AbstractObjectivesThis study was to identify and characterize Decapod iridescent virus 1 (DIV1) in the outbreaks reported in two whiteleg shrimp farms and one black tiger shrimp farm located in northern Taiwan in 2020.MethodsThe histopathology, electron microscopy and polymerase chain reaction (PCR) specific for the DIV1 were used to identify the virus, and the phylogenetic analysis was performed by comparing the major capsid protein gene fragment of DIV1s from Taiwan with reference sequences of the family Iridoviridae.ResultsDIV1 was identified by diagnostic PCR and caused mild mortality (20%) in cultured Penaeus monodon and high mortality (100%) in cultured whiteleg shrimp. Cultured P. monodon was first found to be infected with DIV1 through natural route of infection. Histopathological examination showed dark‐eosinophilic cytoplasmic inclusions in the degenerative cells of targeted hematopoietic tissues. For electron microscopy, a non‐enveloped virus particle was observed from homogenates of mixed target organs through negative staining with a diameter of 112±2 nm. Nucleotide sequences of DIV1 isolates from the Taiwanese outbreak are 100% identical to those from the PRC.ConclusionsBased on the clinical evidence, mortality rates, histopathology, electron microscopy examinations and phylogenetic analysis, it is believed that DIV1 is the causative agent of the outbreak. This is the first report of DIV1 in cultured shrimp in Taiwan. The emergence of DIV1 signals a warning to shrimp aquaculture farmers worldwide.

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. The impact of bTB on global TB control has been underestimated. We adopted the One Health approach to human bTB surveillance in Taiwan. Of 20,972 human TB cases, 202 (1.0%) were bTB, 78.2% were in males, 85.1% were new cases, 83.2% were pulmonary TB, and most were in Central (52.5%) and Southern (24.8%) Taiwan. Only 18.8% of bTB patients had known animal contact. Of the 202 human M. bovis strains, 100% were resistant to pyrazinamide (PZA), 30.2% were concurrently resistant to isoniazid (INH) and 2.0% were multidrug resistant, defined as being resistant to at least INH and rifampin. Whereas, of the 22 animal M. bovis strains, 100% and 22.7% were resistant to PZA and INH, respectively. Seven spoligotypes and 25 mycobacterial interspersed repetitive unit genotypes were identified. The predominant genotype, SB0265, was also prevalent in livestock. Notably, six animal-specific M. bovis genotypes were identified. bTB differential diagnosis and drug resistance detection are crucial for TB control. Comprehensive surveillance and human-animal interface investigations are needed.

Forty-one broodfish of orange-spotted groupers (Epinephelus coioides) were selected to evaluate the effectiveness of a viral nervous necrosis virus (VNNV) and grouper iridovirus (GIV) inactivated bivalent vaccine in grouper broodfish. Real-time quantitative PCR analysis showed that a detection rate of 10.5% (2/19) was found in egg specimens of VNNV and GIV, which carried approximately 1780 copies of GIV viral DNA in the egg specimens from broodfish before vaccination. This confirmed the vertical transmission route of GIV in broodfish. A significant increase of the anti-VNNV serum antibody titer was more than 50% in the high titer level (1:1810 to 1:5120) and 45% in the moderate titer level (1:452 to 1:1280), which were higher than those of the anti-GIV display, with 50% (10/20) in a titer of 1:57 to 1:320 and 40% (8/20) in a titer of 1:452 to 1:1280 one month after the vaccination. This result showed that the VNNV is a highly antigenic virus and can effectively induce neutralizing antibodies better than GIV. In addition, the VNNV and GIV viral copy numbers were 97.1 and 1780 copies per [Formula: see text]g host egg DNA from the broodfish before vaccination, respectively. One month after the vaccination, the viral genomes of VNNV and GIV were undetectable in egg specimens. The results show that immunization can induce the production of specific protective neutralizing antibodies, and the infective antigens can thereby be eliminated by the immunity. The results demonstrate that the specific antibodies of GIV and VNNV induced by vaccination can reduce the risk of vertical transmission of VNNV and GIV in grouper broodfish.

The aim of this study was to clone and express the ORF72 and ORF92 genes of koi herpesvirus (KHV) in a prokaryotic system and to examine the antigenicity of recombinant proteins. Phylogenetic analysis revealed that both ORF72 and ORF92 had 100% homology with KHV-J, and 99% homology with those from KHV-U and KHV-I in nucleotides. This suggests that the KHV isolate in Taiwan is more closely related to the Japanese strain (Asian genotype). In the antigenicity analysis, the crude recombinant ORF72 and ORF92 capsid proteins reacted with the positive sera of the survival fish after a KHV outbreak, indicating that these recombinant capsid proteins might mimic antigens of the wild type KHV to induce an immunological response in the infected host. Our results demonstrated potential for general applicability to serological tests and vaccine development.

A monoclonal antibody (MAb) was generated against the capsid protein (ORF 72) of koi herpesvirus (KHV) isolated from diseased koi Cyprinus carpio in Taiwan. The clone of MAb-B2 was obtained by immunizing mice with whole virus particles and further identified using indirect enzyme-linked immunosorbent assay and Western blot assay. In addition, it detected KHV in KHV-infected cells but not in those of mock-infected cells as demonstrated by indirect immunofluorescence assay. The neutralization test showed that MAb-B2 neutralized KHV. Furthermore, we uncovered that MAb-B2 recognizes the ORF72 of KHV as revealed by liquid chromatography–tandem mass spectrometry and Western blot assays. Additionally, MAb-B2 has been used as a diagnostic tool for detection of KHV in clinical samples by immunohistochemistry. Collectively, our results indicated that MAb-B2 could be used in the development of a diagnostic kit for diagnosis of KHV infections and ORF72 protein of KHV might be a candidate for future vaccine development.

