Protection of chicks against salmonella infection induced by spray application of intestinal microflora in the hatchery

Summary

The efficacy of spray application of intestinal microflora of the adult bird in protection of broilers against salmonella infections was studied in 3 experiments under laboratory and field conditions.

In chicks treated soon after hatching (in the hatchers at approximately 30 per cent hatch or in the chick delivery boxes in the hatchery) with crop‐caecum homogenate or a mixture of aerobically and strict anaerobically cultured intestinal flora, very good results were achieved.

A very obvious protective effect against a high infection dose (3 × 10 c.f.u. S. infantis bacteria per chick) and complete protection against natural infection with different salmonella types was induced.

A significant improvement of growth rate was observed in broilers treated by spray in the hatchery and reared for 7 weeks under field conditions in an environment heavily contaminated with different salmonella types.     

Protection of chicks against salmonella infection induced by spray application of intestinal microflora in the hatchery

The efficacy of spray application of intestinal microflora of the adult bird in protection of broilers against salmonella infections was studied in 3 experiments under laboratory and field conditions. In chicks treated soon after hatching (in the hatchers at approximately 30 per cent hatch or in the chick delivery boxes in the hatchery) with crop-caecum homogenate or a mixture of aerobically and strict anaerobically cultured intestinal flora, very good results were achieved. A very obvious protective effect against a high infection dose (3 X 10(6) c.f.u.S. infantis bacteria per chick) and complete protection against natural infection with different salmonella types was induced. A significant improvement of growth rate was observed in broilers treated by spray in the hatchery and reared for 7 weeks under field conditions in an environment heavily contaminated with different salmonella types.     

Feed can be a source of Campylobacter jejuni infection in broilers

1. The aim was to determine the importance of a contaminated diet as a possible cause of Campylobacter jejuni infection in broilers.

2. This study evaluated the viability of C. jejuni in both starter and finisher diets and the interference from other mesophilic bacteria in this viability.

3. Starter and finisher samples of broiler diet were deliberately contaminated with 3 or 5 log CFU·g^?1 of C. jejuni (NCTC 11351) and then maintained at two different storage temperatures (25°C or 37°C) for 3 or 5 d.

4. C. jejuni survived during this period and, when inoculated at 10³ CFU·g^?1, multiplied with greater proliferation at a storage temperature of 37°C. There was no relationship between the amount of mesophilic bacteria and C. jejuni viability.

5. This study highlights the importance of the diet in the epidemiology of C. jejuni in broilers.     

Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A,serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD).

METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5x10⁹ colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR).

RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non- pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain.

CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

Efficacy of different microbial preparations for controlling Salmonella colonisation in chicks and turkey poults by competitive exclusion.

1. Control by competitive exclusion of intestinal colonisation by Salmonella infantis was studied in domestic chicks and turkey poults given a commercial product developed for use with chickens, compared with two similar preparations containing intestinal microorganisms from turkeys. 2. Each type of material protected both avian species when given orally before challenge; the degree of protection depended at least as much on the type of preparation as its host origin.     

Sources of salmonellae in an uninfected commercially-processed broiler flock.

Cultural monitoring was used to study the incidence and sources of salmonellae in a 4160 bird broiler flock during the growing period, transport and processing in a commercial plant. No salmonellae were isolated from any of 132 litter samples of 189 chickens cultured during the seven-week growing period, even though nest litter samples from four of the eight parent flocks yielded salmonellae and Salmonella worthington was isolated from the meat meal component of the grower ration. On arrival at the plant, 2/23 birds sampled carried S. infantis on their feathers, although intestinal cultures failed to yield salmonellae. Three of 18 processed carcasses samples yielded salmonellae (S. infantis, S. heidelberg, S. typhimurium var copenhagen). The most likely source of these salmonellae was the plastic transport crates, since 15/107 sampled before the birds were loaded yielded salmonellae (S. infantis, S. typhimurium). The crate washer at the plant did not reduce the incidence of Salmonella-contaminated crates, since 16/116 sampled after washing yielded salmonellae (S. infantis, S. typhimurium, S. heidelberg, S. schwarzengrund, S. albany).     

