complexes, I: 1,2; 1,5; 1,6; 1,7 or II: e,n,x; e,n,z15. Through the detection of these antigen gene allele combinations, we were able to distinguish among S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. The assays were useful in identifying Salmonella with O and H antigen gene alleles representing 43 distinct serovars. While the H2 multiplex could discriminate between unrelated H2 antigens, the PCR could not discern differences within the antigen complexes, 1,2; 1,5; 1,6; 1,7 or e,n,x; e,n,z15, requiring a final confirmatory PCR test in the final serovar reporting of S. enterica. CONCLUSION: Multiplex PCR assays for detecting specific O and H antigen gene alleles can be a rapid and cost-effective alternative approach to classical serotyping for presumptive identification of S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.

An optimist's view on limiting necrotic enteritis and maintaining broiler gut health and performance in today's marketing, food safety, and regulatory climate.

The future poultry nutritionist, veterinarian, and husbandryman will have many new regulatory requirements and consumer preferences to navigate in addition to their normal responsibility of raising birds in a cost-efficient and wholesome manner. New challenges include changes to antibiotic use, increased food safety regulations, and more concern over how birds are raised and how to dispose of poultry house waste. All of these new programs and new regulations will alter how we have been raising birds for the last 60 years since the inception of the integrated poultry industry. The most significant change may be the voluntary or regulatory withdrawal of the use of antibiotics in poultry production. In North America, this withdrawal of antibiotic use includes removal of in-ovo antibiotics, performance-improving antibiotics or antibiotic growth promotors (AGP), and the polyether ionophore antibiotics (ionophore anticoccidials).The removal of antibiotics in poultry production may result in welfare concerns due to elevated mortality and less efficient feed conversion, resulting in greater environmental impacts from increased manure production and more use of grain per unit of meat produced. There also may be concerns with greater intestinal disease in the birds resulting in increased numbers of foodborne illness-causing bacteria such as Salmonella sp. or Campylobacter sp. on the carcass. A major impact will be the disease necrotic enteritis (NE). This review will focus on the pathophysiology of NE, the management of the disease, and the additional effects on growth rate, feed efficiency, and body weight that may be associated with NE.

FTA liver impressions as DNA template for detecting and genotyping fowl adenovirus.

The feasibility of using liver impressions on Flinders Technology Associates (FTA filter paper for the collection, inactivation, and molecular analysis of fowl adenovirus (FAV) was evaluated. FAV I European Union (EU) serotype 1 spotted on FTA was shown to be inactivated using specific-pathogen-free (SPF) primary chicken embryo liver cell culture as indicated by absence of cytopathic effect. Sensitivity of the polymerase chain reaction (PCR) test using tenfold dilutions of allantoic fluid from 100 to 10-4 for the detection of adenovirus serotype 1 on FTA cards was determined to be 0.0005 mean tissue culture infectious dose per FTA spot. The stability of the DNA from liver impressions on the FTA was found to be 198 days when stored at -20 degrees C. In a trial, inclusion body hepatitis (IBH) was experimentally reproduced in SPF chickens inoculated with FAV I EU serogroup 1, 4, 8, or 11, which presented weakness, pallor, depression, dehydration, and mortality within 6 days after inoculation. PCR performed on FTA liver impressions from the inoculated birds was able to detect all four viruses, and the nucleotide sequence analysis of the amplified PCR products (1219 bp of the hexone gene) revealed the expected serotypes. In addition to the trial, 55 clinical samples were analyzed from liver impressions on FTA cards, and FAV was detected in 11 of 55 (20%). Sequencing analysis showed that the viruses were EU serotypes 4, 5, 9, and 10. The results demonstrate that FTA filter paper inactivates the FAV I and maintains the DNA template for molecular analysis.