International Poultry Scientific Forum 2007 Intestinal papers

Evaluation of novel bacterial species on intestinal development and microflora.
B. S. Lumpkins*, Y-J. Cho, A. B. Batal, and M. D. Lee, University of Georgia, Athens.
The growing concern of feeding antibiotics to poultry has lead researchers and animal health companies to investigate alternatives. An experiment was conducted to evaluate the effects of an oral inoculation with novel intestinal anaerobes on the development of the intestine. At 0d of age, 500 Cobb male chicks were separated into 4 treatments: a control and 3 test treatments that were orally inoculated with a novel species of Bacteroidaceae, Clostridiaceae, or a combination of the two. Throughout the experiment all birds were fed a corn-soybean meal diet. At 0, 1, 2, 3, 7, 16, and 42d of age performance parameters were measured and samples were taken for morphological and bacterial community analysis. At 42d of age, birds were randomly selected and processed for carcass yield. For bacterial community analysis, community DNA isolated from small intestinal contents were amplified with universal 16s primers. Diversity and compositional changes were assessed using denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP). The performance parameters were similar among all 4 treatments from 0 to 16d of age. At the end of the 42d period the overall weight gain of the birds inoculated with Clostridiaceae was significantly lower than the control birds and the Bacteroidaceae inoculated birds. The birds inoculated with the combination of Bacteroidaceae and Clostridiaceae had increased villi height and goblet cell concentration during the first 3d of age, but after 7d of age there was no overall difference in morphological response between treatments. There was no difference in the carcass yield between treatments. Based on the DGGE analysis, the microbiota populations clustered based on age rather than treatment. Birds inoculated with either Bacteroides or Clostridiaceae had a higher proportion of lactobacilli in the ileum compared to the control birds, based on T-RFLP analysis at 42d of age. The combination of Bacteroidaceae and Clostridiaceae improved intestinal development at young ages, but Clostridiaceae may have negative effects on weight gain during the latter stages of growth. Key Words: Microbiota, Morphological, Anaerobes, Bacterial community, DNA

(Expect more on this topic in the paper by Dr Lee at the Elanco Symposium to be held in Prague on March 27th-29th (Ed.))

Effect of age on intestinal pH of broiler chickens.
J. M. Rynsburger* and H. L. Classen, University of Saskatchewan, Saskatoon, SK, Canada.
Protein digestion has been shown to be relatively poor in young birds therefore it is of interest to identify limiting factors. Initial protein digestion involves hydrochloric acid denaturation of protein and conversion of pepsinogen to its active form in the proventriculus and gizzard. If the production of hydrochloric acid is limited in young broilers, protein digestion may be hampered. A one-way ANOVA was used to study the effect of age on intestinal pH of broiler chickens fed a broiler starter diet. Ross x Ross 308 broilers (150 males) were randomly assigned to 10 battery cages. Using one bird per cage per sample age, the pH of the crop, proventriculus, gizzard, duodenum, jejunum and ileum was measured at 2, 3, 4, 5, 6, 7, 8, 9, 10 and 15 days of age. As the birds got older the crop pH increased while the pH of the proventricular proventriculus and gizzard decreased. The relationship between age and intestinal pH for the duodenum, jejunum and ileum was quadratic.

Age	Crop	Proventriculus	Gizzard	Duodenum	Jejunum	Ileum
2	5.01	5.20	3.49	6.57	6.82	7.74
3	5.51	5.12	3.47	6.37	6.66	7.07
4	5.64	4.78	3.43	6.30	6.44	7.26
5	5.54	4.58	3.50	6.45	6.50	7.1
6	5.42	3.33	3.24	6.42	6.38	6.74
7	6.13	4.85	3.48	6.07	6.3	7.08
8	5.08	4.16	3.30	6.23	6.26	6.9
9	5.56	3.48	3.42	6.35	6.34	7.05
10	6.29	3.56	3.27	6.47	6.42	7.43
15	6.02	3.37	3.27	6.40	6.5	8.15

In conclusion, during the first week of age the pH of the proventriculus and gizzard decreases indicating that the production of hydrochloric acid may not reach optimal levels until some time after hatch. As a result, protein digestion may be hampered during the first week post-hatch.

