Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

Immuno-protective efficiency of feed-based whole-cell inactivated bivalent vaccine against Streptococcus and Aeromonas infections in red hybrid tilapia (Oreochromis niloticus × Oreochromis mossambicus).

Streptococcosis and motile aeromonad septicemia (MAS) are well-known diseases in tilapia culture, which cause mass mortality with significant economic losses. The development of feed-based bivalent vaccines in controlling these diseases has been initiated, however, the mechanisms of immunities and cross-protection in fish remain unclear. This study was conducted to assess the immuno-protective as well as the cross-protective efficacy of a newly developed feed-based bivalent vaccine against Streptococcus and Aeromonas infections in red hybrid tilapia. A total of five groups of fish were vaccinated orally through two different techniques; bivalent vaccine (inactivated Streptococcus iniae and Aeromonas hydrophila) sprayed on feed pellets (BS group); bivalent vaccine (inactivated S. iniae and A. hydrophila) incorporated in feed (BI group); monovalent inactivated S. iniae and A. hydrophila vaccine separately incorporated into feed as monovalent S. iniae (MS group) and monovalent A. hydrophila (MA group); and control group (without vaccine). The feed-based vaccine was delivered orally at 5% of body weight for five consecutive days. The booster doses were given in the same manner on weeks 2 and 6. Serum and skin mucus samples were collected to assess the IgM responses using indirect ELISA. The first administration of the feed-based vaccine stimulated the IgM levels that lasted until week 3, while the second booster ensured that the IgM levels remained high for a period of 16 weeks in the BI, MS and MA groups. The BI group developed a strong and significantly (P < 0.05) higher systemic and mucosal IgM responses against both S. iniae and A. hydrophila and also cross-protective antigen S. agalactiae and A. veronii compared to the BS and control groups. Quantitative real-time PCR results also showed that the relative expressions of IL-8, INF-γ and IgM in the BI immunized fish spleen, head kidney and hindgut exhibited various significant (P < 0.05) rising trends following both the vaccination and the challenge phase. On weeks 10, all fish were challenged through the intraperitoneal route, where relative percent survivals (RPS) of 82.22 ± 3.85% when challenged with S. iniae, 77.78 ± 3.85% when challenged with A. hydrophila and 77.78 ± 3.85% when co-challenged with both S. iniae and A. hydrophila were observed in the BI group, which were significantly higher (P < 0.05) compared to the other groups. The BI group also showed significantly (P < 0.05) higher partial cross-protections following challenges with S. agalactiae (RPS at 60.00 ± 6.67%) and A. veronii (RPS at 57.78 ± 7.70%). This study demonstrated that immunization with feed-based BI vaccine elicited immune responses that were capable of protecting red hybrid tilapia against streptococcosis and MAS.

Haemato-immunological responses and effectiveness of feed-based bivalent vaccine against Streptococcus iniae and Aeromonas hydrophila infections in hybrid red tilapia (Oreochromis mossambicus × O. niloticus).

BACKGROUND: Streptococcosis and Motile Aeromonad Septicemia (MAS) are important diseases of tilapia, Oreochromis spp. and causes huge economic losses in aquaculture globally. The feed-based vaccination may be an alternative to minimize major infectious diseases in tilapia. Thus, this study aims to evaluate the haemato-immunological responses and effectiveness of a newly developed feed-based killed bivalent vaccine against Streptococcus iniae and Aeromonas hydrophila in hybrid red tilapia. A total of 495 hybrid red tilapia of 61.23 ± 4.95 g were distributed into 5 groups (each with triplicate). The fish were immunized orally through bivalent (combined S. iniae and A. hydrophila) spray vaccine (BS group), bivalent formulate vaccine (BF group), monovalent S. iniae vaccine (MS group), monovalent A. hydrophila vaccine (MA group) and unvaccinated as a control group. The vaccine was orally administered on days 0, 14 and 42 applied feed-based bacterin at 5% body weight. The blood and spleen samples were collected from all groups on 7, 21 and 49 days post-vaccination, and also 96 h post-infection to assess their haemato-immune responses. RESULTS: Compared with the unvaccinated group, leukocyte, lymphocytes, monocytes, granulocytes counts in vaccinated groups were significantly (P < 0.05) increased on 21, 49 days post-vaccination and also 96 h post-infection, while erythrocytes, haemoglobin and haematocrit in vaccinated groups were significantly (P < 0.05) enhanced only 96 h post-infection. Additionally, the lysozyme and phagocytic activity and, serum antibody (IgM) were significantly higher (P < 0.05) against S. iniae and A. hydrophila in vaccinated groups compared to the unvaccinated group in the pre- and post-infection. Results from the challenge through co-infection with S. iniae and A. hydrophila showed the relative percent survival (RPS) in BF group was 76.67 ± 4.71%, which had the capacity to induce significant protection (P < 0.05) compared to others groups. CONCLUSIONS: This study demonstrates the bivalent formulate (BF) group could elicit significant non-specific and specific immunological responses with higher protection in hybrid red tilapia. In addition, this newly developed feed-based bivalent vaccination can be a promising technique for effective and large scale fish immunization in the aquaculture industry.

