Investigation of Trehalose Supplementation Impacting Campylobacter jejuni and Clostridium perfringens from Broiler Farming.

In 2006, the European Commission banned the use of antibiotic promoters in animal feed. However, there is a new situation in poultry disease where it is necessary to study feed additives, which can overcome the diseases that were previously controlled through the addition of antibiotics and antimicrobial growth promoters in the feed. Therefore, trehalose was investigated to determine whether it impacts the growth performance and pathogenic bacteria (C. jejuni and C. perfringens) inoculation in broilers. In the first experiment, the tolerance of broilers to the addition of trehalose to their feed was investigated. There was no significant difference (p > 0.05) in body weight changes, daily weight gain, feed intake or feed conversion ratio during the feeding period. Within a 35-day feeding period, it was concluded that a trehalose dosage up to 10% does not exert a negative effect on broiler farming. Moreover, there was no significant difference (p > 0.05) in the broilers' growth performance, as well as C. jejuni and C. perfringens counts in the intestines and feces of broilers observed over a 5-week feeding period. However, Lactobacillus counts significantly increased in these groups with 3% and 5% trehalose supplementation. The findings indicate that trehalose supplementation in the feed cannot directly decrease C. jejuni and C. perfringens counts but may enhance gut health by raising Lactobacillus counts in chicken gut, particularly when enteropathogenic bacteria are present.

Genetic diversity of avian paramyxoviruses isolated from wild birds and domestic poultry in Taiwan between 2009 and 2020.

Avian paramyxoviruses (APMVs) belonging to the subfamily Avulavirinae within the family Paramyxoviridae. APMVs consist of twenty-two known species and are constantly isolated from a wide variety of avian species around the world. In this study, the APMV isolates obtained from wild birds and domestic poultry during 2009-2020 in Taiwan were genetically characterized by phylogenetic analysis of their complete fusion protein gene or full-length genome. As a result, 57 APMV isolates belonging to seven different species were obtained during this period and subsequently identified as APMV-1 (n=17), APMV-2 (n=1), APMV-4 (n=25), APMV-6 (n=8), APMV-12 (n=2), APMV-21 (n=2) and APMV-22 (n=2). Sanger sequencing was performed to provide 22 full-length genome sequences and 35 complete fusion protein gene sequences for the APMV isolates. Phylogenetic analysis showed that the recovered viruses were closely related to Eurasian strains, except five class I APMV-1 and four APMV-4 isolates were related to North America strains. Our findings provided more evidence for the intercontinental transmission of APMVs between Eurasia and North America by wild birds. In addition, according to the criteria of the classification system based on complete fusion protein gene sequences, three novel genotypes within APMV-2, APMV-12, and APMV-22 were identified. Together, this investigation provided a broader perspective on the genetic diversity, evolution, and distribution of APMVs in multiple avian host species sampled in Taiwan.

Phylogenetic analysis of avian paramyxoviruses 1 isolated in Taiwan from 2010 to 2018 and evidence for their intercontinental dispersal by migratory birds.

Avian paramyxovirus 1 (APMV-1), synonymous with Newcastle disease virus (NDV), is a worldwide viral agent that infects various avian species and responsible for outbreaks of Newcastle disease. In this study, 40 APMV-1 isolates collected from poultry, migratory birds, and resident birds during 2010-2018 in Taiwan were characterized genetically. Our phylogenetic analysis of complete fusion protein gene of the APMV-1 isolates revealed that 39 of the 40 Taiwanese isolates were closely related to APMV-1 of class I genotype 1 or class II genotypes I, VI or VII, and one isolate belonged to a group that can be classified as a novel genotype 2 within class I. The fusion protein gene sequences of a branch (former 1d) nested within class I sub-genotype 1.2 were closely related to those isolated from wild birds in North America. Viruses placed in class II sub-genotype VI.2.1.1.2.1 and sub-genotype VI.2.1.1.2.2 were the dominant pigeon paramyxovirus 1 (PPMV-1) circulating in the last decade in Taiwan. All the Newcastle disease outbreak-associated isolates belonged to class II sub-genotype VII.1.1, which was mainly responsible for the present epizootic of Newcastle disease in Taiwan. We conclude that at least five sub/genotypes of APMV-1 circulate in multiple avian host species in Taiwan. One genetically divergent group of APMV-1 should be considered as a novel genotype within class I. Migratory birds may play an important role in intercontinental spread of lentogenic APMV-1 between Eurasia and North America.

