Heterobilharzia americana infection and congestive heart failure in a llama (Lama glama).

The schistosome Heterobilharzia americana infects several mammalian species in the southeastern United States, including horses, but infections have not been reported in camelids. This is a report of H. americana infection in a 6-year-old llama with extensive cardiac pathology and congestive heart failure. Parasite-induced granulomas were widely disseminated and included overwhelming involvement of the lungs and liver. Microscopic lesions in the heart included myofiber degeneration and necrosis, with extensive replacement fibrosis. Polymerase chain reaction amplification and sequencing confirmed the presence of H. americana in the lungs.

Natural Heterobilharzia americana infection in horses in Texas.

The schistosome Heterobilharzia americana infects dogs, raccoons, and other mammals in the southeastern United States. Migration of eggs into the liver results in parasitic granulomas with varying degrees of fibrosis and inflammation. Recently, hepatic parasitic granulomas in horses were shown to be caused by H. americana infection. In the present study, samples of liver from 11 of 12 horses with hepatic granulomas identified at necropsy (n = 11) or surgical biopsy (n = 1) were used for DNA extraction, polymerase chain reaction amplification and sequencing using primers specific for a portion of the H. americana small subunit ribosomal RNA gene. A polymerase chain reaction amplicon of the correct size was produced from the extracted DNA in 8 of the 11 horses. Amplicons from 5 of the 8 positive horses were sequenced and had 100% identity with H. americana. In all but 2 of the 12 horses, Heterobilharzia was not responsible for the primary clinical disease, and the hepatic granulomas were considered an incidental finding.

Mucinous adenocarcinoma and T-cell lymphoma in the small intestine of 2 Vietnamese potbellied pigs (Sus scrofa).

Small intestinal T-cell lymphoma and mucinous adenocarcinoma are rarely reported in the pig, with most lymphomas being of B-cell origin and only a single report of mucinous adenocarcinoma. Two aged Vietnamese potbellied pigs had concurrent T-cell lymphoma and mucinous adenocarcinoma of the small intestine. The lymphomas formed polypoid masses that projected into the intestinal lumen, whereas the mucinous adenocarcinomas were mural masses that bulged from the serosal surface. Immunohistochemically, neoplastic cells within the lymphomas were positive for CD3 and negative for CD79a. Mucicarmine stain highlighted the abundant cytoplasmic and extracellular mucin in the adenocarcinomas.

Localization of antigenic sites of the S glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement.

The S glycoprotein of feline infectious peritonitis virus (FIPV) has been shown to contain the antigenic sites responsible for eliciting both neutralization and antibody-dependent enhancement. To determine the region of S responsible, overlapping DNA fragments spanning the entire S gene were cloned and expressed as fusion proteins by in vitro transcription and translation. Fusion proteins containing relevant epitopes were identified by radioimmunoprecipitation with neutralizing and enhancing FIPV-specific monoclonal antibodies (MAbs). A region spanning residues 509 to 673 reacted with most MAbs tested. Translation in the presence of microsomal membranes did not enhance reactivity, suggesting that glycosylation is not essential for recognition by the MAbs. To localize the antigenic sites further, several MAb-resistant (mar) mutants of FIPV were cloned and sequenced. Amino acid residues that contribute to the neutralizing and enhancing epitopes were localized to two regions, designated A1 and A2, which show partial overlap with the homologous antigenic site A of transmissible gastroenteritis virus. Site A1 contains residues 568 and 591 and is homologous with part of subsite Aa of transmissible gastroenteritis virus. Site A2 contains residues 643, 649, and 656. Double mutations in sites A1 and A2 were found in mar mutants derived from neutralizing and enhancing MAbs 23F4.5 and 18A7.4, while a single mutation in site A2 was found in a mar mutant derived from MAb 24H5.4, which is neutralizing but not enhancing. The data suggest that site A2, which includes residues 643 to 656, is a dominant neutralizing site of FIPV and that sites A1 and A2 may act in concert to induce antibody-dependent enhancement.

Identification of antigenic sites mediating antibody-dependent enhancement of feline infectious peritonitis virus infectivity.

We have previously demonstrated antibody-dependent enhancement of feline infectious peritonitis virus (FIPV) infection of macrophages using both virus-specific antisera and monoclonal antibodies (MAbs) to the spike (S) protein of FIPV. To increase our understanding of this phenomenon, six representative MAbs from a previously documented group of 12 enhancing MAbs were used to identify epitopes that mediate antibody-dependent enhancement of FIPV infectivity. Analysis of the results of kinetics-based competitive ELISA (K-cELISA) among these six enhancing MAbs grouped the epitopes into two clusters. Because transmissible gastroenteritis virus (TGEV) and FIPV are so closely related antigenically, we also conducted K-cELISA experiments between the FIPV MAbs and TGEV S protein-specific MAbs for which the epitopes had previously been mapped to specific sites on the TGEV S protein. Results of these assays indicated that the two FIPV epitope clusters are homologues of the previously defined TGEV S protein sites A and E/F. In addition, two TGEV S protein-specific MAbs also induced antibody-dependent enhancement of FIPV infection of macrophages. This functional cross-reactivity provides further support for the close antigenic relationship between FIPV and TGEV. Our results provide a preliminary localization of several enhancing epitopes within the amino acid sequence of the FIPV S protein.

Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus.

Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26- to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and all but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin G1 subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the N- or M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE.

Monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages.

