A double-antibody sandwich ELISA (DAS-ELISA) was developed for detection of avian influenza virus (AIV) antigen. A monoclonal antibody to the viral nucleoprotein (NP) was used to coat the ELISA plates. A direct DAS-ELISA and an indirect DAS-ELISA were evaluated. In the direct DAS-ELISA, monoclonal antibody to the AIV NP conjugated with horseradish peroxidase was used. The direct DAS-ELISA was evaluated for its sensitivity to detect purified NP; this procedure detected as little as 0.1 ng. In the indirect DAS-ELISA, rabbit NP antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobin were used as primary and secondary antibodies, respectively. The indirect DAS-ELISA was evaluated for its ability to detect the AIV antigen in tracheal and cloacal specimens from turkeys inoculated with AIV. Results of indirect DAS-ELISA were compared with those of conventional virus isolation. Percentage agreement between indirect DAS-ELISA and virus isolation in AIV-positive samples was found to be 76.1% and, in AIV-negative samples, it was found to be 82.1%. These results indicate that the DAS-ELISA might be a viable alternative to virus isolation because of its rapidity, compared with virus isolation.

Naturally acquired turbinate atrophy in rabbits was associated with Pasteurella multocida infection. Several in vitro and in vivo studies were conducted to document toxin production from P. multocida isolates and to determine the relation of toxin to atrophic rhinitis in rabbits. Ten isolates of P. multocida serotype A:12 were obtained from adult New Zealand White rabbits with noninduced atrophic rhinitis. Specific-pathogen-free rabbits inoculated intranasally with isolates of P. multocida developed rhinitis and turbinate atrophy. However, inoculation with filtrates of the same bacteria failed to induce turbinate atrophy. Cytotoxicity was observed in assays, using bovine embryonic turbinate cell cultures with extracts of P. multocida, but not in agar overlay cytotoxicity assays, using bovine embryonic turbinate, bovine embryonic lung, or Vero cell cultures, or in a sandwich ELISA, using monoclonal antibodies to purified P. multocida toxin. Thus, turbinate atrophy was experimentally reproduced in rabbits with isolates of P. multocida, but toxin was only detected in vitro by cell culture assay of P. multocida extracts.

During the spring of the first year of a vaccine study, 57 of 238 calves (24%), in which modified-live Pasteurella haemolytica vaccine (MLV) was injected twice, developed 1 or more abscesses. Abscesses were not observed after multiple visual examinations of 437 calves given killed P. haemolytica bacterin or placebo injections of similar adjuvants used in the vaccine and bacterin. Calves that developed abscesses after the second injection of MLV weighed significantly (P < 0.05) less (on the basis of body weight adjusted for weaning weight) at the second injection than did those that did not develop abscesses. Compared with calves given MLV that did not develop observable abscesses, calves developing abscesses after the second injection of MLV weighed 11.0 and 14.2 kg less, respectively, at 56 days and 112 days after injection, and they had 11.0 kg less gain at 56 days after injection. Abscess prevalence tended to be highest on certain days or at certain locations used for cattle processing, and the prevalence of abscesses increased in cattle processed later on a given day. Abscesses were not observed in 2 other groups of similarly treated calves vaccinated in the autumn or in the subsequent spring.

To study increase of the Salmonella population in the cecum of chickens infected with Eimeria tenella, quantitative changes in mannose residues on the cecal mucosa were investigated. Inhibition of S typhimurium adherence to the cecum by a 2% carbohydrate (D-mannose, D-galactose, L-fucose, alpha-methyl-D-glucoside) in phosphate-buffered saline solution was examined. Only D-mannose had inhibitory effects. Whereas, D-galactose had somewhat enhancing effects on adherence of S typhimurium to the cecal mucosa of uninfected germ-free chickens. In infected and uninfected chickens, D-mannose inhibited adherence of S typhimurium. D-mannose significantly (P < 0.05) increased adherence of Bacteroides sp. In infected and uninfected chickens, D-mannose did not have any effect on adherence of Clostridium perfringens and Bifidobacterium thermophilum. Under microscopic observation, only concanavalin A and Lens culinaris agglutinin, of 8 lectins examined, were recognized as lectin-positive staining lines or spots in the cecal mucosa, indicating presence of mannose residues on the cecal mucosa. In E tenella-infected chickens, lectin-positive staining was seen strongly on the coarse surface of damaged cells and at the bottom of the crypts. These results indicate that coccidial infection may induce increase of mannose residues on the intestinal surface and allow adhesion of more salmonellae to the intestine.