Novel avian metaavulavirus isolated from birds of the family Columbidae in Taiwan.

Avian paramyxoviruses (APMVs) consist of twenty known species and have been isolated from domestic and wild birds around the world. In 2009, the isolate APMV/dove/Taiwan/AHRI33/2009 was isolated from swabs of red turtle doves (Streptopelia tranquebarica) during active surveillance of avian influenza in resident birds in Taiwan, and it was initially identified as paramyxovirus based on electron microscopy. Hemagglutination inhibition assays indicated antigenic heterogeneity of AHRI33 with the known APMV-1, -2, -3, -4, -6, -8, and -9 species, only showing weak but measurable cross-reactivity with APMV-7. Pathogenicity ICPI test revealed that the virus was avirulent for chickens. The AHRI33 virus genome revealed a typical APMV structure consisting of six genes 3'-NP-P-M-F-HN-L-5', and the length of the genome was 16,914 nucleotides, the third longest among the members of the subfamily Avulavirinae. Estimates of the nucleotide sequence identities of the genome between each prototype of APMVs had shown AHRI33 to be more closely related to APMV-7 than to the others, with a sequence identity of 62.8%. Based on topology of the phylogenetic tree of RdRp genes and the branch length between the nearest node and the tip of the branch, AHRI33 met the criteria for designation as distinct species. Together, the data suggest that the isolate APMV/dove/Taiwan/AHRI33/2009 should be considered as the prototype strain of the new species Avian metaavulavirus 21 in the genus Metaavulavirus in the subfamily Avulavirinae.

Influenza B viruses in pigs, Taiwan.

BACKGROUND: Influenza B viruses (IBVs) have never been isolated from natural-infected pigs in clinical cases, although the susceptibility of domestic pigs to experimental IBV infections had been confirmed as well as IBV-specific antibodies were detected from pigs under natural and experimental conditions. OBJECTIVES: We aimed to assess and investigate the activities for infection and circulation of IBVs in pigs. METHODS: Annual active surveys for influenza have been implemented on swine populations in Taiwan since July 1998. Nasal swabs, trachea, lungs, and blood from pigs were tested using virological and serological assays for influenza. Gene sequences of influenza viral isolates were determined and characterized. Preliminary sero-epidemiological data for influenza virus were investigated. RESULTS: Three strains of IBV were isolated and identified from natural-infected pigs in 2014. Genetic characterization revealed the highest identities (>99%) of molecular sequence with the contemporary IBVs belonged to the B/Brisbane/60/2008 genetic clade of Victoria lineage in the phylogenetic trees for all 8 genes. IBV-specific antibodies were detected in 31 (0.2%; 95%CI: 0.1%-0.2%) of 15 983 swine serum samples from 29 (2.8%; 95%CI: 1.9%-3.9%) of 1039 farm visits under annual active surveys from 2007 through 2017. Seropositive cases have been found sparsely in 1-5 of test prefectures every year except 2015 and 2017 as well as scattered loosely over 26 townships/districts of 11 prefectures in Taiwan cumulatively in 11 years. CONCLUSIONS: Influenza B viruse infections from humans to pigs remained sporadic and accidental currently in Taiwan but might have paved potential avenues for newly emerging zoonotic influenza in the future.

Development of a quantitative real-time RT-PCR assay for detecting Taiwan ferret badger rabies virus in ear tissue of ferret badgers and mice.

In 2013, the first case of Taiwan ferret badger rabies virus (RABV-TWFB) infection was reported in Formosan ferret badgers, and two genetic groups of the virus were distinguished through phylogenetic analysis. To detect RABV-TWFB using a sensitive nucleic acid-based method, a quantitative real-time reverse transcription polymerase chain reaction targeting the conserved region of both genetic groups of RABV-TWFB was developed. This method had a limit of detection (LOD) of 40 RNA copies/reaction and detected viral RNA in brain and ear tissue specimens of infected and dead Formosan ferret badgers and mice with 100% sensitivity and specificity. The mean viral RNA load detected in the ear tissue specimens of ferret badgers ranged from 3.89 × 108 to 9.73 × 108 RNA copies/g-organ, which was 111-fold to 2,220-fold lower than the concentration detected in the brain specimens, but 2,000-fold to 5,000-fold higher than the LOD of the assay. This highly sensitive technique does not require facilities or instruments complying with strict biosafety criteria. Furthermore, it is efficient, safe, and labor-saving as only ear specimens need be sampled. Therefore, it is a promising technique for epidemiological screening of Taiwan ferret badger rabies.

