Marvin B. Rhodes

Pathology of Neonatal Calf Diarrhea Induced by a Coronavirus-Like Agent

Lesions induced by a bovine coronavirus-like agent were studied in gnotobiotic and colostrum-fed calves using gross, histologic and electron microscopic procedures. Lesions in gnotobiotic calves were present in the colon, mesenteric lymph nodes and in all segments of the small intestine. Calves killed 4 h after the onset of diarrhea had immunofluorescent epithelial cells on the villi of the small intestine and surface of the colon. Calves killed at 44 h had shortened intestinal villi and cuboidal epithelial cells. The villus-to-crypt ratio in the lower small intestine averaged 1.0 compared with 5.3 in a control calf. Immunofluorescent cells were present at the tips of the villi, and at the surface and in the crypts of the colon. Colostrum-fed calves that had serum-neutralizing antibody titers for the coronavirus-like agent developed diarrhea when inoculated orally with the agent. There was good correlation between histologic, immunofluorescent and electron microscopic findings.

Trypsin and chymotrypsin inhibitors from Ascaris suum.

Abstract Body wall and perienteric fluid of Ascaris suum were extracted with buffer, fractionated, and purified on carboxymethylcellulose. A trypsin inhibitor was eluted between pH 4.5 and 4.8 from perienteric fluid and body wall extract. The latter inhibited 3380 and 5170 μg of trypsin per milligram of protein, respectively. Two distinct chymotrypsin inhibitors were found. One inhibitor was eluted from carboxymethylcellulose between pH 4.3 and 4.6, while the second was eluted between pH 6.3 and 9.0. These inhibited 1930 and 2740 μg of chymotrypsin per milligram of protein, respectively.

STUDIES IN HELMINTH ENZYMOLOGY. IV. IMMUNE RESPONSES TO MALIC DEHYDROGENASE FROM ASCARIS SUUM.

Abstract Purified malic dehydrogenase from Ascaris suum when injected into guinea pigs and swine stimulated the production of specific antibodies, which were demonstrated by enzyme inhibition and immunodiffusion analysis. Fractionation of swine serum on a cellulose ion exchange column resulted in the separation of the inhibiting and precipitating antibodies. Malic dehydrogenases from closely related parasites were inhibited by the antiserum and gave crossreactions in immunodiffusion analyses. However, malic dehydrogenases from swine heart did not react with the antiserum. Guinea pigs receiving injections of malic dehydrogenase from A. suum did appear to receive some protection against migrating A. suum larvae.

Ascaris suum: Hatching of embryonated eggs in swine

Abstract Larvae of the nematode Ascaris suum were recovered in posterior small intestinal and colonie mucosal scrapings of pigs 4 to 29 hr after inoculation of embryonated eggs. Larvae were also recovered in the mucosal scraping from ligated intestinal segments at 4 to 19 hr after injection of embryonated eggs into the segments. Location of the ligated segment in the small intestine did not appear to affect hatching of eggs or migration of the larvae into the mucosal surface. Embryonated eggs hatched in ligated cecums and larvae were recovered in the livers of pigs after 2.5 and 3.5 days. Larvae were recovered in washings from Thiry-Vella intestinal loops at 5 to 23 hr after embryonated eggs were inoculated into the loop. Hatching of eggs occurred in intestinal loops in pigs unexposed to eggs and in pigs given multiple doses of eggs either orally or into the intestinal loop.

Bordetella bronchiseptica and toxigenic type D Pasteurella multocida as agents of severe atrophic rhinitis of swine

Bordetella bronchiseptica and toxigenic type-D Pasteurella multocida were cultured from pigs in each of five herds diagnosed as having severe atrophic rhinitis (AR). B. bronchiseptica alone, P. multocida alone, or both organisms isolated from four herds were inoculated intranasally into 1-week-old gnotobiotic pigs which were necropsied 4 weeks post-inoculation (PI). Nasal turbinate atrophy in B. bronchiseptica-inoculated pigs was moderate to severe, while P. multocida-inoculated pigs had slight to severe atrophy. Pigs inoculated with both organisms had moderate to complete turbinate atrophy. P. multocida was reisolated at necropsy from all pigs receiving the organism except those having no turbinate damage. B. bronchiseptica and P. multocida from a fifth herd were simultaneously inoculated into six naturally farrowed 6-day-old SPF pigs. Necropsy performed 4 weeks PI revealed severe to complete turbinate atrophy. Nasal turbinates were normal for control pigs in both experiments.

