In vitro killing of Pasteurella multocida: the effect of rabbit granulocyte and specific antibody source.

In vitro granulocyte-killing assays were performed to examine the ability of granulocytes from pasteurella-free or immunized rabbits, the in combination with specific immune serum, to kill Pasteurella multocida. Granulocytes from healthy rabbits and from rabbits with P multocida infections were equally competent. Granulocyte source, serum source, and specific antibody titer had no effect on granulocyte phagocytic activity. Moreover, serum containing specific antibody and complement supported the growth of the bacterium. These data suggest that chronic P multocida infections are not attributable to defective granulocytes or lack of serum antibody production.     

Production of Neutralizing Antisera against Viral Hemorrhagic Septicemia (VHS) Virus by Intravenous Injections of Rabbits

Abstract

Rabbit antisera against viral hemorrhagic septicemia virus (VHSV) produced by two immunization procedures were compared for neutralization and immunochemical properties against homologous and heterologous strains. The VHSV isolate used as the immunogen was a member of a serogroup not neutralized by previously available antisera. The results from this study suggested that frequent intravenous (IV) injections of rabbits with viral antigens were superior to adjuvant-mediated, combined subcutaneous and intraperitoneal (SC/IP) injections for the production of neutralizing antisera. All IV injected rabbits produced high neutralization titers against the homologous VHSV isolate but not against an isolate from a different serogroup. The SC/IP injected rabbits had no significant neutralization titers against either the homologous VHSV strain or two isolates of a heterologous VHSV strain. Sera from all injected rabbits reacted in indirect immunofluorescence (IF) assays with either strain; however, the SC/IP injected rabbits produced higher titers against the heterologous VHSV strain by ELISA (enzyme-linked immunosorbent assay). By Western blotting, neutralizing antisera primarily stained the viral glycoprotein (G) whereas the nonneutralizing sera stained all the viral structural proteins equally well. Our results demonstrate that immunization procedures to produce antisera against VHSV in rabbits determine whether the resultant antibodies will have primarily neutralizing or binding capabilities.     

Chromatographic separation and characterization of Pasteurella haemolytica cytotoxin

Biochemical and immunologic properties of the cytotoxin (leukotoxin) produced by Pasteurella haemolytica were examined. Crude, bacteria-free supernatants from logarithmic phase P haemolytica were fractionated, using a series of column chromatographic techniques. Sequential anion exchange chromatography, gel-filtration chromatography, and chromatofocusing resulted in a cytotoxic substance (cytotoxin-C) of approximately 160 kilodaltons (kD), as determined by use of gel-filtration chromatography. Polyacrylamide-gel electrophoresis of cytotoxin-C yielded 3 protein bands with relative mobilities of 0.37, 0.42, and 0.63. On the basis of immunoblotting with a cytotoxin-neutralizing bovine immunoglobulin for antigen detection, the 2 low-mobility bands shared a strong region of immunogenicity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, principal protein constituents of cytotoxin-C were found at 160, 66, 57, and 23 kD. Using immunoblotting with cytotoxin-neutralizing immunoglobulin, strong, distinct reactions with the 66- and 57-kD bands were detected. Immunization of rabbits and mice with cytotoxin-C resulted in sera that reacted strongly with cytotoxin-C in enzyme-linked immunosorbent assays and immunodiffusion assays. The major immunogenic proteins also were detected by use of immunoblotting with anticytotoxin-C sera from rabbits and mice. Postinoculation rabbit sera neutralized crude cytotoxin.     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

Single primer polymerase chain reaction fingerprinting for Pasteurella multocida isolates from laboratory rabbits

OBJECTIVE: To evaluate a rapid polymerase chain reaction (PCR) fingerprinting technique for discriminating among Pasteurella multocida isolates from laboratory rabbits. SAMPLE POPULATION: 33 P multocida isolates from rabbits with clinical pasteurellosis. PROCEDURE: PCR assays were conducted with 2 minisatellites (core sequence and modified core sequence of phage M13) and 2 microsatellites ([GTG]5 and [GACA]4). Each bacterium was assigned to a PCR type for each of the primers used. Boiled bacterial extracts and purified genomic DNA were compared by use of PCR assays for phage M13 and (GACA)4. Plasmids were isolated from each bacterium, and their influence on PCR fingerprint was determined, using boiled extracts as a DNA source. RESULTS: M13 core sequence and M13 modified core sequence yielded 5 and 8 PCR types, respectively. The microsatellites (GTG)5 and (GACA)4 yielded 4 and 9 PCR fingerprint types, respectively. Fingerprint patterns obtained by use of isolated DNA differed from those obtained by use of boiled extracts, although discrimination among P multocida isolates was similar. The presence or absence of plasmids did not affect PCR fingerprints. CONCLUSION: Single primer PCR fingerprinting with minisatellite and microsatellite primers is an efficient and reproducible method for the discrimination of P multocida isolates from rabbits and can be performed directly, using boiled bacterial extracts as a source of template, although more bands were obtained from pure genomic DNA.     

