Analysis of genetic variation of epizootic hemorrhagic disease virus and bluetongue virus field isolates by coelectrophoresis of their double-stranded RNA.

Thirty-two bovine field isolates of bluetongue virus (BTV), 6 field isolates of epizootic hemorrhagic disease virus (EHDV) from deer, 4 BTV prototype serotypes (10, 11, 13, and 17), and 2 EHDV prototype serotypes (1 and 2) were coelectrophoresed, using polyacrylamide gels. Field isolates were obtained from various regions of the United States. Analysis of polyacrylamide gels and scattered plots generated for comparison of migration patterns for different isolates within each serotype of BTV revealed wide variation among the individual segments. The BTV serotypes 10 and 11 had more variation, compared with BTV serotypes 13 and 17, especially for migration of genome segment 5. A definitive correlation was not seen between the double-stranded RNA migration profiles on polyacrylamide gel electrophoresis, geographic origin, herd of origin, or year of collection. One BTV field isolate contained more than 1 electropherotype, with 2 bands at the segment-7 position, and it was further characterized as BTV serotype 11. Segments 2 and 5 of EHDV isolates were more variable in their migration than were the other gene segments. Generally, migration profiles for EHDV double-stranded RNA were more variable, compared with those of BTV isolates. Although a correlation was found between migration profiles and serotype of 2 isolates of EHDV, a study of additional EHDV isolates is required before the diversity of electrophoretic patterns of EHDV can be determined.     

Serotyping of Haemophilus pleuropneumoniae by rapid slide agglutination and indirect fluorescent antibody tests in swine

One hundred and forty-one isolates of Haemophilus pleuropneumoniae from Iowa and Illinois swine were characterized morphologically and biochemically and serotyped by rapid slide agglutination (RSA) and indirect fluorescent antibody (IFA) tests. Hyperimmune antisera were produced in rabbits using inactivated whole-cell suspensions of the reference strains for H pleuropneumoniae serotypes 1 to 7 and strain 202, representing the taxon "minor group." Cross testing of the reference strains and reference antisera indicated the antisera to be essentially serotype-specific, although reactivity of some antisera with heterologous strains was observed. Cultures of the 141 isolates formed adherent or smooth colonies or mixtures of these colony forms. Adherent and smooth colony types were found in all serotypes identified. Microscopic and biochemical characteristics of all isolates were typical of those previously described for H pleuropneumoniae. The overall incidence of H pleuropneumoniae serotypes was serotype 5, 55.3%; serotype 1, 34.0%; serotype 7, 7.8%; and nontypeable, 2.8%. Comparing the 2 test procedures, 87.2% of the isolates could be typed by RSA, and 66.0% could be typed by IFA. Cross-reactions between serotype 4 antisera and serotype 5 and 7 isolates were common with the IFA test. The reactions with serotype 7, but not serotype 5, were eliminated by cross adsorption of serotype 4 antisera. There was good correlation between the 2 test procedures, but RSA was judged to be more specific and sensitive than IFA.     

Serotyping of Haemophilus pleuropneumoniae in the Netherlands: with emphasis on heterogeneity within serotype 1 and (proposed) serotype 9

Four hundred and forty-three Dutch field isolates of Haemophilus pleuropneumoniae were serotyped by rapid slide agglutination (RSA) using specific antisera against serotypes 1 to 5 and against the recently proposed types 6 to 9. The predominant serotypes were 9 (49%) and 2 (32%). Serotypes 1, 3, 5, 7 and 8 were isolated in small numbers: together they accounted for 3% of the total. Five percent of the isolates were not typable either due to autoagglutination or because they were not agglutinated by any of the available antisera. The remaining 49 strains (11%) agglutinated in more than one antiserum and could therefore not be properly classified. Forty-four of these 49 strains agglutinated in both anti type 1 and anti type 9 serum. Antigenic relationships between serotype 1, serotype 9 and isolates reacting with both antisera were studied using immunodiffusion and RSA with adsorbed sera. Serotype 9 strains appeared not to be a homogenous group. Isolates agglutinating exclusively in anti type 9 serum can be divided into two groups: one closely related and another hardly related to serotype 1. Serotype 9 reference strain 13261 belongs to the latter. Type 1 + 9 strains have antigens in common with serotypes 1 and 9, but they also have their own specific antigenic material. Such strains are proposed as a new serotype 10.     

