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A Gram-negative and aerobic bacterium, designated YIM 77875(T), was isolated from a geothermal soil sample collected at Rehai National Park, Tengchong, Yunnan Province, south-west China. Bacterial growth occurred from 37 to 65 °C (optimum 50 °C), pH 6.0-8.0 (optimum pH 7.0) and 0-1 % NaCl (w/v). Cells were rod-shaped and colonies were convex, circular, smooth, yellow and non-transparent. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain YIM 77875(T) belongs to the genus Lysobacter. The 16S rRNA gene sequence similarity values between strain YIM 77875(T) and other species of the genus Lysobacter were all below 94.7 %. The polar lipids of strain YIM 77875(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and five unknown phospholipids. The predominant respiratory quinone was Q-8 and the G+C content was 68.8 mol%. Major fatty acids were iso-C(16:0), iso-C(15:0) and iso-C(11:0). On the basis of the morphological and chemotaxonomic characteristics, as well as genotypic data, strain YIM 77875(T) represents a novel species, Lysobacter thermophilus sp. nov., in the genus Lysobacter. The type strain is YIM 77875(T) (CCTCC AB 2012064(T) = KCTC 32020(T)).

A novel Gram-positive, aerobic, spore-forming, thermophilic actinomycete, designated strain YIM 77521(T), was isolated from a sandy soil sample collected at Rehai National Park, Tengchong, Yunnan province, south-west China. The strain formed branched substrate mycelia and no fragmentation was found. Masses of short, straight or irregular chains of three to eight warty ornamented spores were borne from aerial mycelia. The strain contained meso-diaminopimelic acid in the cell wall and the whole-cell sugars contained mannose, galactose, glucose and ribose. The predominant menaquinones were MK-9(H(4)), MK-9(H(6)) and MK-9(H(8)). The diagnostic polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylmonomethylethanolamine and N-acetylglucosamine-containing phospholipids. The major fatty acids were iso-C(16 : 0), C(17 : 0) 10-methyl and C(18 : 0). The DNA G+C content of strain YIM 77521(T) was 73.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM 77521(T) fell within the radiation of the suborder Streptosporangineae and formed a distinct monophyletic lineage adjacent to the family Streptosporangiaceae with a high bootstrap value. On the basis of combined data from the phenotypic and phylogenetic analyses, strain YIM 77521(T) represents a novel genus and species within the family Streptosporangiaceae, for which the name Thermocatellispora tengchongensis gen. nov., sp. nov. is proposed. The type strain is YIM 77521(T) ( = DSM 45615(T)  = CCTCC AA 2011013(T)).

An orange-coloured, non-spore-forming, motile and coccus-shaped actinobacterium, designated YIM 75677(T), was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan Province, south-west China and its taxonomic position was investigated. Growth of strain YIM 75677(T) occurred at 12-55 °C, pH 6.0-9.0 and NaCl tolerance up to 2 % (w/v). Cells adhered to agar media and were agglutinated tightly together. The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose, mannose and ribose. The predominant menaquinone was MK-9 (H(2)) and the major fatty acids were anteiso-C(15:0) and iso-C(15:0). Mycolic acids were not present. The DNA G+C content of strain YIM 75677(T) was 74.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons clearly revealed that strain YIM 75677(T) represents a novel member of the genus Kineococcus and is closely related to Kineococcus xinjiangensis S2-20(T) (level of similarity, 98.6 %). Meanwhile, the result of DNA-DNA hybridization between strain YIM 75677(T) and K. xinjiangensis S2-20(T) demonstrated that this isolate represented a different genomic species in the genus Kineococcus. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 75677(T) represents a novel species of the genus Kineococcus, for which the name Kineococcus glutineturens sp. nov. is proposed. The type strain is YIM 75677(T) (=CCTCC AA 209075(T) = JCM 18126(T)).

A novel cold-resistant bacterium, designated YIM 016(T), was isolated from a peat bog sample collected from Mohe County, Heilongjiang Province, Northern China and its taxonomic position was investigated using a polyphasic approach. The strain was Gram-positive, aerobic, endospore-forming, motile and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequence clearly revealed that strain YIM 016(T) is a member of the genus Paenibacillus. The strain is closely related to Paenibacillus alginolyticus DSM 5050(T), Paenibacillus chondroitinus DSM 5051(T) and Paenibacillus pocheonensis Gsoil 1138(T) with similarities of 99.0 %, 97.0 % and 96.3 %, respectively. Meanwhile, the low DNA-DNA relatedness levels between strain YIM 016(T) and its closely related phylogenetic neighbours demonstrated that this isolate represents a new genomic species in the genus Paenibacillus. Phenotypic and chemotaxonomic tests showed that growth of strain YIM 016(T) occurred at 4-37 °C, pH 6.0-8.0 and with a NaCl tolerance up to 0.5 % (w/v). The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose and ribose. The predominant menaquinone was MK-7 and the major fatty acids were anteiso-C(15:0) and iso-C(16:0). The DNA G+C content of strain YIM 016(T) was 51.7 mol %. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 016(T) could be clearly distinguished from other species of the genus Paenibacillus. It is therefore concluded that strain YIM 016(T) represents a novel species in the genus Paenibacillus, for which the name Paenibacillus frigoriresistens sp. nov. is proposed. The type strain is YIM 016(T) (= CCTCC AB 2011150(T) = JCM 18141(T)).