Prevalence, antimicrobial susceptibility and plasmid profiling of Vibrio spp. isolated from cultured groupers in Peninsular Malaysia.

BACKGROUND: Numerous prevalence studies of Vibrio spp. infection in fish have been extensively reported worldwide, including Malaysia. Unfortunately, information on the prevalence of Vibrio spp. in groupers (Epinephelus spp.) is limited. In this study, groupers obtained from nine farms located at different geographical regions in Malaysia were sampled for the presence of pathogenic Vibrio spp. and their susceptibility profiles against seven antibiotics. RESULTS: Out of 270 grouper samples, 195 (72%) were detected with the presence of Vibrio spp. Vibrio communis showed highest prevalence in grouper (28%), followed by V. parahaemolyticus (25%), V. alginolyticus (19%), V. vulnificus (14%), V. rotiferianus (3%), Vibrio sp. (3%), V. campbellii (2%), V. mytili (2%), V. furnissii (2%), V. harveyi (1%), V. tubiashii (1%), V. fluvialis (0.3%) and V. diabolicus (0.3%). Assessment on the antibiotic susceptibility profiles of the Vibrio spp. revealed that majority of the isolates were susceptible to tetracycline, streptomycin, erythromycin and bacitracin, but resistance to ampicillin, penicillin G and vancomycin. The mean MAR index of the Vibrio isolates was 0.51, with 85% of the isolates showed MAR index value of higher than 0.2. Results indicate that the Vibrio spp. were continuously exposed to antibiotics. Furthermore, the plasmid profiles of Vibrio spp. showed that 38.7% of the isolates harbored plasmid with molecular weight of more than 10 kb, while 61.3% were without plasmid. During curing process, Vibrio spp. lost their plasmid, but remained resistant to ampicillin, penicillin G, bacitracin and vancomycin while a few isolates remained resistant to erythromycin, streptomycin and tetracycline. The results suggested that the resistance to antibiotics in isolated Vibrio spp. might be due to chromosomal and plasmid borne. CONCLUSIONS: This study demonstrates the prevalence of Vibrio spp. in groupers and the distribution of multidrug resistance strains that could be of concern to the farmers in Malaysia. In addition, data from this study can be further used in fish disease management plan.

Effect of an Oral Bivalent Vaccine on Immune Response and Immune Gene Profiling in Vaccinated Red Tilapia (Oreochromis spp.) during Infections with Streptococcus iniae and Aeromonas hydrophila.

Streptococcosis and aeromonasis inflicted by Streptococcus iniae and Aeromonas hydrophila, respectively, have affected tilapia industries worldwide. In this study, we investigated antibody responses and explored the mechanisms of protection rendered by an oral bivalent vaccine in red tilapia following challenges with S. iniae and A. hydrophila. The results of specific IgM antibody response revealed that the IgM titers against S. iniae and A. hydrophila in the bivalent incorporated (BI) vaccine group were significantly higher (p < 0.05) than those in the bivalent spray (BS) vaccine fish and unvaccinated control fish throughout the experiment. Real-time qPCR results also showed that the gene expression of CD4, MHC-I, MHC-II, IgT, C-type lysozyme, IL-1ß, TNF-α, and TGF-ß remained significantly higher (p < 0.05) than that of the controls between 24 and 72 h post-infection (hpi) in both mucosal (hindgut) and systemic (spleen and head−kidney) organs of BI vaccinated fish. Furthermore, the highest relative expression of the TGF-ß, C-type lysozyme, and IgT genes in the BI vaccinated group was observed in the challenged fish's spleen (8.8-fold), head kidney (4.4-fold), and hindgut (19.7-fold) tissues, respectively. The present study suggests that the bivalent incorporated (BI) vaccine could effectively improve the immune function and activate both humoral and cell-mediated immunities in vaccinated red tilapia following the bacterial challenges.

