A cross-sectional study to investigate the occurrence and distribution of intestinal spirochaetes (Brachyspira spp.) in three flocks of laying hens

A cross-sectional study was conducted on a commercial egg-producing farm with a history of wet litter. A total of 600 fresh caecal faecal samples were obtained from under cages of laying hens in three sheds each containing flocks of approximately 5400 hens. Samples were cultured for intestinal spirochaetes, and growth on the primary isolation plate was observed under a phase contrast microscope and subjected to PCRs specific for the intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli. Spirochaete isolates obtained in pure culture were assessed for their ability to cause haemolysis on blood agar and to produce indole, and were typed using pulsed field gel electrophoresis (PFGE). A 1250 base pair portion of the 16S rRNA gene of three B. intermedia and five unidentified isolates was sequenced, and the sequences compared with those of other Brachyspira species. Overall, 121 (20.2%) of the faecal samples contained spirochaetes as determined by growth on the plate and microscopy. Using PCR on the primary growth from these positive samples, 43 (7.2% overall) were shown to contain B. intermedia, 8 (1.3%) to contain B. pilosicoli, and 70 (11.7%) were PCR negative. Only 24 isolates of B. intermedia and five isolates of unknown species were obtained in pure culture. Comparative analysis of the 16S rRNA gene sequence identified the non-B. intermedia isolates as belonging to the proposed species "Brachyspira pulli". PFGE analysis of the B. intermedia strains identified them as having four major banding patterns. Individual patterns were found in hens from different flocks, suggesting cross-transmission of strains between flocks. No environmental sources of infection were identified. The youngest flock had a significantly lower level of colonisation with B. intermedia than the flock of intermediate age (P = 0.004), suggesting that following initial infection of individual young hens on this farm there was amplification and transmission of infection amongst members of the flock.     

Identification of genes associated with prophage-like gene transfer agents in the pathogenic intestinal spirochaetes Brachyspira hyodysenteriae, Brachyspira pilosicoli and Brachyspira intermedia

VSH-1 is an unusual prophage-like gene transfer agent (GTA) that has been described in the intestinal spirochaete Brachyspira hyodysenteriae. The GTA does not self-propagate, but it assembles into a virus-like particle and transfers random 7.5kb fragments of host DNA to other B. hyodysenteriae cells. To date the GTA VSH-1 has only been analysed in B. hyodysenteriae strain B204, in which 11 late function genes encoding prophage capsid, tail and lysis elements have been described. The aim of the current study was to look for these 11 genes in the near-complete genomes of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. intermedia HB60. All 11 genes were found in the three new strains. The GTA genes in WA1 and 95/1000 were contiguous, whilst some of those in HB60 were not-although in all three strains some gene rearrangements were present. A new predicted open reading frame with potential functional importance was found in a consistent position associated with all four GTAs, located between the genes for head protein Hvp24 and tail protein Hvp53, overlapping with the hvp24 sequence. Differences in the nucleotide and predicted amino acid sequences of the GTA genes in the spirochaete strains were consistent with the overall genetic distances between the strains. Hence the GTAs in the two B. hyodysenteriae strains were considered to be strain specific variants, and were designated GTA/Bh-B204 and GTA/Bh-WA1 respectively. The GTAs in the strains of B. intermedia and B. pilosicoli were designated GTA/Bint-HB60 and GTA/Bp-95/1000 respectively. Further work is required to determine the extent to which these GTAs can transfer host genes between different Brachyspira species and strains.     

Dietary enzyme and zinc bacitracin reduce colonisation of layer hens by the intestinal spirochaete Brachyspira intermedia

Brachyspira intermedia strain HB60 was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 control birds were sham-inoculated with sterile broth. All chickens received an experimental layer diet based on wheat. The infected birds were randomly divided into four groups of 10, with the diet for each group containing either 50 ppm zinc bacitracin (ZnB), 100 ppm ZnB, 256 ppm of dietary enzyme (Avizyme), 1302), or no additive. Birds were kept for 6 weeks after infection, and faecal excretion of B. intermedia, faecal water content, egg numbers, egg weights and body weights were recorded weekly. Control birds remained uninfected throughout the experiment. B. intermedia was isolated significantly less frequently from the groups of experimentally infected birds receiving ZnB at 50 ppm or enzyme than from those receiving 100 ppm ZnB or no treatment. Infected birds had a transient increase in faecal water content in the week following challenge, but no other significant production differences were detected amongst the five groups of birds in subsequent weeks. It was not established how the ZnB at 50 ppm and the dietary enzyme reduced the ability of the spirochaete to colonise, but it may have been by bringing about changes in the intestinal microflora and/or the intestinal microenvironment.     