Selenium sources influence small intestinal characteristics and morphology in reovirus infected broiler chickens.
S. Burgos*1, F. W. Edens1, J. Read-Snyder1, A. Cantor2, and S. A. Burgos3, 1North Carolina State University, Raleigh, 2University of Kentucky, Lexington, 3University of Guelph, Guelph, ON, Canada.
Avian reovirus (ARV) infections have been associated with malabsorption syndrome causing lower weight gains in broiler chickens. The aim of this investigation was to determine if ARV infection with and without selenium -organic and inorganic- affected small intestinal characteristics and morphology. Eggs were obtained from Cobb breeders that had been maintained on isocaloric Torula yeast diets containing either no supplemental selenium, sodium selenite at 0.3 ppm, or organic selenium (SelPlex, Alltech, Inc., Nicholasville, KY, USA) at 0.3 ppm. Chicks hatched from those eggs were placed on Torula yeast broiler diets containing no supplemental selenium, 0.3 ppm sodium selenite, or 0.3 ppm organic selenium similar to their respective parents' diets. On the day of hatch, 60 chicks per dietary selenium treatment were placed into either control or ARV-infected groups in heated metal-growing batteries in separate isolation rooms. Chicks in the ARV-infected groups were given each an oral gavage of 0.2 mcL of ARV-CU98 (104.2 pfu/chick), and control chicks were given the medium only. At 23 d of age, the chicks were weighed, killed by carbon dioxide asphyxiation, and their intestinal tracts were dissected for total and segmental weight and length, with tissue collections for histomorphotography. Data from this 2 X 3 factorially arranged completely randomized experimental design were analyzed using the GLM procedure of SAS. The ARV-CU98 challenge caused more than 15% reduction (p< 0.0003) in body weight and caused 18.4% mortality compared with 6% in controls. ARV-CU98 infection did not affect intestinal length, but infected chicks had more distended and heavier intestines than did controls. Selenium reduced the average intestinal weight in both control and infected birds (P<0.05). Microscopic analysis revealed longer microvilli in selenium-fed control and infected birds, and goblet cell numbers were increased in seleniumfed birds. It was concluded that selenium is necessary to maintain the integrity of the small intestine of both control and ARV-CU98 infected chickens. Key Words: Reovirus, Selenium, Broilers, Small intestine, Morphology

Dietary methionine sources affect intestinal microbial growth in broiler chickens. J. P. Dahiya*1, D. Hoehler2, A. G. Van Kessel1, and M. D. Drew1, 1University of Saskatchewan, Saskatoon SK, Canada, 2Degussa Corporation, Kennesaw, GA.
Previous work in our . laboratory showed that methionine hydroxyanalogue (MHA-FA) is more available to gut bacteria than DLmethionine (DL-Met) and may stimulate the growth of intestinal bacterial populations including Cstudy the effect of . perfringens. An experiment was conducted to various levels of DLchickens. Two cages of 6 birds (14 d -Met or MHA-FA on C. perfringens and other intestinal bacteria in broiler post(control); 2.0, 4.0 or 8.0 g/kg DL-Met or 2.27, 4.54 and -hatch) were assigned to one of 7 different diets containing no added Met 90.4 and 0.8%) of each methionine source. Birds were challenged with C. .08 g/kg MHA-FA, thus providing 3 equimolar levels (0.2, perfringens type A on d killed on d 28. Intestinal populations of C. perfringens, lactobacilli, Streptococcus D and 1 and d 14 to 20, and coli forms were enumerated and necrotic enteritis lesions were scored. There were no significant differences in the growth of various bacterial species in intestinal tract of broiler chickens fed two different methionine sources. However, we observed significantly reduced (P < 0.05) C. perfringens populations in ileum and cecum of birds fed 8 g/kg DL-Met and 9.08 g/kg MHA-FA supplemented diets vs. controlperfringens growth. The coli forms . Also there was a significant interaction (P < 0.05) between methionine source and level for C. and Streptococcus D in ileum were significantly lower in ceca were significantly higher in birds given (P < 0.05) whereas lactobacilli highest levels of either DLtreatments. There were no significant differences in necrotic enteritis -Met or MHA-FA than the other dietary intestinal lesion scores and performance of birds fed different methionine sources or concentrations. The results suggest that the use of low protein diets supplemented with relatively high levels of synthetic amino acids may reduce the risk of clostridial enteritis in broiler chickens. Key WordsMethionine sources, Necrotic enteritis, Broiler chickens :

