Tissue culture isolation, electron microscopic characterization and PCR-detection of a betanodavirus isolated from diseased Asian sea bass.

A viral agent was successfully isolated from Asian sea bass juveniles (Letes calcarifer) exhibiting clinical signs of viral nervous necrosis (VNN) in southern Malaysia on February 2008. Eyes and brains of diseased fish were pooled, extracted and filtered. The filtrates were inoculated on SSN-1 cells and incubated at 25°C. Cytopathic effect (CPE) recognized as rounded cells continued to aggregate and the vacuolation. Electron micrographs of the infected SSN-1 cells revealed icosahedral nucleocapsid virions with 22-28 nm in diameter. Viral harvest was resistant to chloroform and iodine treatments, which indicated that it was naked and contained RNA genome. Identification using reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis of PCR product was conducted and gave a single PCR product at 460 bp in agarose gel, followed by nested PCR with specific primers for PCR products at 220 bp. Partial nucleotide sequence of the nervous necrosis virus coat protein gene showed 94.0-96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia

Development and partial characterization of new marine cell line from brain of Asian sea bass Lates calcarifer for virus isolation

A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses

Development and partial characterization of new marine cell line from brain of Asian sea bass Lates calcarifer for virus isolation.

A new cell line, Asian sea bass brain (ASBB), was derived from the brain tissue of Asian sea bass Lates calcarifer. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB cell line was subcultured more than 60 times over a period of 15 mo. The ASBB cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 25°C with optimum growth at the concentrations of 10% or 15% FBS. Polymerase chain reaction products were obtained from ASBB cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. An isolate of piscine nodavirus from juveniles of marine fish species tested positive by IQ2000 kit for viral nervous necrosis detection and was examined for its infectivity to a fish cell line of ASBB. A marine fish betanodavirus was tested to determine the susceptibility of this new cell line in comparison with commercial highly permissive SSN-1 cells. The ASBB cell line was found to be susceptible to nodavirus (RGNNV genotype), and the infection was confirmed by comparison cytopathic effect (CPE) with commercial SSN-1 and reverse transcriptase-polymerase chain reaction. A nodavirus was further elucidated by electron microscopy, and the virus tested was shown to induce CPE on ASBB cells with significant high titer. This suggests that the ASBB cell line has good potential for the isolation of fish viruses. © 2010 The Society for In Vitro Biology

Isolation and characterization of viral nervous necrosis virus on newly established marine fish cell line and its pathogenicity in non-marine host