Ten multiparous sows were inoculated between 46 and 50 days of gestation with a fetal swine isolate of encephalomyocarditis virus (EMCV) to investigate the ability of the virus to cause transplacental infection and fetal death. Four sows (group 1) were inoculated IM with EMCV MN-25 that had been passaged 4 times on baby hamster kidney-21 line cell monolayers. Two sows were euthanatized at postinoculation (PI) day 23, and the other 2 sows at PI day 44. An additional 6 sows (group 2) were inoculated IM with the same virus that had been passaged 5 additional times in pigs. Two sows were euthanatized at 14 days, and the remaining 4 sows at PI day 28. Clinical signs were not observed in any of the sows, whereas all sows seroconverted to EMCV. In group 1, only 2 of 50 fetuses were mummified. Virus was not recovered, although EMCV antibodies were detected in the 2 mummified fetuses. In group 2, the 2 sows that were euthanatized at PI day 14 had 26 normal fetuses and there was no evidence of fetal infection. However, in the 4 sows euthanatized at PI day 28, 20 of 48 fetuses were mummified, hemorrhagic, or edematous. Encephalomyocarditis virus was recovered from 21 of 48 fetuses. Transplacental infection and fetal deaths in pregnant sows was achieved following infection with EMCV passaged in pigs.

The relation between average duodenal mast cell count, duodenal mucosal mast cell numbers, duodenal connective tissue mast cell numbers, circulating basophil numbers, heterophil-to-lymphocyte ratio, and lesion score were studied to gain an understanding of the events that may lead to intestinal lesion formation associated with hemorrhagic enteritis virus (HEV) infection. Changes in vascular permeability in the duodenum in birds inoculated with HEV were examined, using colloidal carbon and ferritin as vascular markers. Turkeys inoculated with HEV had significantly (P < 0.05) higher duodenal mast cell counts than did noninfected controls. Birds inoculated with HEV had significantly (P < 0.05) more mucosal mast cells than did phosphate-buffered saline solution-inoculated birds. Connective tissue mast cell and basophil numbers were unaffected by viral inoculation. Thermal stress did not have significant effect on lesion severity, but did increase number of birds that developed the characteristic intestinal lesions. The heterophil-to-lymphocyte ratio was significantly (P < 0.05) higher in HEV-inoculated birds, compared with phosphate-buffered saline solution-inoculated controls. Increase in vascular permeability was only detected in HEV-inoculated birds with intestinal lesions. Results indicate that mast cells, and the vasoactive mediators contained within mast cells, may be important in the early manifestation of HEV infection. They also provide a possible mechanism through which biochemical and physiologic changes characteristic of HEV infection can occur.

Pregnant sows, immune against pseudorabies after vaccination, were inoculated at 70 days of gestation either with autologous blood mononuclear cells that had been infected in vitro with pseudorabies virus (PRV) or with cell-free PRV. The infected cells or cell-free PRV were inoculated surgically into the arteria uterina. Eight sows (A to H) had been vaccinated with an inactivated vaccine. The titer of seroneutralizing antibodies in their serum varied between 12 and 48. Five sows (A to E) were inoculated with autologous mononuclear cells, infected either with a Belgian PRV field strain or with the Northern Ireland PRV strain NIA. These 5 sows aborted their fetuses: 2 of them (B and C) 3 days after inoculation, and the other 3 (A, D, and E) 10, 11, and 12 days after inoculation, respectively. Sows F, G, and H were inoculated with a cell-free PRV field strain. They farrowed healthy Utters after normal gestation. Neutralizing antibodies were absent against PRV in the sera of the newborn pigs, which were obtained prior to the uptake of colostrum. The 23 fetuses that were aborted in sows B and C 3 days after the inoculation were homogeneous in appearance and size. Foci of necrosis were not detected in the liver. Viral antigens were located by immunofluorescence in individual cells in lungs, liver, and spleen of 15 fetuses. Virus was isolated from the liver, lungs, or body fluids of 12 fetuses. The 39 fetuses that were aborted in sows A, D, and E between 10 and 12 days after inoculation were of 2 types: 17 were mummified and 22 were normal-appearing. Foci of necrosis were found in the liver of all mummified fetuses and 13 of the normal-appearing fetuses. In fetuses with foci of necrosis in the liver, viral antigens were located in groups of cells in the liver, lungs, and spleen. Virus was isolated from 16 normal-appearing fetuses and from 11 mummified fetuses. Pseudorabies virus was isolated from vaginal excretions of sows A and D until 1 and 2 days after abortion, respectively, and of sows B and C until 4 and 5 days after abortion, respectively. Virus was not isolated from sow E. It was concluded that PRV can reach the uterine and fetal tissues, via infected mononuclear cells, in the presence of circulating antibodies induced on vaccination. This cell-associated spread led to abortion. Cell-free virus did not induce abortion under similar circumstances.

