Adhesion of bacteria to the cecal mucosal surface of conventional and germ-free chickens infected with Eimeria tenella.

When Salmonella typhimurium and Clostridium perfringens were tested in conventional chickens, larger numbers of S typhimurium and C perfringens adhered to Eimeria tenella-infected ceca than to uninfected ceca. In germ-free chickens, S typhimurium and C perfringens adhered to the E tenella-infected cecal mucosa more than to the uninfected cecal mucosa, but fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E tenella-infected ceca than to the uninfected ceca. Many bacteria adhered to the lesions caused by E tenella as observed by scanning electron microscopy. On the basis of our findings, we suggest that infection with E tenella upsets the balance of competitive adherence of bacteria, allowing more colonization of S typhimurium and C perfringens.     

Necrotizing typhlocolitis associated with a spirochete in rheas (Rhea americana).

Necrotizing typhlocolitis was diagnosed in 13 juvenile common rheas (Rhea americana) from three separate of geographically isolated Ohio flocks, with mortality ranging from 25% to 80%. At postmortem examination, a diphtheritic membrane covered ulcerated cecal mucosa. Histologically, cecal sections showed necrosis and granulomatous-to-suppurative inflammation that extended into the submucosa and often surrounded large eosinophilic colonies of bacteria. Warthin-Starry staining showed these colonies to be composed of entangled spirochetes that invaded the submucosa and frequently were present transmurally. Similar organisms were identified by Warthin-Starry staining in the cecum of a juvenile rhea from a fourth flock that histologically had mild lymphocytic typhlitis. Scanning and transmission electron microscopy demonstrated the presence of a spirochete in the ceca. Anaerobic culture yielded a gram-negative, beta-hemolytic spirochete. Coccidia, histomonads, and Salmonella spp. were consistently absent.     

A comparison of the morphologic effects of Serpulina hyodysenteriae or its beta-hemolysin on the murine cecal mucosa.

Studies were carried out to compare the early morphologic changes in the cecal mucosa of mice either infected with Serpulina hyodysenteriae or exposed to the beta-hemolysin of S. hyodysenteriae. Sixty-five 12-24-week-old C3H/HeOuJ mice were infected with S. hyodysenteriae by gastric intubation. Two mice were necropsied every hour for 30 hours following infection. S. hyodysenteriae was isolated from the cecal contents of each mouse at all time points. Macroscopic lesions were first apparent at 14 hours postinfection (PI), and light microscopic lesions were first apparent at 10 hours PI, earlier than has been previously reported. Ultrastructural changes, first evident at 6 hours PI, included disarray and loss of microvilli and terminal web, with dilatation of intercellular spaces. Luminal bacteria were translocated through epithelial cells to the lamina propria, where capillaries exhibited changes indicative of increased permeability. In another experiment, solutions containing between 2,500 and 25,000 hemolytic units of purified S. hyodysenteriae hemolysin were placed within the lumen of surgically closed murine ceca (n = 10); ceca were collected for examination 3 hours following treatment. Ultrastructural changes consisted of loss of microvilli and terminal web and marked vacuolation and exfoliation of epithelial cells. Significant numbers of necrotic and apoptotic epithelial cells were present, and epithelial cells internalized moderate numbers of bacteria. The hemolysin of S. hyodysenteriae induces some of the same early ultrastructural changes in the cecal epithelium of mice as occur following infection with S. hyodysenteriae. Based on the observed bacterial translocation, luminal bacteria also appear to play a unique role in lesion development in this model.     

Dose response and organ invasion of day-of-hatch Leghorn chicks by different isolates of Campylobacter jejuni

Colonization of the ceca and organ invasion by different isolates of Campylobacter jejuni were investigated in day-of-hatch leghorn chicks. This model of Campylobacter colonization of the ceca demonstrates that 1) day-of-hatch birds do not naturally contain cecal Campylobacter, 2) ceca can be colonized with C. jejuni by oral gavage and not by cloacal inoculation; 3) C. jejuni can be recovered from the ceca up until at least 7 days postinoculation, 4) cecal colonization occurs when as little as 10(2) colony-forming units is orally inoculated into chicks, and 5) different C. jejuni isolates vary both in their ability to colonize the ceca and in their ability to invade the liver. These studies demonstrate that we have a working animal model for Campylobacter colonization for day-of-hatch chicks. This animal model is being used to examine intervention strategies such as vaccines by which Campylobacter can be reduced or removed from the food animal.     