Effects of Hyperoxic Rearing Water on Blood Hemoglobin and Hematocrit Levels of Rainbow Trout

Abstract

Rainbow trout Oncorhynchus mykiss cultured under hatchery conditions were exposed to hyperoxia by injection of oxygen into the rearing water. In two separate studies of 67 and 73 d, mean hemoglobin of experimental fish reared at oxygen levels averaging 147–220% saturation was 7.8–8.7% lower than that of control fish reared at mean oxygen saturations of 66–74%. Mean hematocrit of experimental fish was also 3.1–5.7% lower than that of control fish. Fish reared in hyperoxic conditions did not differ in growth, survival, or behavior from control fish. Fish exposed to hyperoxia rapidly adjusted hemoglobin and hematocrit to levels similar to those of control fish after oxygen injection was stopped.     

Intestinal microflora: elimination of germfree characteristics by components of the normal microbial flora.

Strict anaerobic bacteria are the predominant constituents of the intestinal flora of animals and man, the commonly studied aerobic bacteria comprise only a small percentage of the total intestinal flora. Dominating anaerobes include eubacteria, Bacteroides, fusiformis, peptostreptococci, propionibacteria. The intestinal mucosal epithelia is covered with a mucous layer which is extremely rich in anaerobes. Various germfree anomalies may be selectively redressed by various species of the intestinal flora. Obligate and moderate anaerobes are the active strains in elimination of germfree characteristics, in reduction of enlarged cecum in germfree rodents. The metabolic activity of the intestinal bacteria has been discussed.     

Nitrovin and tetracycline: a comparison of their effect on salmonellosis in chicks

The effects of nitrovin and tetracycline on the elimination and persistence of Salmonella infantis in the intestine of broiler chicks were studied. Experiments were also carried out regarding the influence of these drugs on certain bacteria which tend to prevent the establishment of S. infantis in the intestines of chickens.

It was shown that chicks artificially infected with S. infantis and fed for 5 weeks with 20 ppm nitrovin in the food had a lower incidence of salmonellae than those similarly infected and fed with 100 ppm tetracycline or without any drugs in the food.

Ten ppm nitrovin in the food of artificially infected chicks did not prevent pre‐treatment with a culture of intestinal bacteria from having an inhibiting effect on the colonisation of the caeca with S. infantis.

The salmonella inhibiting effect of pre‐treatment of infected chicks with intestinal bacteria was not destroyed when the bacteria were cultured in vitro in the presence of 10 ppm nitrovin, but it was destroyed by both 100 ppm and 20 ppm tetracycline.     

The intestinal flora of the chicken in the period 2 to 6 weeks of age, with particular reference to the anaerobic bacteria

A study has been made of the intestinal flora of chickens aged between 2 and 6½ weeks. No major differences were found when rations containing 9 or 25% fish meal were compared or when two different sources of fish meal were used. Throughout the period investigated, it was confirmed that the lactobacilli are the only group of organisms generally present in the small intestine in numbers exceeding 104/g. Clostridia were regularly found at 102 to 104/g, but Clostridium welchii was isolated from only occasional samples.

In the caeca, the total number of bacteria present at 2, 3, 4 and 6½ weeks was about 1011/g. Of these it was generally possible to isolate more than 20% using an anaerobic roll‐tube technique and oxygen‐free CO2. Many different types of anaerobes were isolated including strictly anaerobic budding bacteria which had not previously been studied. During the growth of the bird the flora changed. Anaerobic streptococci (peptostreptococci) predominated at 2 weeks but gradually decreased so that by 6½ weeks these organisms formed only a small part of the flora. Many of the Gram‐negative non‐sporing anaerobes only appeared between the 4th and 6th weeks.

Throughout the 2 to 6½‐week period all the caecal samples examined contained 108 to 109 anaerobes/g which were able to attack uric acid and sometimes 1010/g. This property was shared by at least one group of peptostreptococci, four groups of Gram‐negative non‐sporing anaerobes, three groups of Gram‐variable or Gram‐positive anaerobic non‐sporing rods and several species of clostridia.     

Infectivity and persistence of an outbreak strain of Salmonella enterica serotype Typhimurium DT160 for house sparrows (Passer domesticus) in New Zealand

AIM: To examine the infective dose, incubation period and disease progression of an isolate of Salmonella enterica serotype Typhimurium definitive type 160 (DT160) originating from a naturally-infected house sparrow (Passer domesticus) during an outbreak of the disease in New Zealand.