Antibody-dependent enhancement of virus infection is a process whereby virus-antibody complexes initiate infection of cells via Fc receptor-mediated endocytosis. We sought to investigate antibody-dependent enhancement of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro. Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells, by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline antiserum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheep erythrocyte serum and an antibody-coated sheep erythrocyte phagocytosis assay. Thirty-seven murine monoclonal antibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific monoclonal antibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal protein A. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.

Bovine viral diarrhea virus proteins and their antigenic analyses.

Bovine Viral Diarrhea Virus (BVDV) polypeptides present in infected cells are the result of the processing of the polyprotein translated from the large single open reading frame of the BVDV genomic RNA. The presence of these proteins in infected cells was studied by radiolabeling under hypertonic conditions and with the aid of radioimmunoprecipitation. The genomic mapping of these polypeptides suggests a complex pattern of processing which involves cellular and viral proteases. The consistent absence of 80k in noncytopathic isolates of BVDV suggests that the processing of the viral polyprotein is different in cytopathic and noncytopathic biotypes of BVDV. The antigenic structure of BVDV was studied with a panel of monoclonal antibodies (MABs) prepared against the Singer isolate of BVDV. Neutralizing MABs were found to bind the 56-58k polypeptide, providing evidence that this glycoprotein is present on the surface of the virion and carries neutralization epitopes. Antigenic analyses with the panel of MABs reveals extensive antigenic heterogeneity among BVDV field isolates. MABs were used to determine the frequency of neutralization escape mutants in stocks of BVDV. Plaque-purified BVDV stocks contain neutralization escape mutants with a frequency of 10(-2.47).

Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus.

A panel of 40 monoclonal antibodies (MAb) specific for bovine viral diarrhea virus (BVDV) was produced, and each MAb was characterized and grouped according to its viral protein specificity, immunoglobulin subclass, virus-neutralizing activity, and immunoreactivity with a large collection of BVDV isolates. The MAb were found to be specific for 1 of 3 sets of related viral-induced proteins found in cells infected with the Singer strain of BVDV. Group-1 MAb were specific for the 80- and 118-kilodalton (kD) proteins of BVDV. Group-2 MAb recognized 3 proteins with molecular sizes of 54, 56, and 58 kD. Group-3 MAb recognized a 43- and a 65-kD protein. The MAb belonged to either the IgG1, IgG2a, IgG2b, IgG3 subclasses or the IgE class of mouse immunoglobulin. All MAb in group 2 were able to neutralize BVDV and had neutralization titers that ranged from 24 to 1,600,000. The reactivity of the MAb with numerous field isolates of BVDV was highly variable. Both cytopathic and noncytopathic biotypes of BVDV were examined and had the same degree of antigenic variation. The greatest degree of variation was detected with group-2 MAb. The data demonstrate that BVDV isolates have a high degree of antigenic variation that is largely confined to the envelope glycoproteins associated with virus neutralization. The results also suggest that antigenic variability of this virus is important in the development and severity of the disease it causes.

Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus.

The relationship between bovine viral diarrhea virus (BVDV) infection and thrombocytopenia was studied in 18 veal calves experimentally infected with BVDV. All calves were free of BVDV, and 13 calves were free of serum neutralizing antibodies to BVDV before virus inoculation. Calves were inoculated at approximately 10 days of age, and platelet counts were monitored over a period of several weeks. Ten additional calves housed in close proximity were kept as uninoculated controls. A profound decrease in platelet counts by 3 to 11 days after inoculation was seen in all calves that had neutralizing antibody titers less than 1:32 before infection. Severe thrombocytopenia (less than 5,000 platelets/microliter) was seen in 12 calves, 11 of which also developed hemorrhages. Necropsy findings in 3 severely thrombocytopenic calves that died included multiple hemorrhages throughout the body. Calves that recovered had increased platelet counts, and in most instances, a corresponding increase in neutralizing antibody titers to BVDV. At 11 days after inoculation, BVDV was detected on platelets by use of immunofluorescence, but evidence of surface-bound immunoglobulin was not found. The results suggest that a nonimmunoglobulin-mediated method of platelet destruction or sequestration develops as a sequela to BVDV infection.

Severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus.

Seven calves between 1 week and 2 months of age were infected with a noncytopathic field isolate of bovine viral diarrhea virus (BDV) in order to evaluate the effect of BDV infection on the concentration of circulating platelets in the blood. All calves were determined to be free of BDV and neutralizing antibodies to BDV before infection. Platelet counts were performed on a daily basis over a 30-day period beginning at the time of infection. By 2 weeks postinfection, all calves showed a significant drop in the number of circulating platelets and a marked hyperplasia of megakaryocytes in the bone marrow. In three of the seven calves, thrombocytopenia was severe (less than or equal to 5,000/microliters) for 1 to 6 days. In two of these three animals, extensive petechial and ecchymotic hemorrhages were observed on all mucosal surfaces and on various internal organs during the period of severe thrombocytopenia. BDV was consistently isolated from the platelets during the early phases of the infection, and viral antigen was occasionally detected on platelets by a fluorescent-antibody assay. The results demonstrate that BDV infection is associated with decreases in platelet numbers and suggest that platelets may serve as carriers of circulating virus.

Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections.

A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56- to 58- and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.

Tricholemmomas in three dogs.

Tricholemmomas characterized by lobules of PAS-positive epithelial cells with a peripheral palisade surrounded by an amorphous eosinophilic band, are described in 3 dogs. This is the second report of this uncommon hair follicle tumour in domestic animals.