Mark S. Chadfield

Comparison of intestinal invasion and macrophage response of Salmonella Gallinarum and other host-adapted Salmonella enterica serovars in the avian host.

The purpose of this investigation was to study the host specific infection of Salmonella Gallinarum in chickens and to determine the contribution of intestinal invasion and macrophage survival in relation to systemic infection in the host. This was carried out by comparing the kinetics of infection of S. Gallinarum to that of other Salmonella host-adapted (S. Cholerae-suis, S. Dublin and S. Typhimurium) and host-specific (S. Pullorum and S. Abortus-ovis) serovars. Establishment of the rate of colonisation in intestinal tissue, bursa and systemic sites was carried out by oral infection in day-old and week-old birds. Salmonella Gallinarum was the only serovar capable of causing systemic infection in chickens, however, general colonising ability in the intestine and bursa demonstrated no apparent selective advantage for S. Gallinarum. Further quantification of gastrointestinal invasion was carried out using ligated loops in the small intestine. Invasion in the jejunum of the chicken intestine over 3h demonstrated that Salmonella Typhimurium invasion was statistically higher (P<0.01) when compared with S. Gallinarum. Specific sites of high lymphoid tissue concentration in the chicken, including the bursa of Fabricius and caecal tonsils, were also targeted in invasion assays to investigate possible areas of tissue tropism. S. Typhimurium demonstrated significantly higher (P<0.01) invasion at these sites when compared with S. Gallinarum. Infection of chicken macrophages with S. Gallinarum did not demonstrate increased multiplication and survival intracellularly when compared with other Salmonella serotypes. The only difference seen was with S. Abortus-ovis, which demonstrated a significantly lower (P<0.05 to 0.001) intracellular survival. Together these data suggest that although S. Gallinarum host specificity in the chicken correlates with systemic infection, intestinal and lymphoid tissue invasion in the bursa and caeca, and macrophage survival does not influence this outcome.

Investigation of the parasitic nematode Ascaridia galli (Shrank 1788) as a potential vector for Salmonella enterica dissemination in poultry.

Abstract During recent years, the level of organically farmed poultry in Denmark has increased. Subsequent investigations have demonstrated an incidence of 64% of Ascaridia galli infections in layers established in organic farming systems. Studies to determine the interaction of Salmonella enterica with the parasitic nematode A. galli associated with poultry were undertaken to establish the significance of A. galli in the dissemination of S. enterica. A. galli was isolated from 40-week-old Lohmann Brown Salmonella-free layers. Worms were subsequently maintained in vitro and exposed to S. e. serovar Typhimurium at concentrations of 105–106 colony forming units/ml for varying times (24–144 h). Eggs were harvested aseptically from the worms and the associations of S. e. Typhimurium in relation both to the eggs and to structures on the surface of the worm were studied, using immunofluorescence, viable counts and in situ hybridisation. Results show attachment of S. e. Typhimurium to the outer coating of the eggs and possible internalisation. Evidence of association of the bacteria with the nematode eggs was further substantiated by establishing Salmonella infection in day-old chicks after dosing them with eggs harvested from parasitic worms infected in vitro with Salmonella.

Characterization of streptococci and enterococci associated with septicaemia in broiler parents with a high prevalence of endocarditis

Increased mortality due to septicaemia, where 29% of the affected birds had developed valvular endocarditis, was observed in a flock of broiler parents affected by myelocytomatosis. Bacterial investigations resulted in isolation of streptococci, the classification of which was unknown according to present taxonomy. Strains were isolated from the liver, spleen, heart and salpinx in clinical cases of septicaemia from the affected flock of Ross broiler parents aged between 26 and 56 weeks. Phenotypic characterization followed by genotypic investigation by ribotyping with HindIII and pulsed-field gel electrophoresis (PFGE) with SmaI demonstrated three ribotypes and six different PFGE profile types, respectively. Ribotype A with variant A1, together with the PFGE profile type represented by the three subtypes Ia, Ib and Ic, dominated the outbreak constituting 85% of the strains investigated, indicating clonality. 16S rRNA sequencing of strains representing this genotype demonstrated the occurrence of a recently described new Streptococcus sp., Streptococcus gallinaceus. Sequence analysis of the other genotypes demonstrated in the outbreak, resulted in identical 16S rRNA sequences to the type strain of Enterococcus faecalis. S. gallinaceus appears to represent a new opportunistic pathogen within the poultry industry.

