Receptor Activator of Nuclear Factor kappa-B Ligand is Not Regulated During Chronic Osteomyelitis in Pigs.

Bone loss is a major complication of osteomyelitis and from numerous in-vitro studies, it has been concluded that the bone lysis is caused by elevated expression of the receptor activator of nuclear factor κB ligand (RANKL), leading to increased osteoclast activity. However, we failed to find any relationship between bone loss and osseous RANKL expression in a porcine model of acute and chronic implant-associated osteomyelitis (IAO) due to Staphylococcus aureus or in chronic osteomyelitis lesions in slaughter pigs. Surprisingly, we found that the expression of RANKL was reduced during chronic bone infections. This is in line with the few studies conducted on human samples. A significant bone loss was observed in IAO lesions and in lesions from slaughter pigs, but with no indication of osteoclast involvement using histochemistry, immunohistochemistry for RANKL, receptor activator of nuclear factor kappa-B, osteoprotegerin and cathepsin K, and high-throughput quantitative real-time polymerase chain reaction on bone tissue from osteomyelitic lesions. A strong inflammatory response was seen in the infected animals and, therefore, we propose proteolytic enzymes induced by inflammation to be a major component of the bone loss. Furthermore, we found a significant upregulation of the IL26 gene in infected animals, which can inhibit RANKL-induced osteoclastogenesis, but has no homologue in mice. This finding emphasises that neither murine models nor in-vitro studies can mirror human disease development completely. The present study emphasises that the interactions between microorganisms, the immune system and bone cells in osteomyelitis are too complex to be accurately represented by an in-vitro model.

The effect of color type on early wound healing in farmed mink (Neovison vison).

BACKGROUND: Individual differences of mink, including color type, are speculated to affect the course of wound healing, thereby impacting wound assessment and management on the farms, as well as the assessment of wounds in forensic cases. In this study, we examined the effect of color type on early wound healing in farmed mink. Full thickness excisional wounds (2 × 2 cm) were made on the back in 18 mink of the color types Brown, Silverblue and Blue Iris. Gross and microscopic pathology of the wounds was evaluated 2 days post-wounding together with degree of wound size reduction, presence of bacteria and blood analyses. RESULTS: Pathological examination on day 2 showed the greatest mean wound size reduction in Brown mink (11.0%) followed by Blue Iris (7.9%) and Silverblue (1.6%). Bacteria were cultured from all wounds, and predominantly Staphylococcus species were recovered in mixed or pure culture. Histopathology from day 2 wounds showed a scab overlying necrotic wound edges, which were separated from underlying vital tissue by a demarcation zone rich in polymorphonuclear leukocytes. Fibroblasts and plump endothelial cells were more numerous in the deeper tissues. Complete blood count parameters were within normal ranges in most cases, however, the mink showed mildly to markedly decreased hematocrit and six mink of the color types Silverblue and Blue Iris showed moderately elevated numbers of circulating segmented neutrophils on day 2. There was a marked increase in concentration of serum amyloid A from day 0 to day 2 in all color types. CONCLUSIONS: We have described differences in early wound healing between mink of the color types Brown, Silverblue and Blue Iris by use of an experimental wound model in farmed mink. The most pronounced difference pertained to the degree of wound size reduction which was greatest in Brown mink, followed by Blue Iris and Silverblue, respectively.

Salmonella prevalence among reptiles in a zoo education setting.

Clinically healthy reptiles may shed Salmonella and therefore act as a potential zoonotic threat. Most people in Northern European countries are rarely exposed to reptiles, but many zoos have education departments where children have direct contact with this group of animals. The objectives of this study were to determine the prevalence and serotype distribution of Salmonella among reptiles in the Education Department (n = 55) at Copenhagen Zoo and compare it to the Zoo's main reptile collection (n = 145) to evaluate the zoonotic risk. Salmonella was isolated from cloacal swabs by selective enrichment, and a single isolate from each positive sample was further identified by biochemical tests and serotyped. The overall prevalence was 35% (69/200) with significant difference between the Education Department (64%, 35/55) and the main reptile collection (23%, 34/145). A total of 28 serotypes were detected. Ten serotypes were isolated from more than one specimen and four from more than one species. Salmonella enterica subsp. enterica serovar Eastbourne was the predominant serotype (32%, 22/69) and was also the serotype isolated from most reptile species (n = 7). Transmission of serotypes from one department to another was very limited indicated by the serotype distribution. Despite the relative high prevalence observed among the reptiles in the Zoo's Education Department compared to the reptiles in the Zoo's main reptile collection, no Salmonella cases have been linked to the Zoo, and Salmonella ser. Eastbourne is very rarely isolated from humans in Denmark. Simple hygienic procedures such as hand washing which is consistently carried out following handling of reptiles at the Education Department may reduce the risk and therefore contribute to this low prevalence.