Safety, efficacy and immunogenicity evaluation of the SAG2 oral rabies vaccine in Formosan ferret badgers.

Since 2013, rabies cases have been reported among Formosan ferret badgers in Taiwan, and they have been shown to be the major reservoirs for Taiwanese enzootics. To control and eradicate rabies, the authorities plan to implement a vaccination programme. Before distributing live vaccines in the field, this study assessed the safety, efficacy, and immunogenicity of SAG2 vaccine on ferret badgers by direct oral instillation. After application of 109 TCID50/dose, no virus was excreted into the oral cavity 1-7 days post-application, and safety was also satisfactorily verified over a 266-day period. Moreover, despite the low level of rabies virus neutralising antibodies induced after vaccination of a 108 TCID50/dose, the efficacy assessment revealed a 100% survival rate (15/15) of vaccinees and an 87.5% fatality rate (7/8) in control animals after a challenge on the 198th day post-vaccination. The immunisation and protection rates obtained more than 6 months after a single vaccination dose demonstrated that SAG2 is an ideal vaccine candidate to protect Formosan ferret badgers against rabies in Taiwan.

Sequence diversity and associated pathogenicity of the hemagglutinin cleavage site of H5N2 avian influenza viruses isolated from chickens in Taiwan during 2013-2015.

The sequence at the hemagglutinin (HA) cleavage site (CS) plays a key role in determining the pathogenicity of avian influenza viruses. Three types of HA CS sequences, QREKR/GL, QRKKR/GL and QRRKR/GL, were previously reported in Taiwanese H5N2 viruses that were isolated from chickens from 2003 to 2013. However, no HA CS sequence was reported for viruses isolated after 2013. This article presents the HA CS sequences and pathogenicity of H5N2 viruses that were isolated from chickens in Taiwan during 2013-2015. Two novel HA CS sequences, QKEKR/GL and KREKREKR/GL, were found in the viruses isolated in 2013 and 2014, and pathogenicity tests showed that the viruses with these novel HA CS sequences are low and high pathogenic viruses, respectively. In contrast, the HA CS sequence QREKR/GL was found in all viruses that were isolated in 2015, and all of these viruses were low pathogenic viruses. After 10 passages in embryonated chicken eggs, a virus strain that was isolated in 2003 evolved into a viral quasispecies that contained at least four distinct types of HA CS sequences. These results highlight the potential of Taiwanese H5N2 viruses to change their pathogenicity and HA CS sequences via mutations. Furthermore, viruses with the HA CS sequence QREKR/GL were more prevalent than others in 2015. These findings are useful for understanding the mechanism of sequence changes at the HA CS and for refining H5N2 virus control measures in Taiwan.

Incidence and Antimicrobial Susceptibility to Clostridium perfringens in Premarket Broilers in Taiwan.

Clostridium perfringens infection causes subclinical and clinical necrotic enteritis in poultry flocks, and it is estimated to result in US$2 billion of losses worldwide every year. The aims of this study were to determine the incidence, toxin types, and antimicrobial resistance levels to C. perfringens isolated from premarket, 5-wk-old, clinically healthy broiler chickens in Taiwan, and to examine the relationships between intestinal lesions and the numbers of C. perfringens in intestinal contents. In total, 435 samples of chicken ileum contents were collected from 98 broiler farms during June 2012 to February 2013. The C. perfringens isolation rate was 9.9% (43/435). The positive rate of tested farms was 29.6% (29/98). All the isolates were C. perfringens type A, only possessing the cpa gene encoding for toxin α. No netB gene encoding NetB toxin associated with necrotic enteritis, and no cpe gene encoding for the C. perfringens enterotoxin causing human intestinal disorder were detected. A quantitative PCR analysis revealed that the mean C. perfringens number in the intestinal contents was 3.9 × 10(6) colony-forming units (CFU)/g, ranging from 6.85 × 10(2) to 1.61 × 10(7) CFU/g. The gross and histopathologic lesions revealed a positive correlation (p < 0.05) between lesion score and C. perfringens number in the ilea of C. perfringens -positive chickens. Antimicrobial susceptibility tests of all C. perfringens isolates indicated that the minimum inhibitory concentration inhibiting 50% of isolates (MIC50) for amoxicillin, bacitracin, chlortetracycline, enrofloxacin, erythromycin, florfenicol, and lincomycin was ≤0.125, 0.5, 128, 0.25, ≥256, 2, and ≥256 µg/ml, respectively. Most of the C. perfringens isolates were susceptible to amoxicillin, bacitracin, and enrofloxacin but resistant to chlortetracycline, erythromycin, and lincomycin. Interestingly, C. perfringens isolated from chickens with severe lesions had higher MIC50 for erythromycin and lincomycin than those isolates from chickens with mild lesions. Conclusively, reductions in both the incidence of C. perfringens infection on farms and the concentrations of C. perfringens in intestines to improve broiler health are still needed in Taiwan.

