Microsphere uptake by the intestine of White Leghorn chickens

A study was conducted to determine the effective size for latex microsphere uptake in the intestine of white leghorn chickens. Three trials were conducted in which ligated intestinal segments of anesthetized 8-wk-old chickens were injected with 0.2-, 0.5-, 2-, 6-, 10-, or 20-mu diameter fluoresceinated latex microspheres. Microspheres were counted in brush border, epithelium, and lamina propria of each intestinal segment, liver, and spleen. After 1 hr, the 0.2-, 0.5-, and 2-mu microspheres were oriented along the brush border of epithelial cells and microsphere uptake into the epithelium and lamina propria was observed in the duodenum, ileum, cecum, cecal tonsil, and colon. Uptake of microspheres of 6, 10, and 20 mu diameter into epithelium and lamina propria was not observed in any intestinal segment. Also, no microspheres of any diameter were observed in sections of liver and spleen to suggest that there was no appreciable entry of microspheres into the bloodstream within 1 hr after administration. The results indicated that uptake of microspheres by the chicken intestine is a size-dependent process with microspheres < or = 2 mu being taken up to an equal extent by most segments of intestine.     

Dynamic surface tension properties of mixed lecithin-cholesterol films related to the respiratory mechanics

Chickens were housed when 1 day old with chickens experimentally infected with Mycoplasma synoviae. Air sacs from 16 of the 2-week-old and eight of the 3-week-old contact-exposed chickens were given gross lesion scores and embedded in glycol methacrylate for slide preparation. Histologic lesions in air sacs with gross scores of 0-2 were mild edema resulting in a two to eightfold increase in air sac thickness, capillary proliferation, and exudate consisting largely of heterophils and necrotic debris. Histologic lesions in air sacs with gross scores of 3 and 4 were marked hyperplasia of epithelial cells and diffuse infiltration of the air sac connective tissue by mononuclear cells. Nine of 10 air sacs with gross scores of 0 had no histologic evidence of inflammation. The most severe histologic lesions were in those air sacs with gross scores of 4. The glycol methacrylate procedure resulted in 2-mum sections with excellent cellular detail.     

CD56bright natural killer cell subsets: characterization of distinct functional responses to interleukin-2 and the c-kit ligand

Horizontal transmission of Salmonella enteritidis and the effect of airflow on spreading were examined in 80 5-wk-old chickens divided into five groups. Sixteen chickens in each group were placed in four cages in a row separated by wire. One among four chickens placed in a cage at the downwind end of the row was inoculated orally with 10(9) colony-forming units of S. enteritidis. Cecal droppings, drinking water, and feed were cultured every day. Horizontal transmission was rapid in the row with low air velocity but slow in the row with high air velocity. However, in another experiment, where the inoculated chicken was situated in a cage upstream in the airflow, horizontal transmission was equally rapid whether the airflow was rapid or slow. Contamination of feed and water never preceded the appearance of positive cecal droppings. These findings suggest that the rapidity of horizontal transmission of S. enteritidis may be affected by airflow patterns.     

Serological and bacteriological observations on experimental infection with Salmonella hadar in chickens

Over the past 3 years the frequency of Salmonella hadar infections has increased in Belgium in both poultry and humans. Therefore, the course of infection with S. hadar in poultry was investigated. One day-old and 4 week-old specific pathogen-free chickens were orally infected with one of two S. hadar strains, SH1 or SH2. Mortality was 6% (SH1) and 17% (SH2) in birds infected at 1 day-old. Chickens infected at 1 day-old with SH2 showed a mild diarrhoea. The S. hadar faecal excretion in birds infected at 1 day-old remained high throughout the experiment until 12 weeks post-inoculation (pi). Faecal excretion was lower in older birds. Antibodies to S. hadar were observed from 2 weeks pi (SH2, infected at 1 day-old) or 4 weeks pi (SH1, both groups; SH2, chickens infected at 4 weeks of age). The percentage of chickens with antibodies was higher after infection at 1 day-old than after infection at 4 weeks of age. In a second experiment 1 day-old chicks were infected with SH1 and autopsied at regular intervals until 42 days pi. SH1 was isolated from the caeca from 3 h pi onwards and from the liver and spleen from 18 h until 14 days pi. Serous typhlitis and omphalitis were the main lesions. The number of macrophages in the lamina propria of the caecal tonsils was slightly increased from 18 h until 2 weeks pi. In the liver, inflammation was observed in the portal triads and in the sinusoids. This study indicates that infections with S. hadar lead to intense colonisation of the gut and extensive faecal shedding. It may also cause invasive infections in 1 day-old chickens.     