METHODS: Thirty-six house sparrows captured from the wild and free of Salmonella spp were divided into six groups of six birds, housed individually, and inoculated orally with phosphate buffered saline (PBS) or 10¹, 10², 10³, 10⁵, 2 × 10⁸ colony forming units (cfu) of the outbreak strain of S. Typhimurium DT160. The birds were observed for 10 days for clinical signs and/or mortality, and faecal samples were collected to determine excretion of S. Typhimurium. The birds were eutha- nised 11 days post-inoculation (p.i.) and a wide range of tissue samples were collected for histopathological examination, and culture and typing of Salmonella spp. Macro-restriction profiling by pulsed-field gel electrophoresis (PFGE) using XbaI was performed for the epidemiological typing of S. Typhimurium DT160 isolates.

RESULTS: Mortality in house sparrows inoculated with S. Typhimurium DT160 was dose-dependent, and 2/6 birds inoculated with ¹⁰⁵ cfu and all six birds inoculated with 2 × 10⁸ cfu died during the study. Infected sparrows displayed few clinical signs, apart from diarrhoea and/or polyuria, fluffed plumage, and sitting on the floor of the cage. Faecal excretion of DT160 occurred briefly in two birds inoculated with 10² cfu and four birds inoculated with 10³ cfu, on most days in five birds inoculated with 10⁵ cfu, and continuously in six birds inoculated with 2 × 10⁸ cfu. DT160 was isolated from the livers of three birds which received 10³ cfu, five birds dosed with 10⁵ cfu, and all six birds given 2 × 10⁸ cfu. Following necropsy, histopathological lesions similar to those seen in the natural disease were observed in the liver or spleen of three birds which received 10³ cfu, and all birds dosed with ≥10⁵ cfu.

CONCLUSION: The results indicate that an isolate of S. Typhimurium DT60 originating from house sparrows in New Zealand is pathogenic to these birds and that the response is dose- dependent. The persistence and excretion of the pathogen may last for at least 10 days. This confirms that sparrows infected with DT160 could be a source of infection to humans and other in-contact animals.     

Salmonella Contamination of the Product and Environment of Selected Canadian Chicken Processing Plants

Of 345 market chicken carcasses received directly from selected processing plants across Canada, six yielded salmonellae. One of the plants submitted 122 carcasses none of which yielded these organisms. A second plant had one Salmonella-contaminated carcass among 20 which were examined. Each of these two plants was subected to detailed bacteriological examinations on four occasions.

In these detailed examinations a total of 175 samples or specimens for culture were taken from a variety of surfaces including vent areas of carcasses, operators' hands, equipment surfaces and from water in tanks of iced birds. Twenty-five (14 per cent) of the cultures yielded salmonellae and all but one of these were either S. oranienburg or S. infantis. Isolations were made during five of the eight series of examinations. The evidence indicated that Salmonella-infected flocks were frequently slaughtered and that Salmonella contamination could become widespread in the plant during processing. The organisms are apparently eliminated from all but a small percentage of the carcasses during processing but opportunities exist for recontamination during subsequent handling.

     

Comparison of the environmental survival characteristics of Salmonella Dublin and Salmonella Typhimurium

To examine possible correlations in bovine Salmonella isolates between environmental survival and serovar-associated epidemiological patterns, bovine field isolates of Salmonella serovars Typhimurium and Dublin (two each) were inoculated into bovine faeces slurry and tested monthly by culture for survival during a six-month period of storage at a variable ambient temperature in a disused animal transporter. Low moisture conditions, where the slurry was dried onto wooden dowels, increased detectable survival of a low-level inoculum by up to five months, compared with wet slurry. A more modest increase of survival time was seen with storage of wet slurry under refrigeration at 4°C. Under both dry and wet conditions, the concentration of culturable Salmonella Typhimurium declined at a slower rate than did that of Salmonella Dublin. Salmonella that was naturally contaminating bovine faeces from farms with Salmonella Typhimurium did not show superior survival times compared with Salmonella Typhimurium that had been artificially inoculated into samples. The differing survival characteristics of the two serovars that was observed in environmental faeces may complement their different modes of infection in cattle. Salmonella Dublin, being a bovine host-adapted strain that establishes chronic infection in some animals, may have less need to survive for a prolonged period outside of its host than does Salmonella Typhimurium.     

