Recent Field Isolates Research Articles

Comparison of the efficacy of autogenous inactivated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccines with that of commercial vaccines against homologous and heterologous challenges

BackgroundThe porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving pathogen of swine. At present, there is a high demand for safe and more effective vaccines that can be adapted regularly to emerging virus variants. A recent study showed that, by the use of a controlled inactivation procedure, an experimental BEI-inactivated PRRSV vaccine can be developed that offers partial protection against homologous challenge with the prototype strain LV. At present, it is however not known if this vaccine can be adapted to currently circulating virus variants. In this study, two recent PRRSV field isolates (07 V063 and 08 V194) were used for BEI-inactivated vaccine production. The main objective of this study was to assess the efficacy of these experimental BEI-inactivated vaccines against homologous and heterologous challenge and to compare it with an experimental LV-based BEI-inactivated vaccine and commercial inactivated and attenuated vaccines. In addition, the induction of challenge virus-specific (neutralizing) antibodies by the different vaccines was assessed.ResultsIn a first experiment (challenge with 07 V063), vaccination with the experimental homologous (07 V063) inactivated vaccine shortened the viremic phase upon challenge with approximately 2 weeks compared to the mock-vaccinated control group. Vaccination with the commercial attenuated vaccines reduced the duration of viremia with approximately one week compared to the mock-vaccinated control group. In contrast, the experimental heterologous (LV) inactivated vaccine and the commercial inactivated vaccine did not influence viremia. Interestingly, both the homologous and the heterologous experimental inactivated vaccine induced 07 V063-specific neutralizing antibodies upon vaccination, while the commercial inactivated and attenuated vaccines failed to do so.In the second experiment (challenge with 08 V194), use of the experimental homologous (08 V194) inactivated vaccine shortened viremia upon challenge with approximately 3 weeks compared to the mock-vaccinated control group. Similar results were obtained with the commercial attenuated vaccine. The experimental heterologous (07 V063 and LV) inactivated vaccines did not significantly alter viremia. In this experiment, 08 V194-specific neutralizing antibodies were induced by the experimental homologous and heterologous inactivated vaccines and a faster appearance post challenge was observed with the commercial attenuated vaccine.ConclusionsThe experimental homologous inactivated vaccines significantly shortened viremia upon challenge. Despite the concerns regarding the efficacy of the commercial attenuated vaccines used on the farms where the field isolates were obtained, use of commercial attenuated vaccines clearly shortened the viremic phase upon challenge. In contrast, the experimental heterologous inactivated vaccines and the commercial inactivated vaccine had no or only a limited influence on viremia. The observation that homologous BEI-inactivated vaccines can provide a more or less standardized, predictable degree of protection against a specific virus variant suggests that such vaccines may prove useful in case virus variants emerge that escape the immunity induced by the attenuated vaccines.

High rate of viral evolution in the capsid protein of porcine parvovirus

In recent years, it has been shown that some parvoviruses exhibit high substitution rates, close to those of RNA viruses. In order to monitor and determine new mutations in porcine parvovirus (PPV), recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analysed. These samples, together with sequences retrieved from GenBank, were included in three datasets, consisting of the complete NS1 and VP1 genes and a partial VP1 gene. For each dataset, the nucleotide substitution rate and the molecular clock were determined. Analysis of the PPV field isolates revealed that a recently described amino acid substitution, S436T, appeared to be common in the VP2 protein in the Austrian, Brazilian and German virus populations. Furthermore, new amino acid substitutions were identified, located mainly in the viral capsid loops. By inferring the evolutionary dynamics of the PPV sequences, nucleotide substitution rates of approximately 10(-5) substitutions per site per year for the non-structural protein gene and 10(-4) substitutions per site per year for the capsid protein gene (for both viral protein datasets) were found. The latter rate is similar to those commonly found in RNA viruses. An association of the phylogenetic tree with the molecular clock analysis revealed that the mutations on which the divergence for both capsid proteins was based occurred in the past 30 years. Based on these findings, it was concluded that PPV variants are continuously evolving and that vaccines, which are based mainly on strains isolated about 30 years ago, should perhaps be updated.