Laboratory and Field Assessments of Oral Vibrio Vaccine Indicate the Potential for Protection against Vibriosis in Cultured Marine Fishes.

Vibriosis is one of the most common threats to farmed grouper; thus, substantial efforts are underway to control the disease. This study presents an oral vaccination against multiple Vibrio spp. in a marine fish with double booster immunisation. The Vibrio harveyi strain VH1 vaccine candidate was selected from infected groupers Epinephelus sp. in a local farm and was formalin inactivated and combined with commercial feed at a 10% ratio (v/w). A laboratory vaccination trial was conducted for seventy days. The induction of IgM antibody responses in the serum of Asian seabass Lates calcarifer immunised with the oral Vibrio harveyi strain VH1 was significantly (p < 0.05) increased as early as week one post-primary vaccination. Subsequent administration of the first and second booster for 5 consecutive days, starting on days 14 and 42, respectively, improved the specific antibody level and reached a highly significant (p < 0.05) value at days 35 and 49 before slightly decreasing from day 56 onwards. Antibody titres of the control unvaccinated group remained relatively stable and low throughout the experimental period. At the end of the 70-day vaccination trial, 23 days post final boost, an intraperitoneal challenge with a field strain of Vibrio harveyi, V. alginolyticus, and V. parahaemolyticus was carried out. Our challenge study showed that oral Vibrio harveyi strain VH1 vaccine candidate could induce significant protection, with an RPS of 70-80% against different Vibrio species. Thereafter, a field trial was conducted in a mariculture farm to study the effect of field vaccination using the oral Vibrio harveyi strain VH1 vaccine candidate. A total of 3000 hybrid grouper juveniles were divided into two groups in triplicate. Fish of Group 1 were not vaccinated, while Group 2 were vaccinated with the feed-based vaccine. Vaccinations were carried out on days 0, 14, and 42 via feeding the fish with the vaccine at 4% body weight for 5 consecutive days. At the end of the study period, the fish survival rate was 80% for the vaccinated group, significantly (p < 0.05) higher than the 65% seen in the control unvaccinated group. Furthermore, the vaccinated fish showed significantly (p < 0.05) better growth performances. Therefore, the oral Vibrio vaccine from the inactivated Vibrio harveyi strain VH1 is a potential versatile vaccine candidate that could stimulate good immune responses and confer high protection in both Asian seabass, Lates calcarifer, and farm hybrid grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus.

Field Efficacy of a Feed-Based Inactivated Vaccine against Vibriosis in Cage-Cultured Asian Seabass, Lates calcarifer, in Malaysia.

Vibrio spp. are important aquaculture pathogens that cause vibriosis, affecting large numbers of marine fish species. This study determines the field efficacy of a feed-based inactivated vaccine against vibriosis in cage-cultured Asian seabass. A total of 4800 Asian seabass, kept in a field environment, were separated equally into two groups (vaccinated and non-vaccinated) in duplicate. Fish of Group 1 were orally administered the feed-based vaccine on weeks 0 (prime vaccination), 2 (booster), and 6 (second booster) at 4% body weight, while the non-vaccinated fish of Group 2 were fed with a commercial formulated pellet without the vaccine. Fish gut, mucus, and serum were collected, the length and weight of the fish were noted, while the mortality was recorded at 2-week intervals for a period of 16 weeks. The non-specific lysozyme activities were significantly (p < 0.05) higher in the fish of Group 1 than the non-vaccinated fish of Group 2. Similarly, the specific IgM antibody levels in serum and mucus were significantly (p < 0.05) higher in Group 1 than in Group 2, as seen in the second week, with the highest level 8 weeks after primary immunization. At week 16, the growth performance was significantly (p < 0.05) better in Group 1 and showed lower bacterial isolation in the gut than Group 2. Despite the statistical insignificance (p > 0.05), the survival rate was slightly higher in Group 1 (71.3%) than Group 2 (67.7%). This study revealed that feed-based vaccination improves growth performance, stimulates innate and adaptive immune responses, and increases protection of cultured Asian seabass, L. calcarifer, against vibriosis.

Prevalence, Antibiotics Resistance and Plasmid Profiling of Vibrio spp. Isolated from Cultured Shrimp in Peninsular Malaysia.

Vibrio is the most common bacterium associated with diseases in crustaceans. Outbreaks of vibriosis pose a serious threat to shrimp production. Therefore, antibiotics are commonly used as preventative and therapeutic measures. Unfortunately, improper use of antibiotics leads to antibiotic resistance. Nevertheless, information on the occurrence of Vibrio spp. and antibiotic use in shrimp, particularly in Malaysia, is minimal. This study aimed to provide information on the occurrence of Vibrio spp., its status of antibiotic resistance and the plasmid profiles of Vibrio spp. isolated from cultured shrimp in Peninsular Malaysia. Shrimp were sampled from seven farms that were located in different geographical regions of Peninsular Malaysia. According to the observations, 85% of the shrimp were healthy, whereas 15% were unhealthy. Subsequently, 225 presumptive Vibrio isolates were subjected to biochemical tests and molecular detection using the pyrH gene. The isolates were also tested for antibiotic susceptibility against 16 antibiotics and were subjected to plasmid profiling. Eventually, 13 different Vibrio spp. were successfully isolated and characterized using the pyrH gene. They were the following: V. parahaemolyticus (55%), V. communis (9%), V. campbellii (8%), V. owensii (7%), V. rotiferianus (5%), Vibrio spp. (4%), V. alginolyticus (3%), V. brasiliensis (2%), V. natriegens (2%), V. xuii (1%), V. harveyi (1%), V. hepatarius (0.4%) and P. damselae (3%). Antibiotic susceptibility profiles revealed that all isolates were resistant to penicillin G (100%), but susceptible to norfloxacin (96%). Furthermore, 16% of the isolates revealed MAR of less than 0.2, while 84% were greater than 0.2. A total of 125 isolates harbored plasmids with molecular weights between 1.0 and above 10 kb, detected among the resistant isolates. The resistant isolates were mediated by both chromosomal and plasmid factors. These findings support the use of surveillance data on the emerging patterns of antimicrobial-resistance and plasmid profiles of Vibrio spp. in shrimp farms. The findings from this study can be used to develop a better disease management strategy for shrimp farming.

Draft genome sequence data of Vibrio harveyi VH1 isolated from a diseased tiger grouper, Epinephelus fuscoguttatus, cultured in Malaysia.

Vibriosis accounts for 66.7% of diseases reported in groupers' cultures and affects almost all stages of growth. The disease could lead up to mortality up to 50% mortality, and it was reported that high stocking density and poor fish handling were among the factors that contributed to the disease dissemination. V. harveyi has been reported to be among the causative agent and has caused acute mortality in cage groupers. In this study, we report the genome of V. harveyi VH1 isolated from a diseased tiger grouper Epinephelus fuscoguttatus, reared in a cage farm located in the coastal area of Langkawi.

Draft genome sequence of myo-inositol utilizing Aeromonas dhakensis 1P11S3 isolated from striped catfish (Pangasianodon hypopthalmus) in a local fish farm in Malaysia.

A hypervirulent pathotype of A. hydrophila (vAh) is responsible for Motile Aeromonas Septicemia (MAS) and causes mass mortalities among farmed carp and catfish species in the USA and China. One unique phenotype for vAh among other A. hydrophila strains is the ability to utilize myo-inositol as a sole carbon source. While screening for Aeromonas isolates from diseased fish that can grow using myo-inositol as a sole carbon source, A. dhakensis 1P11S3 was isolated from the spleen of striped catfish (Pangasianodon hypopthalmus) displaying clinical MAS symptoms from a freshwater farm in Malaysia. Aeromonas dhakensis is also an important pathogen in aquaculture, and in this study, we report the draft genome sequence for A. dhakensis 1P11S3, that utilize myo-inositol as a sole carbon source.

