Experimental infection by Yersinia ruckeri O1 biotype 2 induces brain lesions and neurological signs in rainbow trout (Oncorhynchus mykiss).

Pathological manifestations in rainbow trout (Oncorhynchus mykiss) following experimental waterborne infection with Yersinia ruckeri serotype O1 biotype 2 (strain 07111224) were investigated. Rainbow trout were exposed to 8 × 107  CFU/ml of Y. ruckeri by bath for 6 hr, and mortality was then monitored for 22 days post-infection (dpi). Organs were sampled at 3 dpi and also from moribund fish showing signs of severe systemic infection such as bleeding, exophthalmia or erratic swimming behaviour. Y. ruckeri was observed in the meninges and diencephalon of the brain, and lamina propria of olfactory organ at 3 dpi. At 12 dpi, Y. ruckeri had spread throughout the brain including cranial connective tissues and ventricles and the infection was associated with haemorrhages and an infiltration with leucocytes. Y. ruckeri infection and associated with leucocyte infiltration were observed at 13 dpi. In conclusion, Y. ruckeri strain 07111224 causes encephalitis in the acute phase of infection, which could explain why Y. ruckeri-affected fish show exophthalmia and erratic swimming known as signs of ERM.

Prevalence of Taxa of Pasteurellaceae Among Populations of Healthy Captive Psittacine Birds.

Sixty-two strains of Pasteurellaceae-like bacteria were isolated from the tracheas of 87 clinically healthy psittacine birds in two Danish zoos. The isolates were identified by a combination of rpoB and 16S rRNA gene sequencing and by matrix-assisted laser desorption-ionization time of flight. Twenty-eight strains belonged to the genus Volucribacter or were related to this genus and to the unnamed taxon 34 of Bisgaard, and 28 strains were related to the unnamed taxon 44 of Bisgaard. Four strains were identified as Pasteurella multocida , two isolates were classified with the related taxon 45 of Bisgaard, and a single isolate was classified as Pasteurella sp. The investigation documented an unrecognized reservoir of rarely reported and unclassified or unnamed species of Pasteurellaceae-like bacteria in psittacine birds. The results were in accordance with a recent report on isolation of Pasteurellaceae from diseased psittacine birds, and the investigation documented that the same taxa of Pasteurellaceae-like bacteria can be isolated from apparently healthy birds as well as from diseased birds.

Pasteurellaceae bacteria from the oral cavity of Tasmanian devils (Sarcophilus Harrisii) show high minimum inhibitory concentration values towards aminoglycosides and clindamycin.

UNLABELLED: Threatened by Devil Facial Tumor Disease, the Tasmanian devil populations are vulnerable and decreasing. Additionally, the devils' biting behaviour elevates their risk of acquiring bite wound infections caused by members of the bacterial Pasteurellaceae family that are natural inhabitants of the oral microbiota. In medical management of such bite wounds, antimicrobial susceptibility profiles are crucial. Prior to this investigation, no available data on minimal inhibitory concentration (MIC) values existed. A total of 26 isolates obtained from the oral cavity of 26 healthy Tasmanian devils were tested for their antimicrobial susceptibility by broth micro dilution. Most prominently, high MIC values for clindamycin (≥4 µg ml(-1) ), gentamicin (≥8 µg ml(-1) ) and amikacin (≥32 µg ml(-1) ), were observed for 92, 77 and 73% of the strains tested respectively. This study may be used as a guideline for antimicrobial therapy against bite wound infections caused by Pasteurellaceae originating from the oral cavity of Tasmanian devils. SIGNIFICANCE AND IMPACT OF THE STUDY: Tasmanian devils' aggressive behaviour makes bite wounds in fellow devils and human caretakers a common entity. Pasteurellaceae bacteria are common inhabitants of the oral microbiota of Tasmanian devils and a likely cause of bite wound infections. Here, for the first time, we report antimicrobial sensitivity profiles from a broad collection of Pasteurellaceae isolates obtained from the oral cavity of Tasmanian devils. Low MIC values were observed for the majority of the 22 antimicrobial agents included, yet nearly all strains were tolerant to clindamycin and the aminoglycosides. The work can serve as a guide for clinicians involved in treatment of bite wounds inflicted by devils in animals and humans.

Activation of persistent Streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis.

