Pathogenesis of Erysipelothrix piscisicarius infection in tiger barbs (Puntigrus tetrazona).

Erysipelothrix piscisicarius is an emerging bacterial pathogen and the aetiologic agent of piscine erysipelosis, a recently recognized disease of ornamental fish. However, little is known regarding the dynamics of infection in fish. The purpose of this study was to gain a better understanding of the pathogenesis of piscine erysipelosis in the tiger barb (Puntigrus tetrazona) by investigating tissue tropisms and responses to bacterial dissemination following immersion challenge with a virulent strain recovered from diseased fish. The challenge resulted in 83% mortality by day 16. Erysipelothrix piscisicarius DNA was first detected in the skin using quantitative PCR, and bacteria were visualized in association with microscopic lesions on day 4. By day 8, E. piscisicarius DNA was further detected in intestines, hearts, spleens, gills and skin; parenchymal organs were largely spared. The data suggest a primary cutaneous portal of entry and tropism for collagenous tissues, particularly those within vascular walls. Initial spread occurs directly from the dermis into interstitial areas of skeletal muscle, then centrally to the peritoneum and coelomic cavity following collagenous tissue pathways. Although histopathology revealed widespread bacterial dissemination over time, the severity of skin and muscle lesions with high levels of bacterial DNA identifies these tissues as primary targets of infection.

Description of Erysipelothrix piscisicarius sp. nov., an emergent fish pathogen, and assessment of virulence using a tiger barb (Puntigrus tetrazona) infection model.

A recently described emergent disease of ornamental fish has been associated with an Erysipelothrix species positive for the surface protective antigen (spa) C gene. Whole genome sequencing was performed on five spaC Erysipelothrix isolates from diseased ornamental fish. In addition, these spaC Erysipelothrix isolates were compared to spaA-, spaB- and other spaC-positive Erysipelothrix species isolated from terrestrial and marine mammals, birds and fish using multi-locus sequence analysis (MLSA). The genomes of fish pathogenic spaC isolates were genetically distinct from Erysipelothrix rhusiopathiae, sharing 86.61-86.94 % average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of 31.6-32.2 %, but 99.01-99.11 % ANI and 90.8-91.9 % dDDH values with the uncharacterized spaC-positive Erysipelothrix sp. strain 2 isolated from swine. The findings indicate the spaC-positive fish and swine isolates are conspecific and represent a previously unrecognized taxon. While phylogenies inferred from MLSA sequences confirm this conclusion, slight genetic differences between the spaC fish isolates and swine strain 2 were indicated. Bath immersion challenge trials were conducted using tiger barbs (Puntigrus tetrazona) exposed by immersion to 107 c.f.u. ml-1 of three fish pathogenic spaC Erysipelothrix species, and three spaA and two spaB E. rhusiopathiae isolates as a model of infection. Thirty days post-challenge, cumulative mean percentage survival was 37 % for the spaA, 100 % for the spaB and 13 % for the spaC isolates, revealing differences in virulence among the various spa genotypes in fish. Genetic findings and observed differences in virulence demonstrate the fish pathogenic spaC isolates represent a novel species, for which the name Erysipelothrix piscisicarius sp. nov. is proposed. The type strain is E. piscisicarius 15TAL0474T (=NRRL B-65533T=ATCC-TSD-175T=DSM 110099T).

Acute and Chronic Erysipelothrix rhusiopathiae Infection in Lambs.

