D. J. Caldwell

Effect of Anaerobic Cecal Microflora and Dietary Lactose on Colonization Resistance of Layer Chicks to Invasive Salmonella enteritidis

The effect of oral inoculation with anaerobic cultures of cecal microflora and providing lactose in the feed on colonization resistance to invasive Salmonella enteritidis was evaluated in newly hatched leghorn chicks. Salmonella colonization of the ceca, tissue invasion and organ colonization, horizontal transmission, and seroconversion were significantly decreased (P less than 0.01) in chicks inoculated with cecal flora. The addition of lactose to the feed, in the absence of cecal microflora, failed to provide protection. Dietary lactose enhanced colonization resistance in chicks that were inoculated with anaerobic cultures of cecal flora. The results indicated that establishment of normal cecal flora in layer chicks together with the addition of lactose to the diet markedly increases resistance to cecal colonization and organ invasion, and decreases horizontal transmission of S. enteritidis.

Evaluation of Persistence and Distribution of Salmonella Serotype Isolation from Poultry Farms Using Drag-Swab Sampling

The consistency and persistence of isolation of specific serotypes of Salmonella were evaluated on a 31-farm broiler complex following four complete sampling periods. A total of 25 different serotypes were isolated, with multiple serotypes and were frequently isolated simultaneously from individual farms. When the serotypes recovered from individual farms were further examined, common serotypes were isolated from individual farms on only seven occasions. Furthermore, on only five occasions were multiple serotypes recovered from individual farms on consecutive samplings. A casual association of common serotype isolations among farms with similar flock placement dates was observed in one sampling period. These results indicate little predictability or consistency of Salmonella serotype isolation on individual farms over time.

Predictive value of multiple drag-swab sampling for the detection of Salmonella from occupied or vacant poultry houses.

The results of four independent complete drag-swab samplings of a 31-farm broiler-production complex are described. Samplings of vacant poultry farms (i.e., between flocks) and occupied farms (i.e., containing chickens) were completed in four 3-day intervals beginning in July 1992 and ending in May 1993. During the first two sampling periods, two drag-swab assemblies were dragged through each individual house and pooled into one sample to evaluate overall incidence of Salmonella detection among vacant and occupied houses. During the second two sampling periods, four drag-swab assemblies were dragged through houses to evaluate frequency of Salmonella detection on individual assemblies within each house. Over the four sampling periods, mean isolation frequencies were 42.2% for vacant poultry farms and 76.2% for occupied poultry farms. When four assemblies were dragged in vacant houses and cultured separately, the probability of detecting salmonellae contamination increased with increasing numbers of assemblies, indicating that multiple drag-swab assemblies should be used for sampling either vacant or occupied broiler houses for the presence of Salmonella. On vacant farms, 89.5% of the total isolations occurred in only one house of two-house farms; the remaining 10.5% of farms were positive in both houses. Salmonella isolation from occupied farms occurred in 54.2% of the samplings in only one house, whereas 45.8% of occupied farms were positive in both houses. These data show a higher frequency of Salmonella isolation on multiple swabs in occupied poultry houses, possibly indicating a quantitatively greater presence of Salmonella in these houses.

Evaluation of repetitive extragenic palindromic-polymerase chain reaction and denatured gradient gel electrophoresis in identifying Salmonella serotypes isolated from processed turkeys

The current study was conducted to determine the usefulness of 2 molecular techniques, automated repetitive extragenic palindromic-PCR (REP-PCR) and denaturing gradient gel electrophoresis (DGGE), to identify Salmonella serotypes of poultry origin. Salmonella continues to be a foodborne pathogen of principal concern in the United States. The interspersed conserved repetitive sequence of the bacterial genome and the 16-23S rDNA intergenic spacer region were amplified for REP-PCR and DGGE, respectively. Fifty-four Salmonella isolates from 2 turkey processing plants (A and B) were used for this comparison. Serotypes consisted of Brandenburg, Derby, Hadar, and Typhimurium, with n=6, 21, 12, and 15, respectively. The REP-PCR was fully automated, whereas DGGE was run on an acrylamide gel and the image was captured digitally. Both dendrograms were created using the unweighted pair group method with arithmetic average. There were more variations in percentage similarity in DGGE when compared with REP-PCR. The banding patterns were more distinct and uniform in the REP-PCR group than with DGGE. The results from the REP-PCR were generated within 1 h, whereas the DGGE required approximately 1 d to run. These data suggest that DGGE and REP-PCR are useful tools for identifying Salmonella serotypes isolated from poultry production or processing environments. In addition, REP-PCR is more rapid, may have a higher discriminatory power, but may be less cost-effective than DGGE. However, more research may be needed to validate this argument. Both DGGE and REP-PCR displayed high sensitivity in discriminating among Salmonella serotypes and either method could be considered as an alternative to more expensive and time-consuming conventional antibody-based serotyping methodologies.