Endometritis in horses caused by Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) may be underdiagnosed due to traditional diagnostic methods lacking sensitivity and specificity. We serendipitously identified a bacterial growth medium (bActivate) that appeared capable of inducing growth of dormant S. zooepidemicus, which subsequently allowed detection by standard diagnostics. To assess the effect of bActivate we compared its ability to activate dormant S. zooepidemicus in a group of potentially infected subfertile mares with phosphate-buffered saline (PBS). All mares had to test negative for S. zooepidemicus on a low-volume uterine lavage, be negative on endometrial cytology and without clinical signs of endometritis to be included in the investigation. The mares were instilled with bActivate or PBS in the uterus. Growth of S. zooepidemicus was induced by bActivate in 64% (16/25) and PBS in 8% (1/12) of the mares, respectively (p<0.002). In vitro studies supported that some strains of S. zooepidemicus were able to form persister cells tolerating 32-times of the minimal inhibitory concentration of penicillin compared to normal growing cells. Persister cells had not acquired penicillin resistance, but seemed to tolerate the antimicrobial due to dormancy. This is, to our knowledge, the first description of controlled growth induction of dormant bacteria from a subclinical infection. Moreover we demonstrated how endometritis can origin from a reservoir of dormant bacteria residing within the endometrium, and not only as an ascending infection. Further studies should aim at determining the prevalence of dormant S. zooepidemicus, impact of activation on diagnostic and treatment efficacy, uterine health and mare fertility.

Diagnostic double-guarded low-volume uterine lavage in mares.

Endometritis constitutes a major problem in the management of broodmares; hence, diagnostic tests with a high sensitivity and specificity are highly appreciated. The aim of this study was to compare the results from endometrial, cytologic, and bacteriologic examinations obtained by a newly developed, double-guarded, flushing technique versus standard diagnostic tests, the double-guarded swab and biopsy. The described double-guarded flush technique requires the use of a disposable uterine flushing tube, a sanitary sleeve, a sterile steel speculum, and a 250 mL fluid bag. Endometrial biopsies, swabs, and low-volume lavage samples were obtained from 34 research mares at six different time points in four estrous cycles and were evaluated cytologically and bacteriologically. Endometrial biopsies from the first cycle (n = 34) were examined for the presence of polymorphonuclear neutrophils (PMNs) in the stratum compactum and stratum spongiosum and used as a gold standard for calculation of diagnostic sensitivity and specificity. In all samples, Escherichia coli was most frequently isolated (lavage, 30%; swab, 21%; and biopsy, 12%) followed by ß-hemolytic streptococci (lavage, 11%; swab, 8%; and biopsy, 7%). Positive cytology was less likely to occur when E coli was isolated from the diagnostic tests compared with the growth of ß-hemolytic streptococci. Isolation of pathogens from uterine samples was highly associated with the presence of PMNs in the stratum compactum and straum spongiosum on histology. Using the presence of PMNs in the tissue specimens as the gold standard for diagnosing endometritis, the sensitivity of low-volume lavage culture was 0.75 and the specificity was 0.72. In conclusion, the double-guarded, low-volume, lavage technique was a rapid and accurate method for diagnosing mares with endometritis, and the risk of false-positive samples is considered to be minimal compared with other flushing techniques described.

A major outbreak of Streptococcus equi subsp. zooepidemicus infections in free-range chickens is linked to horses.

Infections of poultry due to Streptococcus equi subsp. zooepidemicus have been rare during the past decades and dissimilarities have been reported as to symptoms and lesions; likewise, the source of serious outbreaks has remained speculative. An outbreak affecting 11,000 free-range chickens at the age of 47 wk is reported. The outbreak manifested itself as acute at the onset and was followed by a chronic stage, resulting in some 80% mortality within 21 wk. Small-colony variants (SCVs) of S. equi subsp. zooepidemicus associated with the chronic phase are reported for the first time, and it is discussed whether SCVs might explain the change in lesions observed. Comparison of partial sequences of rpoB, multilocus sequence typing, and pulsed-field gel electrophoresis of isolates from chickens and horses kept at the farm showed the isolates to be identical and horses a likely source of infection. The present findings underline the importance of protecting free-range chickens from contact with other animals and birds known to host pathogens of importance to poultry.

Effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares.