Pathogenesis of Enteric Septicemia of Channel Catfish,Caused by Edwardsiella ictaluri: Bacteriologic and Light and Electron Microscopic Findings

Abstract

To clarify early events in the pathogenesis of enteric septicemia of catfish, 140 channel catfish Ictalurus punctatus (8–10 months old) were each infected with approximately 1.0 × 10⁹ colony-forming units of Edwardsiella ictaluri by intragastric intubation. Fish were sacrificed at 0, 0.25, 0.5, 1, 3, 6, 12, 24, 48, 72, 96, and 120 h postinfection (PI). Multiple tissue samples at all scheduled sampling times were evaluated by gross observation, light and electron microscopy, and immunohistochemical methods. In addition, at each sampling time, stomach, intestine, trunk kidney, and liver were cultured to quantitate bacteria. Trunk kidney cultures were positive by 0.25 h PI, indicating rapid transmucosal passage. In the intestine, E. ictaluri was seen in contact with the brush border at 0.5 h PI. Also at 0.5 h PI, dilated basilar cells with large intracytoplasmic inclusions were observed adjacent to the basement membrane. From 1 to 3 h PI, occasional necrotic enterocytes were seen on tips of intestinal folds. Proprial leukocytes were rare before 24 h PI but common thereafter. Immunoelectron microscopy showed E. ictaluri in vacuoles within phagocytes as early as 24 h PI in the intestinal mucosa. In other tissues, earliest observed microscopic lesions (48 h PI) consisted of bacteria within vacuoles of phagocytic cells contained within blood vessels. Bacteria were also seen within degenerate vacuoles in enterocytes and hepatocytes at 72, 96, and 120 h PI. This study confirms that E. ictaluri can invade channel catfish within 0.25 h PI by crossing the intestinal mucosa and suggests that the bacterium may have invasion and survival strategies similar to those of other enteroinvasive members of the Enterobacteriaceae.     

Differences in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella enteritidis and other Salmonella serovars

In Experiment 1, mature laying hens were inoculated intravaginally with 10(6) colony-forming units of Salmonella enterica serovar enteritidis (S. enteritidis), Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to compare their abilities to colonize the reproductive organs of chickens and to contaminate eggs. Salmonella enteritidis was more frequently recovered (from 11 of 40 eggs, 27.5%) than the other serovars, and especially the inner shell was contaminated with these organisms in 10 of 40 eggs (25.0%). The contamination rates and the viable counts in cloaca were significantly (P < 0.05) higher in hens inoculated with S. enteritidis than in those inoculated with the other serovars at 4 days postinoculation (PI). In the vagina, the positive rates were 90%-100% in hens inoculated with S. enteritidis, and the viable counts of the organisms in this portion were significantly (P < 0.05) higher than those of the other serovars at 2, 4, and 7 days PI. The ceca were colonized similarly by each serovar at 7 days PI. The spleen and ovary were infected with S. enteritidis in three and one hen, respectively. No Salmonella was recovered from liver and peripheral blood in any hen. Salmonella enteritidis was recovered from other oviductal portions than the vagina (10%-20%), whereas no forming egg was contaminated in the oviduct. In Experiment 2, the in vitro adherence of these six serovars to the vaginal epithelium was compared with vaginal explants. The mean number of S. enteritidis attaching to the secondary villi in the vaginal lumen was significantly (P < 0.05) higher than those of the other serovars. These results suggest that S. enteritidis has a specific advantage over the other Salmonella serovars by its capacity to colonize the vaginal tissues of hens, and this higher affinity of S. enteritidis to the vagina may play a significant role in the production of many S. enteritidis-contaminated eggs.     

Health screening for a translocation of captive-reared tuatara (Sphenodon punctatus) to an island refuge

AIMS: To screen tuatara undergoing translocation from a captive crèche to an island refuge for evidence of health and known diseases, and apply basic epidemiological techniques to assess the significance of disease test results.

METHODS: Tuatara (n=353) were physically examined and samples were taken from a random selection (n=30) for estimated white cell counts, screening for haemoparasites, and culture for Salmonella, Yersinia, Aeromonas and Campylobacter spp. Direct faecal smears were carried out on-site, and faecal floats were later performed to assess levels of endoparasitism with helminths and protozoa (n=69). Modified Ziehl-Neelsen staining was used to screen faecal smears, and positive specimens were further screened using an immunofluorescence antibody (IFA) test for Cryptosporidium oocysts.