Epidemiology and immunoprotection of nephropathogenic avian infectious bronchitis virus in southern China

BackgroundIn last three years, 96 suspected poultry farms from different provinces in China were diagnosed for avian infectious bronchitis (IB) survey. Finally, 221 IBV strains were confirmed by dwarf embryo test and RT-PCR assay. By virus recovery trials, 187 of the isolates caused the birds died or distressed from nephritis, which was accordant with the clinical record.ResultsBased on epidemiology analysis of recent field isolates of nephropathogenic IB in vaccinated farms in China, YL6 strain were used for vaccination and evaluated by antibody titer and challenge tests. The immunoprotection test indicated that the practical application of vaccine based on the recent field strains could finely facilitate controlling the nephropathogenic IB.ConclusionsOur study was aim at setting a guide for safeguard against nephropathogenic IBV-caused disease in China.

Anticoccidial activity of herbal complex in broiler chickens challenged withEimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of different concentrations of the herbal complex of 4 plants (leaves of Azadirachta indica and Nicotiana tabacum, flowers of Calotropis procera and seeds of Trachyspermum ammi) in broiler chickens in comparison with amprolium anticoccidial. Three concentrations (2 g, 4 g and 6 g) of herbal complex were given to the experimental groups once a day and amprolium (at the dose rate of 125 ppm) was given orally in drinking water from the 14th to the 21st days of age. One group was kept as infected, non-medicated control and one as non-infected, non-medicated control. All groups were inoculated orally with 75,000 sporulated oocysts on the 14th day of age except the non-infected, non-medicated control. Among herbal complex medicated groups, the maximum anticoccidial effect was seen in the group medicated with 6 g herbal complex followed by 4 g and 2 g herbal complex medicated groups. Treatment with 6 g of the herbal complex significantly reduced the negative performance and pathogenic effects associated with Eimeria tenella challenge at a level that was comparable with amprolium when using a largely susceptible recent field isolate. In summary, concentration-dependent anticoccidial activity of the studied herbal complex suggests its use as an alternative anticoccidial agent to chemotherapeutic drugs for Eimeria tenella control.

Phylogenetic distribution and predominant genotype of the avian infectious bronchitis virus in China during 2008-2009.

BackgroundThe nephropathogenic avian infectious bronchitis (IB) caused unprecedented economic losses to the commercial chicken industry of China in 2008-2009. To investigate the prevalence of nephropathogenic IB in China, eighty IBV isolates from different provinces during 2008-2009 were identified by dwarf embryo test and RT-PCR.ResultsThe strains were mostly isolated in winter and spring with a wide age range of IB outbreaks, from 4 to 69 days. By the virus recovery trials, 70/80 of the strains resulted in the deaths or distresses of birds from nephritis. To learn more about the molecular evolutionary characteristics of the circulating field strains, the coding region of major spike 1 (S1) protein gene of these strains was RT-PCR amplified and sequenced. Compared to the published representative strains, nucleotides and amino acids sequence analysis indicated that the S1 genes of these strains and the reference strains displayed homologies ranging from 75.1% to 99.8% and from 73.1% to 99.8% respectively. S1 protein of the major pandemic strains contained 540 or 542 amino acids with the cleavage site of HRRRR or RRFRR. Phylogenetic analysis revealed that recent field isolates of IBV in China were mostly belonged to A2-branch (QXIBV-branch) and HN08-branch, only one isolate was belonged to Gray-branch and M41-branch respectively. Most of the 80 strains showed evolutionarily distant from vaccine strains.ConclusionsThe results of this study suggested that nephropathogenic IBVs were mainly A2-like strains in China during 2008-2009.

Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek’s disease virus strains, RB1B and Md5

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens. Recent field isolates of MDV have tended to exhibit increasing virulence, and MDV strains are currently classified into four categories based on their relative virulence. Meq, a putative MDV oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. MDV isolates display distinct diversity and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and subsequently, to observed increases in MDV oncogenicity. In this study, we introduced mutations into the meq gene and used dual luciferase reporter assays to analyze the transcriptional activities of the resulting Meq proteins to determine whether distinct mutations in Meq could be responsible for differences in transcriptional activity among MDV strains. A proline-to-alanine substitution at position 217, the second position of one of the proline direct repeats in the transactivation domain, enhanced the transactivation activity of Meq. In addition, we found that two substitutions at positions 283 and 320 affected transactivation activity. These results suggest that the distinct diversity of and point mutations in the Meq proteins are responsible for differences in transactivation activity among MDV strains.

Assessment of the effectiveness of a combination product of spinosad and milbemycin oxime on the prophylaxis of canine heartworm infection

Three separate randomized, blinded, vehicle-controlled studies were conducted to determine the effectiveness of a single treatment and consecutive monthly treatments of a combination flavored tablet product containing spinosad and milbemycin oxime (MBO) in the prevention of the establishment of heartworm infections in dogs challenged with recent field isolates of the heartworm (HW), Dirofilaria immitis. For each study, dogs were allocated randomly based on pre-treatment body weights to treated or control groups of 10 animals each. Dogs were infected once with infective HW larvae, on Day-30, using either a Michigan isolate or a Georgia (MP3) isolate of D. immitis. Treatments of beef-flavored chewable tablets were administered in two studies one time either on Day 0 or Day 15, and in one study twice (Days 0 and 30, or Days 15 and 45) or 3 times (Days 0, 30 and 60). For the combination product groups, dosages were in the range of 30–45mg/kg (13.6–20.5mg/lb) for spinosad and 0.5–0.75mg/kg (0.2–0.34mg/lb) for MBO. Necropsies for heartworm counts were completed following euthanasia on Day 120 or Day 123.A single treatment with the combination product of spinosad and MBO 30 or 45 days post-inoculation with infective HW larvae was completely effective (100%) in preventing establishment of the Michigan D. immitis isolate, but efficacy against the Georgia MP3 isolate was incomplete, with geometric mean reductions in HW counts relative to vehicle treated controls of 99% reduction of the 30 day infection and a 98.9% reduction of the 45 day old infection. Against this same MP3 isolate, 3 consecutive monthly treatments provided complete prevention (100%) against establishment of D. immitis infections.The combination product of spinosad and MBO provides effective control of canine heartworms. A single treatment at 30 days post infection showed high but incomplete effectiveness against a heartworm isolate that had been shown to be partially refractory to treatment with marketed monthly heartworm preventives. Three consecutive monthly treatments provided complete control, providing support to the recommendation that heartworm prophylaxis should be maintained year round for optimal effectiveness.

Scientific Opinion on safety and efficacy of Clinacox® 0.5% (diclazuril) for turkeys for fattening

The additive Clinacox® 0.5% is a coccidiostat containing 0.5 % of the active substance diclazuril. The recommended dose of 1 mg diclazuril/kg feed was shown to be safe for the target animal with a margin of safety of at least 12. Diclazuril does not have antibacterial properties and consequently no microbial risk for the target species or induction of cross-resistance to clinically relevant antibiotics is expected. The toxicological studies submitted have been previously assessed by the FEEDAP Panel and no new data was found which would require reconsideration of the previous opinion and its conclusion on an ADI of 0.029 mg diclazuril/kg bw (corresponding to 1.7 mg diclazuril/person/day). Therefore, the basis of the assessment of consumer safety is unchanged and the former conclusions remain: compliance with the ADI is achieved without applying a withdrawal period and therefore in principle no MRLs need to be established. The MRL's previously proposed and enforced at the EU level ensure the protection of the consumer regarding the use of Clinacox® 0.5 % at the dose proposed for use in turkeys for fattening. No safety concern would arise for workers handling Clinacox® 0.5 % provided that appropriate protective measures are taken. The use of diclazuril at the dose proposed for turkeys for fattening does not pose a risk for the environment. The results of a battery study and three floor pen studies with recent field isolates and one field trial provided evidence that 1 mg diclazuril/kg complete feed remains effective in controlling coccidiosis in turkeys for fattening.

