Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares.

OBJECTIVE: To characterize clinical, serologic, bacteriologic, cytologic, and pathologic endometrial responses of mares to 2 donkey-origin atypical bacterial isolates resembling Taylorella equigenitalis. DESIGN: Prospective in vivo study. ANIMALS: 10 healthy mares. PROCEDURE: Mares in estrus (2/group) were inoculated by intrauterine infusion with 2 isolates of classic T equigenitalis or 2 isolates of atypical Taylorella sp or were sham-inoculated. Bacteriologic, serologic, clinical, uterine, cytologic, and pathologic endometrial responses were assessed 4, 11, 21, 35, and 63 days after inoculation and on day 111 in mares with positive culture results on day 63. RESULTS: One atypical isolate failed to cause infection. The second atypical isolate and both classic T equigenitalis isolates induced similar transient metritis and cervicitis. Both classic isolates and 1 atypical isolate induced anti-T equigenitalis complement-fixing antibodies detectable at day 11. Classic isolates and an atypical isolate provoked intense neutrophilic endometritis followed by a resolving, subacute, neutrophilic-mononuclear endometrial response. The atypical isolate and classic isolates were recovered from the uterus, clitoral fossa, or clitoral sinus of one or both exposed mares for as long as 111 days. CONCLUSIONS AND CLINICAL RELEVANCE: Atypical Taylorella sp infections should be considered as a differential diagnosis of equine infertility in US-origin mares, even those not exposed to stallions from countries where contagious equine metritis occurs. The origins and prevalence of atypical Taylorella sp infection in US horses and donkeys are undetermined.

Detection of equine antibodies to babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.

Progressive macrodactyly.

A case of progressive macrodactyly in an adult is presented with MRI and CT imaging to illustrate the extent of the deformity.

H2 histaminergic control of inhibition of eating induced by intragastric NaCl in rats.

A role for endogenous histamine and histamine receptor subtypes in mediating the inhibition of eating induced by intragastric (i.g.) hypertonic NaCl was examined in adult male Sprague-Dawley rats surgically equipped with a chronic gastric catheter. The i.g. infusion of 2 mL 900 or 1,800 mOsm/kg of NaCl inhibited: 1) ingestion of pellets in rats eating after 24-h food deprivation; and 2) ingestion of cookies in rats eating without prior deprivation. The H1 receptor antagonists dexbrompheniramine (DXB; 1 mg/kg) and pyrilamine (PYR; 4 mg/kg) did not attenuate the inhibitory effects of i.g. 900 or 1,800 mOsm/kg of NaCl for rats eating pellets and for rats eating cookies. The H2 antagonists cimetidine (CIM; 16 mg/kg) and metiamide (MET; 16 mg/kg) attenuated the inhibitory effects of i.g. 1,800 mOsm/kg of NaCl upon ingestion of cookies, but intracerebroventricular (i.c.v.) infusion (through a chronic indwelling cannula) of 100 microg of CIM did not mimic this effect of intraperitoneal (i.p.) CIM. The i.p. CIM failed to attenuate the inhibition of eating cookies produced by i.p. octapeptide of cholecystokinin (CCK-8; 3 microg/kg). The H3 antagonist thioperamide (TH; 10 mg/kg i.p.) and the H3 agonist R-alpha-methylhistamine (RAM; 3 mg/kg i.p.) did not alter the inhibitory effect of i.g. 1,800 mOsm/kg of NaCl for rats eating cookies. Combined treatments of systemic DXB plus CIM, and DXB plus CIM plus thioperamide (TH) did not reverse the inhibitory effects of i.g. 1,800 mOsm/kg of NaCl upon ingestion of cookies. Finally, i.p. DXB, but not CIM, attenuated the ability of i.g. 900 mOsm/kg of NaCl to increase water intake; conversely, i.p. CIM, but not DXB, attenuated the ability of i.g. 900 mOsm/kg of NaCl to inhibit eating of cookies. These findings demonstrate a double dissociation of effects upon ingestive behavior: H1, but not H2, antagonism attenuates the effect of i.g. hypertonic NaCl on water intake, whereas H2, but not H1, antagonism attenuates the inhibition of eating produced by i.g. hypertonic NaCl. These results demonstrate that different subtypes of peripheral and/or central histamine receptors contribute to different behavioral consequences of postprandial gastrointestinal osmotic loads in rats.

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.

Validation and comparison of three enzyme-linked immunosorbent assays for the detection of antibodies to Cowdria ruminantium infection.

Serological tests for Cowdria ruminantium infection have been hampered by low specificity. Here, an indirect ELISA based on purified antigen, a competitive ELISA using a recombinant major antigenic protein (MAP-1) and an indirect ELISA based on the MAP-1B region of the recombinant MAP-1 were compared. The tests were validated using 3000 sera of ruminants from 14 islands of the Lesser Antilles as well as sequential serum samples from 10 cattle, 17 goats and 10 sheep vaccinated with inactivated C. ruminantium in ISA 50 adjuvant and from 14 goats infected with a virulent culture supernatant. All tests detected significantly higher percentages of positives on Antigua, Guadeloupe and Marie-Galante, where C. ruminantium had been isolated before. Overall specificity calculated with sera from the other 11 heartwater-free islands was 98.1%, 98.5%, and 99.4% for the ELISA based on crude antigen, recombinant MAP-1 and MAP-1B, respectively. Sensitivities observed with sequential serum samples were similar for all tests. Tests based on recombinant antigens, especially the MAP-1B, showed improved specificity, suggesting their use for epidemiological studies in regions where the distribution of cowdriosis is unknown. In addition, the competitive ELISA is useful for studies in wildlife for which species-specific conjugates do not exist.

Development and validation of a competitive enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera.

Serologic screening of avian sera for group-specific antibodies to type A influenza is currently accomplished by using the avian influenza (AI) agar gel immunodiffusion (AGID) test. A competitive enzyme-linked immunosorbent assay (CELISA) was developed using a baculovirus vector, Autographa californica nuclear polyhedrosis virus, expressing the nucleoprotein (NP) gene of A/Ann Arbor/6/60 influenza virus. The recombinant NP was obtained by inoculation of Spodoptera frugiperda (Sf9) insect cells or Trichoplusia ni insect larvae with the recombinant baculovirus. A hybridoma cell line producing monoclonal antibody against influenza virus A nucleoprotein was used to generate mouse ascitic fluid for the CELISA. The nucleoprotein and the monoclonal antibody were used without further purification in a CELISA for detection of avian-origin serum antibodies to type A influenza. The AI AGID and CELISA tests were compared for sensitivity and specificity using 1651 experimental and reference antisera. Samples discrepant in AGID and CELISA test results were further evaluated by the AI indirect fluorescent antibody (IFA), hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI) tests. The results demonstrated a high degree of correlation between the AGID and CELISA test results, with the IFA, HI, and NI tests offering additional support of CELISA test specificity. The CELISA is a rapid, economical, sensitive, and specific serodiagnostic method for screening large numbers of avian sera for antibodies to avian influenza virus.

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 > or = 99.5% 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.

A recombinant antigen from the heartwater agent (Cowdria ruminatium) reactive with antibodies in some southeastern United States white-tailed deer (Odocoileus virginianus), but not cattle, sera.

Recombinant baculovirus techniques were used to express the 260 amino acid carboxyterminal portion of the 32 kilodalton (kDa) major antigenic protein (MAP 1) of Cowdria ruminantium, the heartwater agent, as a fusion protein. The recombinant MAP 1 was fused to an aminoterminal independently antigenic octapeptide sequence (FLAG peptide). Recombinant MAP 1 was used as an immunoblotting antigen to evaluate numerous reference antisera against organisms of the tribe Ehrlichieae. Monoclonal and polyclonal C. ruminantium antibodies, monoclonal anti-FLAG ascites, and antisera to Ehrlichia canis and Ehrlichia chaffeensis reacted with this antigen. Twelve of 79 sera collected 1980 to 1992 from southeastern U.S. white-tailed deer (Odocoileus virginianus) were also unexpectedly immunoblot-positive to MAP 1. These 12 deer sera had, as a group, significantly (P < 0.01) greater anti-E. chaffeensis titers (previously determined) than the sera from MAP 1 immunoblot-negative deer living in the same areas. None of the 262 sera from cattle living in the same areas were immunoblot-positive to MAP 1. All of an additional 50 cervine sera from Michigan (USA), 72 bovine sera from northern U.S. cattle, and 72 sera from Puerto Rican cattle were also immunoblot-negative to MAP 1. Sera from African sheep which were falsely seropositive to authentic MAP 1 were also immunoblot-positive to the recombinant MAP 1. Unidentified Ehrlichia spp. capable of serologic crossreactivity with the heartwater agent appear to be present in some southeastern U.S. white-tailed deer but not cattle. These or related Ehrlichia spp. may also be found elsewhere in the world in non-cervine species.

Construction and insect larval expression of recombinant vesicular stomatitis nucleocapsid protein and its use in competitive ELISA.

The gene encoding the nucleocapsid (N) protein of Indiana 1 serotype vesicular stomatitis virus (VSV-IN1) was transferred into the genome of Autographa californica nuclear polyhedrosis virus (baculovirus) as a full-length non-fusion construct under the control of the polyhedrin gene promoter. Recombinant N protein was obtained from Trichoplusia ni insect larvae inoculated 72-96 h previously with the recombinant baculovirus. Polyclonal antibody (PAB) against VSV-IN1 was produced in mice using VSV-IN1 whole virus antigen concentrated from virus-infected cell culture fluids. The N protein and the PAB were used without further purification in a competitive enzyme-linked immunosorbent assay (C-ELISA) for detection of bovine, porcine, and equine origin serum antibodies against VSV-IN1. A limited number of field origin, experimental, and reference VSV antisera were evaluated using the C-ELISA and with a standard serum neutralization (SN) procedure. Sensitivity of the C-ELISA was comparable to the serotypically homologous SN procedure. Subject to further validation, similar C-ELISA tests for the other VSV serotypes, used in conjunction with the test described here, may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis.

Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4- to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 x g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen.

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.

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.

Mutational analysis of an autoantibody: differential binding and pathogenicity.

We have used site-directed mutagenesis to change amino acid residues in the heavy chain of the pathogenic R4A anti-double-stranded DNA (dsDNA) antibody and have looked for resultant alterations in DNA binding and in pathogenicity. The data demonstrate that single amino acid substitutions in both complementarity determining and framework regions alter antigen binding. Changes in only a few amino acids entirely ablate DNA specificity or cause a 10-fold increase in relative binding. In vivo studies in mice of the pathogenicity of the mutated antibodies show that a single amino acid substitution leading to a loss of dsDNA binding leads also to a loss of glomerular sequestration. Amino acid substitutions that increase relative affinity for dsDNA cause a change in localization of immunoglobulin deposition from glomeruli to renal tubules. These studies demonstrate that small numbers of amino acid substitutions can dramatically alter antigen binding and pathogenicity, and that the pathogenicity of anti-DNA antibodies does not strictly correlate with affinity for DNA.

Detection of a cell line contaminated with hog cholera virus.

Cell lines from the repository of the American Type Culture Collection were examined for possible contamination with bovine viral diarrhea virus. During testing, hog cholera virus (HCV) was detected in the IB-RS-2 D10 porcine kidney cell line. This variant of HCV was avirulent for pigs and seldom induced detectable concentrations of antibody against reference viruses (HCV-Ames or bovine viral diarrhea virus-NY1) in serum of inoculated pigs. Additionally, this variant of HCV did not confer protection to pigs against virulent HCV. The contaminated cell line had been distributed to > 20 laboratories in the United States. The cell line was not used in field studies and has been destroyed.

Isolation of pseudorabies (Aujeszky's disease) virus from a Florida panther.

Pseudorabies virus was isolated in cell culture from the brain tissue of a 3.5-year-old male Florida panther (Felis concolor coryi). The virus was not isolated from other tissues collected at necropsy. Based upon a nested polymerase chain reaction (PCR), the virus was determined to have the classical wild-type virulent genotype, glycoprotein I+ (gI+) and thymidine kinase+ (TK+).