Pathology and biofilm formation in a porcine model of staphylococcal osteomyelitis.

A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), NCTC-8325-4 (a laboratory strain of human origin; n = 3 animals) and UAMS-1 (a human osteomyelitis isolate; n = 3 animals). Two pigs were sham inoculated with saline. At 11 or 15 days post infection the animals were scanned by computed tomography before being killed and subjected to necropsy examination. Osteomyelitis lesions were present in the right hind limb of all pigs inoculated with strain S54F9 and in one pig inoculated with strain NCTC-8325-4. Microscopically, there was extensive loss of bone tissue with surrounding granulation tissue. Sequestrated bone trabeculae were intermingled with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore dependent on the strain of bacteria inoculated and on the formation of a biofilm.

The impact of Staphylococcus aureus concentration on the development of pulmonary lesions and cytokine expression after intravenous inoculation of pigs.

Acute respiratory distress syndrome is a common complication in severe sepsis. In pigs, the lungs play an important role in clearing systemic bacterial infections due to pulmonary intravascular macrophages found specifically in pigs. However, this increases the exposure of the porcine lungs to pathogens and potential injury. The authors propose that increasing the concentration of the inoculum without changing the bacterial dose will lead to severe sepsis with pronounced pulmonary lesions. This could potentially create a risk of cytokine spillover to the circulation, leading to an increased systemic response. Eight Danish Landrace pigs, approximately 10 weeks old, were inoculated twice with a low or once with a high concentration of Staphylococcus aureus. Three pigs were sham-inoculated. The animals were grouped based on macro- and microscopic lung lesions. The mRNA expression of local pulmonary inflammatory markers was compared to protein levels of systemic inflammatory markers. The most severe pulmonary lesions were observed in animals receiving the high S. aureus concentration, indicating that severity of lesions is dependent on inoculum concentration rather than total numbers of bacteria. Furthermore, local mRNA expression of inflammatory cytokines appeared to be dependent on the magnitude and severity of tissue destruction, including the ability to confine the lesions. Increasing mRNA levels of serum amyloid A could be a confident marker of severity of pulmonary lesions. Since no correlation was observed between local and systemic levels of inflammatory cytokines, this finding could indicate an ability of the porcine lung to compartmentalize the local inflammatory response and thus restrict systemic contribution.

Host response to porcine strains of Escherichia coli in a novel pyelonephritis model.

The initial pathology and pathogenesis of pyelonephritis and the influence of different strains of Escherichia coli were investigated in a novel porcine model. Nine female pigs were divided into three groups (A, B and C) and inoculated repeatedly into one renal pelvis with porcine pyelonephritis E. coli strain LK67 (P fimbriae PapG(I)), LK76 (type 1 fimbriae) or LK82 (type 1 fimbriae and P fimbriae PapG(II/III)), respectively. The contralateral kidneys were inoculated with saline and served as controls. Pigs were killed 6h post-inoculation (hpi). Differential leucocyte counts, serum biochemical analyses and measurement of serum concentrations of proinflammatory cytokines and acute phase proteins were carried out at 0, 3 and 6 hpi. Bacteriological evaluation of urine, kidneys, spleen, liver, abdominal swabs and blood samples and gross and histopathological evaluation of kidneys, renal lymph nodes, liver and spleen were performed by quantitative, semiquantitative and/or descriptive methods. Immunohistochemistry was used to identify cells expressing L1 antigen, CD3ɛ, CD4, CD8, CD79αcy and lysozyme, and to identify E. coli and Tamm-Horsfall protein (THP). E. coli was re-isolated from all inoculated kidneys. Gross and microscopical lesions of acute pyelonephritis were demonstrated in all but one kidney inoculated with E. coli, but in none of the control kidneys. Renal parenchymal infiltration with both neutrophils and mononuclear cells, primarily CD3+ T lymphocytes, was observed at 6 hpi. Most T lymphocytes were CD8+. Pigs in group C had the highest mean pathology scores. Neutrophils were the dominant renal leucocyte in this group, while the number of mononuclear cells was at least equal to the number of neutrophils in the lesions of pigs from groups A and B. Kidneys with a high number of E. coli had severe lesions. Systemic spread of E. coli was observed in five pigs. THP was observed interstitially in 89% of the E. coli-inoculated kidneys. In all groups, increased numbers of neutrophils and decreased numbers of lymphocytes and monocytes were shown by differential leucocyte count at 6 hpi, and from 3 to 6 hpi there was a significant increase in C-reactive protein concentration.

Endocarditis-associated brain lesions in slaughter pigs.

Left-sided valvular endocarditis (LSVE) is a common finding in slaughter pigs. The lesion is often associated with renal thromboembolism, but information on embolization to other organs is sparse. This study focuses on the presence and type of endocarditis-associated brain lesions (EABLs). The brains of 20 slaughter pigs with spontaneously arising LSVE and 11 controls were examined by sectioning half of a formalin-fixed brain into 4mm slices for histological examination. The aetiology of the endocarditis was determined by bacteriological and, in some cases, by fluorescence in-situ hybridization examinations. These examinations identified 11 cases of Streptococcus suis, six cases of Erysipelothrix rhusiopathiae, one Streptococcus spp. and two cases that remained aetiologically undetermined. One of the S. suis cases had a dual infection with S. suis in the aortic valve lesions and Streptococcus dysgalactiae subsp. equisimilis in the atrioventricular valve lesions. Renal infarcts were present in eight cases. Focal encephalitis was found in 12 cases, with the number of lesions ranging from one to 11. Most pigs had less than four microscopical lesions. Acute lesions were characterized by focal microabscesses without observable bacteria. Chronic lesions were characterized by astrocytosis and focal accumulation of mononuclear leucocytes. An infarct was observed in one animal. Perivascular inflammation was seen in 14 cases, mostly as two or three lesions, while focal leptomeningitis was found in eight cases. EABLs are therefore common in slaughter pigs with LSVE. The number of lesions per animal is small, which may explain the limited attention paid to this sequela of LSVE. EABLs have rarely been reported in domestic animals and mostly in patients with neurological signs. The frequent occurrence of EABLs in slaughter pigs suggests that this pathology should be investigated in other animal species with LSVE.

Polysynovitis after oligofructose overload in dairy cattle.

Acute bovine laminitis is a systemic disease with local manifestations primarily affecting the claws. However, distension of the tarsocrural joints has been observed after experimental oligofructose overload in dairy heifers as a part of the complex interpreted as acute, clinical laminitis. Therefore, the aim of the present study was to study bovine synovial joints and tendon sheaths after oligofructose overload. Ten dairy heifers received oral oligofructose overload (17 g/kg body weight); four were killed 24h after overload and six after 72 h. Six control heifers received tap water and were killed after 72 or 96 h. Clinical examination included locomotion scoring and palpation of the tarsocrural joints. Ruminal fluid and blood was collected for measurements of pH and hydration status. Total protein concentrations and white blood cell (WBC) counts were determined in synovial fluid collected from tarsocrural joints after death. Synovial joints and tendon sheaths were examined and synovial membranes were studied microscopically. Swabs taken from the synovial cavities were subject to bacteriological culture. Heifers with oligofructose overload developed signs of ruminal and systemic acidosis. Lameness was observed in three of ten heifers 24h after overload and in all remaining heifers after 72 h. Distension of tarsocrural joints was observed from 18 h after overload and peaked at 30 h when all examined joints were moderately or severely distended. The synovial fluid was turbid and protein content and WBC counts were increased at both 24 and 72 h compared with controls. Bacterial culture was negative. Synovial membranes 24 and 72 h after overload had a fibrinous and neutrophil inflammatory reaction that regressed in severity between 24 and 72 h after overload. Heifers subjected to oligofructose overload therefore developed generalized sterile neutrophilic polysynovitis. Focus on this aspect of bovine laminitis may shed new light on the pathogenesis of this complex disease.