Performance improvement with feed additives RepaXol(r), AciXol(tm), and Virginiamycin in broilers challenged with clostridium perfringens. G. Mathis*1, C. Hofacre2, and N. Scicutella3, 1Southern Poultry Research, Inc., Athens, GA, 2University of Georgia, Athens, 3SODA Feed Ingredients, Monaco.
The objective was to evaluate the anticlostridial efficacy of feed additives RepaXol(r), an homogeneus blend of double coated essential oils, AciXol(tm), a blend of organic and inorganic acids (citric, fumaric, malic and ortho-phosphoric) along with the protected essential oils (as in RepaXol(r), encapsulated in the same MICROPEARLS(r), and Virginiamycin, an antibiotic. Groups of 10 birds were weighed and placed into cages on day of hatch. The treatments were nonmedicated, non-challenged (NMNC), nonmedicated, challenged (NMC), RepaXol(r) 100 ppm, challenged, AciXol(tm) 500 ppm, challenged, and Virginiamycin (VIR) 20 ppm, challenged. Birds were challenged at 14 days of age with E. acervulina and E. maxima and on Days 19, 20, and 21 with Clostridium perfringens. Each treatment consisted of 6 replications in a complete randomized block design. Feed in mash form was fed ad libitum throughout the test period. The parameters measured were feed conversion and weight gain (Days 0 to 28 and Day 14 to 28), Necrotic Enteritis (NE) mortality and NE lesion scores. On Day 22, five birds per pen were NE lesion scored. There was a significant improvement of feed conversions and weight gains for both measurement periods with all feed additives compared to the NMC birds. The feed conversions (Day 0-28) of the NMNC was 1.461g, NMC, 1.726, RepaXol(r) 1.586, AciXol(tm) 1.577g, and VIR 1.519. The average live weight gains (Day 0-28) of the NMNC was 0.963 kg, NMC 0.774 kg, RepaXol(r) 0.915 kg, AciXol(r) 0.901 kg, and VIR 0.935 kg. Percent NE mortality was significantly less for the VIR 12 % compared to NMC 33 %. There was no significant difference in percent NE mortality between RepaXol(r) 23 %, AciXol(tm) 22 % and VIR. All feed additive treatments had significantly lower NE lesion scores compared to NMC. This study demonstrated the benefits of adding RepaXol(r) 100 ppm, AciXol(tm) 500 ppm, or Virginiamycin 20 ppm into the feeds of broiler chickens exposed to Clostridium perfringens. Key Words: Clostridium perfringens, RepaXol, AciXol, Virginiamycin, Necrotic Enteritis M62 Herbal products for control of histomoniasis (blackhead disease) in turkeys. R. Hauck*, P. L. Armstrong, L. Fuller, and L. R. McDougald, University of Georgia, Athens. In the absence of approved products for treatment of blackhead disease there is increased interest in the evaluation or use of natural products. Such products are used for other purposes in the feed industry and have good acceptance by producers and consumers. In this study we evaluated the effects of 3 such products which are used for odor management in feed. These products are extracts or essential oils from commonly used herbs. Tests of the products in vitro showed positive results against Histomonas and other protozoa. The test model in turkeys consisted of directly and indirectly exposed birds in battery cages. The products offered little protection against the severe direct infections. However, product 2 alone or in a combination with product 3 had beneficial effects on lesions in the liver and ceca. These results suggest that these products could have benefit in helping birds to resist the spread of histomoniasis within a flock. Key Words: Histomoniasis, Turkeys, Treatment, Essential oils