Immediate postnatal inoculation of a microbial barrier to prevent neonatal diarrhea induced by Clostridium difficile in young conventional and gnotobiotic hares

The cecal content of a healthy young hare (a 10-fold dilution prepared in anaerobiosis) was transferred into gnotobiotic recipient mice. The fecal flora of recipient mice was inoculated into 49 conventional young hares (kept in a closed building) immediately after birth. All the inoculated hares survived until weaning whereas 35% of 48 non-treated conventional hares died of diarrhea. Clostridium difficile and C perfringens were present in all the diarrheal noninoculated hares, whereas these species were present only in 50% of healthy inoculated or noninoculated hares at low concentrations (less than 10(6)/g of feces). A microbial barrier effect against challenge-exposure strains of C difficile and C perfringens was evidenced in the intestinal tract of recipient gnotobiotic mice and young hares. Seemingly, the protective effect of the microbial inoculum was due to antagonisms exerted against C difficile and C perfringens in the intestinal tract of inoculated conventional hares.     

Influence of host lineage on cecal colonization by Campylobacter jejuni in chickens

The resistance to cecal colonization by Campylobacter jejuni was assessed by challenging three crossbred stocks of commercially available broiler chickens. These three stocks, designated A, B, and C, were related as follows: Offspring from four pedigreed grandparent flocks were used as progenitors. Stock B was derived by cross-breeding grandparent 1 with grandparent 3. Stocks A and C were crossbreeds from grandparents 1 and 2 and grandparents 3 and 4, respectively. Campylobacter jejuni were gavaged into 48-hour-old chicks, using the same levels of challenge dose for each of the different chicken stocks. Six days post-challenge, the birds were sacrificed, and cecal contents were plated onto Campylobacter-selective media. Results from two replicate trials with three isolates of C. jejuni indicated that chicken stock A was colonized in only two of 60 ceca, stock B in six of 60, and stock C in 19 of 60 chicken ceca. Statistical analysis of these data indicate that resistance to cecal colonization by C. jejuni was significantly (P less than 0.05) influenced through chicken host lineage.     

Colonization characteristics of Campylobacter jejuni in chick ceca

We report our findings on several parameters influencing cecal colonization of chickens by Campylobacter jejuni. Thirty-five colony-forming units (CFU) of a composite culture of C. jejuni colonized the ceca of one-half of the newly hatched chicks challenged by oral gavage. A challenge dose of 3500 CFU/chick consistently colonized the ceca of all chicks challenged. Challenge doses of approximately 10(5) CFU of C. jejuni per chick resulted in consistent cecal colonization, regardless of whether the birds were challenged 1, 2, or 3 days post-hatch. Four isolates showed consistently strong cecal colonization abilities, whereas two isolates colonized the ceca in only 20 of 122 chicks when given levels of 10(5) CFU per chick. One of these poorly colonizing isolates was repeatedly transferred by fecal-oral passage through chicks; subsequently, this isolate was able to consistently colonize chicks. Competitive exclusion (CE) microflora did not diminish the colonization rates for C. jejuni. Birds treated with five different CE cultures were colonized at a rate of 81 of 84 chicks; control chicks were similarly consistently colonized (45 of 46 chicks).     

Scanning electron microscopy of the cecal mucosa in Eimeria-tenella-infected and uninfected chickens.

Four major types of surface conformation were observed in the ceca of uninoculated control chickens. Spatulate villi were found in the cecal neck region, low ridges in the region where the neck expands, protruding collarlike structures in the mid cecal pouch, and flattened collars in the distal portion. In ceca infected with Eimeria tenella, there was some erosion and sloughing of the mucosal cells. These lesions were slight in the neck region, more severe in the dilated portion, most severe in the midregion, and moderate in the distal area. Oocysts were observed in the mucosal tissue of the cecal pouch.     

Toxicoinfectious botulism in commercial caponized chickens

During the summer of 2003, two flocks of commercial broiler chickens experienced unusually high death losses following caponizing at 3 wk of age and again between 8 and 14 wk of age. In September, fifteen 11-wk-old live capons were submitted to the Iowa State University Veterinary Diagnostic Laboratory for assistance. In both flocks, the second episode of elevated mortality was associated with incoordination, flaccid paralysis of leg, wing, and neck muscles, a recumbent body posture characterized by neck extension, and diarrhea. No macroscopic or microscopic lesions were detected in affected chickens. Hearts containing dotted blood and ceca were submitted to the National Wildlife Health Center in Madison, WI. Type C. botulinum toxin was identified in heart blood and ceca by mouse bioassay tests. Enzyme-linked immunosorbent assay tests on heart blood samples were also positive for type C. botulinum toxin. Clostridium botulinum was isolated from the ceca and genes encoding type C. botulinum toxin were detected in cecal contents by a polymerase chain reaction test. Chickens are less susceptible to botulism as they age, and this disease has not previously been documented in broilers as old as 14 wk of age. Wound contamination by spores of C. botulinum may have contributed to the unusually high death losses following caponizing.     