Typing of heat-stable soluble Haemophilus parasuis antigen by means of agargel precipitation and the dot-blot procedure.

According to Morozumi's and Nicolet's (1986) investigations, a serological classification procedure for H. parasuis and to a certain extent, for H. parasuis-like strains was proposed on the basis of heat-stable cell antigens in the immunodiffusion test. It was possible to classify 72.8% of the investigated strains serologically using this procedure. 7 of 12 serotypes were described for the first time. 60.1% of the classified strains belonged to the already known serotypes SV 1 to SV 5, whereas the new serotypes SV Jena 6 to SV Jena 12 amounted to only 12.7% of the field isolates. The serotypes SV Jena 7 to 9 are represented by H. parasuis-like strains. Unencapsulated strains and isolates of serotype SV 5 dominate in animals with Glasser's disease. The serotypes SV 1, SV 5, Jena 6 and SV Jena 10 proved to be highly virulent in SPF pigs, SV 2 and SV 4 were of medium virulence. The other serotypes were non-virulent. The high specificity in AGPT was not reproducible in the more sensitive dot-blot procedure. This must be taken into account, if the dot-blot is to be used for the classification of serotypes of H. parasuis. The results point to a participation of nonimmunogenic polysaccharides in the detection reaction.     

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.     

Serologic and pathogenetic studies on avian reoviruses isolated in Japan

Eighty-nine avian reoviruses isolated from diseased and clinically normal chickens were classified serologically using antisera against five prototype strains. Eighty-three strains were classified into five serotypes; six strains were untypable. Most of the cytopathogenic strains that produced a clear cytopathic effect (CPE) in chicken embryo fibroblasts (CEFs) were highly pathogenic for chicken embryos (80% or more mortality via the allantoic sac) and for chicks (severe footpad swellings and tenosynovitis). These strains were classified into a single serotype represented by the TS-142 prototype strain. However, 10 strains that could not produce a clear CPE in CEFs showed very low pathogenicity for embryos and chicks, and these strains were serologically different from the TS-142 prototype strain. There was a strong relationship between pathogenicity and serotype. About 17% of the isolates were considered highly pathogenic.     

Virulence and serum-resistance in different clonal groups and serotypes of Yersinia ruckeri.

The virulence in rainbow trout (Oncorhynchus mykiss) of 32 isolates of Yersinia ruckeri, representing a range of biotypes, serotypes, and OMP-types, was examined. Virulence was assayed in fish of average weight 7.7 g by bath challenge for 1 h with approximately 5 x 10(7) cells per ml. Two of the six serotype O1 clonal groups of Y. ruckeri, clones 2 and 5, were virulent, whereas the other four clonal groups, clones 1, 3, 4 and 6, as well as all serotype O2, O5, O6 and O7 isolates examined, were avirulent. Analysis of susceptibility to the bactericidal effect of non-immune rainbow trout serum demonstrated an association between virulence and serum resistance. The virulent serotype O1 clonal groups were serum resistant, whereas the avirulent serotype O1 clonal groups and other serotypes were, with some exceptions, serum sensitive. The fact that some serum resistant isolates were avirulent suggested that other factors may be required for the full expression of virulence. The study also demonstrated that rainbow trout and brook trout (Salvelinus fontinalis) differ in their susceptibility to Y. ruckeri.     

Serotypes of previously unclassified isolates of Erysipelothrix rhusiopathiae from swine in the United States and Puerto Rico

Serotypes of 46 previously unclassified isolates of Erysipelothrix rhusiopathiae from porcine tissues in the United States and serotypes of 31 isolates of the organism from porcine tissues received from Puerto Rico were determined. The 46 isolates from the United States were classified in serotype 21. Four isolates (from Georgia, Minnesota, Ohio, and Oklahoma) were tested and found to be pathogenic for swine. Serotypes 1 (subtypes 1a and 1b), 2, 5, 6, and 21 were found in porcine tissues from Puerto Rico. The relative frequency of the various serotypes was similar to that previously reported in the United States.     