Multiplex RT-PCR for the detection of astroviruses and rotaviruses in poultry. C. Stephens*, M. J. Pantin-Jackwood, and E. Spackman, University of Georgia, Athens.
Viral enteric diseases cause substantial economic loss to the US poultry industry because they lead to decreased weight gain, increased morbidity, increased mortality, and increased production costs from poor feed conversions and increased use of therapeutic anti-microbial treatments. Astroviruses and rotaviruses are among the most commonly detected viruses found in chicken and turkey intestinal samples. Two multiplex RT-PCR tests for the simultaneous detection and differentiation of four avian astrovirus types and avian rotaviruses from feces and intestinal samples from poultry were developed and validated. The multiplex RT-PCR for chicken samples detects Avian Nephritis Virus (ANV), Chicken Astrovirus (CAstV) and Rotavirus. The multiplex RT-PCR for turkey samples detects Turkey Astrovirus 1 (TAstV-1), Turkey Astrovirus 2 (TAstV-2), Avian Nephritis Virus (ANV) and Rotavirus. Assay detection limits for each virus were determined. An evaluation of different sampling times and types for each virus was performed with experimentally infected chickens and poults. The multiplex RT-PCR's developed were successfully used in a survey on enteric viruses circulating in poultry in the United States.

Immunopotentiation of avian heterophils with microbial agonists (Poster). M. Farnell*1, A. Donoghue2, F. Solis de los Santos3, P. Blore3, B. Hargis3, G. Tellez3, and D. Donoghue3, 1Texas A & M University, College Station, 2USDA, Fayetteville, AR, 3University of Arkansas, Fayetteville.
The immune system of neonatal chicks is functionally immature during the first week of life. Researchers have previously demonstrated that the avian humoral response can be increased through the use of probiotics. Although the humoral response provides the chick with an effective mechanism to combat pathogens, sufficient antibody titers are not attained until 7 to 10 d post infection. However, the innate immune system (i.e. heterophils) can respond much more quickly to pathogens. The objective of this study was to determine whether probiotic bacteria can also up regulate heterophil function. Heterophils were isolated from the peripheral blood of neonatal chickens by using a discontinuous density gradient. Oxidative burst and degranulation are bactericidal mechanisms used by heterophils to kill pathogens and were used in this study as indicators of heterophil function. We found that each of the 10 "generally recognized as safe" (GRAS) probiotic isolates (designated G1-11) tested, in vitro, were capable of increasing (P < 0.05) heterophil oxidative burst and degranulation when compared to unstimulated controls. Bacillus subtilis (G3), Lactococcus lactis lactis (G6) and Lactobacillus acidophilus (G8) isolates were determined to elicit the greatest heterophil response in vitro and were subsequently fed to chicks. Phosphate buffered saline (PBS) or one of these three probiotic isolates (~ 2.5 x 108 cfu/chick; 50 chicks/treatment) resuspended in PBS was administered by oral gavage on the day of hatch. Heterophils were isolated from chicks from each of these four treatment groups 24 h post treatment. Significant increases in heterophil degranulation and oxidative burst were observed with the G3, G6 and G8 treated chicks when compared to heterophils isolated from birds with no probiotic treatment. These data suggest that probiotic bacteria can significantly improve heterophil oxidative burst and degranulation in broilers. To our knowledge this is the first study demonstrating a relationship between probiotics and avian heterophil function. Key Words: Chicken, Probiotic, Heterophil, Gastrointestinal tract, Innate immunity