A new cell line (KoG) was developed from the gill of koi, Cyprinus carpio. KoG cells were composed of epithelial-like cells and fibroblast-like cells. The cells were maintained well at 25°C in Leibovitz L15 medium supplemented with 10% fetal calf serum. Chromosome analysis showed that the KoG cells had 43 diploid chromosomes at passage 40. The KoG cells were used to test the susceptibility of a koi herpesvirus isolate using a Taiwanese koi herpesvirus isolate (strain TW BL98). The KHV-infected KoG cells revealed vacuolation in the cytoplasm and marginal hyperchromation in the nucleus, which was confirmed by an indirect immunofluorescence assay and transmission electron microscopy. A multistep growth curve showed the virus reaching the highest titer of 106.2 TCID50/ml at 5 days post inoculation. Sequence analysis targeting the alleles of the two molecular markers indicated that the Taiwanese isolate displayed the I++II+ allele and of the Asian lineage, which was identical to a Japanese strain (Asian genotype variant 1). Collectively, our results demonstrate that the KoG cells may provide a tool for the replication of KHV andthe Taiwanese isolate belongs to a typical Asian genotype. Key words: Taiwan, KoG cell line, koi herpesvirus, phylogenetic analysis.

Three regions of koi herpesvirus (KHV) genomic DNA were compared for 34 samples from Japan, six from Indonesia, two from Taiwan, one from the Philippines, 13 from the Netherlands, one from the UK, one from the USA and one from Israel. The analyzed genomic regions included known PCR-detection targets (SphI-5, 9/5 and the thymidine kinase gene). The KHVs from Asian countries were very homogeneous, although two variants were noted based on a single nucleotide polymorphism. In contrast, seven variants were found in KHVs from outside of Asia, and although closely related to one another, they were clearly distinct from those from Asian. The results suggest that a clear genetic distinction exists between Asian and European (including each single isolate from the USA and Israel) types of KHV, and that unique types of KHV were independently introduced or emerged in the respective geographic locations.

In order to elucidate the possible genetic determinants of resistance to florfenicol and chloramphenicol in porcine Escherichia coli in Taiwan, 600 fecal samples of healthy pigs from 50 different farms were collected from 2003 to 2007. The florfenicol resistance in the isolated E. coli strains doubled from 39.2% in 2003 to 78.3% in 2007. A total of 351 florfenicol-resistant E. coli isolates were isolated from nursery pigs (61.5%), grower-finisher pigs (62.5%), and sows (51.5%). The prevalence of resistance genes, floR, cmlA, cat-1, cat-2 and cat-3, was 82.9, 61.3, 10.8, 3.7, and 0%, respectively. Of the 351 florfenicol-resistant isolates, 184 (52.4%) were positive for both floR and cmlA. Furthermore, the results of efflux inhibitor studies with Phe-Arg-β-naphthylamide showed a 4- to 64-fold decrease in the florfenicol MIC levels. The FloR efflux pump may play a role in phenicol resistance among porcine E. coli isolates in Taiwan. More detailed studies are required to focus on the public health concerns about the spread of antimicrobial resistance from animal food products to humans through the food chain.

Between July 2004 and January 2005, high mortalities (up to 80–100%) were frequently encountered in postlarvae of Macrobrachium rosenbergii in southern Taiwan. Pathologically, eosinophilic intranuclear inclusion bodies (INIs) were found only in the hepatopancreatic tubular epithelial cells of the infected postlarvae from hatchery farms. No lesions could be detected in tissue of ectodermal or mesodermal origin. Interestingly, different lesions were found in sub-adults collected from a grow-out farm. Atrophic changes in abdominal muscles from the fourth to sixth segment and tail fan, associated with a reddish discoloration, were prominent features in these shrimp, but there was no unusual mortality or INI formation. In PCR assays for the detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), yellowhead virus (YHV), M. rosenbergii nodavirus (MrNV) and extra small virus (XSV), only an expected 389-bp product, specific for the IHHNV nonstructural protein gene, was obtained from all postlarvae and sub-adults examined. Positive reactions to in situ hybridization, using a DIG-labelled DNA probe, further confirmed IHHNV as the causative agent. In a comparison of our strains with Taiwanese (GenBank accession no. AY 355306 and AY 355308) and American strains (GenBank accession no. AF218266 and AF273215), nucleotide sequence identities were up to 99.7%. This is the first report concerning natural infection of IHHNV in postlarvae and sub-adults of M. rosenbergii.

investigated in milkfish Chanos chanos, which had a cumulative mortality of up to 66.7% over the course of 1 yr. Gross reddish- or greyish-white nodules appeared on the peritoneal surface, spleen, kidney, liver and gastrointestinal (GI) tract. Epithelioid granulomas with the formation of Langhan's type giant cells were the prominent histopathological changes. Despite large numbers of acid-fast bacilli in the granulomas, neither caseous necrosis nor dystrophic calcification were observed. Using degenerate primers that targeted the heat shock protein 65 kDa gene of Mycobacterium spp., a 441 bp product was amplified. When compared with published sequences, our products were identical to those of Mycobacterium abscessus Type II (GenBank accession number AY603554). This is the first report of M. abscessus infection in milkfish.

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.