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 I4 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 [approximately 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.

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.

A retrospective study of bovine abortions associated with Bacillus licheniformis.

A retrospective study of bovine abortions associated with Bacillus licheniformis is described. The material consisted of 2445 bovine abortions submitted for diagnostics from 1986 through 1993. Initially, B. licheniformis had been isolated from 81 cases. Sections of these cases were re-examined microscopically and immunohistochemically by a PAP technique using a primary antibody against B. licheniformis. Of these abortions, 47 were most likely associated with B. licheniformis as tissue lesions with immunostained bacteria were present in these. In the remaining cases the diagnosis may not have been established due to the lack of sufficient materials, or the isolation of the bacterium was considered to be a result of contamination. In four cases concomitant infections with B. licheniformis and bovine virus diarrhoea virus were present. Abortions caused by B. licheniformis were predominantly seen during the winter months and in late pregnancy. The most common lesions were necrotizing placentitis followed by fetal multifocal suppurative bronchopneumonia. Immunohistochemically, B. licheniformis was demonstrated in association with tissue lesions and intracellularly in trophoblasts. The pattern of bacterial isolations, especially from the placenta, lungs, and abomasal contents, combined with the histological findings points to B. licheniformis abortions as being of haematogenous origin with subsequent transplacental spread to the fetus.

Sensitivity, specificity and predictive value of blood cultures from cattle clinically suspected of bacterial endocarditis.

This study investigated the number of blood culture-positive cattle among 215 animals clinically suspected of having bacterial endocarditis. For animals that were necropsied, the sensitivity, specificity and predictive value of the diagnosis of endocarditis were calculated on the basis of the isolation of the causative bacteria from blood. Furthermore, it was investigated whether the glutaraldehyde coagulation time, total leucocyte count, per cent neutrophil granulocytes, pulse rate and duration of disease could help to discriminate endocarditis from other diseases. Among 138 animals necropsied the sensitivity, specificity and predictive value of blood cultivation were 70.7 per cent, 93.8 per cent and 89.1 per cent, respectively. None of the other measurements could be used to discriminate between endocarditis and non-endocarditis cases.

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.

Abortion and calf mortality in Danish cattle herds.

The aetiology of abortions and calf mortality in 65 Danish cattle herds consisting of both dairy and beef breeds during a 1-year period is described. All observed aborted foetuses, still-born calves, and calves dying before 6 months of age were necropsied, and relevant microbiological examinations were performed. A total of 240 calves and 66 abortions were submitted corresponding to a calf mortality rate of 7%. The abortion frequency could not be calculated. 43% of the calves died at day 0, while 22% were aborted, 15% died during the first week of life, 9% died from 1 to 4 weeks of age, and 11% died at the age of 1 to 6 months. The most common cause was neonatal pulmonic atelectasis (stillbirth) followed by foetal infections, pneumonia, and septicaemia.

Bovine mycotic abortion--a comparative study of diagnostic methods.

The agreement between three diagnostic methods (mycology, morphology, and immunofluorescence staining) was compared two by two in elucidating the aetiological diagnosis in 52 cases of bovine mycotic abortion. Agreement between the three methods was expressed by the kappa coefficient (kappa), and ranked as follows: mycology and immunofluorescence staining (kappa = 0.48); morphology and immunofluorescence staining (kappa = 0.40); mycology and morphology (kappa = 0.28). A. fumigatus was confirmed to be the main cause of bovine mycotic abortion with members of the zygomycetes (order Mucorales) being the second most important group. In two aborted placentas a Candida species was found to be the cause of mycotic placentitis.

A case of guttural pouch mycosis in a horse.

A case of guttural pouch mycosis in an 11-year-old horse is described. The fungus isolated was identified as Emericella nidulans. Housing under bad hygienic conditions without ventilation for three years might have been a predisposing factor.

Bovine mycotic abortion in Denmark.

A survey is given of the occurrence of mycotic infections associated with abortion in Danish cattle. During a period of six years a total of 748 samples of placenta material were examined. Mycotic abortion was demonstrated in 101 cases (14%). The case rate was significantly higher (21%) during the winter months from December through February than during the rest of the year (10%). Abortions occurred as from the 135th day of pregnancy, with maximum in the eighth month, where 45% of all cases were observed. A. fumigatus was demonstrated in 77 cases, Mucor spp. in 11 cases, Absidia spp. in 5 cases, and a mixed infection with A. fumigatus and Mucor spp. in 3 cases. In 5 cases the fungi were demonstrated by microscopic and histological examination, but not by culture. The annual occurrence of mycotic abortion varied from 10% to 24% of cases of abortion examined. The latter figure was recorded in a year following a particularly heavy rainfall in the month of June.

Bovine cryptosporidiosis in Denmark. 2. Cryptosporidia associated with neonatal calf diarrhea.

In recent years cryptosporidia have often been identified in diarrheic calves of under one month old, either as the sole pathogenic agent or in combination with other diarrhea-causing infectious agents. In 203 cases of bovine cryptosporidiosis, recorded over a period of two years, cryptosporidia were present in such great numbers that they would seem to be of causal significance. The calves were from three days to five weeks old. In 111 cases (55%) a monoinfection with cryptosporidia occurred, while in the rest of the cases mixed infections with other agents associated with calf diarrhea were demonstrated, especially rotavirus (61 cases) and coronavirus (32 cases); in addition, mixed infections with ETEC (four cases), septicaemic E. coli (two cases), salmonellosis (11 cases), and BVD and coccidiosis (one case each) were seen. In 87 (10%) of 849 4-21-day-old dead calves a massive occurrence of cryptosporidia was demonstrated. Extreme emaciation was noted in 36 of 122 autopsied calves. Most of these calves were more than 14 days old, and mono-infection with cryptosporidia was demonstrated in two thirds of them. In younger calves, more acute cases of cryptosporidiosis were commoner, often in connection with concomitant infections with rota- or coronavirus.