Following the 2006 outbreaks of the highly pathogenic porcine reproductive and respiratory syndrome, the causative agent was identified as the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). To investigate whether the HP-PRRSV variant continues circulating and accelerating evolution, we sequenced and analyzed the complete genome of the identified HP-PRRSV field strain SD16. The sequence data indicate that the HP-PRRSV variant continues to prevail and accelerate evolution, especially in the nonstructural protein.

Two novel Gram-positive, spore-forming, thermophilic actinomycetes, designated as strain YIM 77501(T) and YIM 77570, were isolated from a sandy soil sample collected at Tengchong National Volcanic Geological Park, Yunnan province, south-west China. Phylogenetic analysis based on the 16S rRNA gene sequences suggested that the two isolates fell within the family Streptosporangiaceae. The strains formed extensively branched substrate and aerial mycelia which carried masses of long, straight or irregular spore chains composed of warty ornamented spores. Cell walls of the two strains contained meso-diaminopimelic acid and glucose, galactose, mannose and ribose were detected as whole-cell sugars. The predominant menaquinones were MK-9(H(4)) and MK-9(H(6)). The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, N-acetylglucosamine-containing phospholipids and phosphatidylinositol, phosphatidylinositolmannosides. The major cellular fatty acids were iso-C(16:0) and 10-methyl C(17:0). The DNA G+C content was 74-76 mol%. On the basis of the morphological and chemotaxonomic characteristics as well as the phylogenetic analysis, these strains represents a novel species of a new genus within the family Streptosporangiaceae, for which the name Thermoactinospora rubra gen. nov., sp. nov. is proposed. The type strain of T. rubra is YIM 77501(T) (=DSM 45614(T) = CCTCC AA 2011014(T)).

A novel Gram-stain positive, aerobic, non-motile, spore-forming actinobacterium, designated YIM 75926(T), was isolated from a soil sample collected at soil forest in Yuanmo county of Yunnan province, south-west China. Its taxonomic position was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that the novel strain YIM 75926(T) belongs to the genus Pseudonocardia and was closely related to Pseudonocardia halophobica DSM 43089(T) (98.1% similarity). Strain YIM 75926(T) had MK-8 (H(4)) as the predominant menaquinone. The whole organism hydrolysates mainly consisted of meso-diaminopimelic acid, mannose, glucose, galactose and arabinose. The major cellular fatty acids were iso-C(16:0) (37.16%) and C(16:0) (12.43%). The DNA G+C content of strain YIM 75926(T) was 70.6 mol%. The resultant phylogenetic trees further showed that strain YIM 75926(T) belong to Pseudonocardia and had a distinct subclade within the evolutionary radiation of the genus Pseudonocardia. On the basis of its comparative analysis of phenotypic and genotypic characteristics, it is proposed that strain YIM 75926(T) represent a novel species of the genus Pseudonocardia, named Pseudonocardia yuanmoensis sp. nov. The type strain is YIM 75926(T) (=CCTCC AA 2011017(T) = JCM 18055(T)).

A novel actinomycete, designated as strain YIM 75980(T), was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan province, south-west China and was subjected to polyphasic taxonomic characterization. The organism produced circular, smooth, red to black coloured colonies comprising coccoid-shaped cells. Colonies on agar medium lacked mycelia and cells adhered to the agar. Strain YIM 75980(T) contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and contained galactose, arabinose and glucosamine as the main sugars in the whole-cell hydrolysates. The predominant menaquinone was MK-9 (H(4)) and the major fatty acids were iso-C(15:0), iso-C(16:0) and C(16:0). The DNA G + C content of strain YIM 75980(T) was 73.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences clearly showed that strain YIM 75980(T) formed a distinct clade within the genus Geodermatophilus and was closely related to Geodermatophilus obscurus DSM 43160(T) (level of similarity, 97.9%). Furthermore, the result of DNA-DNA hybridization between strain YIM 75980(T) and G. obscurus 43160(T) demonstrated that this isolate represented a different genomic species in the genus Geodermatophilus. Moreover, the physiological and biochemical data showed the differentiation of strain YIM 75980(T) from its closest phylogenetic neighbour. Therefore, it is proposed that strain YIM 75980(T) represents a novel species of the genus Geodermatophilus, for which the name Geodermatophilus nigrescens sp. nov. is proposed. The type strain is YIM 75980(T) (=CCTCC AA 2011015(T) =JCM 18056(T)).

The objective of this study was to investigate the function of vimentin in PRRSV infection. Vimentin gene from Marc-145 cells was amplified by RT-PCR, cloned into pET-28a vector and expressed in Escherichia coli BL21(DE3). The expressed vimentin was confirmed by Western blot and purified which was used to immunize BALB/c mice for the production of antibodies. Vimentin and antibodies were tested for blocking PRRSV infection of Marc-145 cells. The binding of vimentin to PRRSV N and GP5 proteins were tested by the ELISA. The results showed that vimentin gene was amplified successfully and expressed as identified by SDS-PAGE and Western blot. Mouse anti-vimentin antibodies were produced with the titer of 10(5). PRRSV infection of Marc-145 cells was blocked partially by vimentin while blocked completely by the antibobies. In addition, vimentin was bound N protein, but not GP5. These results provide additional information on PRRSV entry into Marc-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection compromises the host's innate and adaptive immunity. The aim of this study was to investigate the immune responses of piglets infected with highly pathogenic (HP) PRRSV (HuN4 strain) with or without the immunization with CH-1R attenuated PRRSV vaccine. The response was evaluated for the clinical signs, pathological changes and virus load in immune organs, antibody responses and levels of serum IFN-γ, IL-4 and IL-10. The result showed that in comparison with the piglets received the immunization, the piglets infected with HP-PRRSV alone had the thymus atrophy, decreased serum levels of IL-4 and increased serum levels of IL-10 and INF-γ. These results suggest that elevated IL-10 levels at the early stage of the infection may enhance virus survival and delay the induction of protective immunity, while increased levels of IL-4 induce the effective immune responses and increase the animals' health status., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs., (© 2010 Elsevier B.V. All rights reserved.)