Pathology of udder lesions in sows.

In recent years an increasing frequency of sows with udder lesions has been observed during meat inspection. The frequency and pattern of affected mammary complexes were determined by inspection, palpation, and incision of udders of 640 sows at a local slaughterhouse. From representative lesions, tissue specimens were sampled for histological and bacteriological examination. Moreover, in seven problem cases immunohistochemistry was applied for the identification of Staphylococcus aureus and Arcanobacterium pyogenes. Udder lesions were recorded in 67 sows (10.5%), with mammary complex no. 7 being most frequently affected (39%). In most cases (81%), lesions were restricted to a single mammary complex. Gross lesions were characterized as focal or multiple abscesses, which most often (79%) were accompanied by dermal ulcerations. Based on histological observations and bacterial identification, two groups of lesions were identified: (I) pyogranulomatous lesions of the dermal and subcutaneous tissue caused by S. aureus; and (II) dermal and subcutaneous abscesses dominated by A. pyogenes. In neither group I nor group II lesions, the glandular mammary tissue was involved as demonstrated by immunostaining for cytokeratin, i.e. mastitis was not diagnosed in any of the cases. In conclusion, all udder lesions were caused by skin infections predominantly caused by A. pyogenes and S. aureus resulting in the development of dermal abscesses and pyogranulomatous lesions, respectively.

Ovine fetal necrobacillosis.

The association of Fusobacterium necrophorum with late term abortion in sheep is reported. The bacterium was not culturable, but was identified in five cases by fluorescence in-situ hybridization (FISH) with an oligonucleotide probe specifically targeting 16S rRNA in F. necrophorum. Gross lesions were found in several tissues. Histologically, placental lesions were characterized by locally diffuse infiltration of neutrophils, closely associated with abundant small Gram-negative and FISH-positive rods, thrombosis and necrosis. Lesions in the fetal-maternal interface were multifocal and consisted of villous necrosis and suppurative inflammation. Spread to the fetus from the placenta appeared to occur in two ways. Some fetuses had multifocal necrotizing hepatitis consistent with haematogenous spread through the umbilical vein; further dissemination to other organs occurred. Transplacental spread and infection of the fetus through the amniotic fluid was characterized by development of multifocal suppurative dermatitis and suppurative bronchopneumonia. Localization of FISH-positive bacteria in necrotic lesions was restricted to the periphery. F. necrophorum would seem to have been unrecognized previously as a cause of abortion. The value of culture-independent diagnostic methods is emphasized.

Listeria monocytogenes in horses in Iceland.

Twenty isolates of Listeria monocytogenes associated with five confirmed and four suspected incidents of listeriosis in horses in Iceland were characterised by serotyping, pulsed-field gel electrophoresis and ribotyping. Semiquantitative estimates of the numbers of L monocytogenes were made on faeces from horses with clinical signs of listeriosis and on grass silage fed to them. Large numbers of L monocytogenes were often found in the faeces of horses with severe signs of disease. The 20 isolates could be divided into six genotypes, each incident involving only one genotype. One serovar 1/2a genotype was associated with three confirmed incidents of listeriosis in 1991, 1993 and 1997. In one incident, the same genotype was isolated from the organs of a horse with listeriosis and from the spoiled grass silage fed to it.

The diversity of Listeria monocytogenes strains from 10 Icelandic sheep farms.

AIMS: The purpose of this study was to examine the diversity of Listeria monocytogenes strains from healthy sheep, winter feed and environment of sheep farms in Iceland. METHODS AND RESULTS: A total of 104 L. monocytogenes isolates from animals, winter feed and environment on 10 Icelandic sheep farms were compared by serotyping, ribotyping, and pulsed-field gel electrophoresis with ApaI and AscI. The isolates were divided into 24 genotypes, all identified as serovars 1/2a, 1/2b, or 4b. Nine genotypes were detected on more than one farm. On three of the farms there seemed to be a dominant strain of L. monocytogenes. Isolates from incidents of listeriosis in animals occurring on two of the farms belonged to the genotype most commonly found on the particular farm. Nine of the 24 genotypes found on the sheep farms have been associated with disease in animals and/or humans elsewhere in Iceland. CONCLUSIONS: Certain strains of L. monocytogenes seem to be widely distributed on Icelandic sheep farms. On some farms there appears to be a dominant strain of L. monocytogenes. Incidents of listeriosis in animals may tend to be associated with strains commonly found on the farm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the diversity of L. monocytogenes present in healthy sheep and their environment.