Ehrlichia chaffeensis, the newly recognized agent of human ehrlichiosis, is closely related to E canis, the causative agent of canine ehrlichiosis. Eight pups were inoculated IV with E chaffeensis-, or with E canis-infected DH82 cells, or organisms released from these host cells. Two additional pups served as nonexposed controls. Marked thrombocytopenia was observed in the E canis-infected pups, but not in those infected with E chaffeensis. Homologous serologic response was observed in the E chaffeensis-exposed pups by postinoculation day (PID) 14 and in the E canis-exposed pups by PID 21. Ehrlichia chaffeensis and E canis were reisolated from the respective inoculated pups on each of 8 attempts from PID 7 to 26. One E chaffeensis-exposed pup that was challenge exposed with E canis via blood transfusion, developed fever, anorexia, and thrombocytopenia, suggesting lack of cross protection against E canis.

Canine granulocytic ehrlichiosis was diagnosed in 37 dogs by finding ehrlichial morulae in 0.1 to 26.2% of their blood neutrophils and eosinophils. All 37 dogs had clinical signs of arthritis or muscular stiffness. Titer to Ehrlichia canis was determined in sera from 31 of the 37 dogs; 25 dogs had titer ranging from 1:20 to 1:5,120. In the other 6 dogs, titer to E canis was < 1:10. The most common hematologic abnormality in these dogs, other than rickettsiemia, was thrombocytopenia. Granulocytes infected with ehrlichial organisms were not found in another 10 dogs that had clinical signs of arthritis or muscular stiffness. Of these 10 dogs, 3 had titer to E canis ranging from 1:40 to 1:320. Titer in the other 7 dogs was < 1:10. Ehrlichial morulae were not found in the granulocytes of 18 healthy dogs. Of these 18 dogs, 9 had titer to E canis ranging from 1:20 to 1:5,120. Titer in the other 9 dogs was < 1:10. Titer to Borrelia burgdorferi was determined in dogs with granulocytic ehrlichiosis, arthritic dogs without detected rickettsiemia, and in healthy dogs. Low titer determined by 2 laboratories was considered to be nonspecific reaction in all 3 groups of dogs and, thus, did not indicate that the arthritic disorders were attributable to canine borreliosis.

Inclusion of high-ionic strength buffers helped us to develop a sandwich ELISA to detect hemorrhagic septicemia virus (HSV) in cell culture and infected trout tissue extracts. For maximal sensitivity of 0.1 to 0.2 ng/well/100 microliter or about 10 to 50 TCID50/well/100 microliter, trout extracts were diluted 1:1 and assayed for the earliest synthesized nucleoprotein N. Simultaneous binding of the N protein from HSV in the sample to the wells coated with monoclonal antibody (2D5 against the N protein) and to the peroxidase-labeled monoclonal antibody (2C9 against the N protein) proceeded during a 2-hour incubation at 20 to 22 C (room temperature). The response was linear between 6 to 60 ng/well of purified virus. Monoclonal antibodies used were noncompetitive with each other and reacted with F1, F2, 23.75, and 5 Spanish isolates of HSV, but not with infectious hematopoietic necrosis or infectious pancreatic necrosis viruses. Tissue specimens with low content of HSV virus may now be assayed directly without use of cell culture, rapidly, and with high precision, during the acute phase of the disease in salmonid fishes.