The objective of the present study was to evaluate the effect of immunomodulatory therapy (glucocorticoids (GC) and mycobacterium cell wall extract (MCWE)) on the endometrial gene expression of inflammatory cytokines in susceptible mares with induced infectious endometritis. Endometrial gene expression of pro- and anti-inflammatory cytokines; interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-1 receptor antagonist (ra), acute phase protein (APP) serum amyloid A (SAA) and clinical parameters were evaluated. Five mares were classified as susceptible to persistent endometritis based on their endometrial histopathology and ability to clear an induced uterine inflammation. To investigate the effect of immunomodulatory therapy, the mares were inoculated with 10(5) colony forming units (CFU) Escherichia coli in three consecutive estrus cycles in a modified cross-over study design. Thus, each mare served as its own control and the treatment type was performed in randomized order. The effect of treatment with MCWE (1.5 mg Settle IV), dexamethasone (0.1 mg per kg IV) or no treatment was investigated. All mares were free from uterine inflammation before each E. coli inoculation. Endometrial biopsies were recovered 3, 24 and 72 h post inoculation. Relative gene-expression analyses were performed by quantitative reverse transcriptase PCR (qRT-PCR). Endometrial gene expression of inflammatory cytokines was modulated by administration of GC. Expression of proinflammatory cytokines (IL-1ß, IL-6, IL-8) and SAA was significantly lower in the GC treated group late in the study period (72 h) compared to "no treatment" and MCWE treatment. Increased expression of the anti-inflammatory cytokine IL-10 was observed 3 and 24 h after E. coli infusion and GC treatment. A significant decrease of SAA expression was observed after MCWE treatment compared to "no treatment". MCWE and GC treatment had a significant effect on the clearance of uterine pathogens and number of mares retaining fluid after E. coli infusion. The results of the current investigation suggest that GC is capable of effectively modulating the innate immune response to induced infectious endometritis in susceptible mares.

Classification of organisms previously reported as the SP and Stewart-Letscher groups, with descriptions of Necropsobacter gen. nov. and of Necropsobacter rosorum sp. nov. for organisms of the SP group.

To allow classification of bacteria previously reported as the SP group and the Stewart-Letscher group, 35 isolates from rodents (21), rabbits (eight), a dog and humans (five) were phenotypically and genotypically characterized. Comparison of partial rpoB sequences showed that 34 of the isolates were closely related, demonstrating at least 97.4 % similarity. 16S rRNA gene sequence comparison of 20 selected isolates confirmed the monophyly of the SP group and revealed 98.5 %-100 % similarity between isolates. A blast search using the 16S rRNA gene sequences showed that the highest similarity outside the SP group was 95.5 % to an unclassified rat isolate. The single strain, P625, representing the Stewart-Letscher group showed the highest 16S rRNA gene similarity (94.9-95.5 %) to members of the SP group. recN gene sequence analysis of 11 representative strains resulted in similarities of 97-100 % among the SP group strains, which showed 80 % sequence similarity to the Stewart-Letscher group strain. Sequence similarity values based on the recN gene, indicative for whole genome similarity, showed the SP group being clearly separated from established genera, whereas the Stewart-Letscher group strain was associated with the SP group. A new genus, Necropsobacter gen. nov., with only one species, Necropsobacter rosorum sp. nov., is proposed to include all members of the SP group. The new genus can be separated from existing genera of the family Pasteurellaceae by at least three phenotypic characters. The most characteristic properties of the new genus are that haemolysis is not observed on bovine blood agar, positive reactions are observed in the porphyrin test, acid is produced from (+)-L-arabinose, (+)-D-xylose, dulcitol, (+)-D-galactose, (+)-D-mannose, maltose and melibiose, and negative reactions are observed for symbiotic growth, urease, ornithine decarboxylase and indole. Previous publications have documented that both ubiquinones and demethylmenaquinone were produced by the proposed type strain of the new genus, Michel A/76(T), and that the major polyamine of representative strains (type strain not included) of the genus is 1,3-diaminopropane, spermidine is present in moderate amounts and putrescine and spermine are detectable only in minor amounts. The major fatty acids of strain Michel A/76(T) are C(14 : 0), C(16 : 0), C(16:1)ω7c and summed feature C(14 : 0) 3-OH/iso-C(16 : 1) I. This fatty acid profile is typical for members of the family Pasteurellaceae. The G+C content of DNA of strain Michel A/76(T) was estimated to be 52.5 mol% in a previous investigation. The type strain is P709(T) ( = Michel A/76(T)  = CCUG 28028(T)  = CIP 110147(T)  = CCM 7802(T)).