Polyarthritis caused by Erysipelothrix rhusiopathiae is a relatively common infection in lambs characterized by low mortality and high morbidity. E. rhusiopathiae is a ubiquitous Gram-positive bacterium that is both a commensal and a pathogen of vertebrates. The disease was studied during an outbreak in a Norwegian Spæl sheep flock. In the acute phase, 48 of 230 (20%) lambs developed clinical signs and 4 died (1.7%). One acute case was necropsied and E. rhusiopathiae was cultured from all major organs investigated and from joints. There was a fibrinous polyarthritis, increased presence of monocytes in vessels, and necrosis of Purkinje cells. Sixteen of the diseased animals (33%) developed a chronic polyarthritis. Eight of these lambs were necropsied; all had lesions in major limb joints, and 3 of 8 also had lesions in the atlanto-occipital joint. At this stage, E. rhusiopathiae was cultured only from the joints in 7 of 8 (87.5%) lambs, but by real-time polymerase chain reaction, we showed persistence of the bacterium in several organs. Pulsed-field gel electrophoresis typing of the bacterial isolates indicated that the same strain caused the acute and chronic disease. Five of 6 (83%) chronically affected animals had amyloidosis of the spleen, and 6 of 8 (75%) had amyloidosis of the liver. All chronically affected animals had a glomerulonephritis, and 6 of 8 (75%) had sparse degeneration in the brain. Ceruloplasmin and haptoglobin were significantly increased in the chronically diseased lambs. These results show that chronic ovine erysipelas is not restricted to joints but is a multisystemic disease.

Erysipelas Outbreaks in Flocks of Geese in Poland--Biochemical and Genetic Analyses of the Isolates.

Erysipelothrix rhusiopathiae causes erysipelas in many vertebrate species. Severe disease outbreaks have been noted in many poultry species--chickens, ducks, emus, pheasants, pigeons, and geese. This article describes the biochemical and genetic analyses of six E. rhusiopathiae strains isolated from geese for meat production. The isolates came from birds kept in different poultry houses on one farm, and were collected during two erysipelas outbreaks. We analyzed and compared the isolates by random amplified polymorphic DNA with the use of NK6 primer and pulsed-field gel electrophoresis, with the restriction enzyme SmaI. Biochemical examination was performed with API Coryne test. Analyses showed that the three strains isolated during the first outbreak differed, whereas the three isolates from the second outbreak were identical to one another, but distinct from the isolates from the first outbreak. The results of biochemical and genetic analyses of E. rhusiopathiae strains isolated from geese suggest different sources of infection for the erysipelas outbreaks.

Fatal Erysipelothrix rhusiopathiae septicemia in two Atlantic dolphins (Stenella frontalis and Tursiops truncatus).

We describe gross, histopathologic, ultrastructural, immunohistochemical, and microbiologic features of acute septicemia by Erysipelothrix rhusiopathiae in an Atlantic spotted dolphin Stenella frontalis and an Atlantic bottlenose dolphin Tursiops truncatus. Generalized lymphadenomegaly and widespread hemorrhages were the most consistent macroscopic findings. Tricavitary effusion and icterus were noted in one individual. Histologically, all organs examined showed numerous variably sized bacillary bacterial emboli (Gram-positive; Ziehl-Neelsen-negative), typically associated with systemic congestion, edema, hemorrhages, and fibrinocellular thrombi. These bacteria were frequently intravascular, either extracellular or intramonocytic/macrophagic, and to a lesser extent, free within the interstitium of parenchymal organs. In both cases, microbiological analysis yielded E. rhusiopathiae. A primary anti-E. rhusiopathiae antibody created in mice from one of the strains isolated allowed positive immunohistochemical detection. Electron microscopy and dual immunohistochemistry with lysozyme and MAC387 antibodies confirmed the intramacrophagic location of the bacilli. E. rhusiopathiae, a known multispecies and zoonotic agent, should be considered as a potential etiologic agent in septicemia cases in free-ranging individuals of these dolphin species.

Erysipelas in a numbat (Myrmecobius fasciatus).

An adult, female numbat (Myrmecobius fasciatus) was submitted to the Perth Zoo Veterinary Department for postmortem examination in November 2011. This radio-collared wild numbat had been found dead in the Dryandra Woodland, 191 km southeast of Perth, Western Australia. On external examination, the body condition was good. Three ticks (Ixodes spp.) were found on the thoracic region. The external pouch was contaminated with dirt and palpably flocculent, and the nipples oozed a purulent material. Histopathology showed widespread fibrin thrombi containing bacterial microcolonies within interstitial vessels of the mammary gland with surrounding necrotic tissue. Bacterial microcolonies were present throughout the kidney, intestine, lung, and mammary tissue, and culture produced a moderate growth of Erysipelothrix rhusiopathiae. Although erysipelas has been reported as a cause of morbidity and mortality in marsupials, this is the first report of erysipelas in the order Dasyuromorphia (marsupial carnivores) and highlights the need for ongoing surveillance for causes of disease in wild numbats and species recovery programs.

