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Summary

Monoclonal antibodies were prepared against 40- and 56- to 64-kd antigens of Chlamydia pecorum strain Maeda, which was isolated from a cow with pneumonia. Using the monoclonal antibodies, 5 strains of C pecorum, 25 strains of mammalian and 19 strains of avian C psittaci, 1 strain of C pneumoniae, and 3 strains of C trachomatis were analyzed for immunologic reactivity by use of the indirect immunofluorescent test. Monoclonal antibody analysis revealed immunologic relatedness between C pecorum and mammalian strains of C psittaci, which were completely differentiated from the other avian strains. Bovine strains were distinguished from ovine strains. Antigenic diversity was observed for bovine and ovine strains. Feline- and guinea pig-derived strains were shown to be immunologically different from bovine and ovine strains. Results provide the basis for typing and epidemiologic study of bovine and ovine strains of C pecorum and C psittaci.

Free access
in American Journal of Veterinary Research

Summary

The prophylactic/therapeutic activity of natural bovine fibroblast interferon (BoF-ifn) against bovine rhinovirus infection in calves was assessed. Six calves were each given 8 intranasal inoculations of partially purified BoF-ifn (3.25 × 105 U at 8 am, 11 am, 5 pm, and 8 pm on day 1 and 8 am, 11 am, 2 pm, and 5pm on day 2), and 6 calves were given placebo. All calves were challenge exposed with 105.1 TCID 50) of bovine rhinovirus after the first 2 treatments (6 hours after the first ifn or placebo treatment). Nasal excretion of rhinovirus, ifn concentration in the nasal secretions, and nasal secretion and serum rhinovirus antibodies were measured before and at selected times after calves were inoculated. Interferon-treated calves excreted rhinovirus in their nasal secretions in lesser amounts (mean value, 0.84 log10 TCID 50/ml vs 1.58 log10 TCID 50/ml on postchallenge exposure days 1 and 2; (P < 0.05) and for a shorter duration (P < 0.05) than did placebo-treated calves. No calves developed clinical signs of respiratory tract illness. Rhinovirus antibody titer was not significantly different between ifn- and placebo-treated calves.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To analyze the antigenic cross reactivity of various proteins of strains of Chlamydia pecorum, C psittaci, and C trachomatis.

Samples and Procedures

Strains FC-Stra and LW-613 of C pecorum; strains B577, Fitz-9, and 6BC of C psittaci, and strain LGV-2 of C trachomatis were studied. Strains of C pecorum were propagated in Georgia bovine kidney cells, and other chlamydial strains were propagated in L cells or Georgia bovine kidney cells. Partially purified chlamydial elementary bodies propagated in RAG cells, a BALB/c cell line cloned from a renal adenocarcinoma of BALB/c mice, were used to immunize BALB/c mice for production of monoclonal antibodies. Rabbits were inoculated with yolk sack-propagated, purified elementary bodies to produce polyclonal antisera. The reaction of monoclonal antibodies and polyclonal antisera with chlamydial proteins was analyzed by use of immunoblot techniques.

Results

Two monoclonal antibodies reacted with a 90-kd protein of C psittaci and a 94-kd protein of C pecorum strains. One monoclonal antibody reacted strongly with a 67-kd protein of C pecorum and strain B577 of C psittaci, but weakly with proteins of strains 6BC and LGV-2. Another monoclonal antibody reacted with a 46-kd protein of 2 C pecorum strains and of strain B577 of C psittaci, but not with those of strains 6BC and LGV-2. Two monoclonal antibodies reacted with a 20-kd protein of C pecorum and a 22-kd protein of C psittaci and LGV-2 strains. Polyclonal antisera reacted similarly with the proteins identified by monoclonal antibodies in the various chlamydial strains.

Conclusions

Reactions of several monoclonal antibodies with chlamydial antigens indicated that 67- and 46-kd proteins contain genus- and species-specific epitopes, respectively; a 94-kd protein of C pecorum is homologous to a 90-kd protein of C psittaci and C trachomatis strains; and a 20-kd protein of C pecorum corresponds to a 22-kd protein of C psittaci and C trachomatis. (Am J Vet Res 1996;57:1720–1725)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate the susceptibility of polarized epithelioid human rectal tumor (HRT-18G) cells to bovine coronaviruses (BCV) isolated from enteric (EBCV) and respiratory (RBCV) tract infections.

Procedure

Cells of the G clone of HRT-18 were grown to confluent monolayers on permeable supports, and were directionally infected at the apical and basolateral domains with 3 wild-type BCV strains, RBCV-LSU-94LSS-051-2, RBCV-OK-0514-3, and EBCV-LY138-2, and 1 cell culture-adapted strain, EBCV-L9-80. Sequential cytopathic changes were microscopically monitored. Medium samples for titration of hemagglutinins and viral infectivity were collected directionally from both domains of the infected cell cultures at various intervals.