Evaluation of the chicken crop as a source of Salmonella contamination for broiler carcasses

Much previously published research has focused on the role of cecal and intestinal Salmonella contamination of poultry carcasses within commercial processing plants. Presently, we have evaluated the persistence of experimentally inoculated Salmonella enteritidis in the crops and ceca of commercial broiler chickens during the last week of growth (Weeks 6 to 7) and the presence of crop and cecal Salmonella in 7-wk-old broilers in a commercial processing plant. When broilers were inoculated with 1 x 10(6) cfu S. enteritidis at 6 wk of age by oral gavage, the incidence of crop and cecal contamination was equivalent 2d after challenge (30%), with only 1 of 29 crops contaminated and 0 of 29 ceca contaminated at 7 d following challenge. When broilers were inoculated with 1 x 10(8) cfu S. enteritidis at 6 wk of age by oral gavage, 2 d after challenge the crops and ceca were observed to be 57 and 67% positive for S. enteritidis, respectively. Seven days after inoculation with 1 x 10(8) S. enteritidis, the crops and ceca were 37 and 57% positive, respectively, for the challenge organism. At a commercial broiler processing plant, 286 of 550 crops from three flocks were Salmonella-positive, whereas only 73 of 500 ceca from these flocks were contaminated. Furthermore, data from this plant indicated that the crops were far more likely to rupture than ceca (86-fold) during processing, increasing the possibility of carcass contamination with Salmonella derived from crop contents. The results of these studies suggest that the crop may serve as a source of carcass contamination with Salmonella within some processing plants.     

Efficacy of a live avirulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidis

An avirulent live delta cya delta crp Salmonella typhimurium strain chi 3985 that precludes colonization and invasion of chickens by homologous and heterologous Salmonella serotypes was evaluated for its long-term protection efficacy. Chickens vaccinated orally at 2 and 4 wk of age were assessed for protection against oral challenge with wild-type S. typhimurium and Salmonella enteritidis strains at 3, 6, 9, and 12 mo of age. A comparison of Salmonella isolation from vaccinated and nonvaccinated layers after challenge with S. typhimurium or S. enteritidis showed that delta cya delta crp S. typhimurium chi 3985 induced excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salmonella. The duration of protection lasted for 11 mo after vaccination, at which time the experiment was terminated. S. enteritidis and S. typhimurium were isolated from the yolk, albumen, and shells of eggs laid by nonvaccinated chickens challenged with Salmonella. S. typhimurium caused pathological lesions in nonvaccinated chickens, whereas vaccinated and nonvaccinated chickens challenged with S. enteritidis showed no pathological lesion in the visceral and reproductive organs. Vaccination with chi 3985 prevented transmission of S. typhimurium or S. enteritidis into eggs laid by vaccinated layers with no effect on egg production. To our knowledge, this is the first publication confirming that vaccination with live avirulent Salmonella can induce long-term protection against Salmonella infection in layers.     

Association between in vitro heterophil function and the feathering gene in commercial broiler chickens

We recently showed that in vitro heterophil functional efficiency in commercial broiler chickens is genetically controlled and may be a sex-associated trait. To further characterize the genetic mechanism(s) of heterophil functional efficiency, we wanted to determine whether the feathering gene, present on the Z sex chromosome, contributes to heterophil functional efficiency. Heterophils from two pairs of broiler lines were evaluated; each pair contained a fast feather (FF) (lines A and X) and a slow feather (SF) line (lines B and Y). On days 1 and 4 post-hatch, heterophils isolated from two sets of pure line broilers (A and B, and X and Y) were evaluated for their ability to (1) phagocytize Salmonella enteritidis, and (2) exhibit bactericidal activity against S. enteritidis. On days 1 and 4 post-hatch, heterophils isolated from the FF lines were statistically (P < or = 0.02) more proficient at phagocytizing S. enteritidis than heterophils from SF lines. Bactericidal activity was also statistically (p < or = 0.02) greater on day 1 post-hatch in the heterophils isolated from FF lines compared to heterophils isolated from SF lines. These data indicate that the presence of the FF gene locus on the Z sex chromosome contributes to heterophil function and may contribute to the early innate immune competence of a flock.     