Mannheimia caviae sp. nov., isolated from epidemic conjunctivitis and otitis media in guinea pigs.

Strains T138021-75(T), Pg19 and Pg20 (taxon 25 of Bisgaard) were isolated from guinea pigs and characterized. Strains T138021-75(T) and Pg20 showed identical 16S rRNA gene sequences and were distantly related to the published strain P224 with the highest 16S rRNA similarity of 98.6 %. These two strains showed 97.8 % sequence similarity with the type strain and other strains of Mannheimia glucosida and 97.3 % similarity with the type strain of Mannheimia varigena, but <97 % similarity with all other type strains of the genus Mannheimia, including Mannheimia haemolytica (96.9 %). Phylogenetic analysis of rpoB gene sequences showed that strain P224 had a distant position (89.9 % gene sequence similarity) compared with the three other strains (T138021-75(T), Pg20 and Pg19), which had identical gene sequences. These three novel strains also shared identical recN gene sequences. Phylogenetic analysis of the recN gene sequences showed a close relationship between the three novel strains and strain P224. The DNA-DNA reassociation value between strain T138021-75(T) and P224 was 81.6 % and 40.3 % between strain T138021-75(T) and the type strain of M. glucosida. Based on the DNA-DNA reassociation data, strain T138021-75(T) belonged to a separate species that was closely related to strain P224. Strain P224 differed from strains T138021-75(T), Pg20 and Pg19 in the following phenotypic characteristics: activity of ornithine carboxylase, hydrolysis of glycosides, and acid formation from maltose, dextrin, melibiose and raffinose, as well as reactions for α-galactosidase and ß-xylosidase. Whole genome similarity calculations based on recN gene sequences showed that strains T138021-75(T) and P224 were related at the species level (0.932), whereas 16S rRNA and partial rpoB gene sequence comparisons showed a more divergent position of strain P224 compared with the novel strains, including a different host of isolation. The results showed that the three strains of taxon 25 represent a novel species for which the name Mannheimia caviae sp. nov. is proposed. The type strain, T138021-75(T) ( = CCUG 59995(T) = DSM 23207(T)) was isolated from purulent conjunctivitis in guinea pigs. Previous publications have documented both ubiquinones and demethylmenaquinone to be present in the type strain. The G+C content of the DNA of the type strain has been found to be 41.4 mol% (T(m)).

Observations on the incidence and aetiology of valvular endocarditis in broiler breeders and detection of a newly described taxon of Pasteurellaceae, Avibacterium endocarditidis.

A total of 122 dead broiler breeders randomly selected from a flock showing normal production parameters and covering the age from 44 to 61 weeks were subjected to a comprehensive routine post-mortem examination including examination for lesions of endocarditis. Forty-two hens (34%) showed valvular endocarditis caused by Avibacterium endocarditidis (43%), Enterococcus faecalis (31%), Staphylococcus aureus (5%) and Streptococcus pluranimalium (5%), while growth was not obtained from 17% with the methods used for isolation. Gross lesions associated with the different bacterial pathogens did not allow separation according to pathogens involved. Port of entry and pathogenesis associated with the high prevalence of valvular endocarditis remained speculative. The present findings demonstrated the newly described species of Pasteurellaceae, Avibacterium endocarditidis associated with endocarditis in chickens and confirm previous observations on the prevalence of endocarditis in chickens, partly explaining the slightly increased mortality normally observed in broiler breeders during the last weeks of production.

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1-year period in Lithuania.

AIMS: The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. METHODS AND RESULTS: During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter-positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73.8%) were positive for Camp. jejuni and 17 flocks (40.48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1-year period. CONCLUSIONS: Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm-specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.

Characterization of Pasteurellaceae-like bacteria isolated from clinically affected psittacine birds.