Infection with duck circovirus (DuCV) is associated with growth retardation and developmental problems in farmed ducks. To detect DuCV-specific antibody in duck serum, an indirect enzyme-linked immunosorbent assay (iELISA) method was developed using the recombinant capsid protein antigen prepared by cloning the cap (Cap) gene of DuCV FJ0601 strain into pET-32a (+) vector and expressed in Escherichia coli. Using the optimized iELISA method, DuCV-specific antibodies were detected in 157 (12.96%) of 1211 samples obtained from 17 (89.47%) of 19 meat duck flocks aged from 25 to 40 days and in 89 (22.08%) of 403 samples obtained from 9 (75%) of 12 breeder flocks aged from 14 to 61 weeks. These results indicated that the iELISA method is useful for serological diagnosis of DuCV infection and epidemiological investigation., ((c) 2010 Elsevier B.V. All rights reserved.)

Avian hepatitis E virus (HEV) has been identified in chickens; however, only 4 complete or near-complete genomic sequences have been reported. We found that the near-complete genomic sequence of avian HEV in chickens from China shared the highest identity (98.3%) with avian HEV from Europe and belonged to avian HEV genotype 3.

Diversity of Crenarchaeota was investigated in eight terrestrial hot springs (pH 2.8-7.7; temperature 44-96 degrees C) located in Tengchong, China, using 16S rRNA gene phylogenetic analysis. A total of 826 crenarchaeotal clones were sequenced and a total of 47 operational taxonomic units (OTUs) were identified. Most (93%) of the identified OTUs were closely related (89-99%) to those retrieved from hot springs and other thermal environments. Our data showed that temperature may predominate over pH in affecting crenarchaeotal diversity in Tengchong hot springs. Crenarchaeotal diversity in moderate-temperature (59-77 degrees C) hot springs was the highest, indicating that the moderately hot-temperature springs may provide optimal conditions for speciation of Crenarchaeota.

The clinical symptoms of infectious coryza are multiple and include nasal discharge, facial swelling, lacrimation, and anorexia. In general, the disease is not fatal to chicken; so, in experiments where animals are infected with Avibacterium paragallinarum, there have been debates about conducting the challenge model and evaluating the clinical signs. In this experiment, 150 chickens, aged 30 days, were randomly divided into different groups. Some groups were infected with the 'in-contact' challenge model and others with the artificial intrasinus-injection-route model. The bacterial isolates used were three field isolates of different serogroups of A. paragallinarum, including Hpg-8 (Page serovar A), CCM6075 (Page serovar B) and Hpg-668 (Page serovar C). During this study, a scoring system was used to record the clinical signs of the infected birds and evaluate the pathogenic diversity of the two models. The final results indicated that the 'in-contact' challenge model of the three isolates showed a more reliable representation of the natural infection under field conditions than the artificial intrasinus-injection-route model. Thus, on carrying out animal experiments, the effect of 'in-contact' challenge model is more accurate than the artificial intrasinus-injection-route model., (Copyright 2009 Elsevier B.V. All rights reserved.)

Objective: To estimate seroprevalence of antibodies against the serogroup of epizootic hemorrhagic disease viruses (EHDVs) and describe spatial distribution of antibodies against EHDV among cattle herds in Illinois and western Indiana., Sample Population: 9,414 serum samples collected from cattle in 60 herds over 3 transmission seasons., Procedures: Serum samples were tested for antibodies against EHDV by use of an ELISA. Seroprevalence for 4 zones covering the length of Illinois and parts of Indiana were estimated. A multivariable mixed-effects logistic regression model with a random effect for herd was used to estimate seropositive risk for zone (1 through 4), age (yearling, adult), herd type (beef, dairy), transmission season (2000 to 2002), and zone by year interaction. Isopleth maps of seroprevalence at the herd level were produced., Results: Antibodies against EHDV were detected in 1,110 (11.8%) samples. Estimated seroprevalence in 2000, 2001, and 2002 was 15.3%, 13.4%, and 5.2%, respectively. Seroprevalence was highest in the southernmost zone and lowest in the northernmost zone, but risk of seropositivity for EHDV among and within zones varied by year. Clusters of high seroprevalence in the south, low seroprevalence in the north, and outliers of high and low seroprevalence were detected. Risk mapping revealed areas of higher seroprevalence extending northward along the western and eastern ends of the study region., Conclusions: Seroprevalence of antibodies against EHDV in cattle was higher in the south than north; however, local complexities existed that were not observed in a serosurvey of antibodies against bluetongue virus from the same cattle population.