The impact of Salmonella Enteritidis on lipid accumulation in chicken hepatocytes.

Salmonella enterica serovar Enteritidis (SE) is a public health concern and infected chickens serve as a reservoir that potentially transmits to humans through food. Although SE seldom causes systemic disease in chickens, virulent SE strains can colonize in intestines and lead a persistent infection of the liver. The liver is the primary organ for lipid metabolism in chickens and the site for production and assembly of main components in yolk. We performed a time-course experiment using LMH-2A cells that were infected with SE and co-incubated with ß-oestradiol to evaluate if SE infection affected lipid metabolism and subsequently changed lipoprotein formation for egg yolk. The results indicated that lipid accumulation significantly increased in infected LMH-2A cells while the viability of these cells was only slightly decreased. The mRNA expressions of lipid transportation and most lipogenetic genes including sterol regulatory element binding protein 1, acetyl-CoA carboxylase, fatty-acid synthase, long-chain-fatty-acid-CoA ligase 1, peroxisome proliferator-activated receptor-γ, and very-low-density lipoproteins (VLDLs) II were significantly up-regulated while the expression of lipogenetic-related stearoyl-CoA denaturase 1 was down-regulated. Moreover, decline in lipid transportation of hepatocytes was evidenced by the down-regulation of oestrogen receptor α which promotes VLDLy formation, an increase of intra-cellular accumulation of Apoprotein B (ApoB) protein, and a decrease of cellular excretion of VLDL protein. Conclusively, SE infection could elevate lipid synthesis and reduce lipid transportation in the chicken hepatocytes. These changes may lead excessive lipid accumulation in liver and slower lipoprotein deposition in yolk.

Relationship of bovine TNF-α gene polymorphisms with the risk of bovine tuberculosis in Holstein cattle.

Many studies suggest significant genetic variation in the resistance of cattle and humans to infection with Mycobacterium bovis (M. bovis), the causative agent of zoonotic tuberculosis. TNF-α promotes inflammation and induces apoptosis in response to mycobacterial infection. The aim of the present study was to investigate the influence of single nucleotide polymorphisms of the TNF-α gene on bovine tuberculosis (bTB) susceptibility. We genotyped the TNF-α gene in 74 bTB-infected Holstein cows and 90 healthy control animals. The influence in the exon 3 region of TNF-α polymorphisms on bTB susceptibility was subsequently investigated by association analysis. Our finding demonstrated that the g.27534932A>C polymorphism of the TNF-α is associated with bTB in Holstein cattle. The susceptibility of cattle with the g.27534932A>C genotype compared with the CC genotype was 4.11-fold (95% CI, 1.27-13.36; P=0.02) higher. The g.27534932A>C polymorphism located in exon 3 of the TNF-α gene, and the functional consequence was missense. The deduced amino acid sequence for the protein product revealed an arginine to serine conversion at position 159, which may affect initiation of protein synthesis and disrupt normal TNF-α function that protects animals against mycobacterial infection. A significant association was observed with the A allele as a risk factor for bTB susceptibility (OR, 3.84; 95% CI, 1.21-12.17; P=0.02). In conclusion, this is the first report showing that the g.27534932A>C polymorphism may contribute to TNF-α-mediated bTB susceptibility.

Relationship of bovine NOS2 gene polymorphisms to the risk of bovine tuberculosis in Holstein cattle.

