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.

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.

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.

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.

Identification of Prototheca from bovine mastitis in Denmark.

In the present study, algal isolates recovered during the period 1991 through 1996 from Danish cases of bovine mastitis were characterized and identified. Sixteen isolates of Prototheca were obtained, all of which were identified as Prototheca zopfii according to assimilation pattern, absence of capsules and resistance to clotrimazole.

Detection of galactomannan and complement activation in the pregnant mouse during experimental systemic aspergillosis.

Foeto-placental infections are obtained when pregnant mice are challenged intravenously with conidial suspensions of Aspergillus fumigatus. This experimental model, which is used without any immuno-suppressive pretreatment, can be employed to screen for differences in foeto-placental infectivity of A. fumigatus strains when the number of colony-forming units (CFUs) in conidial suspensions used for infection is from 1 x 10(5) to 1 x 10(6). In the foeto-placental unit, hyphal growth was initiated at the periphery of the placental disc from which infection spread to the central parts of the placenta, the extrafoetal membranes and the foetus. Complement activation was noticed as a consequence of pregnancy and conidial inoculation, but was neither dose-dependent nor related to the extent of infection. Galactomannan was present in the plasma of infected mice and, in contrast to the situation in bovine placental aspergillosis, can be used as a good marker of foeto-placental aspergillosis.

Experimental candidosis in pregnant mice.

Pregnant mice were challenged intravenously with doses of 1 x 10(3)-1 x 10(7) Candida albicans blastospores, and from postmortem histopathology it was found that C. albicans had a propensity for localization in the murine placenta. At the highest dose, blastospores, hyphae and pseudohyphae were randomly located in the foeto-placental units, whereas proliferation of fungi at lower doses started in the rim of the placental disc, after which it spread along Reichert's membrane and/or the splanchnopleure, eventually attacking the foetal skin. These findings are analogous to our previous observations on the nature of Aspergillus fumigatus infection in pregnant mice.

Prevalence of antibiotic-resistant Escherichia coli in Danish pigs and cattle.

The present paper reports on the antibiotic resistance of E. coli isolated from Danish piglets and calves in 1987-1988, and compares the results with similar investigations performed during the periods 1971-1972 and 1977-1978. Rectal swabs from 52 piglets and from 78 calves were examined. All the animals studied harboured resistant E. coli. This is a significant increase compared to the previously conducted investigations. The number of strains having three or more resistance markers did not differ significantly from the previous findings. The spectrum of resistance markers among Danish piglets and calves had increased through all three investigations and resistance to chloramphenicol was still found to be considerable 10 years after the withdrawal of chloramphenicol as a therapeutic drug for farm animals in Denmark in 1978. Certain resistance patterns (sulfonamide + streptomycin, sulfonamide + streptomycin + tetracycline, sulfonamide + streptomycin + tetracycline + ampicillin) were found to be shared by numerous strains, suggesting a genetic linkage of the resistance markers.

Aerogenous infection of microbiologically defined minipigs with Streptococcus suis serotype 2. A new model.

Streptococcus suis serotype 2 is the cause of serious infections in animals and humans, but certain aspects of the infection pathogenesis still remain unclear. In this study an experimental model of aerogenous infection and induction of septicemia with S. suis serotype 2 was established in microbiologically defined Göttingen minipigs. Ten animals were exposed to aerosolized S. suis after previous exposure to mild acetic acid in aerosol. Six of the animals were immunosuppressed with prednisolone acetate on different days. All the animals were monitored clinically until euthanasia on days 6 to 13 after exposure. Necropsy was performed and samples were taken for microbiology, histopathology, and immunohistochemistry Three out of four animals immunosuppressed on days 5 to 7 after exposure developed S. suis septicemia, and S. suis could be detected in the tonsil of the soft palate and/or the nasal cavity of all exposed animals. Thus, using the presented model, local as well as systemic infection with S. suis serotype 2 was established in the Göttingen minipig. Since this breed is defined as free of S. suis and a range of other endemic porcine pathogens, the experimental model could prove useful in the study of this infection.

Mycotic and algal bovine mastitis in Denmark.

A one-year examination of mammary secretions (n = 2,896) from Danish cattle with clinical or subclinical mastitis revealed 45 strains of fungi and algae. The strains originated from 44 mammary secretions of 42 cows in 40 herds. The following species of fungi were identified: Candida catenulata (n = 2), Candida kefyr (n = 6), Candida krusei (n = 17), Candida rugosa (n = 6), Candida tropicalis (n = 3), Candida valida (n = 1), Geotrichum capitatum (n = 5). The algal species Prototheca zopfii was demonstrated in five samples.

Typing of Pasteurella multocida from haemorrhagic septicaemia in Danish fallow deer (Dama dama).