Bovine cryptosporidiosis in Denmark. 1. Prevalence, age distribution, and seasonal variation.

During the period 1.7. 1981-30.6. 1983 the prevalence of infection with cryptosporidia in Denmark was studied by examination of autopsy material from 3358 animals and feces samples from 911 animals. The material was submitted for routine diagnostic examination. Diagnosis of Cryptosporidium infection was based on the detection of oocysts in smears from the mucosa of the small intestine, from colon contents, or from feces samples. Smears were stained by a modified Ziehl-Neelsen technique. Infection with cryptosporidia was demonstrated in 471 specimens, representing 11% of the total of 4269 animals examined. Highest prevalence was found among calves of 4-30 days old, with a peak in the age group 8-14 days (24.8% positive). Low-grade excretion of oocysts was observed in 2-5% of animals older than 1-2 months. No distinct seasonal variation was observed, but there was a tendency for the frequency to be highest in the 2nd quarter of the year and lowest in the 4th quarter.

Numbers of airborne bacteria and fungi in calf houses.

Counts of airborne bacteria colony forming particles (BCFP) and fungi were made at intervals throughout one year in three calf houses using a six stage Andersen Sampler. House 1 was insulated, mechanically ventilated and heated, House 2 was insulated and provided with a controlled natural ventilation system, while House 3 was uninsulated with natural ventilation. Each house contained 36 bought-in bull calves. Every six weeks the 12 oldest calves were removed and replaced by 12 four-week-old calves. The mean count of BCFP was highest in House 2 (101.6 X 10(3) m-3) and lower in House 3 (67.6 X 10(3) m-3). The mean count of aerial fungi was significantly lower in House 3 (40.5 X 10(3) m-3) than in Houses 1 and 2 (119.3 X 10(3) m-3 and 127.1 X 10(3) m-3, respectively). The count of aerial BCFP and fungi showed large seasonal fluctuations, but there was a general trend towards lower counts during the winter period. The mean incidence rate of respiratory disease among the experimental calves was 67.7%. The highest mean incidence rate was recorded in House 2, but differences between houses were not significant. The results are discussed in relation to the environmental requirements for raising of calves, and in the light of the current concept of air hygiene as a major predisposing factor in the web of causation of calf respiratory disease.

Occurrence of enterotoxigenic Escherichia coli in calves with acute neonatal diarrhoea.

Enterotoxigenic Escherichia coli (ETEC) were isolated from 16.4% of diarrheic calves of up to 30 days old. Of 1-2-day-old calves nearly one half (45.0%) harboured ETEC, while this was the case with only 4.9% of 8-day-old calves. Among 206 9-30-day-old calves just three were found to harbour ETEC.

Infection with enterotoxigenic Escherichia coli in calves and protection of the calves by vaccination of the dams.

A report is given of a study on the occurrence of enterotoxigenic Escherichia coli in calves in Denmark. Samples from 1379 1-30-day-old calves were examined and ETEC were isolated from 228 samples (16.5%). While in 1-2-day-old calves nearly one half (47.5%) harboured ETEC, the frequency dropped to 3.4% in 8-day-old calves, and of 165 9-30-day-old calves just one was found to harbour ETEC. A field trial with K99-containing bacterins was performed in 14 herds with ETEC problems. A total of 109 cows were vaccinated with a 4-strain bacterin and 73 with a K99-bacterin, while 114 cows served as controls. Sixteen calves from the 4-strain bacterin group, 6 calves from the K99-bacterin group, and 25 calves from the control group, became diarrheic within their first week of life.

A study of skin diseases in dogs and cat. VII. Ringworm infection.

During 1973 through 1979 a total of 774 specimens from dogs and 227 specimens from cats were submitted for examination for ringworm infection. Ninety-six (12.4%) of the samples from dogs and 66 (29.1%) of the samples from cats were positive by culture (Tables I and II). Microsporum canis accounted for all infections in cats and for 95.8% of the infections in dogs. Four dogs (4.2%) were infected with Trichophyton mentagrophytes. Three fourths of the infections with M. canis were diagnosed during August through January (Fig. 1). Ringworm infections can be diagnosed by direct microscopy of hair and scrapings. Wood's lamp examination, skin biopsy, and culture. Of these, the latter method is the most reliable.

A study of skin diseases in dogs and cats. VI. Microflora of the major canine pyodermas.

From 40 dogs with pyoderma swabs from areas with representative lesions were examined bacteriologically. Staph. aureus was found in 98% of the areas, beta-hemolytic streptococci in 30%, and Gram-negative organisms, mainly Proteus spp., in 30% (Table I). Pure infection with Staph. aureus was found in 55% of the areas. Staph. aureus and beta-hemolytic streptococci were found in 15%, Staph. aureus and Gram-negative organisms in 15%, Staph. aureaus and beta-hemolytic streptococci as well as Gram-negative organisms in 13%, and beta-hemolytic streptococci and Gram-negative organisms in 3% (Table II). Compared to normal and eczematous skin areas, Staph. aureus was found more often, and in greater numbers, in areas with pyoderma, whereas micrococci, alpha-hemolytic streptococci, and Acinetobacter spp. were rarer. Gram-negative organisms such as Proteus spp., E. coli, and Pseudomonas spp. were found with equal frequency in eczema and pyoderma, while beta-hemolytic streptococci were almost exclusively associated with suppurative lesions (Tables IV and V).