The use of in-situ hybridization for the detection of Brachyspira spp. in pigs

Diagnosis of Brachyspira infections in swine and the differentiation of the involved bacteria is time-consuming and in most cases unsatisfactory. Detecting Brachyspira directly in the damaged Brachyspira of the large intestine could provide a direct correlation between histological lesionsa and bacterial growth.In this study we investigated whether in-situ hybridization (ISH) with a digoxigenin-labeled RNA-probe is a suitable method for detecting Brachyspira in the mucosa of the large intestine. Formalin-fixed and paraffin-embedded tissue sections of the large intestine from 78 pigs, which showed macroscopic and histological findings of Brachyspira-associated colitis, were stained with hematoxylin and eosin and Warthin-Starry silver impregnation and subjected to ISH. We used a RNA-probe with a length of 334bp, complementary to a part of the 23S rRNA of all members of the genus Brachyspira. All sections were treated with this anti-sense probe and with a sense control probe. 64 samples (82%) showed clearly positive ISH signals. Thus ISH is a suitable method for detecting Brachyspira directly within the lesions of the large intestine. The quantity of Brachyspira identified by ISH was always lower than by Warthin-Starry staining. Whether this reflects lower sensitivity of the ISH technique, or the fact that other bacteria with morphological similarities to Brachyspira were also stained by Warthin-Starry is unknown as yet. The present investigations provide a basis of further research developing specific probes to distinguish between pathogenic and non pathogenic Brachyspira species and probes detecting other bacteria with morphological similarity to Brachyspira.     

Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems

Several species of intestinal spirochaetes, Brachyspira (B.) alvinipulli, B. intermedia and B. pilosicoli, may cause reduced egg production and faecal staining of eggshells in chickens. The aim of this study was to characterize potentially pathogenic and presumably non-pathogenic Brachyspira spp. from commercial laying hens. Selective culture, phenotyping, PCR and 16S rRNA gene sequencing were used and clinical data were collected. Phenotypic profiles were obtained for 489 isolates and 351 isolates obtained after subculture, and 30 isolates were selected for molecular characterization. Seven isolates were positive by a B. intermedia-specific PCR based on the nox gene, and two were positive in a B. hyodysenteriae-specific 23S rRNA gene based PCR. By comparative phylogenetic analysis in combination with PCR and phenotyping, seven isolates were identified as B. intermedia, eight isolates as B. innocens, five as B. murdochii, and three isolates each as B. alvinipulli and "B. pulli". The remaining four isolates could not be assigned to any presently recognized species. Co-infection with several species or genetic variants of Brachyspira spp. were detected in some flocks and samples, suggesting a high level of diversity. Organic flocks with access to outdoor areas were at higher risk (RR=2.3; 95% CI 1.5-3.6) for being colonized than chickens in other housing systems. No significant differences between colonized and non-colonized flocks were found regarding clinical parameters, i.e. mortality, egg production, faecally contaminated eggshells, and wet litter. Our results show that a combination of traditional laboratory diagnostics, molecular tests and phylogeny is needed for identification of Brachyspira sp. from chickens.     

Tracing the emergence of drug-resistance in coccidia (Eimeria spp.) of commercial broiler flocks medicated with decoquinate for the first time in the United Kingdom