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.     

Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States.

Mannheimia (Pasteurella) haemolytica biotype A serotype1 (A1) is the primary bacterial agent responsible for the clinical signs and pathophysiologic events in bovine pneumonic pasteurellosis. The goal of this study was to determine the prevalence of other serotypes of M. haemolytica biotype A organisms obtained from the upper Midwest diagnostic laboratories. A total of 147 M. haemolytica isolates were collected from Minnesota, South Dakota, and Michigan. Isolates were tested against M. haemolytica antisera obtained from the National Animal Disease Center, Ames, Iowa. Results indicated that M. haemolytica serotype 1 represented approximately 60%, serotype 6 represented 26%, and serotype 2 represented 7% of the total examined isolates. In addition, 7% of the isolates were serotype 9, 11, or untypable. This finding suggests that M. haemolytica serotypes other than serotype 1 can be isolated from the lung lesions of diseased cattle and seem to be capable of causing the pathologic changes observed in the lung with pneumonic pasteurellosis.     

Comparison of phenotypic traits and genetic relatedness of Salmonella enterica subspecies arizonae isolates from a colony of ridgenose rattlesnakes with osteomyelitis.

Reptiles are well-known sources of human Salmonella infections; however, little is known about the ability of Salmonella to cause disease in reptiles. Thirty-seven isolates of Salmonella enterica subspecies arizonae (S. arizonae) were obtained from retrospective and prospective studies of a closed colony of ridgenose rattlesnakes (Crotalus willardi) with osteomyelitis. All isolates (N = 7) from bone lesions were of a single serotype, 56:z4,z23, and this serotype was found on only 1 occasion among 8 other serotypes isolated from 21 cloacal and intestinal samples. The remainder (N = 7) of serotype 56:z4,z23 isolates were from other extraintestinal sites, including liver, ovary, blood, and testis. S. arizonae isolates were susceptible to most antimicrobials, and plasmid profiles did not correlate with serotype or antimicrobial resistance. Isolates of the 56:z4,z23 serotype (N = 14) formed a tight cluster with 95% similarity by XbaI macrorestriction analysis. Individual isolates of serotypes, 56:z4,z23, 38:(k)-z35, and 48:i-z invaded HeLa cells but an isolate of serotype 50:r-z did not. The same individual isolates of serotype 56:z4,z23 and 48:i-z also invaded viper heart cells. The Salmonella InvA gene was detected by polymerase chain reaction (PCR) in all S. arizonae serotypes tested, including 5 serotype 56:z4,z23 isolates and individual isolates of serotypes 48:i-z and 50:r-z. A source or possible explanation for increased virulence of S. arizonae serotype 56:z4,z23 in this unique host has not been found.     

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.     

Genotypic prevalence of apx1V in Actinobacillus pleuropneumoniae field isolates.

A total of 90 Actinobacillus pleuropneumoniae field strains from pigs were serotyped by slide agglutination and analyzed for the presence of the apxIV gene by polymerase chain reaction. Of the 90 isolates serotyped, serotypes 2 (47 isolates) and 5 (25 isolates) were the most common, followed by serotype 6 (10 isolates). Three isolates belonged to serotype 7, and 5 isolates could not be typed. All 90 A. pleuropneumoniae field isolates tested carried the apxIV gene. This gene is species specific rather than serotype specific. Therefore, the ApxIV toxin has potential value for use both in vaccines and in diagnostic tests.     

我国鸭疫里氏杆菌血清型的鉴定

１９９７年１月－１９９８年３月,从北京市２０个商品鸭场自然病死的北京白鸭和河南省与上海市部分鸭场的樱桃谷鸭分离到２７６株鸭疫里氏杆菌,采用凝集试验和琼脂扩散沉淀试验,对其进行了血清型的研究。其中７０株细菌为１型,６４株为２型,其余１４２株怀１,２,型参考菌株的抗血清发生反应。     

Serotyping avian adenoviruses by a microneutralization procedure.