Impact of antigenic and genetic drift on the serologic surveillance of H5N2 avian influenza viruses

BackgroundSerologic surveillance of Avian Influenza (AI) viruses is carried out by the hemagglutination inhibition (HI) test using reference reagents. This method is recommended by animal health organizations as a standard test to detect antigenic differences (subtypes) between circulating influenza virus, vaccine- and/or reference- strains. However, significant discrepancies between reference antisera and field isolates have been observed during serosurveillance of influenza A viruses in pig and poultry farms. The objective of this study was to examine the effects of influenza virus genetic and antigenic drift on serologic testing using standard HI assays and reference reagents. Low pathogenic AI H5N2 viruses isolated in Mexico between 1994 and 2008 were used for phylogenetic analysis of AI hemagglutinin genes and for serologic testing using antisera produced with year-specific AI virus isolates.ResultsPhylogenetic analysis revealed significant divergence between early LPAI H5N2 viruses (1994 - 1998) and more recent virus field isolates (2002 - 2008). Results of the HI test were markedly influenced by the selection of the AI H5N2 virus (year of isolation) used as reference antigen for the assay. These analyses indicate that LPAI H5N2 viruses in Mexico are constantly undergoing genetic drift and that serosurveillance of AI viruses is significantly influenced by the antigen or antisera used for the HI test.ConclusionsReference viral antigens and/or antisera need to be replaced constantly during surveillance of AI viruses to keep pace with the AI antigenic drift. This strategy should improve the estimation of antigenic differences between circulating AI viruses and the selection of suitable vaccine strains.

Development of a highly sensitive real-time RT-PCR protocol for the detection of Classical swine fever virus independent of the 5′ untranslated region

Classical swine fever (CSF) is one of the most severe diseases of pigs, and can cause immense economic losses. Real-time reverse transcription polymerase chain reaction (rRT-PCR) can be used as a sensitive and specific method for detection of Classical swine fever virus (CSFV). Different published protocols are used routinely for CSFV diagnosis. However, almost all these systems use the highly conserved 5' untranslated region (5'UTR) of the CSFV genome as template. For a reliable diagnosis in outbreaks, a confirmatory assay that amplifies a different genome region is advisable. In this study a new CSFV specific rRT-PCR system using the NS5A region as template is described. The assay is multiplexed with a β-actin detection system that is used as an internal control in a single tube assay. The system was validated using recent European CSFV field isolates, dilution series of an in vitro transcribed RNA standard, and a panel of RNAs representing all available Pestivirus species and genotypes. It was shown that the new assay allows reliable detection of CSFV genomes independent of the 5'UTR region. It presents a very useful diagnostic tool, also allowing a 'double check' approach to rule out 5'UTR amplicon contaminations.

Genetic and Antigenic Characterization of Bovine Viral Diarrhea Field Isolates

Bovine viral diarrhea virus (BVDV) is commonly found in cattle and causes significant economic losses. Despite long term and widespread use of commercial modified live vaccines, BVD remains problematic. BVDV are classified into two genotypes, BVDV-1 and BVDV-2, and are further subdivided within the genotype by sequence analysis. Most genetic analysis is performed on the 5'-untranslated region of the genome as it is relatively conserved, however, this region does not code for an antigen. The objective of this study was to characterize recent BVDV field isolates using genetic analysis of the E2 gene in combination with antigenic methods using antisera generated against multiple isolates in a high throughput serum neutralization assay.