Histologic and bacteriologic findings in valvular endocarditis of slaughter-age pigs.

Endocarditis lesions from 117 slaughter pigs were examined pathologically and etiologically in addition to 90 control hearts with cardiac valves. Lesions were located on the valves; however, the lesions had extended to the walls in 21 cases (18%). Lesions predominated on the mitral valve (59%). A total of 28 cases, from which no growth was obtained or a contamination flora was grown, were screened by fluorescence in situ hybridization (FISH) for bacteria (general bacterial probe) and probes specific for Streptococcus suis and Erysipelothrix rhusiopathiae, respectively. Using FISH, an additional 10 cases of endocarditis due to S. suis and E. rhusiopathiae were disclosed. Within lesions, streptococci predominated (53%) followed by E. rhusiopathiae (30%). Distinct features of both the lesions and the shape and localization of bacterial colonies were related to streptococci and E. rhusiopathiae. The propensity for streptococci to be localized on more than 1 valve in single hearts may be because S. suis-infected pigs tend to have been infected for a longer period compared with E. rhusiopathiae. Mineralization of endocarditis lesions was significantly associated with infection by streptococci, and was seen in 71% of the cases, whereas it was present in only 28% of lesions caused by E. rhusiopathiae. In addition, areas with mineralization were significantly correlated to the presence of a granulomatous reaction. Granulomatous endocarditis is likely a result of a foreign body reaction due to dystrophic mineralization. Local proliferation of valvular endothelial cells, found in 9 hearts in the current study, may increase the risk of developing thrombosing endocarditis in pigs.

Proteomic and Transcriptomic Analyses of Swine Pathogen Erysipelothrix rhusiopathiae Reveal Virulence Repertoire.

E. rhusiopathiae is the causative agent of erysipelas in animals and erysipeloid in humans, but its pathogenicity is poorly understood. To identify virulence factors associated with E. rhusiopathiae and screen engineered vaccine candidates, we used proteomics and transcriptomics to compare the highly virulent strain HX130709 with an isogenic avirulent derivative, HX130709a. 1,299 proteins and 1,673 transcribed genes were identified and 1,292 of the proteins could be associated with genes. In a comparison between HX130907 and HX130709a, 168 proteins and 475 genes exhibited differences in regulation level. Among these, levels for 61 proteins and transcripts were positively or negatively correlated. Gene Ontology (GO) analysis suggests that many of the down-regulated proteins in the attenuated strain have catalytic or binding functions. Potential protein-protein interactions suggest that some of the down-regulated proteins may regulate PTS, GMP synthase and ribosomal proteins. Morphological results showed that HX130709 and HX130709a have similar colony and capsule morphology. Growth curves and pyruvate measurements suggest that TCA cycle and saccharide phosphorylation levels were decreased and gluconeogenesis was increased in HX130709a. Our study confirms that SpaA and neuraminidase, but not hyaluronidase and capsule, are associated with virulence in E. rhusiopathiae. We conclude that the virulence of E. rhusiopathiae may be associated with slow reactions of the TCA cycle and down-regulation of selected proteins.

Erysipelothrix rhusiopathiae septicemia in a Laughing kookaburra (Dacelo novaeguineae).

Erysipelothrix rhusiopathiae (E. rhusiopathiae) septicemia was demonstrated in a captive Laughing kookaburra (Dacelo novaeguineae). The bird died after a 2-week period of weakness and weight loss. At necropsy, the bird was emaciated and had reddened and wet lungs. Microscopic lesions were limited to hepatic and pulmonary congestion with focal thrombosis. Erysipelothrix rhusiopathiae was isolated by routine bacterial culture from several organs. Further characterization of the isolate by pulsed-field gel electrophoresis indicated that the isolate has a new genotype pattern 3A(III), which is 91.7% homologous to an E. rhusiopathiae that was isolated from a pig in 2001 and 88% homologous to an isolate recovered in 2000 from a turkey with septicemia. This is the first report of E. rhusiopathiae-induced septicemia in a kookaburra.