伪狂犬病毒SN株、SL株的分离鉴定

对琼扩试验检测为伪狂犬病毒 (PRV )阳性的病料 ,采用MDBK细胞进行病毒分离培养 ,获得了两株PRV ,进而经家兔接种和核酸杂交鉴定为PRV阳性 ,将其分别命名为PRVSN株、SL株。     

Comparison of enzyme-linked immunosorbent assay and immunofluorescence test for determination of anti-Encephalitozoon cuniculi antibodies in sera from rabbits with different clinical and histopathological presentations

Encephalitozoon cuniculi is a microsporidian that infects rabbits resulting in a varied pathology of the nervous, renal, and ocular tissues. Serology based studies using different methods have been conducted indicating a high seroprevalence worldwide in pet rabbits. While qualitative results are not especially helpful in the diagnosis of this infection, quantitative titers can have increased predictive value. In the current study, results of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence test methods were compared using banked samples from rabbits with confirmed infection and clinically normal rabbits. Pearson's analysis found good and significant correlation (R = 0.56, P = 0.0002). Bland Altman analysis demonstrated the methods were similar but not equivalent. The sensitivity ranged from 0.79 to 0.84 and the specificity was 1.00 for both assays. Anti-E. cuniculi IgM titers were also determined by ELISA to be an independent measure from IgG (R = 0.31, P = 0.06) with a sensitivity of 0.24 and specificity of 1.00. In total, these results confirm an agreement in the detection of IgG antibody by two different methods conducted at laboratories in the United States and Austria that are involved in research and clinical diagnostic testing of this agent.     

A rabbit model of non-typhoidal Salmonella bacteremia

Bacteremia is an important cause of morbidity and mortality in humans. In this study, we focused on the development of an animal model of bacteremia induced by non-typhoidal Salmonella. New Zealand White rabbits were inoculated with a human isolate of non-typhoidal Salmonella strain CVD J73 via the intra-peritoneal route. Blood samples were collected at specific time points and at euthanasia from infected rabbits. Additionally, tissue samples from the heart, lungs, spleen, gastrointestinal tract, liver and kidneys were obtained at euthanasia. All experimentally infected rabbits displayed clinical signs of disease (fever, dehydration, weight loss and lethargy). Tissues collected at necropsy from the animals exhibited histopathological changes indicative of bacteremia. Non-typhoidal Salmonella bacteria were detected in the blood and tissue samples of infected rabbits by microbiological culture and real-time PCR assays. The development of this animal model of bacteremia could prove to be a useful tool for studying how non-typhoidal Salmonella infections disseminate and spread in humans.     

Acute Phase Protein Levels in Rabbits with Suspected Encephalitozoon cuniculi Infection

The objective of this study was to evaluate the application of acute phase protein assays for C-reactive protein (CRP), haptoglobin (HP), and serum amyloid A (SAA) in the diagnosis of Encephalitozoon cuniculi (ECUN) infection in pet rabbits. Serum samples from 48 pet rabbits were submitted from veterinary clinics within the United States. Participating veterinarians completed a questionnaire that was used to classify rabbits as either non-ECUN suspect (n = 19) or suspected of having ECUN infection (n = 29). A previously described enzyme-linked immunosorbent assay diagnostic test was used to detect immunoglobulin G (IgG) titers against ECUN. Samples were additionally tested for levels of CRP, HP, and SAA. A nearly 10-fold mean increase in CRP levels was observed in the ECUN-suspect group. This increase was significant (P < 0.05). There was no significant difference in HP or SAA levels between the clinical groups. These data support the use of CRP as an adjunct test in the diagnosis of ECUN infection in pet rabbits.     

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus)

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.     

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.     

Development of a cell culture assay for the quantitative determination of vaccination-induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin

Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.     

兔支气管败血波氏杆菌PCR检测方法的建立及应用

根据支气管败血波氏杆菌菌毛蛋白基因(fimN)序列设计了一对引物,扩增出大小为648bp的目的基因片段,建立了快速检测兔支气管败血波氏杆菌的PCR方法。特异性和敏感性试验表明,该方法对大肠埃希氏菌、金黄色葡萄球菌和巴氏杆菌均无交叉性反应。用该PCR法检测了从杨凌、咸阳采集的50份表现临床症状的兔呼吸道分泌物检出支气管败血波氏杆菌阳性35例,阳性率为70%。同时与传统的细菌检查法、微量凝集法进行了比较,结果显示,该PCR法的检出率是细菌检查法的3.89倍,是微量凝集法的1.94倍。     

Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA)

Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.     