Isolates of Pasteurella multocida ssp. multocida (n = 31) from a Danish population of fallow deer which succumbed to haemorrhagic septicaemia during 1992 1993 and isolates from the palatine tonsils of apparently healthy fallow deer from the same area (n=6) were typed and compared with P. multocida from other sources. Plasmids were net observed in the fallow deer strains and one unique pattern was observed by ribotyping using HindIII and by pulsed-field gel electrophoresis using SanlI as restriction endonuclease. All Danish fallow deer isolates belonged to serotype B:3,4. On restriction endonuclease analysis using HhaI as restriction endonuclease, all had a profile identical to that of a fallow deer isolate from the United Kingdom: profile 0033 of Wilson et al. On restriction endonuclease analysis using HpaII as restriction endonuclease, the Danish fallow deer isolates had a unique profile, designated 0062, which differed slightly from that of a fallow deer isolate from the United Kingdom. P. multocida from other animal species were genotypically different from the fallow deer isolates. It is concluded that a specific clone of P. multocida was responsible for the outbreak of haemorrhagic septicaemia among Danish fallow deer. A carrier rate of 27% was demonstrated among apparently normal animals from the same population.

Enzyme immunohistochemistry with mono- and polyclonal antibodies in the pathological diagnosis of systemic bovine mycoses.

To improve the immunohistopathological diagnosis of systemic bovine mycoses, we have evaluated the utility of antifungal polyclonal and monoclonal antibodies, and peroxidase and alkaline phosphatase staining techniques. A rabbit polyclonal antibody to mannan from Candida albicans was specific for candidosis. The diagnosis of aspergillosis was accomplished using a rat monoclonal antibody to the galactofuran side chains of Aspergillus galactomannan. A murine monoclonal antibody reacting with weakly Con-A binding 41 and 46 kDa somatic antigens from Absidia corymbifera was used for immunostaining of zygomycetic hyphae. Peroxidase antiperoxidase (PAP) and alkaline phosphatase antialkaline phosphatase (APAAP) complexes were visualized using aminoethylcarbazole and fast red substrates. A green staining of PAP reactions with dioctyl sulfosuccinate sodium and 3,3',5,5'-tetramethylbenzidine (DONS/TMB) was effective for the demonstration of fungi in dual and triple infections. Tissue sections of experimentally infected mice were used to determine the sensitivity and specificity of the antibodies. Tissues obtained from 161 bovine mycotic lesions previously studied by indirect immunofluorescence staining were further evaluated using the three antibodies. In all of 45 lesions solely affected by aspergillosis and in three solely affected by candidosis the diagnoses were confirmed by the new evaluation. In 85 of 96 cases of single infections with zygomycetes the diagnosis was confirmed, while none of the antibodies reacted with fungal elements in the remaining 11 lesions. Aspergillus hyphae were detected in all three lesions with dual aspergillosis and zygomycosis, whereas zygomycetic material was confirmed in only two of these cases. A mixed infection of candidosis and zygomycosis in a lymph node was confirmed too. In 13 cases in which a diagnosis had not hitherto been obtained, aspergillosis and zygomycosis were recorded each in three cases.

Mitogen-activated protein kinase-defective Candida albicans is avirulent in a novel model of localized murine candidiasis.

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1 delta/cek1 delta null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 microliter suspension containing 1 x 10(5), 1 x 10(6) and 1 x 10(7) blastopores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1 delta/cek1 delta null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.

Specificity for field enumeration of Escherichia coli in tropical surface waters.

In remote rural areas in developing countries, bacteriological monitoring often depends on the use of commercial field media. This paper evaluates a commercial field medium used for the enumeration of Escherichia coli in different surface waters under primitive field conditions in rural Pakistan. In order to verify the field kit, 117 presumptive E. coli isolates have been tested, finding a specificity of only 40%. By excluding some strains based on colony colours, the calculated specificity could be increased to 65%. Thus, it is suggested that prior to use in a tropical environment, the specificity of any commercial medium used should be tested with representative tropical isolates, in order to increase the specificity.

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.

Summer mastitis in heifers: a bacteriological examination of secretions from clinical cases of summer mastitis in Denmark.

A total of 166 samples of secretions collected from Danish heifers suffering from clinically diagnosed summer mastitis were examined bacteriologically. One hundred and sixty three samples yielded positive findings whereas no growth was obtained from 3 specimens. The majority of samples revealed a mixed flora in which the predominant components were Actinomyces pyogenes, Peptostreptococcus indolicus, a microaerophilic coccus (Stuart-Schwan coccus), Fusobacterium necrophorum, Bacteriodes melaninogenicus and Streptococcus dysgalactiae. Pure cultures were recovered in only 7% of cases. P. indolicus was isolated from 875 of the cases, a microaerophilic coccus from 84%, A. pyogenes from 72%, Fusobacterium necrophorum from 52%, Str. dysgalactiae from 37%, B. melaninogenicus from 33% and various unidentified obligate anaerobic Gram-negative rods from 31%. The results confirm the complex bacterial aetiology of summer mastitis.

Comparative bacteriological studies on summer mastitis in grazing cattle and pyogenes mastitis in stabled cattle in Denmark.