Many studies suggest significant genetic variation in the resistance of cattle and humans to infection with Mycobacterium bovis, the causative agent of zoonotic tuberculosis. The inducible nitric oxide synthase (iNOS which is encoded by the NOS2 gene) plays a key role in the immunological control of a broad spectrum of infectious agents. This study aimed to investigate the influence of genetic variations in the promoter of the NOS2 gene on bovine tuberculosis (bTB) susceptibility. In this study, the NOS2 genes of 74 bTB-infected Holstein cows and 90 healthy controls were genotyped using PCR followed by nucleotide sequencing. Polymorphisms at rs207692718, rs109279434, rs209895548, rs385993919, rs433717754, rs383366213, rs466730386, rs715225976, rs525673647, rs720757654 and g.19958101T>G in the promoter region of the NOS2 gene were detected. The g.19958101T>G SNP produced two different conformation patterns (TT and TG) and the TG genotype was over-represented in the bTB group (20.27%) compared with the control group (2.22%). The TG genotype frequency of the g.19958101T>G variant was significantly higher in bTB cattle than in healthy controls (OR, 11.19; 95% CI, 2.47-50.73; P=0.0002). The G allele of the g.19958101T>G polymorphism was more frequent in bTB group when compared to control group (10.14% versus 1.11%). Furthermore, the G allele was a risk factor for bTB susceptibility (OR, 10.04; 95% CI, 2.26-44.65; P=0.0002). In conclusion, the g.19958101T>G polymorphism of the NOS2 gene may contribute to the susceptibility of Holstein cattle to bTB.

Development of a multiplex Luminex assay for detecting swine antibodies to structural and nonstructural proteins of foot-and-mouth disease virus in Taiwan.

BACKGROUND/PURPOSE(S): Foot-and-mouth disease (FMD) and swine vesicular disease (SVD) are serious vesicular diseases that have devastated swine populations throughout the world. The aim of this study was to develop a multianalyte profiling (xMAP) Luminex assay for the differential detection of antibodies to the FMD virus of structural proteins (SP) and nonstructural proteins (NSP). METHODS: After the xMAP was optimized, it detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus in a single serum sample. These tests were also compared with 3ABC polypeptide blocking enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) methods for the differential diagnosis and assessment of immune status, respectively. RESULTS: To detect SP antibodies in 661 sera from infected naïve pigs and vaccinated pigs, the diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 days postinfection and 8 days postinfection, respectively. Furthermore, the SP and NSP antibodies in all 15 vaccinated but unprotected pigs were detected by xMAP. A comparison of SP and NSP antibodies detected in the sera of the infected samples indicated that the results from the xMAP had a high positive correlation with results from the VNT and a 3ABC polypeptide blocking ELISA assay. However, simultaneous quantitation detected that xMAP had no relationship with the VNT. Furthermore, the specificity was 93.3-94.9% with 3ABC polypeptide blocking ELISA for the FMDV-NSP antibody. CONCLUSION: The results indicated that xMAP has the potential to detect antibodies to FMDV-SP-VP1 and NSP-3ABC and to distinguish FMDV-infected pigs from pigs infected with the swine vesicular disease virus.

In vitro gene expression profile of bovine peripheral blood mononuclear cells in early Mycobacterium bovis infection.

The intracellular parasite Mycobacterium bovis (M. bovis) causes tuberculosis in cattle and humans. Understanding the interactions between M. bovis and host cells is essential in developing tools for the prevention, detection, and treatment of M. bovis infection. Gene expression profiles provide a large amount of information regarding the molecular mechanisms underlying these interactions. The present study analyzed changes in gene expression in bovine peripheral blood mononuclear cells (PBMCs) at 0, 4 and 24 h following exposure to M. bovis. Using bovine whole-genome microarrays, a total of 420 genes were identified that exhibited significant alterations in expression (≥2-fold). Significantly enriched genes were identified using the Kyoto Encyclopedia of Genes and Genomes database, of which the highest differentially expressed genes were associated with the immune system, signal transduction, endocytosis, cellular transport, inflammation, and apoptosis. Of the genes associated with the immune system, 84.85% displayed downregulation. These findings support the view that M. bovis inhibits signaling pathways of antimycobacterial host defense in bovine PBMCs. These in vitro data demonstrated that molecular alterations underlying the pathogenesis of tuberculosis begin early, during the initial 24 h following M. bovis infection.

Molecular epidemiology of porcine reproductive and respiratory syndrome viruses isolated from 1991 to 2013 in Taiwan.

