K. S. Macklin

Evaluation of the efficacy of yeast extract in reducing intestinal Clostridium perfringens levels in broiler chickens

The etiological agent of necrotic enteritis is Clostridium perfringens. Traditionally, necrotic enteritis is controlled with in-feed antibiotics. However, increasing consumer demand for drug-free poultry has fostered the search for nonantibiotic alternatives. Yeast extract contain nucleotides that are immunomodulatory and also essential for cellular functions. An experiment was conducted to evaluate the efficacy of NuPro yeast extract (Alltech Inc., Nicholasville, KY) in reducing intestinal C. perfringens levels in broiler chickens. One hundred ninety-two 1-d-old male broiler chicks were obtained and randomly assigned to 6 treatments in a battery cage trial. Treatment 1 consisted of chicks fed a corn-soybean meal basal diet (BD) without added bacitracin methylene disalicylate or NuPro. Treatment 2 consisted of chicks fed BD into which bacitracin methylene disalicylate was added at 0.055 g/kg. Treatment 3 consisted of chicks fed BD supplemented with NuPro at a 2% level for the first 10 d of the experiment. Treatments 4 (PX), 5, and 6 (PN) consisted of chicks that were challenged with 3 mL of the C. perfringens inoculum (~10(7) cfu/mL) on d 14, 15, and 16 of the experiment and fed diets similar to treatments 1, 2, and 3, respectively. On d 1 and 7 postchallenge, intestinal C. perfringens levels, lesion scores, and alkaline phosphatase activity were assessed. On d 1 postchallenge, C. perfringens level in treatment 5 (2.09 log(10) cfu/g) was lower (P < 0.05) compared with the PX treatment (4.71 log(10) cfu/g) but similar to the PN treatment (2.98 log(10) cfu/g). A similar trend was observed on d 7 postchallenge. NuPro supplementation enhanced alkaline phosphatase activity (P < 0.05) in C. perfringens-challenged chicks and appeared to reduce intestinal lesion scores. Although dietary supplementation of NuPro in the PN treatment reduced C. perfringens levels by 1.73 and 0.68 log(10) cfu/g compared with the PX treatment on d 1 and 7 postchallenge, respectively, these reductions were not significant. Extending the period of NuPro supplementation beyond the first 10 d of life should be considered for achieving significant reduction in intestinal C. perfringensg levels.

The effect of sodium bisulfate on Salmonella viability in broiler litter

Controlling Salmonella populations on commercial broiler grow out farms is a crucial step in reducing Salmonella contamination in processing plants. Broiler litter harbors many species of pathogenic bacteria including Salmonella. Sodium bisulfate has been shown to reduce concentration of bacteria in broiler litter. In experiments 1 and 2, sodium bisulfate was applied to broiler litter at rates that are comparable to what is commonly used by the poultry industry: 22.7, 45.4, and 68.0 kg/92.9 m(2). After application, sodium bisulfate was mixed into the litter. In experiments 3 and 4, sodium bisulfate was applied at 45.4 kg/92.9 m(2) to the surface of the litter. For all experiments, a cocktail of 5 Salmonella serovars was applied to the litter. Ammonia, pH, moisture, and water activity measurements were taken; additionally, total aerobic, anaerobic, enteric, and Salmonella concentrations were determined at 0, 24, and 96 h. In experiments 1 and 2, Salmonella concentrations were higher for treated litter than the control at 24 and 96 h (P < 0.001). In experiments 1 and 2, litter pH was lower for treated litter at 24 and 96 h; lowest pH was observed with the 68.0 kg/92.9 m(2), with a pH of 5.95 (P < 0.001). In experiments 3 and 4, litter pH was lowered for treated litter to 2.1 (P < 0.001). Even this lower pH did not reduce Salmonella concentrations compared with the control (P = 0.05). The decreased litter pH appeared to be responsible for increased viability of Salmonella. This research shows that the lowering of litter pH, which decreases litter ammonia production, could actually lead to an increased survivability of certain bacteria, such as Salmonella.

The effect of vitamin E on cellulitis in broiler chickens experiencing scratches in a challenge model.

Two experiments are described; each experiment contained five treatments with each treatment consisting of a specific diet and vitamin E at 8.82 mg, 41.89 mg, 74.96 mg, 108.03 mg, or 141.10 mg vitamin E per kilogram of feed. Birds were raised with continuous feed containing the various levels of vitamin E available throughout the experiment. At 4 wk of age, the birds were scratched on the breast and placed onto avian cellulitis Escherichia coli-seeded litter. One week later, the birds were euthanatized and lesion presence was noted. There appeared to be a positive correlation between vitamin E and the inhibition of cellulitis formation when the birds were fed a diet containing 74.96 mg vitamin E/kg feed. Conflicting results were seen in the two experiments when the birds were fed 41.89 and 108.03 mg vitamin E/kg feed. Both experiments had a high incidence of cellulitis in birds whose diets consisted of 141.10 mg vitamin E/kg feed.

Multilevel analysis of environmental Salmonella prevalences and management practices on 49 broiler breeder farms in four south-eastern states, USA.