Decoquinate is a quinolone coccidiostat introduced during 1967 as an in-feed prophylactic for broiler chickens. Despite early drug-resistance problems and its age, the drug is still used commercially worldwide. Decoquinate here serves as a valuable model in a field study that addresses the dynamics and economic impact of the development of coccidial resistance to potent synthetic anticoccidial drugs. The results of this unique, hitherto unpublished, study on the initial emergence of resistance of avian coccidia (Eimeria spp.) to a new drug in the field may be of strategic value in the continued use of decoquinate or the introduction of new drugs. The commercial performance of the first 3-5 crops of broilers to be medicated with decoquinate on each of six farms was monitored during 14 months in 1968-1969, supplemented by assessments of the species, population dynamics and decoquinate-resistance of coccidia isolated from each farm. During the rearing of each flock in a single shed on each farm, oocysts were counted in fresh faecal samples collected on three occasions, and the species were identified by their morphology if possible, supported if necessary by the biological characteristics of infections in chickens. E. acervulina was the most common species, followed by E. mitis, E. maxima, E. tenella and E. praecox. E. brunetti occurred rarely, and E. necatrix was not found. Decoquinate-resistance was evident in several species during the rearing of the first decoquinate-medicated crop on each farm, although clinical coccidiosis did not occur. It was concluded that inherently resistant mutants of E. acervulina, E. brunetti, E. maxima, E. tenella, and probably also E. mitis and E. praecox, were selected from field populations by 6 weeks during their first exposure to decoquinate. During up to four more subsequent crops, cycling of resistant parasites stimulated host immunity, which had no obvious adverse impact on commercial performance. There was no apparent seasonal effect. A hypothesis is proposed to explain the sudden and rapid emergence of quinolone-resistance in the coccidia, and why bird health was not thereby compromised in these circumstances.     

Effects of commercial enzyme preparations on egg and eggshell quality in laying hens

1. Four different commercial enzyme products were added to standard commercial layer diets, based on barley, wheat or triticale. 2. Diets were fed to 4 different strains of commercial laying hen: ISA Brown, Hy-Line CB, Tegel SB2 and Tegel HiSex. 3. Diets were given for 5 weeks prior to measurements of egg and eggshell quality. 4. The inclusion of commercial enzyme products in the diets had no effect on egg weight. 5. Positive effects of the enzymes were: improved eggshell breaking strength, shell weight, percentage shell and shell thickness for the barley-based diet and increased eggshell breaking strength for two of the enzymes with the wheat-based diets. 6. Negative effects of the enzyme products were slightly lighter coloured eggshells and reduced albumen quality for the barley-based diet and for two enzymes with the wheat-based diet. 7. The addition of commercial enzyme preparations had no main effect on egg and eggshell quality for the triticale-based diet, except for effects on yolk colour. 8. Yolk colour was lower than control for enzyme 3 in all diets and for enzyme 4 with the barley- and wheat-based diets. 9. Further investigation is required to elucidate the specific enzyme components that influence egg quality.     

Natural presence of Campylobacter spp. in various internal organs of commercial broiler breeder hens

Campylobacter are known to cause acute bacterial gastroenteritis in humans. Poultry products have been implicated as a significant source of these infections. Six experiments were performed to determine whether Campylobacter could be isolated naturally from the primary and secondary lymphoid organs, liver/gallbladder, and ceca of commercial broiler breeder hens. Broiler breeder hens were acquired from different commercial sources during the early, middle, and late lay cycles. The birds were euthanatized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, the thymus, spleen, and liver/gallbladder were aseptically removed prior to removal of the ceca. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. In this study Campylobacter were found in 11 of 43 thymii, eight of 43 spleens, four of 43 liver/gallbladders, and 30 of 43 ceca. Overall, 28 of 53 isolates from the above samples were Campylobacter coli and 25 of 53 isolates were found to be Campylobacter jejuni.     

Assessment of the effects of electromagnetic field modification on egg-laying hens in commercial flocks as indicated by production measures

OBJECTIVE: To assess the effects of electromagnetic (EM) field modification by use of Nufield EM field modification (NEFM) units on egg-laying hens in commercial flocks as indicated by production measures, including hen-day mortality rate (HDMR) and eggs per hen housed (EHH). ANIMALS: 16 commercial flocks of egg-laying hens. PROCEDURE: 5 caged commercial table egg layer flocks (Single Comb White Leghorns) successively housed at the same location during a 6-year period were exposed to NEFM.There were 7 hens/cage (317 cm2 of floor space/bird). At the same site, 11 concurrent non-NEFM-exposed flocks (4 genetically different strains) were sequentially housed. All 16 flocks underwent the same feed and management practices. For each NEFM- and non-NEFM-exposed flock, HDMR and EHH were compared with their respective national breeder goals (BG), defined as the reasonable genetic potential expressed under optimal management and environmental conditions. Furthermore, the HDMRs and EHHs of the NEFM- and non-NEFM-exposed flocks were compared. RESULTS: Mean HDMR and EHH of the NEFM-exposed flocks was 36.9% less and 4.96% greater than the relevant BG, respectively. Mean HDMR and EHH of the non-NEFM-exposed flocks was 12.6% and 0.49% greater than the relevant BG, respectively. Compared with the 11 non-NEFM-exposed flocks, the NEFM-exposed flocks collectively had a 47.6% decrease in HDMR and 1.33% increase in EHH. CONCLUSIONS AND CLINICAL RELEVANCE: Results strongly suggest that application of NEFM in commercial egg-layer flocks improves production measures, which has important welfare implications as well as gross economic advantage.     