Investigations on the pathogenicity of Erysipelothrix rhusiopathiae in laying hens.

Erysipelas was diagnosed in 1998 from 34-wk-old laying hens in a free range flock in Germany. Erysipelothrix rhusiopathiae of serotype 1 was cultured from internal organs of the affected birds. This article describes the pathogenicity of the field isolate of E. rhusiopathiae in experimentally infected specific pathogen-free (SPF) laying hens. Three experiments were performed with SPF chickens inoculated at 17, 27, and 37 wk of age by either intramuscular (IM) or oral route. Inoculated birds were observed for 14 days. The highest mortality rates occurred in older birds, with 100% mortality observed in the 37-wk-old birds inoculated IM, 60% mortality reported in the younger 27-wk-old birds, and no mortality in the 17-wk-old age group. In the orally infected 27-wk-old birds, 40% mortality was detected, whereas no mortality was observed in the oldest birds by the same route. The results of the experiments support the contention that older birds are more sensitive to infection than younger birds and that mortality in laying hens is age related and dependent on the route of infection.

Erysipelothrix rhusiopathiae: an uncommon but ever present zoonosis.

Erysipelothrix rhusiopathiae is an important animal pathogen with a worldwide distribution, yet this zoonotic infection is rarely reported in humans. Three cases of E. rhusiopathiae infection, which illustrate the varied clinical presentations of this pathogen in humans, are presented together with the pathological findings and treatment regimens.

Erysipelothrix septicemia in a little blue penguin (Eudyptula minor).

On June 25, 2002, aquarium veterinarians treated a 5-year-old, male little blue penguin (Eudyptula minor) that was acutely recumbent and dull, with inappetence of 24-hour duration. The penguin died within 10 minutes of presentation despite emergency resuscitation efforts. Gross pathologic findings consisted of pulmonary congestion and intestinal hemorrhage. Histopathologic findings included necrosis of tips of intestinal villi, increased numbers of mononuclear cells in pulmonary interstitium and hepatic sinusoids, and gram-positive bacteria in systemic microvasculature. Transmission electron microscopic examination revealed short gram-positive bacilli located in lumina of glomerular capillaries and in cytoplasm of mononuclear phagocytic cells in the lung and liver. Erysipelothrix rhusiopathiae was recovered from the lung, liver, and intestine by bacteriologic culture. Amplicons from polymerase chain reaction (PCR) tests using Erysipelothrix genus-specific primers and total genomic DNA extracted from formalin-fixed, paraffin-embedded tissue sections of lung and intestine demonstrated 99% nucleotide sequence identity with 16S small-subunit ribosomal DNA of E. rhusiopathiae and E. tonsillarum. The source of infection was speculated to be fish in the diet; however, repeated attempts to detect Erysipelothrix spp. from the mucous layer of food fish using bacteriologic culture and PCR were unsuccessful. This is the first report of erysipelas in a captive aquatic bird. Details of the isolation of E. rhusiopathiae and the application of molecular testing to identify Erysipelothrix DNA in formalin-fixed, paraffin-embedded tissue sections are given.

Pathology of experimental erysipelas in turkeys.

Gross and histopathologic lesions were studied in 10 Broad-Breasted White turkeys with acute erysipelas induced experimentally. The gross pathologic features of the disease conformed very closely with descriptions in the literature (7). Histopathologic evaluation was concentrated on the inoculation site (skin), proventriculus, ileocecal area of the intestine, liver, spleen, kidney, lung, and heart. In all organs examined the dominant histopathologic alteration was vascular damage as evidenced by: generalized congestion, edema, focal hemorrhage, disseminated fibrin thrombi, and numerous bacterial aggregates either within fibrin thrombi or engulfed by cells of the reticuloendothelial system. Degenerative changes or overt necrosis of parenchymal cells were evident in liver, spleen, and kidney. The cellular inflammatory component of acute lesions was minimal. Heterophil and mononuclear leukocytic infiltrates were observed in scarified skin and, in turkeys surviving several days, in livers and hearts.