Organization of the lamina propria mucosae of rat intestinal mucosa, with special reference to the subepithelial connective tissue

Light microscopy, scanning electron microscopy and transmission electron microscopy have been used to delineate the structure and function of the lamina propria mucosae in the rat jejunum. In silver-impregnated sections, the adepithelial surface of the lamina propria mucosae was framed by a sheet of reticular fibers (reticular sheet). Short-term (3-hour) immersion of jejunal tissues in 2 N NaOH solution enabled us to simultaneously view networks of reticular fibrils and fibroblasts residing in the subepithelial connective tissue under a scanning electron microscope. The reticular fibrils, which measured about 40 nm in diameter and were interwoven in dense networks, formed a sheet 2-3 microns thick. In the villi, this sheet contained numerous foramina ranging from 3 to 7 microns in diameter, through which lymphocytes, macrophages, basal extensions of epithelial cells and fat particles traversed. The reticular sheet in the domes of isolated lymphoid nodules was markedly porous, and many lymphocytes migrated into or out of the epithelium through the foramina. The formaina of the reticular sheet may participate in the communication between the intestinal epithelium and the lamina propria mucosae. It was noted that the foramina of the reticular sheet in the villi were surrounded by end feet of the cytoplasmic processes of fibroblasts. In addition, these fibroblasts were combined with lymphocytes or dendritic cells in the lamina propria mucosae.     

Claudication on mastication following bilateral external carotid artery ligation for posterior epistaxis

One-day-old specific-pathogen-free White Leghorn chicks were inoculated orally with Salmonella enteritidis phage type 4 to study adhesion and invasion of the ceca by immunohistochemistry. Positive staining bacilli were associated with the epithelial surface and were present in the lumen of the cecal crypts. They were observed in the interstitial tissue and in the cytoplasm of macrophage-like cells in the lamina propria. A granulomatous nodule containing positive staining bacilli was present in the submucosa of the cecum of one bird at 14 d after inoculation.     

Lamina propria T cell subsets in the small and large intestine of euthymic and athymic mice

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C.B-17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. CD3+ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20-30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the alpha beta lineage in euthymic mice, but only 40% were of the alpha beta lineage in athymic mice. T cells of the gamma delta lineage were thus more frequent than T cells of the alpha beta lineage in the intestinal lamina propria T cells of extrathymic origin. CD4+ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20-30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8+. In intestinal lamina propria T cell populations of athymic mice, the CD8+ T cell population was expanded. Most (60-70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8 alpha + beta- form of the CD8 coreceptor. A fraction of 15-20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were 'double negative' CD4- CD8-. A large fraction of the TCR alpha beta + T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM-1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.     

Gastric cryptosporidiosis in a wild frilled lizard from Australia

An adult male frilled lizard (Chlamydosaurus kingi) was found dead in Northern Territory (Australia). On physical examination it was found to be severely emaciated. At necropsy the stomach was found to be firm with mild thickening of the gastric mucosa. Gastric rugae were prominent and the mucosa was red with a thin layer of dark red mucus on its surface. Light microscopic examination revealed a mild diffuse gastritis with infiltration of the lamina propria by lymphocytes, plasma cells, and heterophils. Large numbers of small, round amphophilic to basophilic (2 to 4 microns in diameter) organisms morphologically consistent with cryptosporidia were seen on the surface of the mucosal epithelium and free in the gastric lumen. The gastric lesions seen in this frilled lizard did not involve atrophy, as previously described in lizards with gastric cryptosporidiosis, and were similar to those described in snakes. The possibility that more than one species of Cryptosporidium parasitizes reptiles could explain the different lesions. This is the first report of cryptosporidiosis in a frilled lizard.     

Epithelial antibiotics induced at sites of inflammation

The role of antimicrobial peptides in epithelial defense is not fully understood. An epithelial beta-defensin, lingual antimicrobial peptide (LAP), was isolated from bovine tongue and the corresponding complementary DNA cloned. LAP showed a broad spectrum of antibacterial and antifungal activities. LAP messenger RNA abundance was markedly increased in the epithelium surrounding naturally occurring tongue lesions. This increase coincided with the cellular hallmarks of acute and chronic inflammation in the underlying lamina propria, supporting a role for epithelial antimicrobial peptides as integral components of the inflammatory response.     

