Isolation, characterization, and experimental infection of Streptococcus gallolyticus subspecies pasteurianus from commercial turkeys with acute septicemia: a pilot study.

Streptococcus gallolyticus (SG) is a Gram-positive cocci found as commensal gut flora in animals and humans. SG has emerged as a cause of disease in young poults between 1 and 3 wk of age. SG is associated with septicemia resulting in acute mortality with no premonitory signs in turkeys. Three SG isolates were obtained from clinical field cases of acute septicemia of commercial turkeys and used in three independent experiments. In Experiment 1, embryos were inoculated 25 d of embryogenesis with varying concentrations of SG1, SG2, or SG3. In Experiment 2, day of hatch, poults were inoculated with varying concentrations using different routes of administration of SG1, SG2, or SG3. In Experiment 3, day of hatch, poults were inoculated with only isolate SG1 using different paths. Poults were randomly selected for necropsy on d 8 and d 15 and sampled to collect spleen, heart, and liver for SG on d 21, the remaining poults were necropsied and cultured. Samples were plated on Columbia nalidixic acid and colistin agar (CNA) (40°C, 18-24 h). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed suspect colonies. Data were analyzed using the chi-square test of independence, testing all possible combinations to determine significance (P < 0.05). Weight data were subjected to ANOVA using JMP with significance (P < 0.05). No differences were found in BW or BWG on d 0, 8, 15, or 22. Splenomegaly, focal heart necrosis, and pericarditis were observed in all groups in experiments 1 through 3. In Experiment 3, only airsacculitis was observed in a negative control in separate isolation (P > 0.05). On d 21 of Experiment 3, increased (P < 0.05) recovery of SG from spleens were observed in co-housed negative controls, as well as poults challenged by oral gavage (P > 0.05 for d 7 and d 14). These results confirm numerous previous studies indicating that SG subsp. pasteurianus is a primary infectious microorganism that causes septicemia in young poults.

Evaluation of the impact of formaldehyde fumigation during the hatching phase on contamination in the hatch cabinet and early performance in broiler chickens.

Commercial hatch cabinet environments promote replication of microorganisms. These pathogenic or apathogenic microorganisms may serve as pioneer colonizers of the gastrointestinal tract (GIT) of poultry. Some of these pioneer colonizers, such as Escherichia coli and Enterococcus spp., are opportunistic pathogens that lead to reduced performance in commercial poultry. Effective hatchery sanitation is imperative to limit contamination of naïve neonatal chicks and poults. Formaldehyde fumigation has been traditionally used to reduce the pathogen load in commercial hatch cabinets. To investigate potential alternatives to formaldehyde fumigation, models to mimic the microbial bloom in a laboratory setting must be utilized. The purpose of the present study was to evaluate the impact of a multispecies environmental challenge model (PM challenge) with and without formaldehyde fumigation during the hatching phase on early performance in broiler chicks. Three experiments were conducted to evaluate microbial contamination in the hatch cabinet environment (air samples, fluff samples), enteric colonization at day-of-hatch (DOH), and 7-day performance. In all experiments, significantly (P < 0.05) more gram-negative bacteria were recovered from the GIT at DOH in the PM challenge control group as compared to the nonchallenged control (NC) group and the formaldehyde-treated group (PM + F). There were no statistical differences in 7-day body weight gain or feed conversion ratio between the PM challenge control group, the NC group or the PM + F group. These data suggest this model could be utilized to evaluate alternatives to formaldehyde fumigation for controlling the microbial load during the hatching phase in a laboratory setting.

Dietary soy galactooligosaccharides affect the performance, intestinal function, and gut microbiota composition of growing chicks.

