Bent Aalbæk

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.

Development of murine monoclonal antibodies for the immunohistochemical diagnosis of systemic bovine aspergillosis

Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions (n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.

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. Tisssues 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.

Some new aspects of the pathology, pathogenesis, and aetiology of disseminated lung lesions in slaughter pigs.

From 40 pigs rejected for human consumption at slaughter due to an apparent presence of pyemic lung lesions (defined as disseminated processes containing pus and/or necrotic material), the lungs, spleen, liver, and kidneys were subjected to an extended macroscopic examination. Several lung lesions were sampled from each animal for histological and bacteriological examination. Samples from the kidneys and spleens were also subjected to bacteriological examination. At gross level, four groups of lung lesions were identified: 1) disseminated foci with contents of pus and/or necrotic material (n=26); 2) disseminated or multifocally located ecchymoses with a central area of fibroplasia (n=9); 3) non‐pneumonic lesions, i.e., disseminated areas of atelectasis (n=1) or haemorrhagic areas developing due to the process of slaughter (n=1); and 4) suppurative lesions without a disseminated distribution pattern (n=3). Histologically, the disseminated suppurative/necrotic foci were identified as: A) abscesses (n=10); B) necrotic lesions (n=6); and C) ectatic or ectatic‐like bronchioles with contents of pus and necrotic material (n=10). The macroscopic observation of disseminated centres of fibroplasia with peripheral ecchymoses (n=9) was confirmed histopathologically. The livers of five pigs contained multiple areas of chronic interstitial fibrosis related to migration of Ascaris suum larvae (“milk spotted liver”). Such hepatic lesions were significantly (p<0.01) related to the simultaneous occurrence of disseminated pulmonary ecchymoses with a central area of fibroplasia. Generally, all lung lesions of each individual animal contained identical monocultures of bacteria following this pattern: Staphylococcus aureus (abscesses); Actinomyces hyovaginalis (necroses); S. aureus, A. hyovaginalis, and Arcanobacterium pyogenes (ectatic and ectatic‐like bronchioles). Areas with fibrosis were sterile or contained bacteria considered to be a result of contamination. Apart from one kidney, from which S. aureus was cultured, all other organs were sterile. It is concluded that difficulties exist in differentiating pulmonary pyemic lesions from non‐pyemic lesions at the gross level. Thus, it was not possible to distinguish between abscesses/necroses and ectatic bronchioles, the pathogenesis of the latter being uncertain. However, the chronic non‐pyemic lesions related to the migration of A. suum larvae should be identified by the absence of pus/necrosis. S. aureus was predominantly isolated from abscesses, whereas, and most surprisingly, A. hyovaginalis was the dominant bacterium isolated from the pulmonary necroses.

Experimental Feline Panleucopenia in the Conventional Cat

Conventional kittens, 12–27 weeks old, were inoculated with cell-cultured feline panleucopenia virus and killed sequentially between day 3 and day 24 after inoculation. All developed a non-fatal mild disease between days 2 and 9, characterized by lymphopenia, neutropenia, listlessness, depression and the development of neutralizing antibodies to the virus. Small intestinal bacterial counts were reduced during the period of maximal clinical disease, presumably a result of decreased food intake. There was involution of the thymus with marked depletion of lymphocytes between days 3 and 12. Depletion of lymphocytes also characterized the lesions in the lymph nodes between days 3 and 8. At the same time crypt lesions with spotty distribution were in the small intestinal and colonic mucosa. The changes were loss of crypt epithelial cells with compensatory attenuation of the remaining epithelium. Electron microscopically, the number and size of microvilli and secretory granules were reduced but there was no change indicating lethal cell injury. There were no virus particles. The findings point to an early and transient cellular damage by the virus. Intestinal alkaline phosphatase activity disappeared from the luminal surface of the attenuated crypt epithelial cells. Otherwise, intestinal alkaline and acid phosphatase activity were not altered in inoculated cats.