Identification of a new intestinal spirochete with pathogenicity for chickens

Two intestinal spirochete isolates obtained from chickens with diarrhea were examined by electron microscopy, biochemical tests, rRNA gene restriction pattern analysis, and multilocus enzyme electrophoresis. One isolate (strain 91-1207/C1) was pathogenicity tested in vivo in chickens. The chicken spirochetes were morphologically indistinguishable from Serpulina innocens and Serpulina hyodysenteriae and phenotypically similar to S. innocens. However, the chicken spirochetes could be distinguished from S. innocens, S. hyodysenteriae, and other swine intestinal spirochetes by rRNA gene restriction pattern analysis and multilocus enzyme electrophoresis. In pathogenicity tests in 1-day-old chicks and 14-month-old hens, chicken spirochete 91-1207/C1 produced pale-yellow, watery cecal contents and mild lymphocytic typhlitis. These findings support the conclusion that avian intestinal spirochetes can be pathogenic to commercial poultry and that the microorganisms are different from intestinal spirochetes that infect pigs.     

Cecal mucosal response to coccidiosis in growing chickens

The cecal mucosal changes of a subclinical coccidial (E. tenella) in chickens was studied by scanning electron microscopy. Oral inoculation was used and the mucosal surface of the ceca was studied. A control group from the same hatch of chickens was sampled simultaneously. The ceca from the infected birds was markedly smaller and contained some hemorrhagic areas. The control birds maintained a relatively smooth continuous epithelium throughout the study. During the infection, early fenestration was seen in the epithelium followed by its disruption. The crypts were easily seen as the disease progressed and in some cases the epithelium became denuded. The infective organism may inhibit replacement of degenerating epithelium.     

Cryptosporidium parvum initiates inflammatory bowel disease in germfree T cell receptor-alpha-deficient mice

Flora-bearing mice with targeted disruption of T cell receptor (TCR)-alpha or -beta genes spontaneously develop intestinal inflammation with features similar to ulcerative colitis in humans. TCR-alpha-deficient mice maintained germfree or colonized with a limited number of intestinal bacteria failed to develop inflammatory bowel disease (IBD)-like lesions. Evidently, inflammation in these mice does not develop spontaneously or result from a generalized antigenic stimulation, but rather requires induction by a heretofore unidentified specific stimulus. We describe the development of IBD-like lesions in germfree TCR-alpha-deficient mice monoassociated with the protozoan Cryptosporidium parvum. Lesions were seen in distal ileum, cecum, and colon and were most severe in the cecum. A prominent leukocytic infiltrate within the lamina propria was a common characteristic of the lesions observed in the C. parvum-infected germfree TCR-alpha-deficient mice. The leukocytic infiltrate was composed of aggregates of B220+ cells, the majority of which expressed surface IgD (ie, conventional B lymphocytes). It has been proposed that antigenic stimulation by a microorganism(s) is needed to initiate intestinal inflammation in TCR-alpha-deficient mice. Our results indicate that a single microbial species, C. parvum, is capable of triggering the development of IBD-like lesions in germfree TCR-alpha-deficient mice.     

B and also T lymphocytes migrate via gut lymph to all lymphoid organs and the gut wall, but only IgA+ cells accumulate in the lamina propria of the intestinal mucosa

In pigs the lymphocytes emigrating from the intestinal wall were collected by cannulating the lymphatics, labeled in vitro using a fluorescent dye and retransfused. The injection of 6.6+/-4.2 x 10(8) cells resulted in a labeling index between 1.5% in intestinal lymph, 0.2% in the spleen and lymph nodes, approximately 0.1% in the intestinal lamina propria and 0.003% in intraepithelial lymphocytes. About 25 % of the injected cells were present in the blood and 1 % was recovered in the lymph. T cells were found in similar proportions in the injected and the recovered cells in the organs (70-80%). The proportion of IgA+ cells among the immigrated cells in the intestinal lamina propria ranged from 5 to 8%, which in absolute numbers was up to 60% of the injected IgA+ cells. T and IgM+ cells did not show a higher accumulation in any organ. These experiments in conventional, unrestrained animals revealed that (1) T cells immigrate into the intestinal lamina propria, (2) preferential migration of IgA+ cells from gut lymph to the intestinal lamina propria is obvious under in vivo conditions and (3) the immigrated IgA+ cells represent a very small population which is difficult to detect when analyzed in relative numbers.     

Accelerated inflammatory bowel disease of TCR-alpha-deficient mice persistently infected with Cryptosporidium parvum

TCR-alpha-deficient mice spontaneously develop inflammatory bowel disease (IBD) at 8-9 mo old. This study characterizes an accelerated form of IBD induced by Cryptosporidium parvum infection. Cryptosporidium parvum-infected TCR-alpha-deficient mice developed IBD as early as 4 wk old when challenged at 1 wk old. The lesions of this accelerated IBD resembled the lesions of spontaneous IBD in TCR-alpha-deficient mice and consisted of a mononuclear cell infiltrate within the intestinal lamina propria and an increased proliferation of enterocytes. The mononuclear cells within the lamina propria consisted of B cells and gamma delta T cells. The distal ileum, cecum, and colon were grossly thickened due to a hyperplastic mucosa and edematous submucosa. The mechanism by which C. parvum infection accelerates development of IBD is presently unclear.     

Nonbursal origin of humoral immunity: immune capacity and cytomorphological changes in chickens bursectomized as 52- to 64-hours-old embryos

Chickens sham-bursectomized as 52- to 64-hours-old embryos (SBx), chickens bursectomized at hatching (NeoBx) and chickens from which the bursal primordium was removed at 52-64 h of embryonation (EBx) were immunized with guinea pig red blood cells when 21 days old. Following the second injection of antigen, EBx chickens were found to elaborate ME-sensitive and ME-resistant hemagglutinins, to produce direct and indirect plaque-forming cells, and to contain lymphocytes bearing a specific bursa (Bu) antigen. These immune properties were more often expressed in sham-bursectomized and neonatally bursectomized chickens. Cytomorphological changes in the spleen, bone marrow and cecal tonsil of EBx chickens were typical for birds lacking the bursa. The number of lymphocytes and plasma cells in the bone marrow and liver of SBx, NeoBx and EBx chickens was very low. These results suggest that a very early embryonic bursectomy of chickens does not abolish humoral immunity completely and does not prevent the differentiation of Bu antigen-bearing lymphocytes.     

Helper activity of chicken V beta 1+ and V beta 2+ alpha beta T cells for in vitro IgA antibody synthesis

Chickens have only two T cell receptor variable beta gene families: V beta 1 and V beta 2 (1). In our previous work we found that IgA production was almost completely suppressed in chickens depleted of V beta 1+ alpha beta T cells by treatment with a TCR V beta 1-specific monoclonal antibody (2), while IgM and IgG production was not affected. Our present results indicate that, in vitro, both V beta 1+ and V beta 2+ chicken cecal tonsil T cells provide help for the differentiation of cecal tonsil IgA B cells, suggesting that the failure of V beta 1+ T cell-depleted chickens to produce IgA is not caused by the inability of V beta 2+ T cells to provide help for IgA production by B cells, but rather by the scarcity of these T cells in mucosal tissues (3), where most IgA responses are induced (4).     

Resistance against Salmonella enteritidis cecal colonization in Leghorn chicks by vent lip application of cecal bacteria culture

Leghorn chicks were treated with cultures of cecal bacteria from adult chickens by crop gavage, upper body spray, or vent lip application on the day of hatch. The chicks were challenged orally with 10(4) Salmonella enteritidis (SE) at 3 d of age and evaluated for SE cecal colonization at 10 d of age. The concentration of volatile fatty acids (VFA) in the cecal contents was determined on the day after culture treatment and at 10 d of age. Compared with controls, SE colonization was significantly decreased in each of the treatment groups. Vent lip application of a single .05-mL drop of cecal bacteria culture resulted in resistance against SE challenge comparable to crop gavage or spray treatment with .5 mL of culture. Resistance to SE challenge was directly associated with the concentrations of total VFA and propionic acid in the cecal contents of the treated chicks on the day after culture treatment. The results indicated that cecal bacteria from adult chickens that increase SE colonization resistance may rapidly become established in the ceca of newly hatched chicks following contact with the vent lips.