Anti-idiotype technique: an alternative approach for immunodiagnosis of bluetongue

In development of a bluetongue alternative immunodiagnostic rest, the polyclonal anti-idiotypic antibodies were generated by the sequential immunization of rabbits with three monoclonal antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies recognize the idiotypes that are located within or near the antigen-combining sites and are associated with both heavy and light chains of the antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies mimic the VP7 antigen by recognizing the anti-VP7 antibodies from cattle and sheep that were infected with various serotypes of bluetongue viruses. The results indicate that the rabbit anti-idiotypic antibodies may be used as surrogate antigen in serological assays to detect the antibodies from different species of animals infected with various serotypes of bluetongue viruses.     

猪链球菌2型及其毒力因子检测多重PCR的建立与应用

根据相关文献设计并合成引物，建立了能同时检测猪链球菌2型（cps）及其重要毒力因子溶菌酶释放蛋白（mrp）和细胞外因子（epf）的多重PCR方法。目的片段的大小分别为885bp（mrp）、675bp（cps）和443bp（epf）。对参考菌株、人工攻毒病料和临床收集病料的检测结果显示，该多重PCR特异性强、敏感性高，可直接从临床病料中检测出猪链球菌2型，并能鉴定其毒力因子表型。     

A comparative study of complement activation

Antisera to sheep erythrocytes (E) were raised in cattle, rabbits, mice, hamsters, guinea-pigs, ferrets, badgers, hedgehogs and fowls. Cross activation of total haemolytic complement (THCA) examined all combinations of sensitized sheep E and normal sera (including human); kinetic assays examined the lysis of E sensitized with rabbit antibodies. From the same species, all combinations of normal serum and xenogeneic E were used to measure total alternative pathway activity (TAPA); TAPA was also activated by rabbit and sheep E in titrations and in agarose gels, and examined kinetically against rabbit E. Ox, rabbit and fowl sera were low in THCA, guinea-pig complement was universally active, while human complement showed marked selectivity; ferret, badger and hedgehog sera were activated to high titres but probably via the alternative pathway. In studies of TAPA an inverse relationship existed between serum complement activities and the activating abilities of E from the same species. The most efficient activators of alternative pathway were E from rabbits and laboratory rodents, while the sera with broadest response were badger, ferret and fowl. Kinetic studies of TAPA showed that initiation of lysis and subsequent completion of lysis could occur with different efficiencies, suggesting these events reflected separate events in complement activation.     

Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.     

Infection of rabbits with bovine immunodeficiency-like virus.

New Zealand white rabbits, which had been prepared for inoculation by intraperitoneal treatment with thioglycollate, were inoculated intraperitoneally with bovine immunodeficiency-like virus (BIV). Infected materials from various sources were used including cultured cells and culture fluids, peripheral blood leukocytes from infected cattle and spleen tissue from previously infected rabbits. Virus isolations and serological responses detected by western blotting provided clear evidence that infections had been established in inoculated rabbits and that the spleen was an important site of BIV infectivity. These results indicate that rabbits may be a useful species when testing for BIV infectivity in materials too toxic or highly contaminated to be inoculated directly into cell cultures. Furthermore, rabbits may also be useful in testing effects of coinfections with other bovine viruses on progression of BIV infection and for the initial evaluation of therapeutic regimens designed to suppress or eliminate BIV infections.     

Isolation and biological characterization of two lipopolysaccharides and a capsular-enriched polysaccharide preparation from Haemophilus pleuropneumoniae

A smooth-type lipopolysaccharide (HpS-LPS), a rough-type lipopolysaccharide (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) were extracted and purified from Haemophilus pleuropneumoniae serotype 5, strain J45, by the use of phenol-water (HpS-LPS) and phenol-chloroform-petroleum-ether (HpR-LPS) techniques. Chemical analysis of the HpS-LPS and HpR-LPS indicated that they contained 0.7% and 4.4% (w/w) 2-keto-3-deoxyoctonate, 11.8% and 10.4% phosphate, 0.8% and 0.8% nucleic acid, and 0.8% and 1.1% protein, respectively. The HpC-PS contained 0.3% 2-keto-3-deoxyoctonate, 1.4% phosphate, 0.2% nucleic acid, and 0.8% protein. With sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the HpS-LPS banded as a smooth-type LPS and the HpR-LPS banded as a rough-type LPS. Electrophoresis of HpC-PS indicated the presence of a broad high molecular weight band. Gelation of Limulus amoebocyte lysate developed at a minimum concentration of 8 ng/ml of HpS-LPS, 0.3 ng/ml of HpR-LPS, and 35 ng/ml of HpC-PS. The lipopolysaccharide preparations provoked a positive dermal Shwartzman reaction in rabbits and swine, a biphasic febrile response in rabbits, and a monophasic response in swine. Responses were typical of endotoxic activity with swine having greater sensitivity than rabbits. The chick embryo 50% lethal dose was calculated to be 7.3 ng for HpS-LPS, 1.6 ng for HpR-LPS, 5.1 ng for the lipopolysaccharide of Escherichia coli 0111:B4; and the HpC-PS was not toxic. In all assays, HpR-LPS was significantly more toxic than was HpS-LPS. The HpC-PS preparation was not toxic, even at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)