Detection of Bovine Herpesvirus-1-Specific IgM Using a Capture Enzyme Immune Assay with Isotype-Specific Monoclonal Antibodies

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7–40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.

Haemonchus contortus: enzymes. III. Glutamate dehydrogenase

Abstract Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti- Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.

Haemonchus contortus: Enzymes: I. Isoelectric focusing of malate dehydrogenase in sucrose and in polyacrylamide gels

Abstract Soluble extracts were prepared from adult male and female Haemonchus contortus and fractionated by DEAE-cellulose chromatography. The initial unadsorbed fraction contained 73% of the total malate dehydrogenase activity present in the crude soluble extract. This was fractionated by isoelectric focusing using LKB Ampholine carrier ampholytes in a sucrose-density gradient column. Seven peaks of malate dehydrogenase were obtained with isoelectric points ranging from 5.9 to 9.0. In addition, six hemoglobin peaks were separated. Composites of fractions from each peak of malate dehydrogenase were then fractionated by gel filtration on Sephadex G-200. Aldolase, malate dehydrogenase, and hemoglobin (when present) were separated from one another. Variations in the pH optimum and the pH optimum curves were noted for the various malate dehydrogenase fractions. In addition, twofold differences in the Michaelis constants were found. The maximum purification of malate dehydrogenase was 22 times compared with that of the crude soluble extract. Maximum purification of the other enzymes investigated was 11 times for aldolase, 3 times for inorganic pyrophosphatase, and 123 times for asparate aminotransferase. Isoelectric focusing experiments performed in polyacrylamide gels resolved the malate dehydrogenase into multiple bands. Polyacrylamide or starch gel electrophoretic analyses failed to give discrete bands of MDH activity.

A study of air quality and respiratory infections in pigs raised in confinement

Abstract Air quality, respiratory disease, and growth rate were followed in four different farrowing and nursery systems. Ammonia levels varied with ambient air temperature, but were within normally accepted levels (25 ppm). These levels of ammonia did not appear to affect the health or performance of the pigs raised in these units. Hydrogen sulfide levels were consistently low. Counts of bacterial colony forming particles (BCFP) varied and the organisms identified were predominantly micrococci. Bordetella bronchiseptica was isolated from nasal cavities of pigs from 3 out of 4 farms. Three of the farms did not have evidence of atrophic rhinitis; pigs farrowed in the last quarter of the test year on one farm from which B. bronchiseptica was isolated developed lesions of atrophic rhinitis. The B. bronchiseptica isolates from the 3 farms were virulent for gnotobiotic piglets. Groups of pigs for slaughter inspection from one farm had lungs with 11–28% pneumonic lesions; these lesions were not typical of mycoplasmal pneumonia.

Ascaris suum: Purification and characterization of an intestinal aminopeptidase

Abstract Semipurified preparations of an aminopeptidase from intestinal extracts of Ascaris suum had low specific activity (.03 units per milligram of protein), a large molecular size, and a limited solubility. Treatment of these preparations with a mixture of chymotrypsin and trypsin resulted in the release of ninhydrin-staining components during the incubation period (6 hours at 37 °C) but the aminopeptidase retained over 90% of its enzymatic activity. The enzyme was now soluble at pH 5 and migrated as a single band by starch-gel electrophoresis after it had been further purified by sucrose-density gradient centrifugation. The purified aminopeptidase was maximally activated by Co 2+ compared to other divalent metallic ions. The enzyme had a pH optimum of 6.8, a specific activity of 2.7 units per milligram of protein, and a calculated Michaelis constant of 2.0 × 10 −4 M . Progressive autolysis of crude intestinal homogenates also resulted in increasing amounts of a component which appeared similar to that of the purified aminopeptidase.