The objective of this experiment was to investigate the effects of the dietary soy galactooligosaccharides (GOS), raffinose and stachyose, on performance, gastrointestinal health, and systemic stress in young broilers. Birds were fed a GOS-devoid diet based on soy protein isolate (SPI) or the SPI diet with 0.9, 1.8, 2.7, or 3.6% added stachyose and raffinose in a ratio of 4:1 at the expense of corn starch. These 5 treatments were administered to 10 replicate cages of 8 birds. Performance was measured weekly and excreta moisture, N retention, apparent metabolizeable energy, and complete blood cell counts were determined at 14 and 21 d. At 21 d, 2 birds per cage were orally gavaged with fluorescein isothiocyanate-dextran (FITC-d) and serum samples were analyzed for FITC-d as a marker of gut leakage. Additionally, intestinal morphology, crop presumptive lactic acid bacteria (LAB) counts, crop and cecal pH, and cecal microbiota via16S rRNA microbial sequencing were evaluated at 21 d. From 0 to 21 d, feed intake increased linearly (P < 0.01) as dietary GOS increased, whereas BWG increased (P < 0.05) quadratically. Feed conversion ratio increased (P < 0.01) linearly as GOS increased. There were linear increases (P < 0.05) in excreta moisture as dietary GOS increased at 14 and 21 d, as well as dose-dependent responses (P < 0.05) in N retention, AME, and AMEn. There was a quadratic increase (P < 0.05) in crop LAB recovery and a linear decrease (P < 0.01) in ceca pH as GOS increased. At 14 d, a linear increase (P < 0.05) in blood heterophil to lymphocyte ratio was observed as dietary GOS increased. Serum concentrations of FITC-d increased quadratically (P < 0.01) to dietary GOS. Increasing levels of GOS influenced alpha and beta diversities and composition of gut microbiota, including the abundance of Ruminococcus and Bifidobacterium. Results from this trial indicate that soy-derived GOS exert dose-dependent effects on nutrient utilization and intestinal health in young broilers.

Development of an environmental contamination model to simulate the microbial bloom that occurs in commercial hatch cabinets.

Microbial blooms that emerge in commercial hatch cabinets consist of apathogenic and pathogenic microorganisms, including Escherichia coli, Enterococcus faecalis, and Aspergillus fumigatus. Objectives of the present study included the development of a multipathogen contamination model to mimic commercial conditions and optimization of sampling methods to quantify bacterial or fungal presence within the hatch cabinet. The pathogen challenge mix (PM) was recreated from select bacterial or fungal isolates recovered from an egg homogenate (EH) derived from the contents of infertile eggs and late embryonic mortalities. Isolates selected for PM included Enterococcus faecalis (∼108 CFU/egg), Staphylococcus aureus (∼107 CFU/egg), Staphylococcus chromogenes (∼107 CFU/egg), Aspergillus fumigatus (∼106 spores/egg), and 2 Escherichia coli (∼108 CFU/egg) isolates. Challenge (100 µL of PM or EH) was administered using a sterile loop to a 28 mm area on the blunt end of the eggshell at day 19 of embryogenesis (DOE). In 3 experiments, microbiological data were collected from environmental hatcher samples (open-agar plate method), fluff samples, postmortem whole-body chick rinse samples, and gastrointestinal tract (GIT) samples to evaluate select bacteria and fungi circulating within the hatch cabinet and colonization of GIT. Cumulative bacterial and fungal recovery from the PM hatching environment from DOE20 to hatch was higher than the nonchallenged group (NC) and EH group at ∼860 and ∼1,730 CFU, respectively. Bacterial recovery from GIT, fluff, and chick rinse samples were similar for the PM and EH group in Exp. 1. However, Aspergillus fumigatus recovery from fluff and chick rinse samples for the PM group was significantly (P < 0.001) higher than the NC and EH group. In Exp. 2 and 3, PM challenge significantly (P < 0.05) increased Gram-negative bacterial recovery from the GIT, fluff and chick rinse samples compared to both the NC and EH group. These data suggest this innovative multispecies environmental contamination model using PM could be utilized to evaluate strategies to mitigate microbial contamination in commercial hatch cabinets in a laboratory setting.

Evaluation of Eimeria adenoeides Preinfection on the Severity of Histomoniasis in Turkeys.