Relationships among strains classified with the ruminant Pasteurella haemolytica-complex using quantitative evaluation of phenotypic data.

The phenotypic relationships among 246 trehalose-negative strains classified under the [Pasteurella] haemolytica-complex in ruminants were investigated by clustering and multidimensional ordinations based upon 79 phenotypic characters. A quantitative evaluation of phenotypic data using a 5-level scoring system is presented permitting a comprehensive utilization of the recorded phenotypic variation among the strains in the analyses. Clustering and ordination analyses display complementary aspects of data which has been clearly demonstrated in this investigation. The main clusters revealed by the numerical techniques could be related to distinctive phenotypic differences and showed an extensive correlation with the recognized biogroups. This classification was based only upon 4 characters (fermentation of L-arabinose, D-sorbitol, glucosides and ornithine decarboxylase). In contrast, there was no obvious interpretation of the clusters formed by using binary scores. Phenotypic subgroups within the recognized biogroups have been described as well as a new, related group of bacteria, tentatively named Bisgaard taxon 36. Quantitative interpretation of phenotypic data seems to represent a promising method for finding relations among affiliated strains of bacteria and to assist in forming hypotheses for subsequent genotypic investigations.

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 haboured 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 + ampi‐cillin) were found to be shared by numerous strains, suggesting a genetic linkage of the resistance markers.

Bovine mammary protothecosis due to Prototheca zopfii

Mastitis due to Prototheca zopfii was diagnosed in three of 28 cows in a dairy herd. As two cows continued to shed algae after 45 days they were slaughtered and organs were examined by cultivation, histology, immunohistochemistry and electron microscopy. Algae were restricted to the mammary glands and regional lymph nodes in which a granulomatous inflammation was seen. Algae were predominantly seen in macrophages but neutrophils also contained organisms. In macrophages both sporangiospores and sporangia were found, suggesting that intracellular proliferation may be responsible for the failure to overcome the infection. Serum samples from all cows were assayed for antibodies against P. zopfii in an enzyme-linked immunosorbent assay (ELISA). Although the highest titre was found in an infected cow the difference between the mean values of the titre in infected and non-infected cows was not significant.

Meat safety consequences of implementing visual postmortem meat inspection procedures in Danish slaughter pigs.

The consequences of a change from a traditional meat inspection procedure, including manual handling, palpation and incision, to an entirely visual postmortem meat inspection procedure in Danish slaughter pigs were assessed by a comparative study of the two methods in 183,383 slaughter pigs. Out of 58 lesion codes (selected with a prevalence ≥5.5 x 10−5), 26 (45 per cent) were assessed either as merely aesthetic or as the healed stage of an earlier lesion and nine (15 per cent) as active, but local processes, occurring only in non-edible tissue. Five lesion codes (9 per cent) were assessed as active, nonabscessal processes occurring in edible tissue, caused by swine-specific pathogens and 10 (17 per cent) were abscessal or pyaemic lesions occurring in edible tissue. Seven lesion codes (12 per cent) may be associated with consumer health hazards (two frequently and five rarely), and one with occupational health hazards. It was estimated that per 1000 carcases, an additional 2 5 with abscessal or pyaemic lesions (in edible tissue) containing Staphylococcus aureus, 4 x 10−4 containing ochratoxin, 0.2 with arthritis due to Erysipelothrix rhusiopathiae, 0.1 with caseous lymphadenitis, 0.7 faecally contaminated with Salmonella species, and 3.4 faecally contaminated with Yersinia enterocolitica would remain undetected as a result of changing from the traditional to the visual inspection procedure. Two valuable reasons for implementing a visual control system are the potential for decreased cross-contamination (no handling, cutting and incision) and reduced inspection costs. The resources released as a result may be reallocated to hygiene and surveillance programmes.

Immunohistochemical Diagnosis of Systemic Bovine Zygomycosis by Murine Monoclonal Antibodies

Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from 14 to 110 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions (n = 140 [∼ 97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis.