Jonathan B. Katz

Detection of Prion Protein in Formalin-Fixed Brain by Hydrated Autoclaving Immunohistochemistry for the Diagnosis of Scrapie in Sheep

4The diagnosis of scrapie has traditionally relied on histopatholog ic demonstration of characteristi c lesions in brain sections. In recent years, however, increasing attention has been given to the use of immunodiagnos tic methods that focus on detection of a specific protein known as the prion protein (PrP). 7 This protein, which is believed to play an essential role in the pathogenesis of scrapie, is present in normal brain as a protease-sensitive isoform (PrP-sen). In contrast, most of the PrP in scrapie brain is protease resistant (PrP-res), probably because of a posttranslational alteration affecting the conformation of normal PrP. An immunohistoch emical (IHC) method to detect PrP-res in periodate, lysine, paraformaldehyde (PLP)-fixed brains from sheep with scrapie has been described. 5 Researchers working on bovine spongiform encephalopathy have recently reported that PrP-res also can be detected in formalin-fixe d brain if tissue sections are autoclaved prior to immunostaining. 1 This novel method of antigen enhancement, termed hydrated autoclaving, was originally devised to improve the immunoreactivity of certain brain proteins. 10 Hydrated autoclaving has not been used to detect other antigens. However, antigen enhancement is believed to result from heat denaturation of the protein, and the same mechanism has been suggested as the basis for antigen retrieval methods that utilize microwave immunohistochemistry. 6 In the present report, we describe diagnosis of scrapie using hydrated autoclaving IHC on formalin-fixe d sheep brains. For comparative purposes, some brains also were tested for PrP-res by western blot assay or by IHC on PLP-fixed brain. Brain samples from 186 scrapie suspect sheep were submitted to veterinary medical colleges, state veterinary diagnostic laboratories, or the National Veterinary Services Laboratories. Additional brain samples from 10 sheep in a scrapiefree flock were used as controls. Tissue sections were cut from paraffin blocks of formalin-fixe d brain stem (usually obex or midmedulla) and mounted on positively charged

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3.

Abstract Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive (P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV.

Diagnostic Analysis of the Prolonged Bluetongue Virus RNA Presence Found in the Blood of Naturally Infected Cattle and Experimentally Infected Sheep

Bluetongue virus (BTV) RNA was detected by the polymerase chain reaction (PCR) in the blood of 24 naturally infected cattle as long as 160 days after the estimated date of infection. Blood samples from these animals and from 10 experimentally BTV-infected sheep, which also exhibited a prolonged hematologic BTV RNA presence, were concurrently evaluated for viral infectivity. Infectivity analyses were conducted using the sentinel sheep inoculation and embryonated chicken egg inoculation procedures. Blood specimens from the experimental sheep 50, 56, 71, and 89 days after BTV inoculation were uniformly negative for viral infectivity despite their uniformly positive status with PCR evaluation. Three collections of blood from the naturally infected cattle at least 100, 135, and 160 days after infection also revealed no recoverable viral infectivity but an initially high and progressively decreasing prevalence of BTV with the PCR technique. These retrospective epidemiologic and prospective experimental approaches were concordant in that both studies demonstrated consistent discrepancies between the viral infectivity and the PCR diagnostic data. The significance of these discrepancies is discussed with respect to Kochs postulates and with respect to the possibility that the biological vector of BTV (Culicoides variipennis) may recover BTV infectivity from PCR-positive but virus isolation-negative blood.

Procedurally Similar Competitive Immunoassay Systems for the Serodiagnosis of Babesia Equi, Babesia Caballi, Trypanosoma Equiperdum, and Burkholderia Mallei Infection in Horses

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.

Clinical, Serologic, and Histopathologic Characterization of Experimental Borna Disease in Ponies

Borna disease was originally described as an equine neurologic syndrome over 200 years ago, although the infectious etiology of the disorder was unproven until the early 20th century. Borna disease virus (BDV) was finally isolated from horses dying of the disorder, and that virus has been used to experimentally reproduce Borna disease in several species of laboratory animals. However, BDV has never been inoculated back into horses to experimentally and etiologically confirm the classic clinical, pathologic, and serologic characteristics of the disease in that species. Three ponies were intracerebrally inoculated with different amounts of BDV and were evaluated clinically, serologically, and neurohistopathologically. All 3 animals developed the clinical signs characteristically described for naturally occurring Borna disease, including ataxia, torticollis, postural unawareness, rhythmic repetitive motor activities, muscle fasciculation, and cutaneous hyperesthesia and hypoesthesia over several body surfaces. Two ponies died after rapid onset of these signs 28–30 days postinoculation. The third animal made a nearly complete clinical recovery. Seroconversion occurred only after the onset of signs and to a marked degree only in the convalescent animal. Virus was recovered postmortem from 2 of the 3 ponies, and a BDV-specific nucleic acid sequence was detectable in all 3 animals using a reverse transcription-polymerase chain reaction procedure. Gross neural lesions were absent, but histopathologically there was generalized intense mononuclear perivascular cuffing, glial nodule formation, and astrocytosis in all 3 brains. Confirming a diagnosis of Borna disease is difficult and perhaps best accomplished using a combination of the clinical, serologic, and histopathologic indicators of this unusual disease supported by positive reverse transcription-polymerase chain reaction findings.

