Paul A. Kapke

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene.

Abstract The 3′ end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into eDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3′ end of the genome. Two potential, nonoverlapping genes were identified within the 3′-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5′ side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3′-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.

Comparison of a Commercial DNA Probe Test and Three Cultivation Procedures for Detection of Mycobacterium Paratuberculosis in Bovine Feces

Diagnosis of paratuberculosis using the IDEXX DNA probe test and 3 methods for cultivation of Mycobacterium paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were positive by 1 or more of the cultivation methods evaluated. Four specimens were DNA probe test positive but were negative by fecal culture. The probe test detected M. paratuberculosis DNA in 62.9% of the specimens positive by a sedimentation culture method, in 56.6% of those positive by a centrifugation culture method, and in 65.4% of the specimens positive by the Cornell culture method. Specificity of the DNA probe test was approximately 97% relative to all culture methods. Generally, the probe test detected M. paratuberculosis DNA in fecal specimens from animals shedding at least 104 M. paratuberculosis colony forming units per gram of feces. Although the probe test did not detect all of the cattle shedding M. paratuberculosis, it was possible to identify cattle shedding the greatest number of organisms in 3 days compared with a minimum of 6 weeks required for positive culture results. The centrifugation method resulted in the most isolations of M. paratuberculosis after 12 weeks of incubation. However, contamination also was greatest when the centrifugation method was used. Contamination was best controlled using the Cornell method. The sedimentation method was the least time consuming and yielded results similar to those of the other 2 methods.

Complement-Dependent Neutralization of Transmissible Gastroenteritis Virus by Monoclonal Antibodies

Monoclonal antibodies (MAb) to each of the 3 major structural proteins of transmissible gastroenteritis virus (TGEV) of swine were compared as to their virus neutralizing activity in the presence or absence of guinea pig complement. MAbs to the peplomer protein had neutralizing activity for TGEV with or without complement and the titers were similar in either case. MAbs to the matrix protein had neutralizing activity for TGEV only in the presence of complement. Antibodies to the nucleocapsid protein were without neutralizing activity with or without complement. High concentrations of guinea pig complement, but not swine complement, had neutralizing activity for TGEV even in the absence of any known TGEV antibodies.

Analysis of restriction endonuclease fragment patterns of DNA from Mycobacterium paratuberculosis

The DNA from 31 isolates and a reference strain of Mycobacterium paratuberculosis was digested individually with restriction endonucleases BstE II and Pst I. DNA fragments were separated by gel electrophoresis and analyzed. The isolates were from 23 American states, Argentina and Nova Scotia. Twenty-seven were isolated from cattle, two from goats and two from sheep. With the exception of one isolate from cattle, all had restriction endonuclease fragment patterns identical to the fragment patterns for the reference strain, M. paratuberculosis ATCC 19698T. These results confirm other reports and indicate that organisms identified as typical M. paratuberculosis isolates are genetically very similar. It may be possible to use restriction endonuclease analysis to differentiate isolates of M. paratuberculosis from other slowly growing mycobacteria. The genetic similarity also indicates that it may be possible to develop a diagnostic probe that is specific for M. paratuberculosis.

Antibody Response in Swine to Individual Transmissible Gastroenteritis Virus (TGEV) Proteins

Transmissible gastroenteritis virus (TGEV) is a coronavirus that produces a life-threatening diarrhea in neonatal pigs. Whereas sows infected with virulent virus produce protective colostral antibody, sows vaccinated with attenuated virus are significantly less effective in passively protecting baby pigs. Therefore, an understanding of the antigenic and biochemical properties of pathogenic TGEV and the pigs’ immune response to these antigens is a critical area of TGEV research. We are in the process of characterizing the porcine immune response to major TGEV antigens in a protein A immunoprecipitation assay. Protein A is known to bind approximately 90% of the swine immunoglobulin isotypes, IgG1. and IgG2, but is less efficient in binding either swine IgM or IgA.1 This study has led to the serological identification of a new TGEV-specific low molecular weight polypeptide.