Ventricular myosin heavy chain isoform expression is altered in vitro in low score normal chickens.

The low score normal (LSN) chicken exhibits a genetic muscle weakness and altered in vitro myogenesis compared to the normal White Leghorn chicken. The ventricular myosin heavy chain isoform has been reported to be the initial muscle-specific contractile protein expressed during myogenesis. The goals of this study were to determine whether altered myogenesis of the LSN satellite cells in culture was accompanied by delayed ventricular myosin heavy chain expression and to further characterize the altered myogenic events exhibited by the LSN chicken. Immunocytochemical and ELISA analyses were employed to document the temporal expression of the ventricular myosin heavy chain during LSN chicken myogenesis. Satellite cells derived from the LSN chicken pectoralis major exhibited lower (P

Serological titers of equine monocytic ehrlichiosis associated with gastro-intestinal disorders and serological follow-up on two endemic farms.

The purpose of this work was to study the association of positive serological titers to Ehrlichia risticii, the causative agent of equine monocytic ehrlichiosis (EME) with gastro-intestinal disorders in hospitalized horses referred to The Ohio State University College of Veterinary Medicine Teaching Hospital (OSU VMTH). In addition, serological titers for E. risticii were monitored in two horse populations with endemic EME for one season to monitor temporal changes in titers. A statistically significant difference was found between the proportion of the total hospitalized horse population presented with a gastro-intestinal disorder during the study period, and study horses with IFA titers > or = 1:80 with these signs (P < 0.05). No such difference was found between the proportion of the total hospital horse population presented with signs of gastro-intestinal disorder, and the study horses with IFA titers of 1:20-1:40 with these signs, suggesting that these titers may not have any clinical significance for EME (P > 0.05). Thirty-eight horses on two farms endemic for EME were tested approximately every 3 weeks, 33 of which were tested serially at least two times. Five of the 38 horses (13.2%) had IFA titers > or = 1:80--two that were positive initially and three that seroconverted during the study; 15 horses' titers fluctuated between negative (IFA titers < 1:20) and exposed titers (1:20 through 1:40); and 18 horses remained negative throughout the study.

Serosurvey of horses with evidence of equine monocytic ehrlichiosis.

In August 1986, an extensive serosurvey for prevalence of IgG and IgM antibodies against Ehrlichia risticii, the causative agent of equine monocytic ehrlichiosis (EME), was performed at 2 Ohio racetracks, River Downs (RD) and Beulah Park (BP). Of 840 horses at RD and 574 at BP, 13 and 20%, respectively, were IgG antibody-positive (by indirect fluorescent antibody test results), with antibody titer ranging from 1:20 to 1:10,240. The titer observed at highest frequency at both racetracks was 1:80. A higher proportion of horses was ill at RD (operating during the summer months) than at BP (winter track). Of ill horses, 41% (24/58) at RD and 58% (11/19) at BP were seropositive. At RD, 70% (589/840) of all horses and 95% (102/107) of IgG seropositive horses had been stabled only at RD during the month prior to testing. Analysis of these sera by use of an ELISA to detect IgM antibody against E risticii antigen indicated that at RD, 42% (57/137) of the seropositive horses were IgM seropositive. At BP, 17% (20/120) of seropositive horses were IgM seropositive. The larger number of IgM seropositive horses at RD indicates that more horses were recently infected at RD than at BP (P = 0.0001). Therefore, at least half the seropositive horses at RD seemed to have acquired the infection at RD. These serosurvey data also indicate that at BP and RD, 78% (85/109) and 91% (111/122) of IgG seropositive horses, respectively, had subclinical infection. At less than or equal to 1:40 titer, there was no difference in seropositive rates between healthy and ill horses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute febrile cerebrovasculitis. A non-spotted fever group rickettsial disease.

In December 1985, a patient was seen with an illness that was clinically compatible with the recently described clinical syndrome of acute febrile cerebrovasculitis, including fever, headache, altered mentation, multifocal neurologic signs, and cerebrospinal fluid pleocytosis. An extensive medical evaluation failed to reveal a cause, until, retrospectively, she was shown to have antibodies to Rickettsia typhi. Detailed serologic analysis with enzyme immunoassays and protein immunoblots indicated that she was infected with a non-spotted fever group Rickettsia, most likely either R typhi or Rickettsia canada. Serum samples from a mouse trapped at her home contained antibody only to R canada. Evaluation of patients with acute febrile cerebrovasculitis in the future should include rickettsial blood cultures to attempt specific identification of the species involved.

Clinical, histopathological, and immunological responses of ponies to Ehrlichia sennetsu and subsequent Ehrlichia risticii challenge.