Syngeneic monoclonal anti-idiotypic antibodies (Mab2s) were generated against idiotypic antibodies to membrane glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) using the sequential immunization method. Six of 12 Mab2s possessed potential internal image characteristics by recognizing a common idiotype on murine and swine anti-GP5 antibodies. Further serological characterization demonstrated that one of the Mab2 (Mab2-5G2) represents internal image anti-idiotope which mimicked the GP5 antigen that inhibited the interaction between idiotypic anti-GP5 antibodies and GP5 antigen, its reaction with the idiotypic anti-GP5 antibody was inhibited by GP5 antigen and detected the common Id on anti-PRRSV antibodies from pigs that were experimentally infected with PRRSV. In addition, Mab2-5G2 identified a soluble protein on MA-104 and porcine alveolar macrophages. These results indicate that Mab2-5G2 may be a useful candidate as an alternative PRRSV serodiagnostic reagent and a useful probe to study PRRSV-cell interaction.

Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

Avian hepatitis E virus (avian HEV) is genetically and antigenically related to human HEV, the causative agent of hepatitis E. To identify the neutralizing epitopes on the capsid (ORF2) protein of avian HEV, four mAbs (7B2, 1E11, 10A2 and 5G10) against recombinant avian HEV ORF2 protein were generated. mAbs 7B2, 1E11 and 10A2 blocked each other for binding to avian HEV ORF2 protein in a competitive ELISA, whereas 5G10 did not block the other mAbs, suggesting that 7B2, 1E11 and 10A2 recognize the same or overlapping epitopes and 5G10 recognizes a different one. The epitopes recognized by 7B2, 1E11 and 10A2, and by 5G10 were mapped by Western blotting between aa 513 and 570, and between aa 476 and 513, respectively. mAbs 1E11, 10A2 and 5G10 were shown to bind to avian HEV particles in vitro, although only 5G10 reacted to viral antigens in transfected LMH cells. To assess the neutralizing activities of the mAbs, avian HEV was incubated in vitro with each mAb before inoculation into specific-pathogen-free chickens. Both viraemia and faecal virus shedding were delayed in chickens inoculated with the mixtures of avian HEV and 1E11, 10A2 or 5G10, suggesting that these three mAbs partially neutralize avian HEV.

Avian hepatitis E virus (HEV) is genetically and antigenically related to human HEV. Vertical transmission of HEV has been reported in humans, but not in other animals. In this study, we showed that avian HEV could be detected in chicken egg-white samples. Subsequently, avian HEV in egg white was found to be infectious, as evidenced by the appearance of viraemia, faecal virus shedding and seroconversion in chickens inoculated with avian HEV-positive egg white, but not in chickens inoculated with HEV-negative egg white. To further assess the possibility of vertical transmission of avian HEV, batches of embryonated eggs from infected hens were hatched, and hatched chicks were monitored for evidence of avian HEV infection. However, no virus was detected in samples collected from the hatched chicks throughout this study, suggesting that avian HEV could not complete the vertical transmission cycle. The possible implications of our findings are also discussed.

Avian hepatitis E virus (avian HEV) is an emerging virus associated with hepatitis-splenomegaly syndrome in chickens in North America. Avian HEV is genetically and antigenically related to human HEV, the causative agent of hepatitis E in humans. In the lack of a practical animal model, avian HEV infection in chickens has been used as a model to study human HEV replication and pathogenesis. A 32 kDa recombinant ORF2 capsid protein of avian HEV expressed in Escherichia coli was found having similar antigenic structure as that of human HEV containing major neutralizing epitopes. To determine if the capsid protein of avian HEV can be used as a vaccine, 20 chickens were immunized with purified avian HEV recombinant protein with aluminum as adjuvant and another 20 chickens were mock immunized with KLH precipitated in aluminum as controls. Both groups of chickens were subsequently challenged with avian HEV. All the tested mock-immunized control chickens developed typical avian HEV infection characterized by viremia, fecal virus shedding and seroconversion to avian HEV antibodies. Gross hepatic lesions were also found in portion of these chickens. In contrast, none of the tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shedding or observable gross hepatitis lesions. The results from this study suggested that immunization of chickens with avian HEV recombinant ORF2 capsid protein with aluminum as adjuvant can induce protective immunity against avian HEV infection. Chickens are a useful small animal model to study anti-HEV immunity and pathogenesis.

There are marked differences in the clinical expression of diseases associated with porcine circovirus type 2 (PCV2) in the field. The objective of this study was to compare the sequences and pathogenicity of PCV2 isolates from field cases with and without PCV2-associated lesions. Forty-two specific-pathogen-free (SPF) pigs were assigned randomly to three groups of 14 pigs each. At 7 weeks of age, group 1 pigs were mock-inoculated with saline, group 2 pigs were inoculated with PCV2-4838 (isolated from a pig with no evidence of PCV2-associated lymphoid lesions) and group 3 pigs were inoculated with PCV2-40895 (isolated from a pig with PCV2-associated lymphoid lesions and disease). The PCV2-4838 and PCV2-40895 isolates shared approximately 98.9 % nucleotide sequence identity across the entire genome. A total of nine amino acid changes in ORF2 and two amino acid changes in ORF1 were identified between the two isolates. PCV2-4838-inoculated pigs had significantly more genomic copy numbers of PCV2 in their sera at 7 days post-inoculation (p.i.) (P<0.0001) and significantly fewer genomic copy numbers at 14, 21 and 28 days p.i. (P<0.05) compared with pigs inoculated with the PCV2-40895 isolate. Microscopic lesions in lymphoid tissues were significantly less severe (P<0.05) and the amount of PCV2 antigen associated with these lesions was significantly lower (P<0.05) in pigs inoculated with PCV2-4838. The results of this study suggest that PCV2 isolates from the USA differ in virulence in an SPF pig model.

