Barry G. Harmon

Diversity and Succession of the Intestinal Bacterial Community of the Maturing Broiler Chicken

ABSTRACT The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of Lactobacillus, with the majority of the rest being related to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). In contrast, Clostridiaceae-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.

Evaluation of Broiler Litter with Reference to the Microbial Composition as Assessed by Using 16S rRNA and Functional Gene Markers

ABSTRACT Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 109 CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.

ISOLATION OF ANTIMICROBIAL PEPTIDES FROM AVIAN HETEROPHILS

Five bactericidal peptides (chicken heterophil peptides CHP1 and CHP2; turkey heterophil peptides THP1, THP2, and THP3) were purified from avian heterophil granules. All peptides were cationic and rich in cysteine, arginine, and lysine. The complete amino acid sequence, consisting of 39 amino acids, was determined for CHP1. This peptide had a molecular weight of 4481 as determined by mass spectrometry. Partial NH2‐terminal amino acid sequences were obtained for the remaining peptides. Both chicken peptides and THP1 shared sequence homology at 22 residues and a cysteine motif which was similar to that of bovine beta‐defensins. THP2 and THP3 were homologous to each other but were not homologous to the other three and had a unique cysteine motif. Peptides CHP1, CHP2, and THP1 killed Staphylococcus aureus and Escherichia coli in vitro, whereas THP2 and THP3 killed only S. aureus in vitro. J. Leukoc. Biol. 56: 661–665; 1994.

Antimicrobial activity of chicken and turkey heterophil peptides CHP1, CHP2, THP1, and THP3.

Abstract Four avian heterophil antimicrobial cationic peptides (Chicken Heterophil Peptides 1 and 2, and Turkey Heterophil Peptides 1 and 3) were evaluated for in vitro microbicidal activity against selected avian pathogens and human pathogens which are harbored by birds. At concentrations of 16-2 μg/ml, all four avian peptides effected a greater than 90% reduction in the survival of Candida albicans, Salmonella enteriditis, and Campylobacter jejuni. None of the peptides, including the known antimicrobial peptide protamine (used as a positive control), were able to reduce the survival of Pasteurella multocida by 90% at the maximum peptide concentration (16 μg/ml) tested. At 16 μ/ml, the turkey peptide THP3 did not effect a 90% reduction in survival of Bordetella avium, Escherichia coli, or Salmonella typhimurium, while all of the other peptides tested were effective at this concentration or less. This peptide, THP3, does not share the same homologous amino acid sequence shared by the other three peptides. Under our experimental conditions, none of the peptides neutralized Infectious Bronchitis Virus, an enveloped coronavirus of chickens.

Fecal shedding of enterohemorrhagic Escherichia coli in weaned calves following treatment with probiotic Escherichia coli

The fecal shedding and pathogenicity of enterohemorrhagic E. coli (EHEC) O26:H11, EHEC O111:NM, and EHEC O157:H7 in weaned calves (8 to 10 weeks of age) were compared with and without treatment with a three-strain mixture of probiotic bacteria (competitive-exclusion E. coli). Three groups of 12 calves were each perorally given a five-strain mixture of one of the EHEC serotypes (10(10) CFU of total bacteria per calf). Seventy-two hours later, six calves from each group were each administered 10(10) CFU of probiotic bacteria. None of the EHEC serotypes caused significant clinical disease, although a few calves developed mild transient diarrhea or pyrexia. Gross or microscopic lesions attributable to EHEC were not detected in control or probiotic-treated calves at necropsy. For probiotic-treated calves given E. coli O157:H7 and for probiotic-treated calves given E. coli O111:NM, fecal shedding was reduced compared with that for untreated calves. For the probiotic-treated calves given E. coli O157:H7, the reductions in fecal shedding on days 8, 12, 14, 16, 20, 22, 28, and 30 after peroral administration were statistically significant (P<0.05). For probiotic-treated calves given E. coli O111:NM, there were statistically significant reductions (P<0.05) in fecal shedding on days 6, 8, 10, and 12. In contrast, there was no reduction in fecal shedding for calves administered E. coli O26:H11 and treated with the probiotic bacteria. In fact, calves in both the treated and the nontreated groups continued to shed large populations of E. coli O26:H11 throughout the 32-day trial. At necropsy, E. coli O157:H7 was isolated from five of six untreated calves and from only two of six probiotic-treated calves. E. coli O111:NM was isolated from four of six untreated calves at necropsy and from two of six probiotic-treated calves. However, E. coli O26:H11 was isolated from five of six untreated calves and from all six probiotic-treated calves. The results obtained in this study indicate that probiotic E. coli substantially reduced or eliminated fecal shedding of E. coli O157:H7 and E. coli O111:NM 8 to 30 days and 6 to 12 days after the administration of the probiotic culture, respectively, and reduced the persistence of E. coli O157:H7 in the gastrointestinal tract at necropsy (31 to 33 days after the administration of the probiotic culture). The probiotic E. coli did not reduce fecal shedding or gastrointestinal persistence of E. coli O26:H11.

Effects of diet on rumen proliferation and fecal shedding of Escherichia coli O157:H7 in calves.