Porcine reproductive and respiratory syndrome virus (PRRSV) was first identified in Taiwan in 1991, but the genetic diversity and evolution of PRRSV has not been thoroughly investigated over the past 20 years. The aim of this study was to bridge the gap in understanding of its molecular epidemiology. A total of 31 PRRSV strains were collected and sequenced. The sequences were aligned using the MUSCLE program, and phylogenetic analysis were performed by the maximum-likelihood method and the neighbor-joining method using MEGA 5.2 software. In the early 1990s, two prototype strains, WSV and MD001 of the North American genotype, were first identified. Over the years, both viruses evolved separately. The population dynamics of PRRSV revealed that the strains of the MD001 group were predominant in Taiwan. Evolution was manifested in changes in the nsp2 and ORF5 genes. In addition, a suspected newly invading exotic strain was recovered in 2013, suggesting that international spread is still taking place and that it is affecting the population dynamics. Overall, the results provide an important basis for vaccine development for the control and prevention of PRRS.

Rabies Virus Infection in Ferret Badgers (Melogale moschata subaurantiaca) in Taiwan: A Retrospective Study.

Fifteen ferret badgers (Melogale moschata subaurantiaca), collected 2010-13 and stored frozen, were submitted for rabies diagnosis by direct fluorescent antibody test and reverse transcription PCR. We detected seven positive animal samples, including some from 2010, which indicated that the ferret badger population in Taiwan had been affected by rabies prior to 2010.

Relationships of bovine ephemeral fever epizootics to population immunity and virus variation.

Bovine ephemeral fever is an arthropod-borne bovine viral disease caused by infection with bovine ephemeral fever virus which belongs to genus Ephemerovirus within the family Rhabdoviridae. In this study, serological data and virological information about the disease and the virus, spanning from 2001 to 2013, were employed to analyze the relationships of bovine ephemeral fever epizootics to population immunity and virus variation. National and regional surveillance data indicated that 2 of the 3 major epizootics and 87% regional outbreaks were associated with lower neutralizing antibody titers and immunity coverage, reflecting the importance of population immunity for the control of bovine ephemeral fever. Phylogenetic analysis and sequence comparison demonstrated that Taiwanese bovine ephemeral fever viruses were >96.0% and >97.6% similar to the East Asian isolates in nucleotide and amino acid sequences, respectively. These analyses supported that the Taiwanese viruses shared the same gene pool with the strains of the other East Asian countries, mainly Japan.

First finding of Southeast Asia topotype of foot-and-mouth disease virus in Kinmen, Taiwan, in the 2012 outbreak.

Foot-and-mouth disease virus, a member of genus Aphthovirus within the family Picornaviridae, affects cloven-hoofed animals, causing foot-and-mouth disease characterized by vesicle development. The Southeast Asia topotype, one of the topotypes within serotype O of the virus, is prevalent in some Asian countries, but had not previously been found in Taiwan. The topotype was first found in pigs in Kinmen Island, Taiwan, in 2012 and identified by nucleotide sequence comparison and phylogenetic analysis. Outbreaks were reported at 4 farms, resulting in the culling of 628 pigs and 1 cattle. Pigs were the only species infected during the outbreak. The incursion of Southeast Asia topotype into Taiwan implies the expansion of the topotype in East Asia.

A complete molecular diagnostic procedure for applications in surveillance and subtyping of avian influenza virus.

INTRODUCTION: The following complete molecular diagnostic procedure we developed, based on real-time quantitative PCR and traditional PCR, is effective for avian influenza surveillance, virus subtyping, and viral genome sequencing. METHOD: This study provides a specific and sensitive step-by-step procedure for efficient avian influenza identification of 16 hemagglutinin and 9 neuraminidase avian influenza subtypes. RESULT AND CONCLUSION: This diagnostic procedure may prove exceedingly useful for virological and ecological advancements in global avian influenza research.

Comparison of primer sets and one-step reverse transcription polymerase chain reaction kits for the detection of bluetongue viral RNA.

Bluetongue virus is the etiological agent of bluetongue, one of the most important insect-transmitted animal diseases in the world. To establish a feasible diagnostic procedure for detecting the viral RNA, seven commercially available one-step RT-PCR kits in combination with three primer sets were evaluated. Results of this study showed remarkable differences in analytical sensitivity between the examined RT-PCR kits. In addition, it was found that a World Organization for Animal Health-recommended primer set may not be effective in detecting most BTV RNA.