In Southeast Asia, the intensification of aquaculture industry has led to the occurrence of various infectious diseases. Virus diseases in particular, affecting especially the larval and juvenile stages, are the most suppressing factors for sustainable production of high value fish species such as seabass (Lates ca/carifer). Inevitable, because of lack of sensitive methods that could be used for diagnosis and the inadequacy of pathogen control, the aquaculture industry will continue to be plagued by these diseases. Currently, viral nervous necrosis (VNN) is one of fish diseases listed by the Office International des Epizooties (OlE) as a notifiable disease in the production of marine fish worldwide. Thus the main objective of the present study was to determine the role of VNN virus (VNNv) as a pathogen in the Malaysian aquaculture industry, with emphasis on virus isolation in susceptible cell line, virus identification using polymerase chain reaction (PCR) and identification of characteristic microscopy lesions in VNNv infections. A new cell line designated as Asian Seabass Brain (ASBB-1) was derived from the brain tissue of seabass cultured in Malaysia. This cell line was maintained in Leibovitz L-15 media supplemented with 10% fetal bovine serum (FBS). The ASBB-1 cell line was sub-cultured more than 60 times over a period of 15 months. This cell line consists predominantly of fibroblastic-like cells, which are able to grow at temperatures between 20°C and 30°C with an optimum temperature of 25°C. At 25°C, the growth rate of these cells increased as the proportion of FBS increased from 5 to 20% with optimum growth at 15% or 10% FBS. The ASBB-1 cell line was characterized by karyotyping, and chromosome number distributions were different with subcultures P20 and P50 producing 48 and 46 chromosome peaks respectively. The ASBB-1 cells showed 90% viability after recovery from one year storage in liquid nitrogen. The ASBB-1 cells up on characterization by peR using primer sets of microsatellite markers of Asian seabass (Lates ca/carifer) were shown to be similar to the cells from tissues of seabass at 250 bp. The VNNv isolate derived from seabass was tested positive for VNN by the 102000 Kit®. The isolate designated UPM08-1 M was used in the cell line infectivity study. The susceptibilities of ASBB-1 cell line isolate and the highly permissive commercial SSN-1 cells to UPM08-1 M were compared. The results showed that ASBB-1 cell line was susceptible to VNNv (RGNNV genotype) with typical cytopathic effect (CPE) manifesting mainly as rounding-up at1 day post infection (dpi), severevacuolationwithin3-5dpi and complete detachment within 7dpi. The VNNv-induced CPE was further elucidated by electron microscopy (EM). Under EM, the ASBB-1cellsexhibitedvacuolated degeneration with presence of viral inclusion-like bodies. The CPE of VNNv on ASBB-1cells was producedat a virus titer of 109 . 5 TCID50/ml. This indicated that the ASBB-1 cell line is highly susceptible for use in the isolation of VNNv. Biophysical and biochemical characterization of VNNv isolate was determined by heat treatment, UV irradiationand the stability under effect of chemical disinfectants. The VNNv isolate showed susceptibility to heat treatment at 60°C within 30 minutes with no viable virus after 1 hour. UV irradiation at an intensity of 440 mWrcm2 resulted in a reduction in virus titer after 8 minutes. The virus appeared relatively resistant to changes in pH ranging between 2 to 11 after 1 hour incubation at 25°C. Treatment with 2% formalin was not totally effective even after 6 hours. Iodine did not inactivate the virus. Molecular characterization of the tissue culture-propagated virus using RT-PCR and nested peR, showed positive amplifications of genome size of 460 bp and 220 bp respectively. The 220 bp nucleotides from the T4 region of the coat protein gene was sequenced and the phylogenetic analysis resultsshowed close resemblance between the UPM08-1 M strain andSBNNV and GPNNV2strains of Malaysianisolates.The local isolate of UPM08-1 M also showed phylogenetic similarity with other strains from South East Asia, while showed relatively distant similarity with other VNNv strains from the Middle East. Cold water VNNv strains isolated from Norway, Canada and USA were distant phylogenetic relations to the UPM08-1 M strain. Experimental infectivity study was also performed using fresh water guppy fish (Poecilia reticulata) as a model. Guppies were intra nasally infected with 0.1 ml of the UPM08-1M strain at a titre of 109.5 TCID5010.1ml. Although the artificial infection resulted in slight mortality (11%) occurring within 14 days post inoculation, the VNN-specific lesions such as necrosis and vacuolation in the target organs of brain and retina were evident. The presence of virus in infected brain and retina tissues was confirmed by transmission electron microscopy (TEM). The VNNv from experimentally infected guppy was successfully reisolated in ASBB-1 cells inoculated with homogenate of brain and retinas of the survivors. There was gradual increment of virus titer in the guppy throughout the experimental period. The results indicated that guppy fish could be infected with VNNv isolated from diseased seabass. In summary, the study successfully isolated VNNv using the ASBB-1cell line. Complete CPE of this cell line was observed within 5 - 7 dpi after infection with VNNv at producing considerable higher titer of 1095TCID5010.1ml. Thus ASBB-1 cells can be recommended for VNNv (RGNNV genotype) isolation and will be an important tool for future conduct of fish health assurance programs in the aquaculture industry. The study also showed that RT-PCR followed by nested- PCR is a sensitive technique for identification of VNNv isolated from sea bass. It was also shown that guppy fish could be experimentally infected with VNNv, thus can be used as a virus-infection model

Conventional and Molecular Detection of Newcastle Disease and Infectious Bursal Disease in Chickens

Putra Malaysia (UPM) .A total of 187 sick and dead chickens (63 broilers and 124 layers) of different ages (1 week to >15 weeks) were collected from 12 selective poultry farms (4 broilers and 8 layers). Clinically, 7 (14.89%) of 63 affected broiler and 27 (30.68%) of 124 affected layer chickens were diagnosed as Newcastle disease (ND) whereas, 11 (23.4%) of 63 affected broiler and 6 (4.82%) of the 124 affected layer birds were diagnosed as IBD on the basis of clinical history, clinical signs and postmortem findings. Virus isolation from field samples was performed by inoculating each suspected sample into 10-day-old chicken embryos. Out of 34 ND suspected field samples, 26 (5 broilers and 21 layers) were positive for NDV isolation and 11 (8 broilers and 3 layers) of 17 IBD suspected field samples, were positive for IBDV isolation. For confirmatory diagnosis, virus detection was confirmed by serological tests (HI and AGID) and RT-PCR assay. Out of 34 clinically diagnosed ND field samples, 20 (5 broiler and 15 layer) were positive by RT-PCR assay and 15 (10 broiler and 5 layer) of 17 IBD suspected field samples, were positive by both AGIDT and RT-PCR assay. Of the 26 HA positive NDV suspected AF, 19 (4 broilers and 15 layers) were positive by both HI and RT-PCR assay whereas, 10 (7 broilers and 3 layers) of 11 IBDV isolation positive tissue suspension were positive by both AGIDT and RT-PCR assay in the laboratory. Therefore, it may be concluded that serological (HI and AGIDT) and molecular (RT-PCR) techniques which allow rapid identification of most of samples are the reliable, sensitive, specific and more accurate methods to detect the viruses for the confirmatory diagnosis of diseases