Active serologic surveillance programs to detect avian influenza viruses (AIVs) in table egg-laying chickens have been initiated by several states as a response to the economic threat posed by these viruses. Most outbreaks of avian influenza in domestic poultry are caused by mildly pathogenic AIVs. In the study reported here, infection by an H6N2 AIV was used as a model of mildly pathogenic AIV infections in egg-type chickens. The total number of eggs laid by 5 control hens was 619 or 0.904 eggs/day/hen, whereas the total number laid by 10 infected hens was 1,018 or 0.743 eggs/day/hen. The difference in egg production between the 2 groups was not statistically significant (P = 0.38). Anti-influenza antibodies were monitored by use of an agar gel immunodiffusion test and an ELISA for a period of 20 weeks after inoculation. Antibodies in serum developed sooner, peaked at higher levels, and remained at higher levels than did antibodies found in egg yolk, as indicated by ELISA results. For infected chickens, the correlation between serum and egg yolk ratios was 0.66. Serum samples would appear to be preferable to egg yolk samples for surveillance programs intended to identify chicken flocks that may have been infected by an AIV weeks or months before samples are collected.

A Western blot immunoassay (WBI) was developed as a confirmatory test for 2 commercial Mycoplasma hyopneumoniae (Mhyo) ELISAs. The WBI detected at least 5 antigen bands (150, 130, 74, 70, and 30 kDa) from Mhyo whole membrane proteins that were not present in the antigens prepared from M. hyorhinis and M. hyosynoviae. Among discrepant sera from vaccinated pigs (n = 17) and field samples (n = 91) assayed by WBI: 1) 2 of the ELISA-positive samples reacted with all 5 antigens bands; 2) all blocking ELISA-positive samples (n = 53) bound 150-, 130-, 74-, and 70-kD antigen bands; and 3) all indirect ELISA-positive samples (n = 55) bound 150-, 130-, and 30-kD antigens. We conclude that the WBI targeting the top 4 antigen bands is a useful confirmatory test for samples initially screened using the commercial Mhyo ELISAs.

Avian hepatitis E virus (avian HEV) was recently discovered in chickens from the USA that had hepatitis-splenomegaly (HS) syndrome. The complete genomic sequence of avian HEV shares about 50 % nucleotide sequence identity with those of human and swine HEVs. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, but the B-cell epitopes in the avian HEV ORF2 protein have not been identified. Nine synthetic peptides from the predicted four antigenic domains of the avian HEV ORF2 protein were synthesized and corresponding rabbit anti-peptide antisera were generated. Using recombinant ORF2 proteins, convalescent pig and chicken antisera, peptides and anti-peptide rabbit sera, at least one epitope at the C terminus of domain II (possibly between aa 477-492) that is unique to avian HEV, one epitope in domain I (aa 389-410) that is common to avian, human and swine HEVs, and one or more epitopes in domain IV (aa 583-600) that are shared between avian and human HEVs were identified. Despite the sequence difference in ORF2 proteins between avian and mammalian HEVs and similar ORF2 sequence between human and swine HEV ORF2 proteins, rabbit antiserum against peptide 6 (aa 389-399) recognized only human HEV ORF2 protein, suggesting complexity of the ORF2 antigenicity. The identification of these B-cell epitopes in avian HEV ORF2 protein may be useful for vaccine design and may lead to future development of immunoassays for differential diagnosis of avian, swine and human HEV infections.

Mycoplasma hyopneumoniae (Mhyo) causes mycoplasmal pneumonia, an economically important disease of swine. Serodiagnosis of Mhyo is based on the current available commercial enzyme immunoassays for detection of swine antibodies against Mhyo, which are the indirect enzyme-linked immunosorbent assay (ELISA) and the blocking ELISA (B-ELISA). Because of the limited information available for these ELISAs, these 2 assays were compared by testing 347 serum samples collected from vaccinated pigs at 0, 13, 28, 43, and 62 days postimmunization (DPI), 50 samples from nonvaccinated pigs, and 1,013 field serum samples. The results of comparison study showed that the specificity for both ELISAs was 99.2% generated from 139 non-vaccinated negative samples. The sensitivities for indirect ELISA generated from samples collected from animals that received the vaccine at DPI 13, 28, 43, and 62 were 0%, 95.7%, 88.4%, and 92.6%, respectively, whereas the sensitivities for B-ELISA were 0%, 98%, 100%, and 97%, respectively. The overall agreement of 96.7% and 80.3% was generated between 2 ELISAs from negative and vaccinated pigs and from field samples, respectively.

A biotinylated 3ABC recombinant protein was developed and used in a competitive ELISA (cELISA) to detect foot-and-mouth disease virus (FMDV) antibodies in cattle, sheep and pigs. In this report, we describe the cloning and expression of 3ABC protein in Escherichia coli cells as fusion protein with 6xHis and biotin. This cELISA uses streptavidin to capture bacterially expressed and in vivo biotinylated 3ABC antigen. The antigen capture strategy provides a simple and reliable method, which does not require purification of recombinant antigen before the serological assay. An hyperimmune guinea pig antiserum produced against purified 6xHis-3ABC was used as competitor in the test. The potential use of this cELISA for the identification of antibodies induced by FMD virus infection from those induced by vaccination is discussed.