The objectives of this study were to evaluate whether a preinfection of Eimeria adenoeides (EAD) or Eimeria tenella (ET) could affect the severity of subsequent histomoniasis in turkeys (Experiment 1) and if previous exposure to EAD infection, when a single or multiple inoculations of EAD were administered with sufficient time for complete cecal recovery, would affect the severity of HM incidence and lesions (Experiment 2). In Experiment 1, 200 poults were assigned to 1 of 5 groups, as follows: unchallenged negative control, positive challenge control inoculated with 105 HM, EAD at 500 oocysts/bird and Histomonas meleagridis (HM), EAD at 2500 oocysts/bird and HM, or ET at 9 × 106 oocysts/bird and HM. ET and EAD were inoculated on day 15 and HM on day 20. In Experiment 2, the trial consisted of two different challenge ages to evaluate short- or long-term EAD effects before HM challenge. Poults (n = 260) were assigned to either early-HM-challenged groups (HM on day 19 challenge control or EAD at 2500 oocysts/bird on day 14 with HM on day 19) or late-HM-challenged groups (HM on day 35 challenge control, EAD at 2500 oocysts/bird on day 14 and HM on day 35, or EAD at 100 oocysts/bird every 2-3 days during the first 3 weeks and HM on day 35). An unchallenged negative-control group was used for both the early- and late-challenge phases in Experiment 2. Mortalities were recorded, and surviving poults were scored for histomoniasis-related hepatic and cecal lesions. In Experiment 1, preinfection with both doses of EAD reduced the mortality as well as the cecal and hepatic lesions caused by histomoniasis. In Experiment 2, neither short- nor long-term preinfection with EAD had an effect on histomoniasis-related mortality or lesions. Differences between Experiments 1 and 2 may be due to the level of infection caused by the prechallenge with EAD and the resulting destruction of cecal tissue.

Evaluación de la preinfección por Eimeria adenoeides sobre la severidad de la histomoniasis en pavos. Los objetivos de este estudio fueron evaluar si una preinfección por Eimeria adenoeides (EAD) o Eimeria tenella (ET) podría afectar la severidad de la histomoniasis subsequente en pavos (Experimento 1); y si la exposición previa a la infección por E. adenoeides, cuando se administraron una o varias inoculaciones de E. adenoeides con tiempo suficiente para la completa recuperación cecal, afectaría la gravedad de la incidencia y las lesiones de Histomonas meleagridis (Experimento 2). En el Experimento 1, se asignaron 200 pavipollos en cinco grupos, de la siguiente manera: control negativo no desafiado, control de desafío positivo inoculado con 105 de H. meleagridis, un grupo con E. adenoeides a 500 ooquistes/ave e H. meleagridis (HM), otro grupo con E. adenoeides a 2500 ooquistes/ave y H. meleagridis, o E. tenella a 9×106 ooquistes/ave y H. meleagridis. Se inocularon E. tenella y E. adenoeides el día 15 y H. meleagridis el día 20. En el Experimento 2, el ensayo consistió en dos edades de exposición diferentes para evaluar los efectos de E. adenoeides a corto o largo plazo antes del desafío con H. meleagridis. Los pavipollos (n = 260) se asignaron a los grupos de desafío temprano con H. meleagridis (H. meleagridis en el día 19 en el grupo control de desafío o E. adenoeides con 2500 ooquistes/ave el día 14 y con H. meleagridis en el día 19) o los grupos de desafío tardío con H. meleagridis (H. meleagridis en el día 35 del control de desafío, E. adenoeides a 2,500 ooquistes/ave el día 14 y H. meleagridis en el día 35, o E. adenoeides con 100 ooquistes/ave cada 2-3 días durante las primeras 3 semanas y H. meleagridis en el día 35). En el Experimento 2, se utilizó un grupo de control negativo no desafiado para ambas fases de exposición temprana y tardía. Se registraron la mortalidad y los pavipollos supervivientes se asignaron puntuaciones en cuanto a lesiones hepáticas y cecales relacionadas con histomoniasis. En el Experimento 1, la preinfección con ambas dosis de E. adenoeides redujo la mortalidad, así como las lesiones cecales y hepáticas causadas por histomoniasis. En el Experimento 2, ni la preinfección a corto ni a largo plazo con E. adenoeides tuvo un efecto sobre la mortalidad o las lesiones relacionadas con la histomoniasis. Las diferencias entre los Experimentos 1 y 2 pueden deberse al nivel de infección causado por el desafío previo con E. adenoeides y la destrucción resultante del tejido cecal.

Evaluation of live-attenuated Histomonas meleagridis isolates as vaccine candidates against wild-type challenge.