Actinomyces hyovaginalis associated with disseminated necrotic lung lesions in slaughter pigs.

Lungs from 26 slaughter pigs with a diagnosis of pyaemic lung lesions (disseminated necrotic lesions and abscesses), as determined at post-mortem meat inspection, were subjected to a thorough examination, including re-evaluation of gross pathology and histopathological and microbiological examination of samples from multiple lesions. The pulmonary lesions, which appeared identical on gross inspection, could be divided into three histopathological types, namely (1) abscesses, (2) circumscribed (contained) necrotic lesions, and (3) bronchiolar ectasias. Some characteristic relations between bacterial species and histopathological type were demonstrated. Thus, abscesses were dominated by Staphylococcus aureus infections, and circumscribed (contained) necrotic lesions were dominated by infections with an Actinomyces species, identified as Actinomyces hyovaginalis by 16S rRNA gene sequencing. Actinomyces hyovaginalis was demonstrated in 23% of all cases, pointing to this organism as an important agent of disseminated lung lesions of pigs. Furthermore, a characteristic pyogranulomatous reaction with a central area of necrosis was found to be associated with this infection.

Early pathogenesis and inflammatory response in experimental bovine mastitis due to Streptococcus uberis.

A generally similar clinical response was observed in six lactating Holstein-Friesian cows after intramammary inoculation with approximately 10(7) colony-forming units of Streptococcus uberis. Increased concentrations of serum amyloid A (SAA) were measured in both milk and serum taken 6 and 11h after inoculation, respectively. In contrast, increased concentrations of haptoglobin were detected after 10h of infection, in milk only. In the blood, tumour necrosis factor-alpha (TFN-alpha) was detected (0.503 ng/ml) in only one animal, at the time of euthanasia (10h after infection). Interferon-gamma (IFN-gamma), like haptoglobin, was not detected in blood. Parallel to the development of inflammation and influx of inflammatory cells into the udder tissue, a marked decrease in the number of monocytes and neutrophils in blood was observed. Bacteria were found both intracellularly (macrophages and neutrophils) and within the lumen of ducts and alveoli. Lesions developed progressively in an ascending manner and became widespread throughout the mammary gland in less than 8h. The parallel development of inflammation and increased concentrations of SAA and haptoglobin in milk points to these acute phase proteins as potential diagnostic markers for the early detection of S. uberis -associated mastitis.

Preston and Park-Sanders protocols adapted for semi-quantitative isolation of thermotolerant Campylobacter from chicken rinse.

Human campylobacteriosis has become the major cause of foodborne gastrointestinal diseases in several European countries. In order to implement effective control measures in the primary production, and as a tool in risk assessment studies, it is necessary to have sensitive and quantitative detection methods.Thus, semi-quantitative detection of thermophilic Campylobacter spp. in 20 naturally contaminated chicken rinse samples was carried out using the two most common standard protocols: Preston and Park-Sanders, as proposed by Nordic Committee on Food Analysis (NMKL) and International Standard Organization (ISO), respectively. For both protocols, the chicken rinse samples were prepared in 500 ml buffered peptone water, as recommended in the ISO protocol no. 6887-2. The results indicated that the Preston protocol was superior to the Park-Sanders protocol in supporting growth of Campylobacter spp. In conclusion, the established semi-quantitative assessment using Preston broth could be useful in monitoring the outcome of control programs or quantitative risk assessments.

Bacterial colonization and invasion in pigs experimentally exposed to Streptococcus suis serotype 2 in aerosol.