A two‐part serial survey of 49 broiler breeder farms was conducted in four south‐eastern states: Arkansas, Alabama, Georgia and North Carolina. Broiler breeder farms from three to five broiler company complexes in each state were visited on two separate occasions to document management practices and perform environmental sampling for Salmonella prevalence estimation. Salmonella was detected in 88% of the broiler breeder houses that were sampled and was identified on all 49 farms enrolled. Many management characteristics were consistent across the different states and companies. Multilevel analysis was used to evaluate management characteristics as risk factors for Salmonella prevalence and to estimate the proportion of variance residing at the different hierarchical sampling levels. Management characteristics associated with increased Salmonella prevalence included treatment of the flock for any disease, having dusty conditions in the house, having dry conditions under the slats and walking through the house more than one time per day to pick‐up dead birds. After adjusting for state as a fixed effect, the percentages of variance in Salmonella prevalence occurring at the complex, farm, visit, house and individual sample levels were 5.2%, 6.8%, 11.8%, 2.8% and 73.4%, respectively. The intraclass correlations for samples collected from the same house; for samples from different houses during the same visit; for samples from different visits to the same farm; and for samples from different farms in the same complex were as follows: 0.27, 0.24, 0.12 and 0.05, respectively.

Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

Low Prevalence of netB and tpeL in Historical Clostridium perfringens Isolates from Broiler Farms in Alabama.

SUMMARY The discovery of novel Clostridium perfringens toxins NetB and TpeL has initiated questions regarding their role in the pathogenesis of disease. However, data showing the prevalence of these genes in C. perfringens populations are limited to certain geographical areas. If netB and tpeL are important virulence factors for disease worldwide, one would expect to find these genes in isolates from other regions as well. To address this hypothesis, C. perfringens isolates collected from Alabama broiler farms over 15 yr ago were toxin genotyped using PCR. Each isolate was screened for netB and tpeL; the major lethal toxin genes cpa, cpb, etx, and ia; and the enterotoxin gene cpe. Results of the assay showed all isolates presumed to be C. perfringens were genotypically type A, cpe negative except for one broiler litter isolate, which was genotypically type C. Only two isolates were positive for netB. Similarly, only two isolates were positive for tpeL, one of which was also netB positive. The low incidence observed for netB and tpeL indicates that these genes are not significant virulence factors for the sampled population. RESUMEN Baja Prevalencia de los genes netB y tpeL en aislamientos históricos de Clostridium perfringens de granjas de pollo de engorde en Alabama. El descubrimiento de las nuevas toxinas NetB y TpeL de Clostridium perfringens ha iniciado preguntas con respecto a su papel en la patogénesis de la enfermedad. Sin embargo, los datos muestran que la prevalencia de estos genes en las poblaciones de C. perfringens se limitan a ciertas áreas geográficas. Si netB y tpeL son factores importantes de virulencia de la enfermedad en todo el mundo, se esperaría encontrar estos genes en los aislamientos de otras regiones también. Para evaluar esta hipótesis, se genotipificaron mediante PCR, los genes que codifican las toxinas de aislamientos de C. perfringens que fueron recolectados hace más de quince años atrás de granjas de pollo de engorde de Alabama. Cada cepa se analizó para netB y tpeL; los genes principales de toxinas letales cpa, cpb, etx, e ia y el gene cpe de la enterotoxina. Los resultados del ensayo mostraron que todos los presuntos aislamientos de C. perfringens eran genotípicamente del tipo A, negativos para cpe, con excepción de un aislamiento de la cama de una parvada de pollo de engorde, que era genotípicamente del tipo C. Sólo dos aislamientos fueron positivos para netB. Del mismo modo, sólo dos aislamientos fueron positivos para tpeL, uno de los cuales también fue netB positivo. La baja incidencia observada para netB y tpeL indica que estos genes no son factores de virulencia importantes en la población muestreada.

Use of a Multiplex PCR for the Detection of Toxin-Encoding Genes netB and tpeL in Strains of Clostridium perfringens

Some studies have shown that the NetB toxin may be an important virulence factor of Clostridium perfringens associated necrotic enteritis in poultry. Additionally, research has shown that strains of C. perfringens positive for both the netB gene and a second toxin-encoding gene, tpeL, appear to be more virulent than strains with only netB. In the past, detection of these genes has been performed relatively inefficiently using two single locus PCRs. This report describes a novel multiplex PCR developed to detect netB and tpeL simultaneously in C. perfringens strains isolated from cases of necrotic enteritis in broilers, providing a more efficient diagnostic tool in the screening of strains for these genes.

