Jens Christensen

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.

Investigations on the carrier rate of Pasteurella multocida in healthy commercial poultry flocks and flocks affected by fowl cholera

Twenty flocks of web-footed birds (Pekin and Muscovy ducks and geese) and eight flocks of chickens raised under intensive management were examined for the presence of carriers of Pasteurella multocida. Five hundred and seventy-eight web-footed birds and 240 chickens from healthy flocks, as well as from flocks affected by fowl cholera, were investigated. A total of 135 isolates (80 from healthy flocks and 55 from flocks affected by fowl cholera) were obtained from the pharyngeal and cloacal mucosae after mouse passage (134 isolates) and culture in selective medium (one isolate). Thirty-five percent (7/20) of the flocks of web-footed birds and 38% (3/8) of chicken flocks had birds carrying P. multocida in the pharynx and/or cloaca. Birds from flocks affected by fowl cholera carried P. multocida at a significantly higher prevalence in the mucosa of the cloaca (P < 0.001) compared with the pharynx, while the opposite was observed in birds from healthy flocks. Extended phenotypic characterization confirmed the presence of P. multocida ssp. multocida, P. multocida ssp. septica and P. multocida ssp. gallicida in the flocks examined. P. multocida ssp. gallicida was exclusively isolated from Pekin ducks, while P. multocida ssp. multocida and P. multocida ssp. septica were obtained from chickens as well as web-footed birds. Each flock was shown to be infected by a single phenotypic clone, but some clones were found in more than one flock. A different clone was found in each of four outbreaks of fowl cholera on one of the farms in the preceding 2 years. Two genotypic and phenotypic clones each of P. multocida ssp. multocida and P. multocida ssp. septica were found. This observation indicated that outbreaks are usually clonal and that elimination of P. multocida from infected farms is possible. The results suggest that healthy poultry, in addition to convalescent carriers, may also be carriers of P. multocida. However, the virulence of P. multocida isolates and resistance of carriers to clinical infection needs to be examined. This is the first report of isolation of P. multocida from the cloacal mucosa of apparently healthy domestic poultry. Sampling of the cloaca appeared to be more sensitive for detecting carriers of P. multocida. Although selective medium was used only to a limited extent, the results suggested that mouse inoculation was a more efficient method of isolating P. multocida from poultry than the use of selective media.

Genomic lineage of Salmonella enterica serotype Gallinarum

Forty-eight strains of Salmonella enterica serotype Gallinarum of biotypes Gallinarum and Pullorum were characterised by three chromosomally based typing methods. The patterns obtained were compared with those of strains of eight other serotypes of Salmonella of O serogroup D. The same PvuII and PstI IS200 patterns were commonly observed among strains of both biotypes and the three SmaI ribotypes of serotype Gallinarum strains differed in only one or two bands, supporting the view that members of these two biotypes are closely related. The same IS200 patterns were also commonly observed among strains of serotype Enteritidis, indicating its evolutionary relationship with serotype Gallinarum. NotI pulsed-field gel electrophoresis (PFGE) patterns divided strains into 24 types. Based on a similarity analysis, two clusters were formed. One contained the majority of biotype Gallinarum strains and two atypical strains of Pullorum; the other contained strains of biotype Pullorum and an otherwise typical strain of biotype Gallinarum. Two atypical strains of biotype Pullorum remained unclustered by PFGE analysis. The grouping of strains differed according to the typing method used, but the majority of strains within each of the biotypes Gallinarum and Pullorum were very similar by the chromosomal markers analysed.

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.

Relationships among Pasteurellaceae isolated from free ranging chickens and their animal contacts as determined by quantitative phenotyping, ribotyping and REA-typing.

One hundred and forty-three Pasteurella spp. strains and 10 unclassified strains obtained from free ranging poultry, dogs and cats were investigated by extended phenotypic characterization. One hundred and forty-nine of these strains were selected for further studies using ribotyping and REA-typing to evaluate the role of dogs and cats in Pasteurella multocida transmission. Seven and six type strains were included for comparison in phenotyping and genotyping, respectively. Eleven clusters and six unclustered strains were revealed by phenotyping. Ribotyping outlined 12 clusters and six unclustered strains. A correlation between clusters obtained by phenotyping and ribotyping was demonstrated which indicated that a genetic basis exists for clusters outlined by quantitative evaluation of phenotypic data. Similarities and differences in hosts, phenotype, ribotype, and zone of isolation were demonstrated among Pasteurella strains investigated. Isolates of P. multocida from ducks were shown to be clonal by both phenotyping and ribotyping. These strains were identical to one of the chickens strains. REA-typing, however, showed that the chicken strain was different underlining that exchange of clones of P. multocida between avian species rarely happens under village conditions. Management practise in the villages suggest the potential for exchange of P. multocida between poultry and animals kept in contact. The present findings, however, did not indicate that clones of P. multocida are widely exchanged between poultry and other animal species, even though close contact exists. In the present investigation exchange of clones of P. multocida was only demonstrated among animals belonging to the same species. Caution is drawn to the use of ribotyping as the sole method for epidemiological typing and tracing of P. multocida. The present results also underline the importance of proper phenotyping in the identification of P. multocida and related species.