Replication of Infectious Bronchitis Virus in the Chicken Mesenchymal Stem Cells

ABSTRACT The susceptibility of the chicken mesenchymal stem cells to infectious bronchitis virus was characterized after twenty consecutive passages in chicken mesenchymal stemm cells. Virus replication was monitored by cytopathic observation, indirect immunoperoxidase, and reverse transcription polymerase chain reaction. At 72 h post-infection (p.i.) in third passage, the cytopathic effect was characterized by rounding up of cell, monolayer detachment, intracytoplasmic brownish colouration was readily observed by from 24h p.i in third passage, and at all times the extracted viral RNA from IBV-infected monolayers was demonstrated by reverse transcription polymerase chain reaction. Tissue culture effective dose 50 was used to measure virus titration performed on chicken mesenchymal stem cells and the titres in twenty passages was 108.6 TID 50 /ml. The results obtained in this study suggested that the chicken mesenhymal stem cells can be used for adaptation IBV and may be considered a step forward for the use of these cells in the future for IBV vaccine production

Longitudinal Analysis of Antimicrobial Resistance among <em>Enterococcus</em> Species Isolated from Australian Beef Cattle Faeces at Feedlot Entry and Exit

Enterococcus faecium are commensal bacteria inhabiting the gastrointestinal tract of animals and humans and an important cause of drug-resistant nosocomial infections. This longitudinal study aimed to determine whether changes in the antimicrobial resistance (AMR) phenotype and genotype occurred among Enterococcus spp. isolated from cattle rectal samples obtained at the entry to and exit from an Australian feedlot. The samples obtained at the feedlot induction yielded enterococci (104/150; 69.3%), speciated as E. hirae (90/104; 86.5%), E. faecium (9/104; 8.7%), E. mundtii (3/104; 2.9%), E. durans, and E. casseliflavus (1/104; 1.0% each). AMR was observed to lincomycin (63/104; 60.6%), daptomycin (26/104; 25.0%), nitrofurantoin (9/104; 8.7%), ciprofloxacin (7/104; 6.7%), tetracycline (5/104; 4.8%), tigecycline (4/104; 3.9%), and quinupristin/dalfopristin (3/104; 2.9%). From the rectal swab samples collected at the abattoir from the same animals (i.e., the feedlot exit), the enterococci recovery was significantly higher (144/150; 96.0%), with a marked shift in species distribution dominated by E. faecium (117/144; 81.3%). However, the prevalence of AMR to individual antimicrobials remained largely static between the entry and exit except for the increased resistance to nitrofurantoin (77/144; 53.5%) and quinupristin/dalfopristin (26/144; 18.1%). Overall, 13 AMR genes were observed among the 62 E. faecium isolates. These included aac(6′)Ii, aac(6′)-Iid, and ant(6)-Ia (aminoglycosides); eatAv, lnu(G), vat(E), msr(C), and erm(B) (macrolides, lincosamides, and streptogramins); efmA (fluoroquinolones); and tet(45), tet(L), tet(M), and tet(S) (tetracyclines). The results confirm the presence of fluoroquinolone- and streptogramin-resistant enterococci in cattle faeces at the feedlot entry in the absence of antimicrobial selection pressure. E. faecium, exhibiting increased nitrofurantoin resistance, became the dominant Enterococcus spp. during the feeding period

Antimicrobial susceptibility and molecular characteristics of Mycoplasma bovis isolated from cases of bovine respiratory disease in Australian feedlot cattle

To date, antimicrobial susceptibility has not been reported for Australian Mycoplasma bovis isolates. This study determined minimal inhibitory concentrations (MICs) for 12 different antimicrobials against Australian M. bovis isolates and used whole genome sequencing to screen those showing high macrolide MICs for point mutations in target genes. Most lung tissue/swab samples from bovine respiratory disease cases (61/76, 80.3%) tested positive for M. bovis. A set of 50 representative isolates (50/61, 82.0%) that showed adequate growth, was used for MIC testing. Uniformly, low MIC values were confirmed for enrofloxacin (≤ 4 μg/mL), florfenicol (≤ 8 μg/mL), gamithromycin (≤ 2 μg/mL), spectinomycin (≤ 4 μg/mL), tetracycline (≤ 8 μg/mL), tiamulin (≤ 4 μg/mL), and tulathromycin (≤ 0.5 μg/mL). A small proportion (10%) of isolates exhibited high MICs (≥ 32 μg/mL) for tildipirosin, tilmicosin, tylosin, and lincomycin, which were above the epidemiological cut-off values for each antimicrobial (≥ 4 μg/mL). These isolates, originating from three Australian states, underwent whole genome sequencing/multilocus sequencing typing and were compared with the reference strain PG45 to investigate mutations that might be linked with the high macrolide/lincosamide MICs. All five belonged to ST52 and two macrolide associated mutations were identified within the 23 S rRNA gene (A2058G in two sequenced isolates and G748A in all sequenced isolates). Four additional 23 S rRNA gene mutations did not appear to be linked to macrolide resistance. Whilst the majority of Australian M. bovis isolates appear susceptible to the tested antimicrobials, emerging macrolide resistance was detected in three Australian states and requires continued monitoring