RESULTS: There was no evidence of external parasites on any of the animals examined and only one animal had a gross abnormality. All estimated white cell counts were in the range 2.8– 17.5 × 10⁹/L. No haemoparasites were observed. There were no enteric pathogens cultured, indicating the intestinal carriage of these bacteria in the tuatara was 9.4%. Of the 69 individual faecal samples examined, 12 (17%) had unidentified coccidial oocysts, 21 (30%) had nematode ova of various kinds, and 12 (17%) had intestinal carriage of motile protozoa consistent with Trichomonas spp and another unidentified organism. Nineteen (28%) tuatara had acid-fast oocysts present; however, IFA staining failed to detect any Cryptosporidium oocysts.

CONCLUSIONS: Our understanding of the diversity of gastrointestinal endoparasites affecting tuatara is inadequate as many of the parasite ova seen could not be identified. This is the first record of tuatara as a host for Trichomonas spp of protozoa in the gastrointestinal tract.     

Prevention of the growth of Salmonella infantis in chicks by the flora of the alimentary tract of chickens

The effect on the growth of Salmonella infantis in the caeca of chicks pre‐treated with either the cultured flora of the alimentary tract of an adult chicken, horse faeces or bovine rumen fluid was studied. Forty 2‐d‐old chicks were inoculated orally with S. infantis. Pre‐treatment with the cultured flora of the alimentary tract of an adult chicken as well as fluids from the alimentary tract prevented the colonisation of the caeca by S. infantis. The numbers of salmonella isolated from the caeca of all chicks with no pre‐treatment were more than 10⁷ per g. Bovine rumen fluid and horse faeces were ineffective in preventing the caeca of chicks being colonised by S. infantis.     

Hot air treatment for surface decontamination of table eggs experimentally infected with Salmonella,Listeria, and Escherichia coli

Hot-air pasteurization was investigated in the EU-funded project “Reducing Egg Susceptibility to Contaminations in Avian Production in Europe (RESCAPE)” as an innovative treatment for surface bacterial decontamination of table eggs. Possible side effects of the treatment on egg quality traits were also studied. The decontamination power of hot air was evaluated over 1 month on shell eggs that were experimentally inoculated with Salmonella enteritidis, Escherichia coli, or Listeria monocytogenes. The S. Enteritidis and L. monocytogenes populations on the surfaces of treated eggs showed a significant reduction compared with untreated eggs. No statistically significant results were obtained comparing E. coli loads on treated and untreated eggs. No detrimental effects on quality traits either immediately after treatment or after 28 days of storage at 20°C were recorded.

     

Surveillance of Francisella tularensis in surface water of Kurdistan province,west of Iran

BackgroundThe etiologic agent of tularemia, Francisella tularensis, is transmitted to humans via ingestion of contaminated water or food, arthropods bite, respiratory aerosols, or direct contact with infected animals body fluids or tissues. In the current study, due to the importance of water in transmitting the disease and the report of the disease in different regions of Iran, surface water of Kurdistan province were evaluated for the presence of F.tularensis.Materials and MethodsSampling was carried out in five-counties of Kurdistan province. Sixty-six specimens of surface water were collected. The detection was carried out by targeting ISFtu2 and fopA genes using TaqMan real-time PCR. Moreover, the samples were both cultured and inoculated into NMRI inbreed mice. Spleens of inoculated mice and bacterial isolates were tested by TaqMan real-time PCR.ResultsDespite the lack of isolation of F. tularensis, the results of the molecular testing indicate the presence of bacteria in surface water. Molecular positivity of one sample (1.51%) was confirmed using a real-time PCR for both ISFtu2 and fopA genes. Moreover, 4.54% of the samples were positive for ISFtu2.ConclusionSince the in vitro isolation of bacteria from environmental samples is associated with a very low success rate and depends on various environmental parameters, the use of molecular techniques for monitoring of the bacteria in the contaminated areas is fully recommended.     

The role of rpoS,hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H₂O₂, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGΔ3 (SGΔrpoSΔhmpΔssrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGΔ3 mutant did not cause any mortality after inoculation with either 1 × 10⁶ or 1 × 10⁸ colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGΔ3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGΔ3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGΔ3 could be a promising candidate for a live Salmonella vaccine against FT.