Use of multilocus enzyme electrophoresis to distinguish clinically important strains of Staphylococcus intermedius from the skin of dogs

AIMS: To use multilocus enzyme electrophoresis to determine the genetic structure of Staphylococcus intermedius from normal skin of dogs and those isolated from a variety of disease conditions and to distinguish clinically important strains in dogs. METHODOLOGY: The diversity amongst 129 isolates of S intermedius from the skin and mucosa of 32 healthy dogs and 120 isolates from diseased sites in 120 individual dogs was examined using multilocus enzyme electrophoresis. Associations among ETs were examined to determine the diversity of isolates. RESULTS: Twenty two ETs were distinguished comprising 21 containing isolates from diseased sites and 11 containing isolates from normal dogs. The majority of isolates (171 of 249; 69% were located in two ETs (ET1 and ET 4), that were not distinguishable phenotypically. ET 1 contained 94 isolates (54 isolates from healthy dogs and 40 isolates from diseased sites) and ET 4 contained 77 isolates (46 from healthy dogs and 31 isolates from diseased sites). Further, 77.5% of isolates from healthy dogs were present in ET 1 and ET 4 and 59% of isolates from diseased dogs belonged to the same two ETs. There was only a small difference in genetic diversity among isolates taken from healthy dogs (11 ETs; H = 0.182) and those isolates taken from clinical specimens from diseased dogs (21 ETs; H = 0.218). Of the 21 ETs from diseased sites, ET 16 contained all six isolates from Staphylococcal Scalded Skin Syndrome in racing Greyhounds. CONCLUSIONS: The small difference in genetic diversity between isolates from the skin and mucosa of healthy dogs and isolates from various diseases, as well as the presence of the majority of isolates in two ETs, is consistent with the role of S intermedius as an opportunistic pathogen. The confinement of all Staphylococcal Scalded Skin Syndrome isolates within one ET is confirmation of this entity as a distinct disease of dogs.     

The specific antibody to Brachyspira aalborgi in serum obtained from a patient with intestinal spirochetosis

Serum obtained from a patient histopathologically diagnosed as intestinal spirochetosis was investigated serodiagnostically by agglutination test. B. aalborgi which is a human intestinal spirochete reacted strongly with the human serum, while B. pilosicoli which has potential pathogenicity to humans reacted with the serum, but as strongly and its titer was different than the other three species. On the other hand, intestinal spirochetes (Matsumoto isolates) were isolated from the biopsy samples of the patient. The morphological, biochemical, and genetic characteristics of the isolates were very similar to those of B. aalborgi. Furthermore, the protein profiles of the Matsumoto isolates were also similar to those of B. aalborgi but were different than those of B. pilosicoli and B. hyodysenteriae. The reaction profiles of the Matsumoto isolates in immunoblotting were relatively similar to those of B. aalborgi except for a 74 kDa band but were different from those of B. pilosicoli and B. hyodysenteriae. Therefore, we identified the Matsumoto isolates as B. aalborgi and diagnosed the patient with a B. aalborgi infection.     

The shape of the response curve relating protein intake to egg output for flocks of laying hens

1. Two experiments were conducted to test the hypothesis that, as dietary protein concentration is increased, increments of response gradually diminish as egg output approaches an asymptotic value; and that the resulting response curve is consistent with a model (the “Reading model”) which assumes that the shape of the curve is a function of individual variation in body weight and potential egg output.

2. Each experiment involved a total of 3 888 laying pullets. Nine diets, with crude protein contents ranging from 87 to 212 g/kg, were used in the first experiment and ten diets, with protein contents ranging from 81 to 215 g/kg, in the second experiment. Diets were denned in terms of their calculated contents of essential amino acids and the same, well‐balanced amino acid profile was used at all protein contents within one experiment. Supplementary experiments were conducted to identify the limiting amino acid (s) in the experimental diets.