Frequency of Isolation of Salmonella from Protective Foot Covers Worn in Broiler Houses as Compared to Drag-swab Sampling

In this study we tested the hypothesis that the frequency of Salmonella isolation from protective foot covers worn in individual broiler production houses would compare favorably to isolation rates obtained from conventional drag-swab methods. Salmonella was detected with equal frequency from protective foot covers and drag-swab assemblies on nine individual broiler farms over three separate sampling periods. Salmonella was detected in 13 of a total 27 individual samplings by culturing the protective foot covers, whereas positive detections occurred in 16 of a total 27 samplings when using the drag-swab method. Of the total number of houses identified as positive, these frequencies were unaltered when evaluated against the flock status of each farm at the time of sampling. Both methods were equally as likely to detect Salmonella when houses were either vacant (awaiting the placement of the next flock) or occupied (currently housing chickens of any age). In addition to highlighting the development of a potential new Salmonella monitoring technique, this study reinforces our current understanding regarding the importance of stringent biosecurity practices on poultry farms.

The effect of dietary protein level on performance characteristics of coccidiosis vaccinated and nonvaccinated broilers following mixed-species Eimeria challenge

A series of experiments were conducted to investigate the effect of starter diet protein levels on the performance of broilers vaccinated with a commercially available live oocyst coccidiosis vaccine before subsequent challenge with a mixed-species Eimeria challenge. Data indicated that an increasing protein concentration in the starter diet improved broiler performance during coccidiosis vaccination. Prechallenge performance data indicated that vaccination could decrease BW and increase feed conversion ratio. The time period most important for the observed effects appeared to be between 13 and 17 d of age. This reduction in performance parameters of vaccinated broilers compared with nonvaccinated broilers was eliminated by the conclusion of the experiments (27 d) in the diet groups with higher protein. Vaccination was effective at generating protective immunity against Eimeria challenge, as evidenced by increased (P < 0.05) BW gain, improved feed conversion, reduced postchallenge mortality, and reduced lesion development in vaccinated broilers compared with nonvaccinated broilers. These observations support numerous other reports that confirm live oocyst vaccination can be used effectively as a preventive against avian coccidiosis in commercially reared broilers. More important, these findings suggest that reduced protein concentration of starter diets can lead to significant losses in broiler performance when using a vaccination program to prevent coccidiosis.

Comparison of Prophylactic or Therapeutic Dietary Administration of Capsaicin for Reduction of Salmonella in Broiler Chickens

Abstract In three experiments the effects of prophylactic or therapeutic dietary inclusion of capsaicin, the pungent component of peppers, were evaluated as a nonantibiotic alternative for reduction of Salmonella in broiler chickens through culture and morphologic assessment of cecal tissue. Expt. 1 evaluated the effects of 0 or 10 ppm purified capsaicin (CAP) in the starter phase (days 1–16) on chicks challenged with Salmonella Enteritidis (SE) on day of age. Therapeutic inclusion of 10 ppm purified CAP increased (P < 0.05) liver/spleen (L/S) and ceca positive results for SE. In Expt. 2, capsaicin oleoresin (CO) was included in the finisher diet (days 30–37) at 0, 5, or 20 ppm with SE challenge on day 31. Inclusion of 5 ppm CO increased ceca positive results for SE, and a linear decrease in cecal lamina propria thickness of SE-challenged birds was observed with increased CO concentration in the diet. Expt. 3 evaluated prophylactic CO treatment at 0, 5, or 20 ppm in starter, grower, and finisher diets for resistance to SE or Salmonella Typhimurium (ST) challenge on day 14 or 29. With challenge on day 14, 5 and 20 ppm prophylactic CO feeding reduced ceca SE positive results by 37% and 26%, respectively, and ST culture rate was reduced similarly with 5 ppm CO. Lamina propria thickness of the ceca increased with 5 ppm CO feeding in SE-challenged birds, whereas a decrease was observed in nonchallenged birds fed 5 ppm CO. Challenge on day 29 of birds fed 20 ppm CO resulted in reduced L/S positive results for SE. Lamina propria thickness decreased with 5 ppm CO and SE or ST challenge compared with nonchallenged birds fed 5 ppm. An increase was observed in ST- or SE-challenged birds fed 20 ppm CO compared with nonchallenged birds fed 20 ppm CO. No differences were observed in mast cell number in either Expt. 2 or 3. These data provide evidence that prophylactic or therapeutic dietary capsaisin differentially affects broiler susceptibility to Salmonella.