AIMS: The aim of the present investigation was to identify and characterize Pasteurella-like isolates obtained from clinically affected psittacine birds. METHODS AND RESULTS: A total of 37 isolates from psittacine birds tentatively classified with the family Pasteurellaceae were characterized phenotypically. The genetic relationship was investigated by sequencing of partial rpoB and 16S rRNA genes for selected isolates. The results obtained were compared with the data from 16 reference strains. Nine isolates were identified as Gallibacterium spp., 16 as Volucribacter spp. or Volucribacter-like, while 11 isolates were classified as taxon 44 of Bisgaard. A single isolate was identified as Pasteurella multocida. CONCLUSIONS: Characterization of Pasteurellaceae by traditional methods is often inconclusive because of inconsistent reactions and phenotypic diversity. For the same reason, genotyping is essential to allow proper classification as demonstrated in the present study. SIGNIFICANCE AND IMPACT OF THE STUDY: Limited information exists on the isolation and significance of Pasteurellaceae associated with clinically affected psittacine birds showing signs of digestive and/or respiratory disorders. The present investigations demonstrated that these organisms are widely distributed among clinically affected birds, but isolation of these taxa cannot be unambiguously correlated with the symptoms observed.

Hemagglutinating activity of Gallibacterium strains.

In the present study, the hemagglutinating activity of seven reference strains, and nine Mexican and three Danish field isolates, of Gallibacterium was investigated by using fresh erythrocytes of 19 different types including chicken (broiler, rooster, layer hen), turkey, pigeon, quail, duck, Harris's hawk (Parabuteo unicinctus), house finch (Carpodacus mexicanus), cow, sheep, horse, dog, rabbit, pig, and human (groups A, B, AB, and O; Rh+). Agglutination was observed for broiler chicken, layer hen, quail, rabbit, and pig erythrocytes with a subset of Gallibacterium strains, whereas most tested strains agglutinated rabbit erythrocytes. Transmission electron microscopic examination of a hemagglutinating strain demonstrated a close interaction between the bacterial and erythrocyte surfaces. The results indicate that some Gallibacterium strains are able to agglutinate avian or mammalian erythrocytes, or both. However, the mechanisms enabling hemagglutination are not known and will be addressed in future studies.

Tissue distribution of haemolytic Gallibacterium anatis isolates in laying birds with reproductive disorders.

Gallibacterium anatis biovar haemolytica has been suggested to have a causal role in peritonitis and salpingitis in chickens. Therefore, the aim of this study was to investigate the occurrence of G. anatis biovar haemolytica in chickens with reproductive disorders. One hundred and forty one birds from 31 layer flocks were submitted for necropsy and the following organs were examined for bacteria: choana, trachea, lung, heart, liver, spleen, ovary, oviduct, duodenum and cloaca. Examination for Escherichia coli was included as it can induce reproductive disorders. G. anatis was isolated in pure culture from the reproductive tract of affected birds in six of the 31 flocks while E. coli was obtained in pure culture from 10 of them. Both G. anatis and E. coli were isolated from the reproductive tract of 14 of the 31 flocks. The genetic diversity of the Gallibacterium isolates was assessed by amplified fragment length polymorphism on a subset of 83 isolates. Generally, each flock was infected with a single clone, which could be isolated from various sites in the birds. However, in two flocks, the majority of birds yielded positive samples from the internal organs, indicating that these particular clones may be more invasive. The findings support previous suggestions that G. anatis biovar haemolytica is associated with infection of the reproductive tract of chickens, making it a likely cause of lowered productivity and an animal welfare concern.

Genetic diversity of Gallibacterium isolates from California turkeys.

The aim of the present study was to investigate the genetic diversity of Gallibacterium isolates recovered from lesions in turkeys. Gallibacterium has been isolated from various bird species including turkeys, but no large investigations have yet been made to characterize isolates from turkeys genetically. We therefore genotyped 53 Gallibacterium isolates obtained from turkeys between 1998 and 2004. Fifty isolates originated from 29 different flocks in California and the remaining three came from three German turkey flocks. All were recovered from birds with lesions, mainly in the upper respiratory tract. Five chicken isolates from California and five Gallibacterium reference strains were also included. Amplified fragment length polymorphism analysis demonstrated substantial genetic diversity among the Gallibacterium isolates. However, we also demonstrated that some Gallibacterium clones were present in consecutive rotations at the same farm during the entire 6-year observation period and were present in different flocks from different farms. Similarly, the same clone was identified from two of the three German flocks. Further investigation of the spread of Gallibacterium between turkey flocks, including infections acquired from chickens or wild birds, should be carried out.

Host-specific bacterial lineages in the taxon 2 and 3 complex of Pasteurellaceae.