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.

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.

Occurrence of Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania

Investigation was done to determine the presence of Pasteurella multocida and related species in free ranging chickens and ducks, dogs, cats and pigs in three climatic zones (cool, warm and hot) of rural Morogoro, Tanzania. A total of 153 isolates of P. multocida ssp. multocida and related species were obtained by direct culture on blood agar, selective medium and mouse inoculation. P. multocida ssp. multocida was isolated from 0.7% of chickens and 7% of ducks. In dogs and cats, P. multocida ssp. multocida was isolated from 1 and 68%, respectively. One isolate of Pasteurella gallinarum was isolated from a duck. Other species obtained were; P. multocida ssp. septica, Pasteurella stomatis and taxon 16 from dogs and cats, while Pasteurella dagmatis and Pasteurella canis were found in dogs only. Prevalence of P. multocida ssp. multocida was significantly higher (P<0.01) in ducks of the warm zone (22%) than in ducks of other zones (0%). No significant difference was observed between the prevalence of P. multocida ssp. multocida in chickens of the warm zone (2%) and chickens of the cool and hot zones (0%). Extended phenotypic characterization revealed phenotypic similarities between two isolates from chickens and the duck strains. Mouse inoculation appeared to be more sensitive in detecting P. multocida ssp. multocida than blood agar and selective medium. Direct culture on blood agar recovered most of the isolates from dogs. This study has demonstrated for the first time the presence of P. multocida and related species in the village free ranging chickens, ducks, dogs and cats in Tanzania. Other non-classified Pasteurella spp. were also observed in the study, but further characterization is required before the final classification can be made. This paper reports for the first time the isolation of unclassified Pasteurella from dogs and cats in Africa. The results implies that fowl cholera might be occurring in free ranging poultry, and dogs and cats kept in contact might serve as sources of P. multocida to chickens and ducks. Subsequent applications of molecular techniques to analyse the epidemiological relatedness of clones isolated from different host species is indicated.

Pasteurella multocida Infection in Heterophil-Depleted Chickens

Abstract The present study was aimed at elucidating the role of heterophil granulocytes during the initial infection with Pasteurella multocida subsp. multocida in chickens. Chickens (17 and 19 wk old) were depleted of their heterophil granulocytes by 5-fluorouracil treatment. When the heterophil blood counts were significantly reduced, the birds were inoculated intratracheally with 1.8–4.3 × 104 colony-forming units of P. multocida. Twelve, 24, or 48 hr postinoculation, the birds were euthanatized and examined for macroscopic and histologic lesions in the lungs. Bacterial invasion was determined by culture of P. multocida from the spleen. Recruitment of heterophils into the respiratory tract during infection was found to contribute considerably to the lung lesions in chickens and was found to mediate tissue damage, possibly allowing a more rapid systemic spread of P. multocida. However, during progression of the infection, the heterophil-mediated necrosis in chickens seemed to stimulate giant cell demarcation of infected lung tissue, which coincided with the clearance of P. multocida from the spleen, thus hampering further invasion. Consequently, heterophil activation plays a dual role for the outcome of a P. multocida infection in chickens, where it initially seems to promote invasion and systemic spread but subsequently helps limit the infection by giant cell formation and bacterial clearance.

Serum resistance of Pasteurella multocida in avian and porcine sera, and comparative virulence investigations of selected serum-sensitive and resistant strains in chickens.

Growth in serum of Pasteurella multocida and related species in chicken, turkey, duck and pig sera were compared, and selected serum-resistant and serum-sensitive strains were inoculated into 18-week-old layers. Eighty-seven field strains of Pasteurella spp. and nine reference strains representing different clones defined by restriction endonuclease analysis (REA) profiles were used in the study. Serum activity was measured by changes in the optical density (OD) of the serum after inoculation and incubation at 41°C for chicken, turkey and duck serum and 39°C for pig serum. Serum activity was measured by comparison with previously determined serum-resistant (P-1059) and serum-sensitive (CU vaccine) strains, and classified into highly serum-resistant, moderately serum-resistant and serum-sensitive. Strains of the same REA type were found to have identical growth curves and the same maximum OD values when tested in serum from the same host species. Turkey serum was shown to be less inhibitory to a wide range of P. multocida strains than chicken, duck and pig sera. Serum-resistant strains were demonstrated among avian as well as mammalian strains. Among the avian strains, the proportion of serum-resistant strains was higher in outbreak strains than in strains from apparently healthy carriers. Removal of the capsule from selected strains by hyaluronidase treatment failed to change the serum activity. The most severe lesions in experimentally infected chickens were produced by a serum-resistant strain; however, lesions were also found in chickens infected by serum-sensitive strains, indicating the involvement of multiple factors in the virulence of P. multocida . Further investigations on serum resistance are indicated in order to relate other host and bacterial factors responsible for the development of fowl cholera.