Evaluation of a Competitive Exclusion Culture and Megan Vac 1 on Salmonella Typhimurium Colonization in Neonatal Broiler Chickens

The present study was designed to evaluate the effect of individual or simultaneous application of 2 products, a competitive exclusion culture (CEC) or Megan Vac 1 (MV), for bioefficacy in reducing Salmonella Typhimurium (ST) cecal colonization in broiler chicks following experimental challenge. In experiment 1, CEC and MV were applied to day-of-hatch broiler chicks, and chicks were experimentally challenged with ST approximately 48 h later. In control chicks, ST was recovered at a level of 3.84 log₁₀ cfu/g following direct plating of cecal contents, and 12 of 19 (63.15%) cultured individual ceca were positive following selective enrichment. In chicks receiving CEC by spray application on day of hatch, a numerical reduction in ST recovered from cecal contents (2.63 log₁₀ cfu/g) and a significant reduction (P < 0.05) in recovery of ST from cultured ceca following selective enrichment (4 of 18, or 22.2%) was observed when compared with controls. Significant reductions in ST cecal colonization in chicks treated with MV alone were not observed in experiment 1. In experiment 2, chicks received day-of-hatch spray application of CEC alone, MV alone, or CEC and MV as a combined application immediately prior to or within 24 h of chick placement. When chicks were experimentally challenged with ST 48 h posthatch, significant reductions (P < 0.05) in cecal colonization by ST were observed with each experimental group when compared with nontreated controls. These data suggest that both commercially available products, alone or in combination, are efficacious in reducing cecal colonization in broiler chicks challenged with ST.     

Cecal spirochetosis caused by Brachyspira pilosicoli in commercial turkeys

Spirochetes that were identified as Brachyspira pilosicoli were present in the ceca of 7.5- to 18-wk-old turkeys with cecal spirochetosis and typhlitis. The identity of B. pilosicoli was confirmed on the basis of ultrastructural morphology of the cecal epithelium adherent microbes, immunohistochemical staining with a Brachyspira genus-specific monoclonal antibody, and amplification of a B. pilosicoli species-specific 16S ribosomal RNA (rrs gene) sequence by using the polymerase chain reaction and DNA obtained by laser-capture microdissection of the epithelium-adherent microbial fringe. To the author's knowledge, this is the first report of B. pilosicoli in the ceca of turkeys.     

Clostridium difficile-associated cecitis in guinea pigs exposed to penicillin

Penicillin treatment resulted in lethal hemorrhagic cecitis in seven of eight guinea pigs. Cecal contents at necropsy from all seven animals contained a cytopathic toxin which was neutralized by Clostridium sordellii and C difficile antitoxins. Bacteriologic cultural examinations of these specimens yielded penicillin-sensitive strains of C difficile which produced a similar or identical cytotoxin in vitro. Stools obtained before penicillin administration and cecal contents from control animals lacked a cytotoxin and cultures failed to yield C difficile. Intracecal injection of cell-free supernatant of C difficile broth cultures reproduced the lesion noted with penicillin treatment. These results implicate C difficile as an agent of penicillin-induced lethal hemorrhagic cecitis in guinea pigs.     

Natural and experimental infection of neonatal calves with Clostridium difficile

Clostridium difficile toxins were associated with calf diarrhea in a recent retrospective study; however, no causal relationship has been prospectively investigated. This infection study tested whether the oral inoculation of neonatal calves with a toxigenic strain of C. difficile (PCR-ribotype 077) results in enteric disease. Fourteen 6-24 h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4 x 10(8) +/- 3.5 x 10(8) cfu) (n = 8) or sterile culture broth (n = 6). Calves were euthanized on day 6 or after the onset of diarrhea, whichever came first. Fecal and intestinal samples were blindly cultured for C. difficile, and tested for its toxin A/B (C. difficile TOX A/B II ELISA, Techlab). PCR-ribotyping was used to compare inoculated and recovered isolates. Diarrhea was observed in all control calves and 3/8 of inoculated calves (p = 0.03), but it did not occur in calves that tested positive for C. difficile toxins. Fecal toxins were identified only from two controls. PCR-ribotyping confirmed the presence of C. difficile PCR-ribotype 077 in samples of all inoculated calves, but not from controls. The identification of five other PCR-ribotypes in 3/8 (37.5%) and 2/6 (33.3%) of inoculated and control calves, respectively, indicated early natural infection (< or = 24h of age). Five of 14 cecal samples had C. difficile (p = 0.01). In conclusion, the oral administration of C. difficile PCR-ribotype 077 to neonatal calves resulted in fecal/intestinal colonization but not in detection of toxins, or signs of enteric disease. Further studies are required to investigate the clinical relevance of C. difficile in calves.     