Development and Evaluation of a Recombinant Antigen, Monoclonal Antibody-Based Competitive ELISA for Heartwater Serodiagnosis

Cowdria ruminantium is the etiologic agent of heartwater, a tick-transmitted foreign animal disease with considerable potential for entrance into the USA. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect serologic responses to C. ruminantium infection. The cELISA utilized a recombinant form of the C. ruminantium major antigenic protein (MAP-1) as the antigen and an anti-MAP-1 monoclonal antibody as the competing indicator reagent. Experimental antisera to C. ruminantium and a wide variety of related ehrlichial organisms were used to evaluate cELISA reactivity. Only sera against C. ruminantium, Ehrlichia canis, E. chaffeensis, and a recently discovered cervine ehrlichia-like organism reacted positively in the cELISA. Specificity of the cELISA was 299.5% in a survey of 1,774 southeastern US and Puerto Rican slaughter cattle sera but was only 85% in a group of 79 hunter-killed white-tailed deer (Odocoileus virginianus) from the southeastern USA. Reference true-positive and cELISA false-positive sera were further analyzed by end point titrations using the cELISA and by indirect fluorescent antibody (IFA) tests for reactivity with C. ruminantium, E. canis, and E. chaffeensis antigens. True heartwater-positive sera were significantly more reactive using the cELISA and C. ruminantium IFA procedures (P < 0.05), whereas false-positive sera were significantly more reactive with the antigens used in the E. chaffeensis IFA procedure (P < 0.05). A group of sera from 210 field-origin ruminants residing on known or potentially heartwater-endemic Caribbean islands revealed a substantial (12.4%) prevalence of cELISA-positive specimens. The cELISA is a relatively specific serodiagnostic test for heartwater in cattle and could be used to monitor for possible introduction of the disease into the USA. The cELISA may also be an excellent tool for monitoring the success of an ongoing Caribbean Amblyomma tick eradication program designed to eliminate the biological vector responsible for the perpetuation and spread of this dangerous foreign animal disease.

Assessment of Western Immunoblotting for the Confirmatory Diagnosis of Ovine Scrapie and Bovine Spongiform Encephalopathy (BSE)

Ovine scrapie and bovine spongiform encephalopathy (BSE) PrP and PrP were detergent extracted from 1.0-g specare members of a group of transmissible, genetically influimens as previously described. Nearly all brains were enced, neurodegenerative diseases known variously as unconventional slow virus infections, prion diseases, or spontested in triplicate using separate brain stem, midbrain, and cerebellum samples. To adapt WI to a diagnostic laboratory giform encephalopathies. 4-7,14 Laboratory diagnosis of these environment, several technical modifications were made. Each human and animal diseases has, until recently, depended tissue was homogenized in 20 ml of 25 mM TRIS buffer, solely upon neurohistopathologic examination and laborapH 7.4, containing 10% (w/v) N-lauroyl sodium sarcosinate tory rodent inoculation. The histopathologic approach is often inconclusive, particularly with autolyzed specimens,

In vitro assessment of viral antigen content in inactivated aluminum hydroxide adjuvanted vaccines

Antigen capture enzyme immunoassays (ELISA) were developed to assess the antigenic content of inactivated aluminum hydroxide (AH) adjuvanted porcine parvovirus, pseudorabies, and infectious bovine rhinotracheitis vaccines. Reference preparations of these viruses were constructed as a basis for comparison. Because AH-associated ELISA interference was largely circumvented, the need for isotopic or complex antigen-adjuvant desorption methods was eliminated. A 4-parameter logistic model related optical density to vaccine dilution. High correlation coefficients (r) were routinely achieved with commercial monovalent and polyvalent vaccines, and reference preparations. The procedure quantified antigen in both aqueous and AH-associated phases. The method may be generally applicable as a partial substitute for in vivo vaccine potency testing by allowing in vitro estimation of inactivated viral antigenic content in AH adjuvanted vaccines.

Serodiagnosis of equine piroplasmosis, dourine, and glanders using an arrayed immunoblotting method.

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The effect of the virus-serum incubation period upon vaccinia virus serum neutralization titers

Rabbit and human vaccinia virus immune sera were titrated by the plaque reduction serum neutralization (SN) method. Titers determined following a 2-h, 37 degree C virus-serum incubation period were not significantly different from those determined following an 18-h incubation period (2-h, 37 degree C incubation followed by a 16-h, 4 degree C period). However, control virus plaque counts decreased significantly after the longer incubation period. Numerous factors affecting SN titer estimation are reviewed. Standardization of SN test conditions may be useful in comparative potency evaluation of vaccinia-vectored recombinant DNA viral vaccines.