A recently developed porcine model for aerogenous infection with Streptococcus suis serotype 2 was applied in a study of the phases of bacterial colonization and initial invasion. Eighteen pigs were exposed to aerosolized S. suis serotype 2 after pre-exposure to mild acetic acid in aerosol. The animals were killed consecutively within the first six days after challenge. After death, all animals were necropsied and examined by bacteriology, histopathology, and immunohistochemistry. Systemic infection was established in four out of 18 animals exposed to S. suis serotype 2. All systemically infected animals developed clinical signs and lesions typical of the infection. In four additional animals, subclinical infection was demonstrated by re-isolation of S. suis from the palatine tonsil. However, in all 18 challenged animals, immunohistochemistry demonstrated S. suis serotype 2 antigen in the palatine and/or nasopharyngeal tonsils. In all four systemically infected animals, S. suis serotype 2 antigen was also found in the mandibular lymph node. These observations point towards the tonsils as possible portals of entry for S. suis serotype 2 with subsequent lymphogenous spread. Thus, the present findings parallel the proposed pathogenesis for S. suis serotype 1 infection in pigs.

Streptococcus suis serotype 2 infection in pigs: new diagnostic and pathogenetic aspects.

In a study aimed at improving the diagnosis and elucidating the pathogenesis of Streptococcus suis serotype 2 infection in pigs, a combination of bacterial culture and histopathological and immunohistochemical examination was applied to a range of tissues from 42 naturally infected pigs with typical macroscopical lesions. By culture, 21 pigs (50%) were shown to be systemically infected with S. suis serotype 2; seven (17%) were infected with S. suis serotype 7, two with other bacteria, and 12 yielded no bacterial pathogens. The highest isolation rate for S. suis serotype 2 was obtained from the lateral cerebral ventricles and other regions of the brain, whereas the bacterium was only rarely isolated from the liver or spleen. Immunohistochemically, a diagnosis of S. suis serotype 2 infection was obtained in two of 12 (17%) animals from which no pathogens had been cultured. Moreover, immunohistochemistry differed from culture in revealing a greater number of positive tissue specimens. The microanatomical distribution of bacteria pointed toward the pharyngeal and palatine tonsils as principal portals of entry. Furthermore, S. suis serotype 2 bacteria were frequently identified immunohistochemically in the regional lymph nodes of the upper respiratory tract, possibly reflecting primary lymphogenous spread from the tonsils.

Evaluation of the pathology, pathogenesis and aetiology of auricular elephantiasis in slaughter pigs.

Ears from slaughter pigs with auricular elephantiasis (n = 24) and the corresponding lymph nodes (lnn.) (n = 26) were grossly, histopathologically and microbiologically examined. Immunostaining for IgM, IgG, Cd3epsilon and bacterial antigens of Arcanobacterium pyogenes and Staphylococcus aureus was performed by indirect enzyme-based techniques. Ears were variably thickened depending on the sampled area (basis, centre and apex). However, at all locations the thickness, the length from basis to apex and the weigh of whole ears with elephantiasis were significantly increased (P < 0.01). The corresponding lnn., that is, ln. parotideus superficialis and profundus, had also increased significantly (P < 0.01) in volume. Histopathologically, lesions of the ears and the corresponding lnn. revealed changes characterized by diffuse fibrosis intermingled with multiple pyogranulomatous foci containing asteroid bodies. In the majority of lesions, four distinct zones due to different cellular infiltrates encircled the central core of the asteroid bodies. In several lesions, the pyogranulomatous foci were contained within the lymph vessels. Immunohistochemically, only the bacterial antigen of S. aureus was detected within the cytoplasm of the macrophages and/or in the asteroid bodies of the ears (41.5%) and in the regional lnn. (30.8%). An abundant number of IgM, IgG and CD3epsilon-positive cells were present in all the pyogranulomatous lesions, whereas a positive IgG-staining was observed only in a single asteroid body. Thus, porcine auricular elephantiasis is a chronic pyogranulomatous inflammation that is frequently positive for S. aureus and is lymphogenically spread. Therefore, the lesions of the ears with auricular elephantiasis and the corresponding lnn. should be termed auricular botryomycosis and botryomycotic lymphadenitis, respectively. Moreover, as the disease is observed frequently in slaughter pigs it must also be considered according to the welfare of the animals and in relation to post-mortem meat inspection.