A total of 143 secretions from clinical cases of summer mastitis (SM) in grazing cattle and from 89 cases of pyogenes mastitis (PM) in stabled cattle were examined bacteriologically. The typical bacteriological finding was a mixed flora in which the predominant organisms were Actinomyces pyogenes (SM-70%, PM-85%), Peptostreptococcus indolicus (54%, 54%), a microaerophilic coccus (Stuart-Schwan coccus) (26%, 25%), Fusobacterium necrophorum biovar B (22%, 12%), Bacteroides melaninogenicus (20%, 9%) and Streptococcus dysgalactiae (21%, 5%). All except six cases occurred in non-lactating animals or within three weeks after parturition. The majority of animals (about 90%) had only one quarter affected and no differences in quarter distribution were observed between the two groups.

Summer mastitis in heifers: studies on the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus and Bacteroidaceae in clinically healthy cattle in Denmark.

With the aim of investigating the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus, Bacteroides melaninogenicus ss. levii and Fusobacterium necrophorum, and thus the potential for development of summer mastitis, clinically healthy Danish Holstein-Friesian heifers due to calve in the autumn were sampled from the teat tip, the conjunctiva and the oral cavity at 2-6 week intervals from 1979 to 1981. The overall isolation rates of F. necrophorum, P. indolicus and B. melaninogenicus ss. levii, in order of significance, were significantly higher during the pasture period whereas no differences in isolation rates of A. pyogenes between housed and pastured animals were detected. F. necrophorum was recovered almost exclusively from the oral cavity, P. indolicus and A. pyogenes occurred most frequently in samples from the teat skin, whereas isolates of B. melaninogenicus ss. levii were evenly distributed between conjunctiva and teat tip samples. A distinct seasonal pattern of the isolation rates of summer mastitis pathogens was recorded, which corresponded closely to the seasonal activity of symbovine insects, in particular the headfly Hydrotaea irritans (Fallén). However, the high proportion of clinically healthy bacterial carriers as compared with the incidence of clinical disease strongly suggests that as yet unknown contributing or triggering factors, apart from the mere presence of the relevant bacterial species, are required for the establishment and development of clinical summer mastitis.

Immunohistochemical identification of aetiological agents of systemic bovine zygomycosis.

The reactivity of a panel of rabbit hyperimmune antisera raised against somatic antigens of three zygomycetes, Absidia corymbifera, Rhizopus oryzae and Rhizomucor (Mucor) pusillus was assessed by crossed immunoelectrophoresis (XIE) and indirect immunofluorescence (IIF) staining of experimentally infected murine tissues. Despite a widespread cross-reactivity of the neat antisera, heterologous absorption rendered antisera monospecific as assessed by XIE. Heterologous absorption also rendered the antisera against R. oryzae and R. (Mucor) pusillus specific by IIF, whereas heterologous absorption of Abs. corymbifera antiserum did not abolish reactivity with R. oryzae. The reactivity of the heterologously absorbed antisera and a murine monoclonal IgG1 antibody against Abs. corymbifera enabled zygomycetes within bovine lesions to be identified. Of 113 zygomycotic lesions of 95 cattle examined, 82 (72.5 per cent) showed a pattern of reactivity compatible with Abs. corymbifera, 21 (18.6 per cent) with R. oryzae, and 5 (4.4 per cent) with R. (Mucor) pusillus. Fungi in four lesions reacted with the monoclonal antibody only, and fungal elements in one lesion showed no reactivity at all. Each of the bovine lesions contained only a single fungal species, but one animal was infected by Abs. corymbifera in the rumen and by R. oryzae in the other stomach compartments. Apart from being the main cause of systemic bovine zygomycosis in Denmark, Abs. corymbifera also seems to be the most pathogenic due to its frequent haematogenous and lymphatic spread (87.8 per cent) as compared with R. oryzae (1.4 per cent) and R. (Mucor) pusillus (5.4 per cent).

The occurrence of fungi in bovine tissues in relation to portals of entry and environmental factors.

The occurrence of fungi in tissue specimens from 72 cattle was examined by culture, histopathology and indirect immunofluorescence staining (IIF). Groups of 12 animals each had been fed either concentrate or roughage and had been housed either in tie stalls, on slatted floors or on deep bedding. Specimens were obtained from the lung, omasum and Peyer's patches of the ileum and corresponding lymph nodes. Both hyphae and spores were made visible by IIF and by combination of IIF, morphology and conventional staining it was possible to differentiate between aspergilli and zygomycetes. In the lungs, aspergilli were detected at the same rate by morphology and IIF, whereas zygomycetes were found nearly twice as often by IIF than by culture. Fungi in pulmonary tissue were most frequent in cattle tied or kept on deep bedding (P < 0.01) as assessed by IIF. Within lymph nodes only spores were found, and Aspergillus fumigatus was the predominant species. Lesions devoid of fungi, especially ulcerations, were observed on the edges of the largest omasal laminae with a notable preference for the aboral third. The localization and histopathology suggested a reflux of acid abomasal contents to be the pathogenic principle. Granulomas with centrally located plant material were found more frequently in cattle fed roughage than in cattle fed concentrate (P = 0.01) and were differentiated from mycotic granulomas.