3. Treatments were applied for ten weeks, starting at 30 or 32 weeks of age. Average egg output on the highest protein diets was 57.0 g/bird d in the first experiment, which used White Leghorn pullets, and 53.7 g/bird d in the second experiment, which used a crossbred brown‐egg stock.

4. The response curves obtained showed diminishing returns and, in the second experiment, a clear plateau was reached. The Reading model was fitted and compared with a two‐straight‐line model and with curves derived from quadratic and inverse polynomial equations. Only the Reading model proved satisfactory for the interpretation of the data, as judged by goodness of fit and estimation of the optimum amino acid input. Although alternative models may provide a good fit when dealing with a single set of data, none of the alternatives is consistently satisfactory when considering the results of more than one experiment.     

The efficacy of three commercial Mycoplasma gallisepticum vaccines in laying hens

The efficacy of three commercial Mycoplasma gallisepticum (MG) immunizing agents-a bacterin, a recombinant fowlpox-MG vaccine, and a live F-strain vaccine-was compared in specific-pathogen-free hens in egg production. Three groups of 25 chickens were vaccinated with one of the vaccines at 10 wk of age and 25 birds were not vaccinated. At 25 wk of age (and approximately 50% egg production), 20 birds from each of the three vaccinated groups and 15 nonvaccinated controls were challenged with virulent R-strain via aerosol; the birds were necropsied and evaluated at 10 days post-challenge. The MG bacterin and live F-strain vaccinations were both protective and resulted in significant differences in air sac lesions, tracheal lesions, and ovarian regression compared to the nonvaccinated controls and the recombinant fowlpox-MG vaccine (P < or = 0.05). The evaluation of ovarian regression is a useful method of testing the efficacy of MG vaccines in laying hens.     

The use of selected plasma enzyme activities for the diagnosis of fatty liver-hemorrhagic syndrome in laying hens

Profiles of plasma enzymes were compared in two strains of single comb white leghorn laying hens, a normal commercial strain and strain UCD-003, which is highly susceptible to fatty liver-hemorrhagic syndrome. Plasma activity of lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) averaged 194 +/- 27, 4.0 +/- 2.8, 146 +/- 20, 1.0 +/- 1.0, and 1041 +/- 268 U/liter, respectively in normal birds. Activities of LDH, GDH, AST, and ALT, but not CK, were significantly higher in UCD-003 than in normal hens. A bimodal distribution of activities of all enzymes was found in the UCD-003 hens, with some birds showing activities comparable with those of the normal hens and others with values that were 2-10 times greater than those found in normal hens. These results are consistent with the extensive hepatic lesions observed in the UCD-003 strain of birds. Average gross hemorrhagic scores from visual inspection (scale of 0-3) were 0.28 +/- 0.45 in normal birds and 1.63 +/- 0.94 in the UCD-003 birds. Even though no clear relationship was found between plasma enzyme activities and the extent of liver hemorrhage in individual birds, the UCD-003 hens consistently had average values significantly higher for plasma enzymes that indicate liver damage. The results suggest that measurement of enzyme activities indicative of liver damage in birds, particularly AST, LDH, and GDH, is a valuable tool in the diagnosis of fatty liver-hemorrhagic syndrome in a flock of layers.     

The growth of Treponema hyodysenteriae and other porcine intestinal spirochaetes in a liquid medium.

A new simple method for the preparation of a liquid medium containing rabbit serum for the propagation of Treponema hyodysenteriae and other porcine intestinal spirochaetes is described. The medium, when dispensed in shallow layers and sealed under 10 per cent CO2 in nitrogen, had a redox potential not greater than -125mV and an initial pH of about 6.9 when buffered with bicarbonate. Growth of T hyodysenteriae developed more rapidly and viable counts reached higher levels at 42 degrees C than at 37 degrees C. Viable counts increased at least 10,000-fold after two to five days' incubation, depending on the temperature. Growth could be initiated from small inocula that failed to produce colonies on blood agar. Using a 1 per cent inoculum, the medium supported the growth of two strains of T hyodysenteriae through 10 serial passages.