Quantitative comparison of intestinal invasion of zoonotic serotypes of Salmonella enterica in poultry

The aim of the present study was to compare the invasion of selected zoonotic Salmonella serotypes of poultry in an in vivo chicken intestinal loop model and also in vitro in epithelial cell cultures. Invasion was measured relative to a reference strain, Salmonella Typhimurium 4/74 inv H201::Tn phoA . Two serotypes demonstrated intracellular log 10 counts that differed significantly from all other serotypes tested: Salmonella Enteritidis PT4 being 1.5 log 10 colony forming units (CFU) (31-fold) higher, and Salmonella Tennessee being 0.7 log 10 CFU (fivefold) lower than the reference strain ( P h 0.0001). A group of serotypes, which can be vertically transmitted, showed significantly higher intracellular counts (fourfold to eightfold) than the reference strain. The group included S. Typhimurium 4/74, S. Typhimurium DT104 (poultry and porcine isolates), S. Enteritidis PT1, S. Enteritidis PT6, S. Enteritidis PT8, and Salmonella Berta. The serotypes Salmonella Hadar, Salmonella Virchow, S. 4,12:b:-, S. Typhimurium DT41, and Salmonella Infantis, most of which are considered horizontally transmitted, did not show significantly different intracellular counts from the reference strain. Results from the cell culture invasion studies agreed with the in vivo data, with the exception of S. Berta and the poultry isolate of S. Typhimurium DT104.

An epidemiological study of Salmonella enterica serovar 4, 12:b:- in broiler chickens in Denmark

Epidemiological investigations of isolates of Salmonella enterica serovar 4, 12:b:- were carried out to establish particular molecular markers to assign isolates to a common origin. Plasmid profiling demonstrated that over 50% of 291 isolates, obtained between 1991 and 1996, were plasmid-free. The remaining isolates exhibited a common trend in plasmid content of 105 and 2kb. Although no specific correlation to any particular source within the poultry industry was discernible using plasmid analysis, there were indications of clonality with local divergence. Ribotyping with EcoRI demonstrated limited discriminative potential as 96% of the isolates expressed a common profile. Ribotyping with HindIII failed to further differentiate the isolates. IS200 (PstI) typing and PFGE (NotI and XbaI) afforded some degree of further discrimination with selected isolates. Each technique produced four profiles, but dominant profiles were also apparent. Eighteen variables were selected for multivariate logistic regression analysis in order to identify risk areas associated with broiler flocks within the industry. An increased risk for S. 4, 12:b:- infection was only associated with the feedmills used. Random effects at the house and/or farm level were also found to be statistically significant. Of the 16 feedmills associated with the isolation of 4, 12:b:-, six were deemed to be significant risk factors.

Characterization of Small-Colony Variants of Enterococcus faecalis Isolated from Chickens with Amyloid Arthropathy

ABSTRACT In this study we report the isolation and characterization of normal-sized and small-colony variants of Enterococcus faecalis from outbreaks of amyloid arthropathy in chickens. Postmortem examinations of 59 chickens revealed orange deposits in the knee joints, typical for amyloid arthropathy. Bacterial cultures from 102 joints and 43 spleens exhibited pure (n = 88) and mixed (n = 11) cultures of normal (n = 60) and pinpoint (n = 28) colonies of E. faecalis. Pulsed-field gel electrophoresis of 62 isolates demonstrated seven different band patterns with at most two band size variations, and multilocus sequence typing demonstrated two different sequence types, sharing six out of seven alleles, suggesting a close evolutionary relationship between isolates obtained from four outbreaks. In addition, all isolates were clonally related to an amyloid arthropathy reference strain from The Netherlands, previously shown to be globally dispersed. Initial investigation of the isolated small-colony variant phenotype revealed no difference in whole-cell protein profiling between normal and pinpoint colonies. However, the pinpoint colony isolates appeared to be more virulent in an in vivo challenge model in chickens than their normal-sized-colony counterparts. In addition, pinpoint morphology and associated slow growth were expressed without reversion after in vitro and in vivo passage, suggesting a genuine altered phenotype, and in some instances normal colonies converted to pinpoint morphology postinfection. In conclusion, small-colony variants of E. faecalis are described for the first time from veterinary clinical sources and in relation to amyloid arthropathy in chickens.

Characterization of Enterococcus hirae Outbreaks in Broiler Flocks Demonstrating Increased Mortality Because of Septicemia and Endocarditis and/or Altered Production Parameters

Abstract In Denmark, increased problems associated with streptococci and enterococci have been observed in broilers and broiler parent flocks, resulting in increased mortalities, uneven flocks, and subsequent downgrading and increased condemnations. Postmortem lesions associated with recent outbreaks due to Enterococcus hirae have been accompanied or dominated by septicemia and endocarditis. As a result of infection at an early age and relatively low mortality rates, outbreaks are not always clearly defined and may go unnoticed or may be attributed to poor chick quality. For the same reasons, the pathogenesis and epidemiology of observed outbreaks has only remained speculative. Four separate outbreaks associated with E. hirae infections in broiler flocks occurring between 1998 and 2002 have been investigated. Two of the outbreaks indicated evidence of two separate clones, with 89% and 79% of isolates involved in the individual outbreaks belonging to a single pulsed-field gel electrophoresis (PFGE) profile, respectively. Another outbreak (outbreak 4) demonstrated clear clonality, with all isolates demonstrating affinity to one of two PFGE profiles that differed by only two bands. However, all three outbreaks demonstrated a different clone. The remaining outbreak was nonclonal, with isolates distributed between six separate PFGE profiles. One of the outbreaks (outbreak 4) was descended from a parent broiler flock previously associated with an outbreak of Streptococcus gallinaceus; the flock also exhibited septicemia and endocarditis. Initial indications suggested the possibility of vertical transmission of S. gallinaceus to the current broiler flock, causing infection. By extended phenotypic characterization and subsequent genetic characterization, including 16S rRNA sequencing, all strains from the four outbreaks were confirmed as E. hirae. This investigation highlights the problems associated with characterizing enterococci infections in broiler flocks.