Auto-anti-idiotypic antibodies (Aab-2s) specific for antibodies against envelope glycoprotein GP5 were previously identified in 10 of 12 pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). In this study, we report in addition the induction of Aab-2s specific for antibodies against matrix (M) and envelope glycoprotein GP5 antigens in 38 of 47 pigs experimentally infected with PRRSV. Two sets of Aab-2s were induced in the sera, one at 21 days post-infection (DPI) and peaked on 35 DPI and another at 49 DPI and peaked on 77 DPI. The Aab-2s induced at an early stage were from non-virus carrier pigs as defined when they were euthanized on and after 77 DPI. In contrast, the Aab-2s induced at a later stage were from virus carrier pigs. These Aab-2s possessed the characteristics of internal image Ab-2s and recognized shared idiotypes on swine and murine antibodies to the GP5 and M antigens of PRRSV. This study further demonstrates that, during the antibody responses to PRRSV infection, Aab-2s specific for both anti-M and anti-GP5 antibodies were induced. More importantly, the early and late Aab-2s possessed different idiotype-binding specificities. By detecting and characterizing these Aab-2s, it may be possible to identify whether pigs infected with PRRSV will clear the virus or become virus carriers.

A commercial indirect swine influenza virus (SIV) H1N1 enzyme-linked immunosorbent assay (ELISA) was compared with the hemagglutination inhibition (HI) assay by testing 72 samples from experimentally infected pigs and 780 field samples of undefined SIV status. The HI assay was performed using SIV isolates A/Swine/IA/73 for H1N1 and A/Swine/IA/8548-1/98 for H3N2. The ELISA used an SIV isolated in 1988. The results showed that HI and ELISA detected an antibody in 11 and 6, respectively, of 72 serum samples collected from pigs experimentally infected with a 1992 SIV isolate (A/Swine/IA/40776/92). The presence of antibodies in these experimental samples was confirmed by HI tests in which all 72 samples were positive against the homologous virus, a more recent H1N1 SIV isolate (A/Swine/NVSL/01) supplied by National Veterinary Services Laboratories, Ames, Iowa, and a 1999 H1N1 isolate currently used in a commercial vaccine. On testing 780 field samples, an overall agreement of 85.5% was generated between the HI and ELISA. This study demonstrated that the ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against SIV. However, a new SIV isolate representing current SIV strains circulating in the field is needed to replace the older isolates used in the HI and ELISA to increase the test accuracy for serodiagnosis of SIV.

The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated. Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription. Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity. After three immunizations, vaccinated chickens were completely protected from virulent IBV infection. These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.

Auto-anti-idiotypic antibodies (Aab-2s) were detected from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The Aab-2s were specific against the idiotypic antibodies (Ab-1s) to the envelope glycoprotein GP5 of PRRSV and were detected from serum samples collected between 21 and 98 days post-infection (DPI). Serological characterization indicated that the Aab-2s recognized the idiotype located within or near the antigen-combining sites of the anti-GP5 antibodies, which was shared by both mouse MAb anti-GP5 and swine polyclonal antibodies. The fact that the Aab-2 inhibited the anti-GP5 antibodies from binding to PRRSV and that they were detected at different time periods in pigs that cleared the infection prior to 98 DPI versus pigs in which virus was detected at 98 DPI suggests that Aab-2 antibodies may play a role in immunity to PRRSV infection.

Embryonated chicken eggs (ECE) and the Madin-Darby canine kidney (MDCK) cell line were compared for isolation of swine influenza virus (SIV) from nasal swabs and tissue samples. Samples originated from 30 pigs experimentally inoculated with 2 x 106 to 2 x 10(7) embryo infectious dose 50% (EID50)/mL of swine influenza strain A/Swine/Indiana/1726/88 (H1N1). The results were analyzed with McNemar's chi-squared test for symmetry. The results indicated that more samples were SIV-positive with ECE than with tissue culture (P < 0.001), suggesting that ECE remains the system of choice for isolation of SIV. It is recommend that routine use of both SIV isolation systems will increase the sensitivity of detection of virus shedding by considering the differences in growth and tropism of diverse SIV strains.

A competitive enzyme-linked immunosorbent assay (C-ELISA), based on a truncated E2 recombinant protein of the Alfort/187 strain of classical swine fever virus (CSFV) and a specific monoclonal antibody M1669, was evaluated using 2,000 sera from clinically healthy pigs in Canada (a CSFV-free country) and sera from experimentally infected pigs. The relative specificity and sensitivity of the C-ELISA were 100% and 86%, respectively, at a cutoff of 25% inhibition using negative and positive pig sera, as defined by the neutralizing peroxidase-linked assay (NPLA). A kappa value of 0.91 was obtained, indicating an excellent level of agreement between the NPLA and the C-ELISA. When sera from 120 infected pigs were used in the test at > or = 21 days postinfection, the sensitivity of the C-ELISA and the kappa value increased to 97% and 0.98, respectively. This C-ELISA will be useful when a large number of samples must be tested, as could occur during a disease outbreak or for surveillance or prevalence studies.

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.

In September 1999, unusually high mortality rates in white-tailed deer and California bighorn sheep occurred in the southern Okanagan Valley. Necropsy and histopathologic findings were compatible with epizootic hemorrhagic disease (EHD); the presence of virus was not demonstrated. Subsequent serologic and polymerase chain reaction assays on sentinel cattle suggested an EHD virus incursion.