Repeated serial in vitro passage of Histomonas meleagridis, the etiological agent of histomoniasis (blackhead) of turkeys, was demonstrated to markedly achieve attenuation and reduction of virulence as compared to the original wild-type isolate. Four experiments were performed to evaluate the route (oral vs. intracloacal) and age (day-of-hatch vs. d 14) for administration of attenuated H. meleagridis isolates as vaccine candidates against homologous or heterologous wild-type challenge. Attenuated H. meleagridis were developed from 2 different strains (Buford strain originating in Georgia; PHL2017 strain originating in Northwest Arkansas). Buford P80a (passage 80, assigned as isolate lineage "a" following repeated passage) was selected as the primary vaccine candidate and was evaluated in Experiments 1-3. Experiment 4 evaluated selected candidates of attenuated PHL2017 (P67, P129) and Buford (P80a, P200a, P138b, P198c) strains against Buford wild-type challenge. As has been demonstrated previously, wild-type H. meleagridis cultures administered orally after 1 day of age were not infective in the current studies, but infection with wild-type cultures could be induced orally at day-of-hatch. Infection was effectively achieved via the intracloacal route at day-of-hatch and in older turkeys (d 21, d 28-29, d 35). Intracloacal inoculation of turkeys with the attenuated passaged isolates as vaccine candidates at d 14 was shown to produce significant (P < 0.05) protection from mortality, reduction in body weight gain, as well as reduction in hepatic and cecal lesions in these experiments following challenge with either the homologous wild-type isolate or from a wild-type strain obtained years later from a geographically disparate area of the United States. Inoculation with the attenuated H. meleagridis isolates at day-of-hatch, either orally or cloacally, did not produce significant protection against subsequent wild-type challenge. While offering significant protection with minimal vaccine-related negative effects, the protection from cloacal vaccine administration was neither significantly robust nor encouraging for industry application using the methods evaluated in the present manuscript since mortalities and lesions were not completely reduced which could thereby potentially allow transmission from residual infection and shedding within a flock.

Research Note: Evaluation of quinine as a chemoprophylactic candidate against histomoniasis in turkeys.

Histomoniasis, also commonly referred to as blackhead disease, is caused by the protozoan parasite Histomonas meleagridis. Since the removal of nitarsone in 2015, no approved prophylactics are available for mitigating histomoniasis. Disease incidence and high mortalities are frequently associated with turkey flocks, although infection of broiler breeders also occurs. Quinine is a naturally occurring alkaloid with antimalarial properties. In vitro assays have shown strong antihistomonal properties of quinine, leading to our hypothesis that quinine inclusion within the feed could prevent histomoniasis in turkeys. Selected concentrations of quinine were included within a turkey starter diet to evaluate effects on body weight gain (BWG), liver lesions, cecal lesions, and mortality of H. meleagridis-challenged turkeys. On day-of-hatch, poults were randomly assigned to either the basal diet or a quinine diet. Groups consisted of a non-challenged control (NC; basal diet), 0.022% quinine + challenge, 0.067% quinine + challenge, 0.2% quinine + challenge, or a positive-challenged control (PC; basal diet). On d 10, challenged groups were intracloacally inoculated with 105H. meleagridis cells/turkey, and lesions were evaluated on d 21 post-infection. Individual body weights were recorded on d 0, d 10, and d 31 to calculate the pre-challenge and post-challenge BWG. No significant differences (P > 0.05) were observed between the d 0 to 10 pre-challenged BWG between quinine treatment diets and the basal diet. Similarly, no differences (P > 0.05) were observed in post-challenge d10-31 BWG of the quinine dietary treatments as compared to the PC. Cumulative mortalities, liver lesions, and cecal lesions related to histomoniasis were not reduced (P > 0.05) in any of the quinine treatment groups as compared to the PC. Although quinine successfully reduced H. meleagridis cells in vitro, results from the in vivo experiment indicated no reduction in histomoniasis severity as evidenced by similar lesions and mortality as the PC. Taken together, these data indicate that quinine inclusion within the feed at these concentrations and under these experimental conditions was not efficacious in the prevention or treatment of histomoniasis.

Research Note: Application of an Escherichia coli spray challenge model for neonatal broiler chickens.