Efficacy of commercial swine influenza vaccines against challenge with a recent European H1N1 field isolate

This study examines the immunogenicity and efficacy of four commercial swine influenza (SI) vaccines against challenge with a recent European H1N1 virus, Sw/Gent/112/07. The vaccines contained different H1N1 strains showing between 77% and 95% genetic homology with the haemagglutinin (HA) of the challenge virus. Four groups of 10 pigs each received a double vaccination, with a 4-week interval, with one of the vaccines; a fifth group served as unvaccinated controls. All pigs were challenged 3 weeks after the second vaccination intratracheally with 10 5.0 EID 50 of Sw/Gent/112/07. Sera were examined in haemagglutination inhibition (HI) tests against the homologous vaccine H1N1 strains, the challenge virus and a panel of five recent H1N1 isolates. Pigs were euthanized at 24 or 72 h post-challenge and virus titres were determined in right and left lung halves. Two vaccines, in which the H1N1 strains showed a genetic homology of 93% and 89% to Sw/Gent/112/07, significantly reduced virus replication. The vaccine containing an H1N1 strain with 95% homology to Sw/Gent/112/07, did not offer significant protection, neither did it induce the highest HI titres. In general, pigs with HI antibody titres ≥20 against Sw/Gent/112/07 were virologically protected against challenge. HI titres against other viruses, however, differed compared to the challenge virus and between viruses. Our data clearly show that the genetic homology with the challenge virus is not the ultimate predictor for SI vaccine performance. The true reason for the differences in vaccine potency remains obscure because other factors, such as the antigen dose and/or the adjuvant, also differed between the vaccines.

5'-UTR-based phylogenetic analysis of Classical swine fever virus isolates from India

Classical swine fever (CSF) caused by Classical swine fever virus (CSFV) is a globally significant disease of pigs. Genetic typing of CSFV isolates can help in understanding the epidemiology of disease and trace down the source of outbreak. 5'-UTR sequence analysis and subsequent genetic classification of nine CSFV field isolates from India indicated that 3 isolates belonged to genotype 2.1 and were closely related to European CSFV strains. The remaining 6 isolates belonged to genotype 1 that contained old and new strains. However, the genotype 2.1 group consisted of recent field isolates only. The study showed circulation of both genotypes 1 and 2.1 in north-eastern part of India.

Epitope mapping of canine distemper virus phosphoprotein by monoclonal antibodies

The gene for phosphoprotein (P) of CDV encodes three different proteins, P, V, and C. The P protein is involved in viral gene transcription and replication. In the present study, we produced MAbs against a unique domain of the CDV-P protein, from aa 232 to 507, and determined their antigenic sites. By immunizing BALB/c mice with the recombinant P protein-specific fragment, we obtained six MAbs. Competitive binding inhibition assays revealed that they recognized two distinct regions of the P protein. Western blot analysis and immunofluorescence assays using deletion mutants of the unique C-terminus of the CDV-P protein revealed that all MAbs recognized a central short region (aa 233-303) of the CDV-P protein. In addition, linear and conformational epitopes have been determined, and at least four antigenic sites exist in the P protein central region. Furthermore, four of the MAbs were found to react with the P protein of recent Japanese field isolates but not with that of the older CDV strains, including a vaccine strain. Thus, these MAbs could be clinically useful for quick diagnosis during the CDV outbreaks.

Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain (“MAV”, Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain “Lausanne” and its attenuated field derivative strain “6918”. The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to “Lausanne” and “6918”. Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.

A Deletion Within Glycoprotein L of Marek's Disease Virus (MDV) Field Isolates Correlates with a Decrease in Bivalent MDV Vaccine Efficacy in Contact-Exposed Chickens