Diagnosis by polymerase chain reaction of Erysipelas septicemia in a flock of ring-necked pheasants.

A flock of 810 pheasants experienced 6.2% mortality over 6 days. Affected birds were weak and lethargic for up to 24 hr before death. Examined birds were thin, and gross lesions consisted of thick opaque crops and cecal cores. Histologically, there was capillariasis of the crop and multifocal ulcerative typhlitis with Heterakis spp. infection, and numerous systemic intravascular monocytes were filled with clusters of blue rod-shaped organisms. The organisms were gram-positive bacilli by Brown and Brenn staining and ultrastructural analysis. Liver bacterial cultures were negative for pathogenic bacteria. Erysipelas septicemia was diagnosed by an Erysipelothrix species-specific polymerase chain reaction method with the substrate DNA isolated from formalin-fixed, paraffin-embedded liver.

Disseminated intravascular coagulation in chickens inoculated with Erysipelothrix rhusiopathiae.

In a first experiment, 28 specific pathogen-free chickens aged 3 weeks showed clinical signs 1 to 5 days after intramuscular inoculation with Erysipelothrix rhusiopathiae. Twelve of 28 birds died 2 to 4 days after inoculation. Macroscopically, the liver, spleen and kidneys were seen to be enlarged and congested. Histologically, fibrinous thrombus formation, seen in the hepatic sinusoids, renal glomerular capillaries and small pulmonary blood vessels, was a characteristic feature. In addition, the liver showed marked congestion, increase of mononuclear cells and heterophils in the sinusoids, hyperplasia of sinusoidal lining cells, and vacuolar changes in hepatic cells. The spleen showed fibrinous exudation of the lymphoid follicles and ellipsoids with lymphocytic depletion, and hyperplasia of ellipsoidal reticular cells. There was oedema, congestion and cellular infiltration in the interstitium of the kidney. The bursa of Fabricius and thymus showed marked lymphocytic depletion. In a second experiment, the blood chemical values (uric acid, glutamic-oxalacetic transaminase, lactate dehydrogenase and gamma-glutamyl transpeptidase) of birds inoculated intramuscularly with E. rhusiopathiae were significantly higher than those of uninfected controls. The blood prothrombin times and activated partial thromboplastin times of the inoculated group were significantly greater than those of the control group. The pathological and haematological findings demonstrated that E. rhusiopathiae induced disseminated intravascular coagulation in the chickens.

Naturally occurring erysipelas in rats.

Erysipelothrix rhusiopathiae was isolated from the main limb joints of two Sprague Dawley rats affected by spontaneous lesions of chronic fibrinopurulent polyarthritis, endocarditis and mycocarditis.

Erysipelothrix rhusiopathiae, serotype 17, septicemia in moose (Alces alces) from Algonquin park, Ontario.

Erysipelothrix rhusiopathiae septicemia was diagnosed in three of four moose found dead in Algonquin Provincial Park, Ontario, Canada, in the spring of 1989. Type 17 E. rhusiopathiae was isolated from liver, lung, kidney, and lymph nodes of affected animals, which were in poor body condition, and suffering hair loss associated with tick (Dermacentor albipictus) infestations. Microscopic lesions consisted of mild, multifocal, necrotizing myocarditis, sarcocystosis, and lymph node atrophy. The bacterium may have gained entry to these animals via ingestion of, or percutaneous exposure to, contaminated water, or possibly by the bites of ticks. Malnutrition and tick infestation may have predisposed the animals to infection by this opportunistic pathogen.

Isolation of Erysipelothrix rhusiopathiae from a red-tailed hawk (Buteo jamaicensis) with a concurrent pox virus infection.

Erysipelothrix rhusiopathiae was isolated from the spleen, liver, lung, heart, kidney, and skin of a red-tailed hawk (Buteo jamaicensis) which had a concurrent avian pox virus infection. The hawk had been housed on a farm with domestic turkeys, providing a possible source of the E. rhusiopathiae.