Avian pathogenic Escherichia coli (E. coli) is an opportunistic pathogen often introduced to neonatal chicks during the hatching process. This commensal bacterium, particularly as a pioneer colonizer of the gastrointestinal tract, can have substantial implications in the rearing of poultry because of reduced flock performance. In order to mimic the effects of the natural bacterial bloom present during the hatch, a seeder challenge model was developed to expose neonatal chicks to virulent E. coli. On day 20 of embryogenesis, selected early hatched chicks (n = 18/hatcher) were briefly removed and sprayed challenged with saline (vehicle) or E. coli at 1 × 107 colony-forming unit (CFU)/chick (exp 1) and 2.5 × 107 CFU/chick (exp 2). These challenged chicks were returned to the hatcher to serve as seeders to transmit the pathogen to the indirect challenged or contact chicks (n = 195/hatcher). For two 7-d experiments, the efficacy of transmission was evaluated via enteric bacterial recovery, body weight gain (BWG), and mortality. For exp 1 and exp 2, significantly (P < 0.0001) more gram-negative bacteria were recovered from the seeder and contact gastrointestinal samples than the negative control samples on day of hatch. In addition, there was a reduction (P < 0.05) in 7-d BWG and significantly (P < 0.0001) higher mortality in the contact-challenged chicks than the negative control chicks in both exp 1 and exp 2. These data suggest that this challenge model could be used to evaluate different methods of controlling the bacterial bloom that occurs in the hatching environment.

Development of a wild-type Escherichia coli environmental bloom model to evaluate alternatives to formaldehyde fumigation in broiler chicken hatch cabinets.

Horizontal transmission of opportunistic Escherichia coli during hatch can have detrimental effects on early performance, particularly as pioneer colonizers. Commercially, formaldehyde is often applied in the United States to combat the bacterial bloom that occurs inside of the hatching environment. The purpose of these experiments was to develop a replicable E. coli horizontal challenge model to evaluate alternatives to formaldehyde sanitation applied to the hatching environment. In experiment 1, two trials were conducted for 2 wild-type (WT) E. coli isolates (isolate 1 [I1] or isolate 2 [I2]) to determine the appropriate in ovo challenge dose and day of embryogenesis (DOE) for challenge administration. In experiment 1 trial 1, the most appropriate inoculation dose and time point were determined to be 102 cfu/embryo on DOE 19. Experiment 1 trial 2 evaluated whether placement of seeder (direct-challenged) embryos with contact (indirect-challenged) embryos during hatch affected contact hatchability. Trial 2 showed no differences in hatchability between groups. A 7-day experiment (experiment 1 trial 2) was conducted to evaluate the effects of I1 or I2 on horizontal transmission, gram-negative bacterial (GNB) recovery from the gastrointestinal tract (GIT), and impact on BW gain (BWG). Compared with the negative control, seeder, and contact chicks challenged with I1 or I2, we observed increased (P < 0.05) GNB recovered from GIT on the day of hatch. There was a marked (P < 0.05) reduction in 7-day BWG between the I1 indirect-challenged group and the negative control group. To further validate the model, 2 7-day trials (experiment 2, experiment 3) were conducted to evaluate the effects of formaldehyde fumigation on coliform recovery from the hatching environment and on early performance using I1 for the challenge. Isolate 1 positive control hatchers had increased levels of circulating coliforms compared with the negative control and formaldehyde-treated hatchers, although there was no significant impact on performance induced by challenge or formaldehyde treatment in experiment 2 or experiment 3. These data provide a potential model for investigations related to horizontal transmission of WT E. coli at a low dose on DOE 19 to promote simulated commercially relevant bacterial blooms under laboratory conditions.

Research Note: Evaluation of deoxycholic acid for antihistomonal activity.