Bursal anti-steroidogenic peptide (BASP): modulation of mitogen-stimulated bursal-lymphocyte DNA synthesis

The present study examined the effects of bursal anti-steroidogenic peptide (BASP) on mitogen-induced DNA synthesis in bursa-derived B-lymphocytes in short-term culture. Partially purified extracts of chicken bursa of Fabricius tissue, containing BASP, significantly (P < 0.05) reduced DNA synthesis in bursal-lymphocytes exposed to increasing concentrations of phorbol 12,13-dibutyrate (PDB). Following these initial observations, BASP, further purified from bursal extracts using sequential rpHPLC fractionation, was observed to reduce (P < 0.05) both B-lymphocyte PDB-stimulated DNA synthesis and ovarian granulosa cell progesterone biosynthesis with bioactivity observed at similar retention times in each assay, suggesting that each bioactivity may be due to the same or similar molecules. A similar BASP-enriched fraction was not effective in altering basal levels of DNA synthesis in chick embryonic kidney cells. Subsequently, BASP was further purified by several sequential chromatographic methods including: C-18 rpHPLC (preparative rpHPLC followed by a semi-preparative rpHPLC column), cation exchange chromatography, molecular sieve HPLC chromatography, and SDS-PAGE. Biologically active material was observed at approximately 29 or 34 kDa. Protein concentration was determined and bioactivity was evaluated. Anti-proliferative effects of this partially purified BASP on bursal-lymphocytes was observed at concentrations as low as 1.6 micrograms ml-1, with complete suppression of mitogen-stimulated DNA synthesis observed at approximately 25 micrograms ml-1. This partially purified BASP was also efficacious for attenuation of ovarian granulosa cell progesterone biosynthesis at concentrations as low as 0.4 microgram ml-1, with complete suppression of gonadotrophin-stimulated progesterone biosynthesis observed at approximately 0.8 microgram ml-1. While BASP is efficacious for attenuation of both granulosa cell steroidogenesis and bursal-lymphocyte proliferation, these data suggest that BASP is much more potent with regard to anti-steroidogenic activity.

Guar Meal Diets as an Alternative Approach to Inducing Molt and Improving Salmonella Enteritidis Resistance in Late-Phase Laying Hens

Induced molting of laying hens is a practice used by commercial egg producers to increase the productive lifetime of their flock. However, the conventional method of inducing molt, which involves removal of feed, water, or both as well as a reduction in photoperiod to less than a natural day has drawn criticism due to animal welfare and food safety concerns. The objective of this study was to explore the efficacy of diets containing high levels of guar meal (GM) in inducing molt and reducing susceptibility to Salmonella Enteritidis colonization in late-phase laying hens. Late-phase (68 wk old) Lohmann laying hens were either full-fed standard laying hen diets (nonmolted control), induced to molt by feed withdrawal, or full-fed standard laying hen diets containing 20% GM with or without 250 units/kg of mannanase Hemicell supplementation. On the fourth day of treatment, all hens were orally challenged with SE (1.65 x 10(7) cfu). Hens were killed and evaluated for Salmonella colonization and differences in organ weights 5 d postinoculation. Salmonella Enteritidis present in crop, liver, ovary, and cecal contents were significantly reduced by feeding GM with enzyme supplementation compared with feed withdrawal hens. No significant differences were observed in reproductive tract weights of molted groups, although a difference in liver weight was detected. Results indicate that feeding diets containing 20% GM are as effective as complete feed withdrawal with respect to inducing molt with the added benefit of improved resistance to Salmonella Enteritidis colonization and translocation.

Molecular analysis of Salmonella serotypes at different stages of commercial turkey processing

Salmonella isolates were collected from 2 commercial turkey processing plants (A and B) located in different US geographical locations. Isolates recovered at different stages of processing were subjected to 2 genotype techniques [PAGE and denatured gradient gel electrophoresis (DGGE)] to determine their usefulness for Salmonella serotyping. Primers used for PCR amplification were to a highly conserved spacer region located between the 16S and 23S rDNA genes. Sampling sites at plant A were 1) postscald, 2) pre-inside-outside bird wash, 3) post-IOBW, and 4) postchill with 30, 44, 36, and 12 Salmonella isolates recovered, respectively. Plant B had an additional site and these locations were 1) prescald, 2) postscald, 3) pre-inside-outside bird wash, 4) post-IOBW, and 5) postchill with 16, 54, 24, 35, and 24 Salmonella isolates recovered, respectively. In plant A, 4 different Salmonella serotypes were identified: Derby, Hadar, Montevideo, and Senftenberg. In plant B, 10 serotypes were identified: Agona, Anatum, Brandenburg, Derby, Hadar, Meleagridis, Montevideo, Reading, Senftenberg, and Typhimurium. Salmonella Derby was predominant in plant A (83%), whereas Salmonella Typhimurium was the most common serotype recovered in plant B (39%). Genotype analyses of the Salmonella serotypes were expressed in dendrograms with comparisons interpreted as percentage similarity coefficients. Both PAGE and DGGE were able to distinguish serotype band patterns. However, DGGE was more discriminating than PAGE. Isolates of the same serotypes were grouped together on the dendrogram of band patterns generated by DGGE. In contrast, PAGE failed to group all like serotypes together on the corresponding dendrogram. The results of the study suggest that genotyping techniques can be very useful in discriminating Salmonella serotypes collected from the processing plant environment of commercial poultry production. These molecular techniques may offer more cost-effective means to identify Salmonella serotypes from large numbers of isolates and with more immediate results than those currently achieved with conventional typing techniques.