T-RFLP analysis of the gastrointestinal flora of broilers affected and unaffected with gangrenous dermatitis. K. Bos*1, T. Neumann1, D. Ritter2, and T. Rehberger1, 1Agtech Products, Inc., Waukesha, WI, 2Mountaire Farms of Delaware, Inc., Millsboro, DE.
The gastrointestinal (GI) tract of a commercial broilers harbors a dense and metabolically active microbial community. This GI community has been shown to play a large role in animal performance and health status. Recently, gangrenous dermatitis (GD) has reemerged as a significant concern for poultry producers in the United States. One theory states that GD comes from pathogenic bacteria present in the GI community. GD can be treated with antibiotics, but the effects of these antibiotics on the GI microbial community are poorly understood. Classically, traditional plating methods are used to characterize microbial populations. Unfortunately, these techniques are limited in their ability to detect only cultivable microorganisms. The objective of this study was to use terminal restriction length polymorphism (T-RFLP) to: assess the GI microbial community in GD affected and unaffected birds both with and without antibiotic treatment, and determine bacteria that are unique to each health status. GI tracts from broilers with and without GD symptoms in the same house were collected from farms in Delaware. Genomic DNA was isolated from the mucosal bacteria of the duodenum, jejunum, and ileum of each bird, and analyzed by T-RFLP to characterize the GI communities. Comparisons of terminal restriction fragment (TRF) patterns from the affected and unaffected birds indicated which TRFs appeared to be associated with diseased or healthy status. GD treatment with antibiotics altered the microflora within the GI community in a similar manner in both affected and unaffected birds. These bacterial changes are different than the changes seen in the affected and unaffected birds that had not received an antibiotic treatment. The preliminary identification of the bacteria associated with GD indicated by the TRFs are Clostridial spp., Mycoplasma spp. or phytoplasmas, and Pasteurella or Actinobacillus spp. While the bacteria associated with a nondiseased health status are Lactobacillus spp. Overall, T-RFLP has been an effective tool to monitor changes within the GI community of broilers both affected and unaffected by GD, with and without antibiotic treatment. Key Words: T-RFLP

RAPD comparison of Clostridium perfringens isolated from cases of necrotic enteritis and gangrenous dermatitis. T. Neumann*, S. Dunham, J. Skalecki, and T. Rehberger, Agtech Products, Inc., Waukesha, WI. Clostridial diseases have become a major concern in today's poultry industry. Accompanied by high mortality and reduced efficiency they often inflict a heavy economic burden on producers. Necrotic enteritis and gangrenous dermatitis are among the most common clostridial diseases observed in broiler chickens. The anaerobic, spore-forming bacterium Clostridium perfringens is recognized as the causative agent of necrotic enteritis and has also been implicated in gangrenous dermatitis. This opportunistic pathogen is capable of producing a myriad of extracellular toxins and enzymes that degrade host tissues and are responsible for the necrotic lesions observed. RAPD PCR is a technique that can be used to generate a molecular fingerprint of a bacterial species or strain. These fingerprints can then be compared to determine the relationship among the organisms. The purpose of this study was to use RAPD PCR to determine if strains of C. perfringens isolated from cases of necrotic enteritis and gangrenous dermatitis are similar. Fourteen broilers showing signs of gangrenous dermatitis were collected for microbiological analysis from an East coast production system. Twenty-two broilers with necrotic enteritis from both East coast and West coast farms were also examined. A total of 153 C. perfringens isolates were recovered for the study. All of the isolates were confirmed as type A by a multiplex PCR that amplifies the four major toxins (alpha, beta, epsilon and iota). Fingerprints generated by RAPD PCR were used to construct a dendrogram in order to study the relationship among the strains. Necrotic enteritis isolates from East and West coast sites clustered separately from gangrenous dermatitis isolates. However, East coast necrotic enteritis isolates were more related to East coast gangrenous dermatitis isolates than West coast necrotic enteritis isolates. These results show that geographic location is an important source of genomic variability in strains of C. perfringens type A. Key Words: Necrotic, Enteritis, Gangrenous, Dermatitis, Clostridium