A microneutralization procedure, using chicken kidney cell monolayers as an indicator system, was developed and applied to the serotyping of isolates characterized as avian adenoviruses. The method was determined to be reproducible, since coefficients of variation were low for 12 replicate titrations of homologous reagents of 9 prototype avian adenoviruses. Prototype reagents were specific according to results of reciprocal end point-neutralization tests and comparison of antigenic relatedness, using results obtained by previous researchers. Forty-two avian adenovirus isolates were classified into 6 serotypes by one-side end point-neutralization tests against antiserums made to 9 prototype avian adenoviruses. An additional 20 isolates were antigenically related to prototype viruses, but they could not be specifically types with the typing criteria. Different serotypes were isolated from birds having similar clinical diagnostic signs and lesions of disease.     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Serologic and virologic evidence of bluetongue virus infection in cattle and sheep in Mexico

Three independent 1-year studies were conducted during 3 consecutive years to better define the prevalence of bluetongue virus (BTV) infection in Mexico. Serologic data were obtained by use of agar-gel immunodiffusion for identification of BTV group-reactive antibodies, and virologic data were obtained by virus isolation. Samples were obtained from sheep in 6 states over a 1-year period, with 9% seropositive; samples were obtained from cattle in 11 states during the same 1-year period, with 35% seropositive. Two years later, samples were obtained from cattle in 4 additional states, with 69% seropositive. Virus isolation was conducted on pooled blood samples obtained from cattle in 7 states. Six virus isolates were recovered and included 2 isolates each of BTV serotypes 11 and 13 and 1 isolate each of serotypes 10 and 17. All virus isolates were partially characterized by electrophoretic analysis of genomic RNA migration profiles (electropherotypes) in polyacrylamide gels. All Mexican isolates of BTV differed considerably in electropherotype profile, as compared with their respective US prototype strain of the same serotype. Such differences appeared to be much more extensive than those described to exist between numerous California isolates of the same serotype.     

Recombinant DNA probe for serotype-specific identification of bluetongue virus 17

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.     

Analysis of southern Ontario Actinobacillus (Haemophilus) pleuropneumoniae isolates by restriction endonuclease fingerprinting.

Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonuclease fingerprinting (REF) analysis using the enzymes BamHI and HindIII. Restriction fragments were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Except for serotypes 1 and 9, reference strains of A. pleuropneumoniae serotypes 1 to 10 had clearly distinguishable REF profiles. Analysis of REF profiles of southern Ontario field isolates revealed limited heterogeneity amongst isolates of serotype 1 or serotype 5. The REF profiles of the serotype 7 isolates studied showed greater variation. Heterogeneity could not be correlated with the presence of plasmids nor with antibiotic resistance. Limited heterogeneity could also be detected amongst REF profiles of A. pleuropneumoniae isolates recovered from a closed herd suggesting that there is a small amount of genetic variation within clonal populations.     

Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping

During the period of 2001-2003, a total of 591 Actinobacillus pleuropneumoniae field isolates from the Czech Republic were serotyped with a high occurrence of cross-reactions. The cross-reactions were observed in 416 isolates. Most frequently, in 401 isolates (67.9%), cross-reactions with antisera specific for serotypes 9, 11, and/or 1 were observed. Two additional molecular methods, ribotyping and restriction analysis of PCR amplified apxIVA gene (PCR-REA), were therefore used for detailed characterisation of A. pleuropneumoniae. In this subsequent analysis, reference strains representing serotypes 1-12 and 25 field isolates showing the most frequent serotype cross-reactions were examined. PCR-REA enabled all reference strains to be distinguished except for the strains of serotypes 9 and 11. Ribotyping distinguished all reference strains except two pairs of serotypes: 3 versus 6, and 9 versus 11, respectively. Field isolates with serotype cross-reactivity 9, 11, and/or 1 could not be differentiated by either of these methods.