The aim of the investigation was to determine the genetic relationship of a phenotypically diverse strain collection of epidemiologically unrelated strains from the taxon 2 and 3 complex of Bisgaard isolated from different hosts. A total of 325 isolates belonging to the taxon 2 and 3 complex of Bisgaard was characterized phenotypically in 82 characters. The genetic relationship among a subset of 60 isolates was investigated by amplified fragment length polymorphism (AFLP). The isolates were selected aiming at including the broadest diversity with regards to phenotype and host spectrum. The results suggested a statistically clear association between AFLP clusters and the host species families Columbidae (pigeon, dove), Anatidae (duck, goose) and Psittacidae (parrot, parakeet, budgerigar), respectively. This association was further supported by results from previous whole cell protein profiling and DNA:DNA hybridization studies. In conclusion, it appears that distinct genetic lineages within the taxon 2 and 3 complex of Bisgaard have evolved specificity for host bird species of different families. The observed host specificity of taxon 2 and 3 organisms may be used in future diagnostics and studies elucidating aspects of pathogenicity and epidemiology associated with the different lineages and their respective hosts.

Clonal stability of Pasteurella multocida in free-range layers affected by fowl cholera.

Detailed longitudinal studies of the genetic stability of Pasteurella multocida ssp. multocida, the cause of fowl cholera, have not previously been carried out. Consequently, the aim of the present study was to provide detailed information on the genetic stability and diversity of P. multocida ssp. multocida in poultry flocks over time, enabling new insights into the molecular epidemiology of this important poultry pathogen. Longitudinal investigations of the rate and causes of mortality were carried out on two free-range layer farms (A and B) over a period of 11 months. The total mortality of two flocks, A1 and A2, on farm A were 62 and 91%, respectively, while the total mortality of a single flock B1 on farm B was 6%. Postmortem examinations were performed on 708 layers from flocks A1 and A2 and in 159 from flock B1. Fowl cholera was the main cause of mortality on both farms. Pasteurella multocida isolates recovered from layers on both farms were characterized phenotypically and genotypically, and 322 isolates were identified as P. multocida ssp. multocida. The genetic diversity of 99 isolates from farm A and 31 from farm B was characterized by restriction endonuclease analysis and amplified fragment length polymorphism analysis. The isolates on each farm had a unique restriction endonuclease analysis and amplified fragment length polymorphism analysis type, suggesting a single introduction of a successful clone. Furthermore the clone on farm A was identical to clones previously isolated from outbreaks in the avifauna of Denmark in 1996 and 2001 and in Sweden in 1998. This study provides convincing evidence for the clonal stability of outbreak clones of P. multocida.

Reproduction of sepsis and endocarditis by experimental infection of chickens with Streptococcus gallinaceus and Enterococcus hirae.

This study describes experimental infections in 4-week-old chickens inoculated intravenously with approximately 10(8) colony-forming units Streptococcus gallinaceus strain CCUG 42692T (C13156) or Enterococcus hirae strain DSM 20160 (C17410). Birds were necropsied following death and obvious clinical signs of disease or were euthanized weekly after infection for up to 4 weeks. At necropsy, lesions included splenomegaly, hepatomegaly, valvular and/or mural endocarditis. Cardiac lesions included focal necrotizing myocarditis and/or yellow-white vegetative valvular endocarditis or greyish proliferations associated with the mitral valves in 35% (6/20) and 79% (19/24) of birds infected with S. gallinaceus and in 20% (4/20) and 55% (12/22) of birds infected with E. hirae via the brachial and jugular veins, respectively. S. gallinaceus was reisolated from heart valves in 45% (9/20) and 75% (18/24) and E. hirae in 35% (7/20) and 73% (16/22) after inoculation via brachial and jugular veins, respectively. Both challenge strains were also isolated from liver, spleen, bone marrow and hock joints. A significant difference between the infections with the two strains was seen only with reisolation of E. hirae from hock joints (P < 0.007). Significant differences were apparent between the two inoculation routes only with E. hirae, where infection via the jugular vein was associated with higher culture positive isolations from the heart (P = 0.029), bone marrow (P = 0.002) and hock joints (P < 0.001) compared with the brachial vein. Birds injected with sterile phosphate-buffered saline were negative for culture of the challenge strains and no lesions were observed in these controls. The results confirm that both S. gallinaceus and E. hirae can cause endocarditis in experimentally infected chickens.