Deoxycholic acid (DCA) is a naturally occurring secondary bile acid that originates from intestinal bacterial metabolic conversion of cholate, a primary bile acid. Deoxycholic acid was shown to have antihistomonal properties in vitro, leading to our hypothesis that DCA inclusion within the feed might prevent histomoniasis. Selected dietary concentrations of DCA were evaluated for effects on body weight gain (BWG), lesions, and mortality of turkeys challenged with wild-type Histomonas meleagridis (WTH). Treatments consisted of non-challenged control (NC; basal diet), 0.25% DCA diet + challenge, 0.5% DCA diet + challenge, 1% DCA diet + challenge, and a positive-challenged control (PC; basal diet). All groups were fed a basal starter diet until day 7, at which time DCA diets were administered to the respective groups. On day 14, 2 × 105 WTH cells/turkey were intracloacally administered. H. meleagridis-related lesions were evaluated on day 13 post-challenge. Pre-challenge day 0 to 14 BWG was higher (P ≤ 0.05) in the 0.25% DCA group than in the 1% DCA group. There were no significant differences in pre-challenge day 0 to 14 BWG between any of the other groups. No significant differences in mortalities from histomoniasis occurred in the DCA groups as compared to the PC group. No H. meleagridis lesions or mortalities were observed at any time in the NC group. Presence of H. meleagridis-related liver lesions was higher (P ≤ 0.05) in the 0.5% DCA group as compared to the PC group. Using the same controls and experimental timeline, an additional group was included to evaluate a biliogenic diet formulated with 20% whole egg powder to encourage endogenous bile acid production. The biliogenic diet had no statistical impact on pre-challenge day 0 to 14 BWG, but did not reduce H. meleagridis-related mortalities or lesions after the challenge. Taken together, these data suggest that DCA inclusion within the feed at these concentrations and under these experimental conditions does not prevent histomoniasis.

A feed restriction milieu for Pekin meat ducks that may improve gait characteristics but also affects gut leakiness.

In a previous study, we demonstrated that a 15% feed restriction (FR) during the first 2 wk after hatch could improve gait in Pekin meat ducks, but did result in reduced breast mass. We hypothesized that feed restriction after day 5 following muscle satellite cell development would allow the full growth of the breast meat. To accomplish this goal, 300 1-day-old ducklings (Maple Leaf Farms Inc.) were randomly allocated to 1 of the 3 groups (n = 4 pens, 25 ducks per pen): (1) Control group fed to ad libitum; (2) 85% daily feed intake from days 1 to 14 (FR 85% 1-14); 85% daily feed intake from days 5 to 14 (FR 85% 5-14). All ducks were vaccinated with inactivated Salmonella enteritidis on day 14 and boosted on day 26. The FR 85% 5-14 did show similar production standards to controls, and improved gait characteristics (P < 0.05). To determine if the partial feed restriction would have an impact on intestinal epithelial tight junction integrity, we treated ducks on days 7, 14, 21, 28, and 35 with 8.32 mg/kg FITC-d in water per os and blood samples were obtained via the tibial vein 1 h later. Serum samples were analyzed for presence and quantification of FITC-d. Feed restriction elicited a significant increase in FITC-d permeability at all points of evaluation. Anti-S. enteritidis specific IgY responses were assessed by ELISA from serum collected at 14 D, 28 D, and 35 D. Although all ducks showed an increase humoral immune response to the S. enteritidis, both feed restricted groups showed reduced IgY production compared to ad lib controls. Our data suggest that although the FR 5-14 feed restriction paradigm may reduce gait abnormalities without affecting production rates, some challenges exist due to increased gut leakiness or decreased acquired immune activity. Future studies will look at altering the feed restriction milieu to ameliorate these challenges.

Research Note: Evaluation of boric acid as a chemoprophylaxis candidate to prevent histomoniasis.

Histomoniasis, caused by the protozoan parasite Histomonas meleagridis, is a disease to which turkeys are especially susceptible. Currently, no chemoprophylaxis compounds are available to mitigate this disease. Boric acid (BA) exhibits antifungal, antiseptic, and antiviral properties and has been used in the treatment of yeast infections. Based on these characteristics, an experiment was conducted to evaluate whether BA might be an efficacious prophylaxis against challenge with wild-type H. meleagridis (WTH). On day-of-hatch, poults were randomly assigned to either a basal control diet or a BA diet (basal diet + 0.2% BA). Groups consisted of a nonchallenged control (NC; basal diet), 0.2% BA + challenge (BA; 0.2% BA diet), and a positive-challenged control (PC; basal diet). On day 21, challenged groups were intracloacally inoculated with 2 × 105 WTH cells/turkey, and lesions were evaluated on day 14 postchallenge. Individual body weights were recorded on day 0, 21, and 35 to calculate the prechallenge and postchallenge body weight gain (BWG). The BA group resulted in lower prechallenge day 0 to day 21 BWG (P = 0.0001) than the NC group. Postchallenge day 21 to day 35, BWG was also lower (P = 0.0503) in the BA group than the PC group. No differences between the BA and PC groups were detected for mortalities associated with histomoniasis. Moreover, liver and cecal lesions were not statistically different between the BA and PC groups. Taken together, these data suggest that BA was not efficacious in the prevention or reduction of histomoniasis disease severity when provided at 0.2% dietary concentration under these experimental conditions.

