Sanjay Kapil

Defining the Cellular Target(s) of Porcine Reproductive and Respiratory Syndrome Virus Blocking Monoclonal Antibody 7G10

ABSTRACT We produced a monoclonal antibody (MAb) (7G10) that has blocking activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, we identified the components of the 7G10 MAb-bound complex as cytoskeletal filaments: vimentin, cytokeratin 8, cytokeratin 18, actin, and hair type II basic keratin. Vimentin bound to PRRSV nucleocapsid protein and anti-vimentin antibodies showed PRRSV-blocking activity. Vimentin was expressed on the surface of MARC-145, a PRRSV-susceptible cell line. Simian vimentin rendered BHK-21 and CRFK, nonsusceptible cell lines, susceptible to PRRSV infection. These results suggest that vimentin is part of the PRRSV receptor complex and that it plays an important role in PRRSV binding with the other cytoskeletal filaments that mediate transportation of the virus in the cytosol.

Infectious Bovine Rhinotracheitis, Parainfluenza-3, and Respiratory Coronavirus

A number of viruses have been proven to be primary respiratory pathogens of cattle. Viruses may play an important role in making cattle susceptible to secondary respiratory bacterial pathogens. Epidemiology, pathogenesis, laboratory diagnosis, and important properties in infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI-3), and bovine respiratory coronavirus (BRCV) are described in this article.

Canine Distemper Spillover in Domestic Dogs from Urban Wildlife

Canine distemper virus (CDV) causes a major disease of domestic dogs that develops as a serious systemic infection in unvaccinated or improperly vaccinated dogs. Domesticated dogs are the main reservoir of CDV, a multihost pathogen. This virus of the genus Morbillivirus in the family Paramyxoviridae occurs in other carnivorous species including all members of the Canidae and Mustelidae families and in some members of the Procyonidae, Hyaenidae, Ursidae, and Viverridae families. Canine distemper also has been reported in the Felidae family and marine mammals. The spread and incidences of CDV epidemics in dogs and wildlife here and worldwide are increasing.

Viral Diseases of New World Camelids

The increased popularity and population of New World camelids in the United States requires the development of a broader base of knowledge of the health and disease parameters for these animals by the veterinary livestock practitioner. Although our knowledge regarding infectious diseases of camelids has increased greatly over the past decade, the practice of camelid medicine is a relatively new field in North America, so it is important to seek out seasoned colleagues and diagnostic laboratories that are involved in camelid health treatment and diagnosis.

Enteric Coronavirus Infection in a Juvenile Dromedary (Camelus Dromedarius)

A case of an enteric coronavirus infection in a 6-week-old dromedary calf is described. The animal had diarrhea for 5 days and died despite symptomatic treatment. Numerous viral particles, approximately 140 nm in diameter, with club-like projections were detected in the feces by electron microscopy. These characteristics were consistent with a coronavirus. Immunohistochemical reactivity with 2 antigenic group II coronavirus-specific antibodies confirmed the presence of viral antigen in colonic epithelial cells. The death of the animal was attributed to a neutrophilic and emphysematous colitis that likely was caused by an infection with a Clostridium sp.

Detection of bovine immunodeficiency virus antibodies in cattle by Western blot assay with recombinant gag protein

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.

Comparison of Immunohistochemistry, Electron Microscopy, and Direct Fluorescent Antibody Test for the Detection of Bovine Coronavirus:

Bovine coronavirus (BCV) is 1 of the major causes of calf diarrhea and has also been implicated in respiratory infections of young calves and winter dysentery of adult cattle. Currently, transmission electron microscopy (TEM), direct fluorescent antibody (DFA), and enzyme-linked immunosorbent assay (ELISA) techniques are considered standard methods for the diagnosis of BCV infection. However, these techniques are not useful if fresh tissues and intestinal contents are not available for examination. The detection of viral antigens in formalin-fixed, paraffin-embedded tissues using immunohistochemistry (IHC) is a suitable alternative. In the present study, 166 tissue specimens were tested by IHC for the presence of BCV. These tissues were from animals whose feces were positive for rotavirus and/or coronavirus by TEM. Some of these samples were also tested by DFA. Thus, TEM, DFA, and IHC were compared for the detection of BCV. There was 56% agreement among the 3 methods (overall kappa = 0.368). When IHC was compared with TEM, 78% agreement was observed (kappa = 0.475). Similarly, IHC and DFA had 64% agreement (kappa = 0.277). These kappa values indicate a moderate degree of agreement between IHC and TEM; agreement between IHC and DFA was fair. The results of this study indicate that IHC may be a suitable adjunct for the detection of BCV because of its simplicity, ease of use, and relatively close correlation with TEM results.

Characterization of bovine coronavirus isolates/from eight different states in the USA

Abstract Bovine coronavirus isolates from eight different states of the USA were compared for their antigenic properties and susceptibility to hygromycin B. Antigenic differences were observed among the isolates in a one-way hemagglutination-inhibition (HI) test using a polyclonal antiserum against the Mebus bovine coronavirus isolate. Differences were observed on isoelectric focusing among viral proteins with isoelectric points between 4.45–4.65. Most of the BCV isolates were susceptible to hygromycin B (0.5mM) whereas a few hygromycin B resistant isolates were also found.

Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay†

ABSTRACT An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. Thegag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein fromEscherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

Characterization of a small plasmid (pMBCP) from bovine Pseudomonas pickettii that confers cadmium resistance

This is the first report of isolation of Pseudomonas pickettii from a normal adult bovine duodenum. This organism was one of several bacteria isolated as part of a study to examine cadmium resistance genes (cad(r)) for use in generating transgenic plants to reclaim cadmium-contaminated soils in Kansas. P. pickettii containing a plasmid of 2.2kb (designated pMBCP) grew in Luria-Bertani broth and agar containing up to 800 microM of cadmium chloride and was resistant to 16 antibiotics. Curing the organism of plasmid revealed that antibiotic resistances were not plasmid-mediated. Low-level cadmium resistance was conferred by the plasmid because uncured organism grew significantly better (P<0.05) at 55 microM compared to cured organism. Both plasmid and chromosomal DNA were probed by DNA-DNA hybridization for the presence of known cadmium resistance genes (cadA, cadC, and cadD from Gram-positive (Staphylococcus aureus), but none were detected. The plasmid had one restriction site each for BamHI, PstI, SmaI, and XhoI; two sites each for HincII, SacI, and SphI; and multiple sites for AluI and XcmI. DNA sequence analyses of the cloned and original plasmids showed a GC content of greater than 60% and no homology to any published sequences in the GenBank, European Bioinformatics Institute, or Japanese Genome Net databases. The DNA sequence is contained in GenBank accession number AF144733. Thus, pMBCP offers low-level cadmium resistance to P. picketttii.