Litter Beetle (Alphitobius diaperinus Panzer) Counts in Broiler Houses Treated with a Range of Insecticides

Abstract Hess, J.B., Macklin, K.S. and McCrea, B.A. 2008. Litter beetle (Alphitobius diaperinus Panzer) counts in broiler houses treated with a range of insecticides. J. Appl. Anim. Res., 33:127–131. Litter beetles (Alphitobious diaperinus Panzer) remain an important poultry production pest on several fronts. They have been shown to damage poultry houses and harbor pathogens that affect people and poultry. This trial examined beetle counts in commercial broiler houses treated with l of 2 insecticide treatments, applied dry or wet. Three levels of dry (12.20, 15.26 and 18.31 g/m2) and one level of liquid application of an organophosphate (Rabon) and pyrethroid (Tempo) dry (1% dust) and wet (20% Wettable powder) were compared to an untreated control house across one growout. Six pvc pipes (2.5 cm wide by 25 cm long) were filled with a rolled up piece of chick paper. They were placed in each house for 3 d period during brooding, at 3 wk of age and prior to marketing the birds. The tubes were placed in plastic bags and refrigerated for 24 h prior to counting larval and adult litter beetles. All treatments reduced larval and adult litter beetles in relation to the untreated control house. The organophosphate did not reduce adult beetle numbers as quickly as pyrethroid, but showed good control by the 3rd week. As expected, the 15.26 and 18.31 g/m2 application rates of the organophosphate reduced beetle numbers, while the 12.20 g level did not control beetles as well. Products applied dry showed better control than those applied as a liquid, with larvae levels increasing as the flock aged in the liquid treatment. The products tested controlled litter beetles at suggested application rates, with dry applications showing better control than liquid applications.

Detection and Isolation of Infectious Laryngotracheitis Virus on a Broiler Farm After a Disease Outbreak

SUMMARY A broiler farm in North Alabama suffered a mild infectious laryngotracheitis (ILT) outbreak, as determined by clinical disease and PCR. The poultry integrator sought help to control further outbreaks in subsequent flocks. Samples were collected from various areas of the poultry houses on the farm over an 8-wk period. The first sampling was conducted 8 days after the infected farm was depopulated; the second was conducted 2 days prior to subsequent flock placement; and the third was conducted when the new flock was 5 wk of age. Samples were examined for ILT virus (ILTV) DNA by real-time PCR and virus isolation in embryos. The infected houses were cleaned, disinfected, heated, litter composted, and curtains replaced after the first sampling and prior to placement of the next flock. Samples from all periods were positive for ILTV DNA. However, the number of positive samples and crossing point values indicated a decrease in the amount of viral DNA, while virus isolation in embryos was successful only on the first sampling. The subsequent flock was vaccinated against ILTV by in ovo route using a commercial recombinant vaccine. Cleaning and sanitation after the disease outbreak reduced the amount of ILTV on the farm and together with in ovo vaccination of the new flock may have prevented a recurrence of another ILT outbreak. RESUMEN Nota de Investigación—Detección y aislamiento del virus de la laringotraqueítis infecciosa en una granja de pollos de engorde después de un brote. Una granja de pollos de engorde en el norte de Alabama sufrió un brote leve de laringotraqueitis infecciosa (ILT), tal como se determinó por la enfermedad clínica y por las pruebas de PCR. El integrador avícola solicitó ayuda para controlar nuevos brotes en parvadas posteriores. Se recolectaron muestras de diferentes áreas de las casetas en la granja durante un período de ocho semanas. El primer muestreo se realizó nueve días después de que la explotación infectada fue despoblada, la segunda se llevó a cabo dos días antes de que se introdujera la parvada siguiente, y la tercera se llevó a cabo cuando la nueva parvada tenía cinco semanas de edad. Se examinaron las muestras para detectar el ADN del virus de la laringotraqueítis infecciosa aviar por PCR en tiempo real y por el aislamiento del virus en embriones de pollo. Las casas infectadas fueron limpiadas, desinfectadas, calentadas, y se realizó el compostaje de la cama, las cortinas fueron reemplazadas después de la primera recolección de muestras y antes de la introducción de la siguiente parvada. Las muestras de todos los tiempos de muestreo fueron positivas para la presencia de ADN del virus de la laringotraqueítis. Sin embargo, el número de muestras positivas y los valores de ciclos umbrales indicaron una disminución en la cantidad de ADN viral, mientras que el aislamiento del virus en los embriones fue exitoso sólo en la primera toma de muestras. La parvada posterior ha sido vacunada contra laringotraqueítis in ovo con una vacuna recombinante comercial. La limpieza y saneamiento después del brote de la enfermedad redujo la cantidad del virus de laringotraqueítis en la granja y junto con la vacunación in ovo de la nueva parvada, se pudo haber prevenido la recurrencia de otro brote de laringotraqueítis.

Potential of Vitamin E and Zinc-Amino Acid Complex for the Reduction of Cellulitis in Broilers

Abstract Downs, K.M., Norton, R.A., Macklin, K.S. and Hess, J.B. 2003. Potential of vitamin E and zinc-amino acid complex for the reduction of cellulitis in broilers. J. Appl. Anim. Res., 23: 25–32. A research study was conducted to determine the effects of dietary supplementation with vitamin E and a zinc-amino acid complex (Zn-AA) on the incidence of cellulitis in broiler chickens. Vitamin E and Zn-AA supplementation did not influence body weight, but subtle feed conversion and mortality treatment differences were observed. Furthermore, a trend in cellulitis lesion reduction was indicated through combined supplementation of vitamin E and Zn-AA. The results of this study indicate an overall positive effect from higher dietary vitamin E and Zn-AA level on cellulitis incidence.