Research Note: Evaluation of dietary administration of sodium chlorate and sodium nitrate for Histomonas meleagridis prophylaxis in turkeys.

Histomoniasis is currently a re-emerging disease of major significance for many commercial turkey and broiler breeder production companies because of the unavailability of drugs or vaccines. The protozoa Histomonas meleagridis (HM) requires the presence of enteric microflora to promote the disease. The objectives of this research note were to evaluate the effect of dietary administration of sodium chlorate (SC) and sodium nitrate (SN) in vitro and in vivo for HM prophylaxis in poults. A total of 128 day-of-hatch female poults obtained from a commercial hatchery were wing-tagged and randomly assigned into 1 of 4 experimental groups: negative control (NC), positive control, dietary inclusion of SC (3,200 ppm) and SN (500 ppm). Poults from groups SC and SN started on their respective diets on day 12. All groups, except the NC, were challenged with 2 × 105 HM on day 19. Controls were fed a basal diet, identical to the treatment diets but not supplemented with SC or SN. Body weight gain (BWG) was determined weekly, starting on day 1 until day 28, and postchallenge morbidity and mortality were recorded. On day 28 of age, all surviving poults were lesion scored for hepatic and cecal lesions. Ceca and distal ileum were collected on day 28 for bacterial recovery on selective media for total aerobic, lactic acid bacteria, or gram-negative bacteria. The addition of SC and SN in the in vitro growth of HM greatly reduced the growth of the protozoa after 20 h of incubation when compared with the control nontreated group (P < 0.05). However, dietary supplementation of SC and SN had no effect against HM in vivo, as was demonstrated by BWG, the severity of lesions in the liver and ceca or bacterial recovery of treated poults when compared with the positive control group.

Evaluation of in ovo Bacillus spp. based probiotic administration on horizontal transmission of virulent Escherichia coli in neonatal broiler chickens.

This study evaluated the effect of in ovo Bacillus spp. base probiotic (BBP) administration on hatchability, Gram-negative bacteria (GNB) recovery, performance, and microbiota composition in 2 independent trials using a virulent E. coli seeder challenge model. In each trial, one hundred and eighty 18-day-old embryos were allocated into 1 of 2 groups: Control and treated group (inoculated with 107 BBP). On day 19 of embryogenesis, seeder embryos (n = 18) were inoculated with 4.5 × 104E. coli/mL+272 µg/mL tetracycline and segregated into mesh hatching bags. Twelve chicks per group were euthanized at hatch and at day 7 to evaluate the gastrointestinal composition of total GNB or total aerobic pasteurized bacteria. Also, in trial 2, ceca content from five chickens at day 7 were collected to evaluate microbiota composition. Embryos inoculated with BBP showed a significant (P < 0.05) reduction in the total number of GNB at day-of-hatch (DOH) and day 7. Probiotic treatment increased BW at DOH and day 7, and BW gain (days 0 to 7) when compared with Control chickens. Proteobacteria phylum was significantly reduced, while the Firmicutes was significantly increased by the BBP as compared to the Control (P < 0.05). At family level, Enterobacteriaceae was significantly decreased, while the Lachnospiraceae was significantly elevated in the BBP as compared to the Control group (P < 0.05). The genus Oscillospira was significantly enriched in the BBP group, whereas the unidentified genus of family Enterobacteriaceae in the Control group (P < 0.05). The BBP group increased the bacterial species richness, although there was no significant difference between treatments (P > 0.05). Interestingly, beta diversity showed a significant difference in bacterial community structure between Control and BBP groups (P < 0.05). The results of the present study suggest that in ovo administration of a BBP can reduce the severity of virulent E. coli horizontal transmission and infection of broiler chickens during hatch. The reduction in the severity of the transmission and infection by the BPP might be achieved through alterations of microbiota composition and its community structure.