Calves inoculated with Escherichia coli O157:H7 and fed either a high-roughage or high-concentrate diet were evaluated for rumen proliferation and fecal shedding of E. coli O157:H7. Calves fed the high-roughage diet had lower mean rumen volatile fatty acid concentrations and higher rumen pH values than did calves fed the high-concentrate diet. Despite these differences in rumen conditions, the calves fed the high-roughage diet did not have greater rumen populations of E. coli O157: H7 and did not exhibit increased or longer fecal shedding compared with the calves fed the high-concentrate diet. Two calves shedding the highest mean concentrations of E. coli O157:H7 were both fed the high-concentrate diet. There was a significant (P < 0.05) positive correlation between fecal shedding and rumen volatile fatty acid concentration in calves fed a high-concentrate diet. The effects of diet on E. coli O157:H7 proliferation and acid resistance were investigated using an in vitro rumen fermentation system. Rumen fluid collected from steers fed a high-roughage diet, but not from steers fed a high-concentrate diet, supported the proliferation of E. coli O157:H7. Rumen fluid from steers fed a high-concentrate diet rapidly induced acid resistance in E. coli O157:H7. The impact of diet on fecal shedding of E. coli O157:H7 is still unclear and may depend on dietary effects on fermentation in the colon and on diet-induced changes in the resident microflora. However, rapid development of acid tolerance by E. coli O157:H7 in the rumens of calves fed high-concentrate diets, allowing larger populations to survive passage through the acidic abomasum to proliferate in the colon, may be one factor that influences fecal shedding in cattle on feed.

Fecal Shedding and Rumen Growth of Escherichia coli O157:H7 in Fasted Calves

Nine weaned calves aged from 8 to 12 weeks were fitted with rumen cannulas and were inoculated by cannula with 10(10) CFU of a five-strain mixture of nalidixic acid-resistant Escherichia coli O157:H7. Six calves were fasted for 48 h on days 15 and 16 and days 22 and 23 after inoculation. Samples of rumen contents and feces were obtained daily to enumerate E. coli O157:H7 populations and to determine rumen volatile fatty acid (VFA) concentrations and rumen pH. Fasting resulted in a marked decrease in rumen VFA concentrations from a mean of 135 mmol/liter before the fast to a mean of 35 mmol/liter during the second day of the fast. However, there was no correlation between daily VFA concentration and daily rumen or fecal numbers of E. coli O157:H7 in any of the calves. Fasting generally had no significant effect on the rumen or fecal numbers of E. coli O157:H7. The exception was a single fasted calf that experienced a 3-log(10) CFU/g increase in fecal shedding during and after the first fast. Despite the consistent changes in VFA concentrations in fasted calves, the fluctuations in rumen numbers of E. coli O157:H7 in the rumen of fasted calves were minimal. At the end of the experiment, E. coli O157:H7 was detected in either the rumen or omasum in two of three control calves at necropsy and in either the rumen or reticulum in five of six fasted calves. E. coli O157:H7 was detected in the colon in two of three control calves and in six of six fasted calves at necropsy. These results suggest that in cattle already shedding E. coli O157:H7, feed withdrawal and the associated changes in rumen pH and VFA concentrations have little effect on fecal shedding and rumen proliferation of E. coli O157:H7.

Pathogenicity of enterohemorrhagic Escherichia coli in neonatal calves and evaluation of fecal shedding by treatment with probiotic Escherichia coli.

The pathogenicity and fecal shedding of enterohemorrhagic Escherichia coli (EHEC) O26:H11, O111:NM, and O157:H7 were compared in calves (< 1 week of age) with or without prior treatment with probiotic bacteria (competitive exclusion E. coli). Three groups of 12 to 14 calves were used for these treatments. Half of the calves in each group were perorally administered 10(10) CFU of probiotic bacteria per calf, and, 2 days thereafter, 10(8) CFU of a five-strain mixture with one of the three EHEC serotypes per calf were administered to each calf. None of the EHEC serotypes caused clinical disease,and neither gross nor microscopic lesions attributable to EHEC were detected in control or probiotic-treated calves at necropsy. In calves administered E. coli O157:H7, fecal shedding was greatly reduced (> 6 log10 CFU/g) by 8 days after administration, and there was no significant difference (P > 0.05) in fecal shedding of E. coli O157:H7 between probiotic-treated and untreated control groups at that time. In contrast, control calves perorally administered E. coil of serotypes O111:NM or O26:H11 continued to shed substantial populations (10(2.1) to 10(6) CFU/g of feces and 10(2.5) to 10(4.9) CFU/g of feces, respectively) throughout 7 days postadministration of EHEC. In both groups administered either E. coli O111:NM or O26:H11, significantly less (P < 0.05) EHEC was isolated from feces at 7 days postadministration of EHEC and at necropsy from theprobiotic-treated group than from the untreated control group. Overall, neonatal calves shed in the feces from 1 to 7 days following peroral administration of EHEC greater populations of E. coli O111:NM and O26:H111 than E. coli O157:H7. In addition, treatment of calves with probiotic E. coli reduced fecal shedding of E. coli O111:NM and O26:H11 in most calves.

Variation among Pathologists in the Histologic Grading of Canine Cutaneous Mast Cell Tumors with Uniform Use of a Single Grading Reference

Ten veterinary pathologists independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors using the Patnaik classifications. The degree of agreement in grading among the pathologists was compared with the degree of agreement among the same pathologists in a previous study, in which each pathologist used the reference for grading that he/she uses routinely. Mean agreement improved significantly from 50.3% to 62.1% with uniform use of the Patnaik classifications (P = 0.00001), suggesting that there is value in uniform application of a single grading scheme for canine cutaneous mast cell tumors. Agreement among pathologists was still not 100%, suggesting that a more objective grading scheme should be developed and that other histologic indicators of prognosis should be investigated.

Variation among pathologists in histologic grading of canine cutaneous mast cell tumors.

Ten veterinary pathologists at 1 veterinary institution independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors (MCTs). There was significant variation among pathologists in grading the MCTs (P < 0.001). The probability of assigning a low grade was significantly higher for the pathologists in this study who use a published reference for histologic grading of canine cutaneous MCTs that allows subcutaneous MCTs or MCTs with mitotic figures to be included in the low-grade category (P < 0.0001 and P < 0.0001, respectively).