Evaluation of broiler performance as affected by acidifier and zinc bacitracin under field conditions. C. Lueckstaedt, T. Steiner, and M. Mohnl*, BIOMIN GmBH, Herzogenburg, Austria.
Due to recent concerns about residues in animal products and bacterial resistance to antibiotics, alternatives to in-feed antibiotics (AGP) are required. Various feed additives such as organic acids have been studied already. Many scientists reported that the inclusion of organic acids in the diet can enhance growth performance and modulate intestinal microbiota. Ban on antibiotic growth promoters creates significant opportunity for feed acidification was stated recently in a report from Frost & Sullivan. An objective of the study was therefore to test a well balanced acidifier on a sequential release medium against a commercially available AGP (Zinc Bacitracin). The trial was conducted under field conditions in Argentina. The aim of the trial was to test a well balanced acidifier (2 kg per t of feed) against a commercial piglet diet containing Zinc Bacitracin in a dose of 0.35 kg / t. Feed and water were available ad libitum. The performance of two groups of chicken (30,000 birds in total) was evaluated. Each pen consisted of 15,000 randomly selected male and female birds (Ross line 308). They were kept in pens of 150 m length x 10 m width. The trial period lasted for 42 days, during that period pre-starter feed was given from day 1 to day 14, feed was fed from day 15 till 28 days, while the finisher feed was provided until day 42. After 42 days broilers in the acidifier treated group were numerically heavier (2236 g vs. 2223 g for the positive control), even though this difference was not statistically significant (P>0.05). The G:F was also not statistically different (P>0.05) between the two treatments (0.51 and 0.53 for acidifier and Zinc Bacitracin, respectively). Mortality in the Zinc Bacitracin treated group was slightly reduced (2.8% vs. 3.1% for the acidifier group) but again without any statistical significance. It can be therefore concluded, that a well defined acidifier can successfully replace an AGP, such as Zinc Bacitracin, in broiler production under Argentinian conditions. Key Words: Acidifier, Zinc Bacitracin, Broiler, Antibiotic replacement

Authors	Institution(s)	Title (link)
B. S. Lumpkins, Y-J. Cho, A. B. Batal, and M. D. Lee,	University of Georgia, Athens.	Evaluation of novel bacterial species on intestinal development and microflora.
J. M. Rynsburger and H. L. Classen	University of Saskatchewan, Saskatoon, SK, Canada.	Effect of age on intestinal pH of broiler chickens.
S. Burgos, F. W. Edens, J. Read-Snyder, A. Cantor, and S. A. Burgos	North Carolina State University, Raleigh, University of Kentucky, Lexington, University of Guelph, Guelph, ON, Canada	Selenium sources influence small intestinal characteristics and morphology in reovirus infected broiler chickens
J. P. Dahiya, D. Hoehler, A. G. Van Kessel, and M. D. Drew	University of Saskatchewan, Saskatoon SK, Canada, Degussa Corporation, Kennesaw, GA.	Dietary methionine sources affect intestinal microbial growth in broiler chickens
G. Mathis, C. Hofacre, and N. Scicutella, .	Southern Poultry Research, Inc., Athens, GA University of Georgia, Athens, SODA Feed Ingredients, Monaco	M59 Performance improvement with feed additives RepaXol(r), AciXol(tm), and Virginiamycin in broilers challenged with clostridium perfringens.
C. Stephens, M. J. Pantin-Jackwood, and E. Spackman,	University of Georgia, Athens.	Multiplex RT-PCR for the detection of astroviruses and rotaviruses in poultry.
M. Farnell, A. Donoghue, F. Solis de los Santos, P. Blore, B. Hargis, G. Tellez, and D. Donoghue,	Texas A & M University, College Station, USDA, Fayetteville, AR, University of Arkansas, Fayetteville.	Immunopotentiation of avian heterophils with microbial agonists (Poster).
K. Bos, T. Neumann, D. Ritter, and T. Rehberger	Agtech Products, Inc., Waukesha, WI, Mountaire Farms of Delaware, Inc., Millsboro, DE	T-RFLP analysis of the gastrointestinal flora of broilers affected and unaffected with gangrenous dermatitis (Poster).
T. Neumann, S. Dunham, J. Skalecki, and T. Rehberger	Agtech Products, Inc., Waukesha, WI	RAPD comparison of Clostridium perfringens isolated from cases of necrotic enteritis and gangrenous dermatitis.(Poster)
C. Lueckstaedt, T. Steiner, and M. Mohnl	BIOMIN GmBH, Herzogenburg, Austria.	Evaluation of broiler performance as affected by acidifier and zinc bacitracin under field conditions.(Poster)