We examined the functional role of a naturally occurring deletion within the glycoprotein L (gL) gene of Marek's disease virus (MDV) field isolates. We previously showed that this mutation incrementally increased the virulence of an MDV in contact-exposed SPF leghorn chickens, when chickens shedding this virus were co-infected with herpesvirus of turkeys (HVT). In our present study, we examined this mutation using two stocks of the very virulent plus (vv+)MDV strain TK, one of which harbored this deletion (TK1a) while the other did not (TK2a). We report that TK1a replicating in vaccinated chickens overcame bivalent (HVT/SB1) vaccine protection in contact-exposed chickens. Treatment groups exposed to vaccinated chickens inoculated with a 1:1 mix of TK1a and TK2a showed decreased bivalent vaccine efficacy, and this decrease correlated with the prevalence of the gL deletion indicative of TK1a. These results were also found using quadruplicate treatment groups and bivalently vaccinated chickens obtained from a commercial hatchery. As this deletion was found in 25 out of 25 recent field isolates from Delaware, Maryland, North Carolina, Pennsylvania, and Virginia, we concluded that there is a strong selection for this mutation, which appears to have evolved in HVT or bivalently vaccinated chickens. This is the first report of a mutation in a vv+MDV field strain for which a putative biological phenotype has been discerned. Moreover, this mutation in gL has apparently been selected in MDV field isolates through Marek's disease vaccination.

Adegboye, D.S.; Rasberry, U.; Halbur, P.G.; Andrews, J.J. and Rosenbusch, R.F. (1995): Monoclonal antibody-based immuno-histochemical technique for the detection of M. bovis in formalin-fixed, paraffin embedded calf lung tissues. J. Vet. Diagn. Invest. 7, 261-265 Ayling, R.D.; Baker, S.E.; Peek, M.L.; Simon, A.J. and Nicholas, R.A.J. (2000): Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against

Adegboye, D.S.; Rasberry, U.; Halbur, P.G.; Andrews, J.J. and Rosenbusch, R.F. (1995): Monoclonal antibody-based immuno-histochemical technique for the detection of M. bovis in formalin-fixed, paraffin embedded calf lung tissues. J. Vet. Diagn. Invest. 7, 261-265 Ayling, R.D.; Baker, S.E.; Peek, M.L.; Simon, A.J. and Nicholas, R.A.J. (2000): Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against

Characterization of a Chicken Embryo-Adapted H9N2 Subtype Avian Influenza Virus

The low pathogenic avian influenza (LPAI) caused by H9N2 virus has generated economic losses in the Ko- rean poultry industry since 1999, but field isolates could not be used for killed oil emulsion vaccine directly because of low productivity in embryonated chicken eggs (ECE). Therefore, we established a vaccine strain, KBNP-0028, by passage through ECE with allantoic fluid showing the highest volume and titer. KBNP-0028 possessed 158 N-glycan on hemag- glutinin (HA) as its parent strain, but acquired a 16 amino acid deletion in the stalk region of neuraminidase (16-del NA) as a result of balancing with the glycosylated HA. KBNP-0028 was sufficiently productive in ECE to be compared with the A/Puerto Rico/8/34 (PR8) strain and immunogenic enough to induce high antibody titers. The antibodies inhibited hemagglutination of recent field isolates as highly as they inhibited KBNP-0028. Genome analysis revealed that KBNP- 0028 carried no known mutation relevant to virulence in mammalian. In conclusion, KBNP-0028 is a promising vaccine candidate for prevention of the LPAI in the Republic of Korea.

<em>Culicoides imicola</em>, vecteur du virus de sérotype 8 de la fièvre catarrhale ovine