Results

Polarized epithelioid HRT-18G cells from apical domains had maximal susceptibility to infection with the EBCV and RBCV strains, and those from basolateral surfaces had minimal susceptibility. Titers of hemagglutinins and infective progeny BCV reached 1,280 hemagglutinin units and 4.2 × 108 plaque-forming units/ml for apical samples, but were minimal for basolateral samples. Asymmetric virus release occurred through the apical surfaces of the HRT-18G cells by 12 hours after infection when cell fusion as a sign of cytopathic changes began. When cells were infected basolaterally, progeny virions released from apical surfaces reinfected the target cells from the apical domains and induced cytopathic changes were delayed about 12 hours, compared with changes detectable in apically exposed cultures.

Conclusions

EBCV and RBCV, isolated from cattle, had marked tropism for polarized epithelioid HRT-18G cells. Entry of BCV into the polarized HRT-18G cells was effected maximally through the apical domains and minimally through the basolateral domains. Release of progeny BCV occurred preferentially from the apical domains. (Am J Vet Res 1997;58:1120–1124)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate the antigenic diversity of lipo-oligosaccharides of Haemophilus parasuis.

Procedures

Immunoblot assays were done with monoclonal and polyclonal antibodies on whole-cell lysates. Individual colonies of H parasuis strains H 54, H 53, and H 128 were tested for reactivity with lipo-oligosaccharide-specific monoclonal antibodies after a single passage on chocolate agar, and colonies of strain H 54 were analyzed after 10 passages. Colony blot tests were used to screen H parasuis strains for spontaneously occurring antigenic variation in their lipo-oligosaccharides.

Results

Eight H parasuis strains were separated into 4 lipo-oligosaccharide serovars on the basis of immunoblot reactions with 3 polyclonal rabbit antisera. Nine monoclonal antibodies against lipo-oligosaccharides of a lipo-oligosaccharide-serovar I strain reacted with all tested serovar I strains but failed to react with other H parasuis strains.

Conclusions

Variations in the antigenic reactivity after 1 or 10 passages on chocolate agar were not observed. The serovar I lipo-oligosaccharide strains included virulent as well as avirulent H parasuis strains, indicating that these epitopes do not correlate directly with virulence properties of H parasuis. (Am J Vet Res 1996;57:63-67)

Free access
in American Journal of Veterinary Research

Summary

A technique for detection of bovine viral diarrhea virus (bvdv) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of bvdv and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral rna in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of bvdv by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.

Free access
in American Journal of Veterinary Research

SUMMARY

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and the morphologic features and ultrastructure of intra-cellular inclusions. Amplified chlamydial ompA dna fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (rb) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet rb were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 rb, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the rb as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.

Free access
in American Journal of Veterinary Research

Summary

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (cmp) of noninfected cells were labeled with biotin (b-cmp), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (eb) were reacted with the b-cmp extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound b-cmp. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound b-cmp. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 eb and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull. The reason for the 2 types of responses to infection remains to be determined.

Free access
in American Journal of Veterinary Research

SUMMARY

Latency and reactivation of pseudorabies virus in swine was studied. Thirty-one pigs were assigned to 5 groups and were given 1 of 4 vaccines; 10 remained unvaccinated controls. All pigs were then challenge exposed with a sublethal dose of virulent pseudorabies virus. One hundred one days after challenge exposure, all pigs were treated with dexamethasone to reactivate the virus. Virus-positive tonsil and nasal mucus isolates were recovered from 29 of the 31 pigs over a 12-day period. Frequency and duration of virus-positivity were significantly (P < 0.05) and consistently lower among vaccinated pigs than among the unvaccinated controls. It was concluded that vaccination before challenge exposure had little or no effect on the rate of establishment of virus latency, but that vaccination reduced shedding after subsequent reactivation of the virus.

Free access
in American Journal of Veterinary Research

SUMMARY

The survival of pseudorabies virus in an aerosol was studied under different environmental conditions of temperature and relative humidity. Pseudorabies virus decayed logarithmically with mean half-lives of 17.4 (85% relative humidity, 22 C), 18.8 (25% relative humidity, 22 C), 27.3 (85% relative humidity, 4 C), 36.1 (55% relative humidity, 22 C), and 43.6 (55% relative humidity, 4 C) minutes. Virus survival was significantly improved in environments at 55% relative humidity, compared with those at 85% relative humidity (P = 0.017). Rates of survival were improved in environment at 4 C in comparison with those at 22 C. Results suggest that, under the best conditions of this study, the infectivity of pseudorabies virus in an aerosol decreases by 50% in < 1 hour.

Free access
in American Journal of Veterinary Research