Ostriches were inoculated with a laboratory-derived highly pathogenic avian influenza (HPAI) virus of emu origin, A/emu/TX/39924/93 (H5N2) clone c1B. The aim of this study was to evaluate the pathogenicity of this isolate for ostriches and assess the ability of routine virological and serological tests to detect infection. Avian influenza virus (AIV) was isolated from cloacal and tracheal swabs from 2 to 12 days post-infection. AIV was also isolated from brain, thymus, eyelid, spleen, ovary/testis, liver, air sac, proventriculum, duodenum, caecal tonsil, heart, pancreas, kidney, nasal gland and lung. Virus isolation was also possible from swabs of the luminal surfaces of the cloaca, jejunum, lower ileum, bursa of Fabricius, trachea and bone marrow. Birds seroconverted as early as 7 days post-infection. This study suggests that HPAI virus of emu origin replicates extensively in infected ostriches without causing significant clinical disease or mortality.

An IgM-capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of recent infection of bluetongue virus (BTV) in cattle. The test is based on the use of biotinylated capture anti-bovine IgM antibodies bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of BTV VP7 antigen and a VP7 antigen-specific monoclonal antibody. The IgM-capture ELISA was compared with the competitive ELISA by testing serum samples from groups of calves infected experimentally with five USA and 19 South Africa serotypes of BTV. The IgM-capture ELISA was able to detect bovine anti-VP7 antibodies from all animals infected with the 24 BTV serotypes at 10 days post-infection, whereas the competitive ELISA was not. When the detectable IgM diminished after 40 days post-infection by the IgM-capture ELISA, the IgG anti-VP7 antibodies remained high. The IgM-capture ELISA is sensitive and can be applied for the detection of recent infection of BTV in cattle.

The sequence encoding a truncated E2 glycoprotein of the Alfort/187 strain of classical swine fever virus (CSFV) was expressed in Escherichia coli using the pET expression system and the recombinant product purified by Ni-NTA agarose affinity chromatography. The antigenicity of this recombinant protein was demonstrated by immunoblot using anti- CSFV-specific antibodies. A monoclonal antibody was produced against the truncated E2 protein and used as competitor in an ELISA for the detection of antibodies to CSFV. Specific antibodies were demonstrated by competitive ELISA (C-ELISA) as early as 21 days post-infection (dpi) in experimentally infected pigs. Seroconversion was demonstrated by C-ELISA and neutralising peroxidase-linked assay (NPLA) in all infected animals by 4 weeks. No cross-reaction with antibodies to bovine viral diarrhoea virus (BVDV) was seen in the C-ELISA using sera from experimentally infected pigs. The C-ELISA is not intended as a substitute for the NPLA. However, it is expected it will be useful for monitoring and prevalence studies. It will also assist in testing a large number of samples in the event of an outbreak., (Copyright 2001 Harcourt Publishers Ltd.)

Bluetongue virus was isolated from a sentinel herd in British Columbia. Virus isolation was by intravenous inoculation of embryonated chicken eggs and subculture in BHK-21 cells. The cytopathic agent was identified as bluetongue virus by electron microscopy and the immunoperoxidase test. The serotype was identified as serotype 11 by virus neutralization.

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.

Trade in animals and animal products has reached global proportions and so too has the threat of infectious diseases of veterinary importance. The Manual of Standards for Diagnostic Tests and Vaccines, published by the Office International des Epizooties (OIE), contains chapters on infectious diseases that may cause various degrees of socio-economic, public health, and/or zoo-sanitary consequence. These chapters cover the major diseases of cattle, sheep, goats, horses, pigs, poultry, lagomorphs and bees. A number of factors are considered when qualifying animals and animal products for international trade including epidemiological, clinical and testing parameters. Of particular note and relevance is a strong international movement to standardize the test methods and reference reagents in order to promote harmonization of testing and facilitation of trade. There is message here that is directed to those of us involved in the development and application of test methods for infectious disease diagnosis. Serological test methods have been and still remain the mainstay of diagnostic methods prescribed for trade. More than ever, there is a need to observe and apply international guidelines for the development and validation of serological test methods. There is also a need to develop international standard reagents for use in the calibration of test methods and the production of national and working standards. In the future, veterinary diagnostic testing laboratories involved in trade may also require a form of international accreditation unique to their specialty. This presentation describes the current developments in international standardization of test methods and reference reagents.

Two groups of emus were experimentally inoculated with a low and high pathogenic strain of avian influenza virus (AIV), type A to determine the virus susceptibility, pathogenicity, shedding and seroconversion. Emus were found susceptible to infection with AIV, with virus shedding detectable in tracheal and cloacal swabs between 3 and 10 days post-infection. Only the birds infected with the highly pathogenic viral isolate showed a brief period of mild clinical signs associated with infection. Virus recovered from the infected emus was found to be of similar pathogenicity to that of the virus inoculum. All the birds seroconverted by 10 days post-infection, as determined by haemagglutination inhibition, agar gel immunodiffusion and competitive ELISA assays. This study suggests that emus are similar to wild waterfowl in their response to AIV infection, in that they are susceptible and will replicate and shed the virus, but do not show any marked clinical signs of infection.

Emu antibody responses to avian influenza virus (AIV) infection were evaluated by the competitive enzyme-linked immunosorbent assay (C-ELISA), agar gel immunodiffusion (AGID) and hemagglutination inhibition (HI) tests. All birds infected with AIV H5N1, H5N3, or H7N7 developed antinucleoprotein (NP) antibodies as early as 7 days postinfection as detected by the C-ELISA. The responses lasted 49 days for the emus receiving H5N3 and at least 56 days for emus receiving the other two viruses. By evaluating 50 emu field serum samples, the C-ELISA was found more sensitive than the AGID test for the detection of anti-NP antibodies. This study indicates that emus experimentally infected with AIV developed antibody responses that can be detected by C-ELISA, AGID, and HI tests. The results from this and our previous studies demonstrate the use of the C-ELISA as a substitute for the AGID test in a routine serodiagnostic screening for detection of antibodies to AIV infection in multiple avian species.