Based on its abundance near livestock and wide geographical distribution, one of the most important Culicoides virus vector species of orbiviruses is the Afro-Asiatic species C. (Avaritia) imi­cola. It is considered a proven vector of bluetongue virus (BTV) and is one of the world’s most widely distributed Culicoides spe­cies. Strong correlations were found between the presence of C. imicola and outbreaks of BT and African horse sickness (AHS) in Africa and BT in Southern Europe. In 2006, BT serotype 8 (BTV- 8) was responsible for the first outbreaks ever of BT recorded in Northern Europe. Outbreaks first appeared in the Netherlands and Belgium, and subsequently spread to Germany, France, and Luxemburg. The virus apparently overwintered in Northern Europe and, during 2007-2008, spread across the whole of Europe. The outbreaks in Northern Europe happened in the absence of C. imicola. Members of the Culicoides (Culicoides) pulicaris and C. (A.) Obsoletus complexes have been implicated in BTV transmission. In the present study, the oral susceptibil­ity of C. imicola and other South African livestock associated Culicoides species to various isolates of BTV-8 were determined by artificial feeding. During summer 2008, a Belgium and a recent (2004) South African field isolate of BTV-8 were fed to Culicoides collected at two localities in South Africa. Adult Culicoides midges were collected at the Agricultural Research Council; Onderstepoort Veterinary Institute (ARC-OVI) and at Clarens, in a cooler mountainous area of South Africa. At ARC-OVI the dominant species was C. imicola and at Clarens it was C. (A.) bolitinos. The South African field isolate was fed at con­centrations of 5.8 and 7.8 log10TCID50/mL in the blood meal and the Belgium isolate at 6.8 log10 TCID50/mL. The following summer (2009) the same South African (6.8 log10 TCID50/mL) and Belgium (6.8 log10 TCID50/mL), a Dutch (4.8 log10 TCID50/ mL) and a Spanish (6.1 log10 TCID50/mL) isolates of BTV-8 were fed to Culicoides species collected at ARC-OVI. In the same season, the South African and Belgium isolates were also fed to midges collected at Clarens. Virus recovery, after an incubation period of 10 days at 23.5°C in C. imicola, was low for all iso­lates for both years. The South African isolate (5.8 log10 TCID50/ mL) and the Spanish isolate of BTV-8 could not be recovered from 420 and 492 midges tested after incubation. The recov­ery rate for the other isolates ranged from 2/403 (0.5%) (2008: SA isolate) to 1/683 (0.1%) (2009: SA isolate). These relatively low recovery rates make direct comparison between isolates problematic. Virus recovery from C. bolitinos fed on the South African isolate (7.8 log10 TCID50/mL) was significantly higher than any of the other isolates. During 2008 the Belgium iso­late could be recovered from 4/229 (1.7%) C. bolitinos tested. Other Culicoides species from which BTV-8 could be isolated included C. (Meijerehelea) leucostictus, C. (A.) gulbenkiani and C. (unplaced) angolensis. Viral concentrations in infected C. imi­cola ranged from 0.7 to 2.4 and from 0.7 to 3.4 log10 TCID50/ mL in C. bolitinos. These results are in agreement with previous studies indicating a similarly low susceptibly in C. imicola for the reference strain of BTV-8. The relatively low oral susceptibility to near refractory status of C. imicola as determined for some of the isolates in this study will easily be compensated for by the high abundance of C. imicola in South Africa. Cumulative laboratory oral susceptibility results from South Africa indicate a consistently higher susceptibility in C. bolitinos for BTV as well as a multi-vector potential for BTV as well as AHSV. The unique biology of potential vector competent Culicoides species emphasizes the complex epidemiology of these diseases.

Serological Studies of Infectious Bronchitis Vaccines against Japanese Field Isolates of Homologous and Heterologous Genotypes

The genetic diversity of the partial S1 gene involving the hyper variable region for infectious bronchitis (IB) vaccine strains in Japan were compared with those of IB virus isolated from the field in Japan. Field isolates have mainly been classified into three major genotypes, JP-I, JP-II and JP-III, since 2003; however, the 4/91 genotype was detected from recent field isolates in Japan. The virus neutralization (VN) activity with vaccine immunized serum was investigated to evaluate the protective effects of vaccines against Japanese field isolates. In the results of the VN test, antiserum immunized with the GN and C78 (JP-I), TM-86w and Miyazaki (JP-II) and 4/91 (793B) vaccine strains could neutralize a high rate of field isolates of homologous genotype (75% of field isolates of JP-I, 100% of that of JP-II and 100% of that of 793B, respectively). For field isolates of JP-III, even though there are no homologous genotype vaccine strain, some strains of JP-III were neutralized with immune serum from vaccine strains of the heterologous genotype. In this study, a correlation between serological property and genotype was found for JP-I, JP-II and 793B. Our results suggested that an effective vaccine could be predicted in accordance with the genotype of field isolates.