Ehrlichia risticii has a close antigenic relationship to E. sennetsu. Sera of ponies experimentally infected with E. risticii, the etiologic agent of Potomac horse fever, consistently reacted with E. sennetsu, a human pathogen, in indirect fluorescent-antibody (IFA) testing, while human E. sennetsu convalescent serum reacted with E. risticii by IFA testing and immunoferritin labeling of cells infected in vitro. Two ponies injected intravenously with live E. sennetsu did no develop clinical illness. Subsequent injection with live E. sennetsu did not develop clinical illness. Subsequent injection with live E. risticii also did not induce any disease, in contrast to two control ponies given E. risticii without prior exposure to E. sennetsu. Both controls developed fever, anorexia, depression, dehydration, and diarrhea, which are typical clinical signs of Potomac horse fever, and had characteristic lesions of enteritis and lymph node histiocytosis at postmortem examination. E. sennetsu-exposed ponies had normal gastrointestinal morphologies and lymph node hyperplasia. Ponies primed with E. sennetsu before E. risticii challenge developed high titers of immunoglobulin G antibody which reacted against both E. sennetsu and E. risticii antigens by IFA testing. The most prominent antigenic polypeptide in Western (immuno-) blot analysis of sera collected from ponies primed with E. sennetsu before subsequent challenge with E. risticii was present in lysates of both Ehrlichia species and had an apparent molecular mass of 44 kilodaltons. This band was not prominent in Western blots performed with sera of ponies injected with E. risticii alone. Thus, injection of E. sennetsu protects ponies from clinical and pathological manifestations of the disease induced by injection with E. risticii. Immunologic cross-reactivity of the two organisms with IFA testing and strong immunologic recognition by ponies of the 44-kilodalton antigen common to the two organisms may be related to the development of protective immunity against E. risticii.

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.

Evidence of paratuberculosis in Ohio's white-tailed deer, as determined by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed and used to detect antibodies to Mycobacterium paratuberculosis in serum samples obtained in December of 1983 from 954 hunter-killed white-tailed deer (Odocoileus virginianus) in 13 Ohio counties. Positive or negative status was determined by calculating a signal-to-noise ratio, a ratio between the optical density of the test serum and negative reference sera; a ratio of greater than or equal to 3.0 was considered positive. Twenty-four samples (2.5%) were found to be assay positive, using this method. A statistically significant difference among age groups was found, with those less than or equal to 6 months of age having a lower proportion of positives. Differences by sex were not observed. To determine the validity of the ELISA in deer, serum samples from 46 fallow (Dama dama) and axis deer (Axis axis) harvested from a known infected population were tested by ELISA and agar-gel immunodiffusion. The agar-gel immunodiffusion test showed evidence of exposure of the deer to M paratuberculosis or a related antigen. The ELISA closely approximated the prevalence of paratuberculosis infection as previously determined by fecal culture in this population. As a result of these tests, it was concluded that free-ranging Ohio deer have been infected with M paratuberculosis or exposed to a closely related antigen.

Serodiagnosis of La Crosse virus infections in humans by detection of immunoglobulin M class antibodies.

Sera from 92 humans with illnesses clinically compatible with those caused by California serogroup virus infections were tested for antibody to La Crosse (LAC) virus by using the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC ELISA), the IgG ELISA, and the hemagglutination inhibition (HI), complement fixation and serum dilution-plaque reduction neutralization tests. On the reported day of onset of illness in 18 individuals, 94% had IgM antibody, 50% had neutralization antibody, 33% had HI antibody, and 11% had IgG antibody. Neutralization, HI, and IgG antibody prevalence rates increased thereafter, whereas IgM antibody prevalence remained high (92% 2 or more weeks after the onset of illness). It was concluded that the MAC ELISA is a sensitive test for the presence of antibody to LAC virus. The sensitivity of the MAC ELISA and the rapidity with which it can be performed appear to provide a powerful tool for the clinically relevant serodiagnosis of LAC virus infections in humans.

Rapid separation of IgM from whole serum using spun column chromatography.

Spun columns were used to separate IgM from serum samples by exploiting molecular weight and size difference between IgM and other serum antibodies. Miniature gel chromatography columns were prepared in 3 ml syringe barrels. A polyacrylamide liquid chromatography gel, Bio-Gel P-200, was chosen to exclude proteins with molecular weights greater than 200,000. IgM, 900,000 MW, was excluded while IgG, 150,000 MW, was retained in the gel column. Centrifugation of the serum-loaded columns in test tubes accomplished the separation in one step by eluting a void volume equal to the sample volume from each column into a holding tube. IgM recovery in the eluent exceeded 96% of the total serum IgM in the pre-column sample. No IgG was detected in the eluent. IgM separated from La Crosse encephalitis immune human serum retained immunological activity in a viral neutralization test. Spun column chromatography is eminently suitable for diagnostic laboratories as more than 100 sera may be fractionated in one day using inexpensive materials and a low-speed centrifuge equipped with a swinging-bucket rotor.

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.