A competitive enzyme-linked immunosorbent assay (C-ELISA) employing a baculovirus-expressed recombinant nucleoprotein and a monoclonal antibody was developed for the detection of antibodies to type A influenza virus nucleoprotein. The performance of the C-ELISA was evaluated by testing 756 chickens, 1123 turkeys, 707 emus, and 1261 ostriches, for a total of 3847 serum samples. Relative to the agar gel immunodiffusion (AGID) test, the C-ELISA had a sensitivity of 100% for all four species. The C-ELISA's sensitivity relative to the hemagglutination-inhibition (HI) test results was 100% for chicken, turkey, and emu and 96.2% for the ostrich serum samples. More than 90% of the AGID-negative/C-ELISA-positive serum samples were found positive by HI for at least one influenza serotype. The specificity of C-ELISA relative to AGID ranged from 85.5% to 99.8% for sera collected from these species. These results indicated that the C-ELISA was more sensitive and more specific than the AGID test and as sensitive and as specific as the HI test. The C-ELISA has the potential to replace the AGID test for screening sera from avian species, including ratites, for detection of antibodies to type A influenza virus.

An antigen-capture enzyme immunoassay (EIA) was developed to detect classical swine fever virus (CSFV) antigen directly from 10% w/v tissue suspension. The assay, based on the sandwich principle, uses a biotinylated monoclonal antibody bound to streptavidin-coated microplates as the capture system and a swine anti-CSFV antibody and rabbit anti-swine HRPO-conjugate as the detector system. The antigen-capture EIA was compared with conventional virus isolation and polymerase chain reaction (PCR) for detection of CSFV in tissues. The ability of the antigen-capture EIA to discriminate classical swine fever (CSF) from bovine viral diarrhea and African swine fever viruses was also tested. The assay was shown to detect 21 different strains of CSFV and was unreactive with tissues from uninfected animals. Signal to noise (S/N) ratios were calculated from the EIA absorbance values. Readings from samples positive by virus isolation (n = 47) averaged a S/N ratio of 5.34. In contrast, samples negative by virus isolation (n = 96) demonstrated a mean S/N ratio of 0.16. At S/N cut-off value of 1.0, all samples that yield virus isolation and PCR negative result were negative in the antigen-capture EIA. Compared with virus propagation in tissue culture using PK15 cells (followed by indirect peroxidase assay detection) and PCR, the EIA had a specificity of 98.7% and a sensitivity of 91.4%. The EIA is simple, can be performed in 4 h and lends itself to automation for screening of tissues sample from pigs suspected of CSFV infection.

The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV.

The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

Three groups of anti-idiotypic antibodies (anti-Id or Ab2), designated RAb2-A, RAb2-B1, and RAb2-B2, were isolated from rabbit antiserum raised against three monoclonal antibodies (mAbs) (M1875, M1877, and M1886) specific for the bluetongue virus core protein, VP7. RAb2-A was specific for the idiotype of M1875. RAb2-B1 and RAb2-B2, isolated through the M1877 and M1886 affinity columns, respectively, were directed against the common idiotype that is shared by M1877 and M1886 and therefore classified in the same population (RAb2-B). Further characterization revealed that the two anti-Id populations, RAb2-A and RAb2-B, were significantly different. RAb2-A was an Ab2 beta type of anti-Id since (i) its reaction with M1875 was inhibited by the antigen; (ii) it inhibited the M1875-VP7 interaction; and (iii) it elicited anti-VP7 antibody response in Balb/c mice. In contrast, RAb2-B may represent an Ab2 alpha type of anti-Id since its reactions with M1877 or M1886 were not inhibited by the antigen, even though it inhibited mAbs from binding to the antigen. These results indicated that RAb2-A and RAb2-B represent two distinct populations of anti-Ids to anti-VP7 mAbs with similar epitope specificity.

A subpopulation of rabbit polyclonal anti-idiotypic antibody (anti-Id) was previously produced to a murine monoclonal antibody (mAb) (M1875) specific for the bluetongue virus core protein VP7. In this report, mimicry of VP7 by this anti-Id (designated RAb2-A) was functionally analyzed through immunization of Balb/c mice with RAb2-A or purified VP7. Animals immunized with RAb2-A were able to produce an M1875-like Ab3 antibody response with idiotype and epitope specificity resembling that of M1875 without subsequent exposure to the nominal antigen. This conclusion was supported by experiments showing that the RAb2-A-induced Ab3 antibodies (i) reacted specifically with the immunizing anti-Id; (ii) were capable of binding VP7; (iii) inhibited M1875 from binding to VP7; and (iv) inhibited M1875 from binding to RAb2-A. Similarly, mice immunized with purified VP7 also produced antibodies that exhibited characteristics such as idiotype and epitope specificity in common with M1875. No antibody response to VP7 was detected in control groups of mice immunized with either normal rabbit IgG or BHK-21 cell components. Therefore, it can be concluded that rabbit anti-Id RAb-2-A mimics an M1875-defined VP7 epitope sufficiently to function as a surrogate antigen for inducing an anti-bluetongue virus response.