Preliminary studies on development of a novel subunit vaccine targeting Clostridium perfringens mucolytic enzymes for the control of necrotic enteritis in broilers.

Necrotic enteritis (NE) is a pervasive enteric disease responsible for large scale economic losses within the global poultry industry. The etiologic agent of NE is Clostridium perfringens (CP), an opportunistic pathogen that utilizes numerous extracellular toxins and glycoside hydrolases (GH) as key virulence and nutrient acquisition factors. Notably, some GH, mucinases, degrade components of mucin in the gastrointestinal tract as an energy source. Targeting this mechanism may serve to reduce the incidence of disease associated with CP. Two experiments were completed that evaluated mucinase vaccine targets sourced from conserved peptide sequences of carbohydrate binding module 32 of CP mucinases. In experiment 1, 37 antigen peptides were synthetically generated and used to produce hyper-immune sera, which was then evaluated for ability to obstruct CP growth in vitro. Total CFU of CP were measured at 4, 6, and 8 h incubation to determine growth rate. Peptides 4, 5, 22, 24, and 30 were selected for further in vivo testing based on conservation or the ability to inhibit CP growth by over 50% at 6 and 8 h. In experiment 2, the aforementioned peptides were conjugated to an agonistic, CD40-targetting antibody and evaluated in vivo. Broilers were given an Eimeria maxima and CP in order to induce NE and assess vaccine efficacy. Treatments included a non-vaccinated non-inoculated control, non-vaccinated inoculated control (NVIC), vaccination with peptide 4, 5, 22, 24, or 30 (VP4-VP30), or a combination of all 5 peptides (MC). There was a significant increase (P < 0.05) in the percent change in BWG relative to NVIC for vaccination with peptide 22 and MC of 18.54 and 17.43%, respectively. MC vaccinated group had the lowest lesions with a mean score of 0.63 ± 0.18. These results suggest the MC combination was the most successful in alleviating overall performance losses associated with NE-infected broilers and encourage future testing of MC in the development of an NE vaccine.

Development of a novel in ovo challenge model for virulent Escherichia coli strains.

During the hatching process, chicks are exposed to opportunistic and/or pathogenic organisms, such as virulent or avirulent Escherichia coli. Virulent E. coli strains have not been feasible for induction of neonatal colibacillosis via in ovo challenge due to high embryonic mortality. In this manuscript, we describe the addition and co-administration of the bacteriostatic antibiotic tetracycline to a virulent E. coli challenge culture, improving hatchability and livability of seeder chicks while allowing robust horizontal transmission in the hatching cabinet to contact chicks. Experiment 1 consisted of 3 trials. Experiment 1, trial 1 was conducted to determine an effective ratio of E. coli challenge and tetracycline dose to be utilized in the seeder model. Trials 2 and 3 were conducted to evaluate the transmission of E. coli from seeder to contact chicks. Experiment 2 consisted of 3 independent 7-D trials where body weight gain (BWG), mortality, and selected enteric bacterial recovery were evaluated. In trials 1 to 3, significantly (P < 0.05) more Gram-negative bacteria were recovered from whole gut samples (GIT) vs. negative controls on day of hatch, from both seeder and contact chicks. At day 7 in trial 1, contact chicks had significantly (P < 0.05) more Gram-negative bacteria recovered from the GIT than the negative control, but not in trials 2 and 3. Presumptive lactic acid bacterial recovery was elevated in contact and seeder chicks compared to the negative control in all 3 trials. Contact challenge caused a significant (P < 0.05) reduction in BWG in 2 out of 3 trials at day 7, and there was a significant (P < 0.05) increase in mortality as compared to the negative controls in all trials. These data suggest that co-administration of a virulent E. coli strain with tetracycline allows for hatch of direct challenged chicks and effective horizontal transmission to contact chicks during the hatching process, as evidenced by reduced day 7 performance and altered selected enteric bacterial recovery.