Geno- and phenotypic diversity of avian isolates of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis) and associated diagnostic problems.

ABSTRACT Recently, strains of Streptococcus bovis were reclassified as Streptococcus gallolyticus. In the present study we describe for the first time an outbreak of S. gallolyticus in a broiler flock. Mortality during the first week was normal (<1%), with a final total mortality at the end of production reaching 4.3%. Specific symptoms were not observed. Postmortem pathology demonstrated enlarged and light spleens and livers accompanied by multifocal irregular necroses surrounded by a hemorrhagic zone. In addition, these birds suffered from arthritis and osteomyelitis. Strains isolated from liver and spleen lesions showed clonality as demonstrated by pulsed-field gel electrophoresis. Compared to strains representing previously derived phylogeny, including the S. bovis-S. equinus complex, the 16S rRNA-derived phylogeny of the strains investigated in this study demonstrated a paraphyletic group (S. gallolyticus) well separated from two monophyletic groups: (i) S. equinus-S. bovis plus S. infantarius and (ii) S. alactolyticus plus S. intestinalis. According to information in GenBank, none of the strains included from the two monophyletic groups have been isolated from birds. Further biochemical analyses, including tannase activity, identified for the first time avian isolates belonging to S. gallolyticus subsp. gallolyticus. However, these investigations also demonstrated a clear heterogeneity with pigeon isolates.

Pathology, Tissue Metalloproteinase Transcription and Haptoglobin Responses in Mice after Experimental Challenge with Different Isolates of Pasteurella multocida Obtained from Cases of Porcine Pneumonia

Pasteurella multocida is a major cause of porcine pneumonia, but the pathogenesis of the disease is poorly defined. The aim of this study was to further understand the host response to infection by use of a mouse model of P. multocida pneumonia. Twenty female mice were divided into four groups (n=5). Three groups were infected with one of three isolates of P. multocida isolated from clinical cases of chronic porcine pneumonia with necrotizing, suppurative and non-suppurative lesions, respectively. The fourth group served as uninfected controls. Mice were killed 24 h postinfection and samples were collected for bacteriology, histopathology and in-situ hybridization for detection of P. multocida. Measurements of expression of genes encoding matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) in lung tissue and quantification of serum haptoglobin concentration were performed. P. multocida was found in the lung and spleen. Lung lesions were characterized by deposition of fibrin in alveoli and bronchioles, perivascular oedema, suppuration and necrosis. The cellular infiltration was mainly of neutrophils. Splenic neutrophilic infiltration was also evident. Minor differences in the severity and nature of lesions were seen according to the isolate of P. multocida used for infection. Intranasal infection of mice can therefore be used to evaluate the host response and lesions caused by P. multocida obtained from porcine pneumonic infections. The inflammatory response in this model is associated with increased tissue expression of genes encoding MMP9, TIMP1 and serum haptoglobin concentration.

Application of molecular methods for identification of strains classified as Salmonella enterica serovar 6, 7:-:- by conventional serotyping.

An increased prevalence of Salmonella enterica serovar Tennessee (6, 7: z 29 :-) was observed in broiler flocks in Denmark in 1994 and a parallel increase in the prevalence of Salmonella enterica serovar 6, 7:-:- was demonstrated, albeit at a lower level. Plasmid profiling and ribotyping revealed similar genotypes and it was speculated that serovar 6, 7:-:- could represent a non-motile variant of Salmonella Tennessee. Re-testing of the Salmonella 6, 7:-:- isolates demonstrated the presence of flagella through positive motility. All isolates but one demonstrated motility using both tube tests and light microscopy of overnight broth cultures. Molecular characterization indicated that all but two isolates previously classified as Salmonella 6, 7:-:, were isolates of Salmonella Tennessee and Salmonella Infantis, exhibiting reduced motility. Re-serotyping and multiplex polymerase chain reaction analysis for the phase 2 gene fljB demonstrated variants of Salmonella Infantis (6, 7: r: z 49 ) expressing the R-phase antigen (Rz 49 ) and possessing the gene for normal phase 2 antigen H: 1, 5. One of the two undefined strains demonstrated genotypic identity with a Salmonella Livingstone reference strain. The remaining putative 6, 7:-:- strain could not be identified and was genuinely non-motile. Diagnostic procedures performed initially were thus insufficient to differentiate between the different levels of motility and also resulted in mis-serotyping. As similar observations were made with two of 14 isolates received from a foreign laboratory, this may represent a general diagnostic problem.