Biological control of Salmonella typhimurium in young chickens

The effect of dietary lactose and anaerobic cultures of cecal microflora of mature chickens on the colonization of young broiler chickens by Salmonella typhimurium was evaluated. Newly hatched chicks were given either no treatment (controls), anaerobic cecal cultures, lactose (2.5%) in the drinking water, or both anaerobic cultures and lactose. Chicks were challenged per os at 3 days of age with either 10(6) or 10(8) S. typhimurium resistant to nalidixic acid and novobiocin. On day 10, the cecal contents of the chicks were examined for S. typhimurium, pH, short-chained volatile fatty acids (VFAs), undissociated VFAs, and lactic acid. Chicks given either lactose alone or cecal anaerobes alone had significantly (P less than 0.05) fewer S. typhimurium recovered from their ceca than the controls. Chicks given the combination of dietary lactose and cecal anaerobes had significantly fewer S. typhimurium recovered from their ceca than the chicks given dietary lactose or cecal anaerobes alone. Chicks given lactose had significant (P less than 0.05) increases in the lactic acid concentration of their cecal contents. Increased lactic acid concentrations were directly correlated to decreased cecal pH values and caused a reduction in the total concentration of VFAs but a significant (P less than 0.05) increase in the undissociated form of some VFAs.     

Effect of feeding selected carbohydrates on the in vivo attachment of Salmonella typhimurium in chick ceca.

Two-day-old chicks were orally inoculated with 1 ml of Salmonella typhimurium (10(5) colony-forming units/ml) and divided into four groups. Three groups were fed 2.5% carbohydrates starting on day 1 (arabinose, galactose, and lactose), while the fourth group served as the control. Ceca were obtained from each group at 7, 14, and 21 days. At the end of 14 days, all three carbohydrates statistically reduced Salmonella recovery. However, lactose failed to reduce recovery between day 14 and day 21. Arabinose and galactose continued to show significant reductions of recovery through 21 days. No statistical difference was found between Salmonella recovery from whole ceca (with cecal material) and inverted ceca (washed free of cecal material).     

Pyogranulomatous typhlitis and hepatitis of market turkeys.

Pyogranulomatous lesions in the liver and cecum were observed in groups of 7-to-8-week-old market turkeys. Necropsy revealed multifocal lesions in the liver and the presence of cecal cores that, in some cases, distended the cecum to the point of rupture. Mortality was associated with the ruptured ceca. Two successive flocks also succumbed to these lesions. An epizootiological study was performed to determine the cause and/or initiation of lesions leading to the formation of cecal cores.     

Comparison of prophylactic or therapeutic dietary administration of capsaicin for reduction of Salmonella in broiler chickens

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.     

Immunization of young chickens by trickle infection with Eimeria tenella.

Immunization of chickens was attempted with low levels of Eimeria tenella oocysts (50 oocysts per day) over the first 1 or 2 weeks of life--the "trickle infection" (TI) method. When chickens were immunized by TI at 0-13 days of age, no cecal lesions and a reduced number of oocysts in ceca were observed after challenge at 17 days of age. TI at 0-6 days of age conferred better protection against challenge with E. tenella than did TI at 7-13 days. However, cecal lesions were observed in almost all of these chickens. These findings indicated that TI for 2 weeks (0-13 days of age) provided better immunity than TI for 1 week (0-6 or 7-13 days of age).     

Cecal and hepatic granulomas in chickens associated with Heterakis gallinarum infection

Heterakis gallinarum infection was detected in the ceca of chickens with cecal and hepatic granulomas found at slaughter. The chickens were diagnostic submissions from four small backyard flocks in Saskatchewan. Detailed dissection of some cecal granulomas in one flock and pepsin digestion of cecal granulomas in the three other flocks demonstrated the presence of the parasite within granulomas in three of the flocks. A possible causal relationship between Heterakis gallinarum and the cecal and hepatic granulomas is discussed.