Microsatellite Characterization of Malaysian Mahseer (Tor spp.) for Improvement of Broodstock Management and Utilization.

In this study, a mixture of Tor tambra and T. tambroides with unknown genetic background were collected from 11 localities in Malaysia for broodstock development and sperm cryo-banking. This study aims to assess the microsatellite (simple sequence repeat, SSR) variation, genetic diversity, genetic differentiation, level of gene flow, population structure, genetic relatedness and their demographic aspects among these Tor populations, in addition to establishing their SSR profile by employing 22 SSR markers via fragment analysis. Total genomic DNA was extracted from 181 samples (91 cryopreserved milt samples and 90 scale samples of live broodfish). Results showed the Tor spp. collection retained their genetic variation but exhibited excessive homozygosity among individuals within population. Moderate genetic differentiation was shown among the populations, with highly significant (p < 0.001) fixation indices (FST, FIS and FIT). A low gene flow over all loci (Nm 1.548) indicates little genetic variation transfer between populations. The genetic structures of all the populations were successfully resolved into four main clusters by an unweighted pair group method with arithmetic mean (UPGMA) dendrogram generated based on Nei's genetic distances. The population structures based on principal coordinates analysis (PCoA) and the Bayesian model also suggested four distinct clusters following geographical regions and eight closely related populations. This study provided a useful baseline reference for better genetic management and utilization of the Tor spp. stocks in their breeding and conservation programmes.

Comparative Pathogenicity of Aeromonas spp. in Cultured Red Hybrid Tilapia (Oreochromis niloticus × O. mossambicus).

The genus Aeromonas has been recognised as an important pathogenic species in aquaculture that causes motile Aeromonas septicaemia (MAS) or less severe, chronic infections. This study compares the pathogenicity of the different Aeromonas spp. that were previously isolated from freshwater fish with signs of MAS. A total of 124 isolates of Aeromonas spp. were initially screened for the ability to grow on M9 agar with myo-inositol as a sole carbon source, which is a discriminatory phenotype for the hypervirulent A. hydrophila (vAh) pathotype. Subsequently, LD50 of six selected Aeromonas spp. were determined by intraperitoneal injection of bacterial suspension containing 103, 105, and 107 CFU/mL of the respective Aeromonas sp. to red hybrid tilapias. The kidneys, livers and spleens of infected moribund fish were examined for histopathological changes. The screening revealed that only A. dhakensis 1P11S3 was able to grow using myo-inositol as a sole carbon source, and no vAh strains were identified. The LD50-240h of A. dhakensis 1P11S3 was 107 CFU/mL, while the non-myo-inositol utilizing A. dhakensis 4PS2 and A. hydrophila 8TK3 was lower at 105 CFU/mL. Similarly, tilapia challenged with the myo-inositol A. dhakensis 1P11S3 showed significantly (p < 0.05) less severe signs, gross and histopathological lesions, and a lower mortality rate than the non-myo-inositol A. dhakensis 4PS2 and A. hydrophila 8TK3. These findings suggested that myo-inositol utilizing A. dhakensis 1P11S3 was not a hypervirulent Aeromonas sp. under current experimental disease challenge conditions, and that diverse Aeromonas spp. are of concern in aquaculture farmed freshwater fish. Therefore, future study is warranted on genomic level to further elucidate the influence of myo-inositol utilizing ability on the pathogenesis of Aeromonas spp., since this ability correlates with hypervirulence in A. hydrophila strains.

Draft genome sequencing data of a pathogenic Pantoea stewartii subspecies stewartii strain SQT1 causing bronzing disease of jackfruit in Malaysia.

A Gram-negative bacterium, Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii) has been recognized as the causative agent for jackfruit bronzing disease in Malaysia. Here, we report the whole genome sequencing dataset of P. stewartii subsp. stewartii strain SQT1 isolated from local infected jackfruit. The paired-end libraries with an insert size of 350 bp was subjected to the Illumina Hiseq 4000, generating a genome size of 4,783,993 bp with a G+C content of 53.7%. A total protein of 4,671 was identified including virulence factors, resistance factors and secretion systems. Pantoea stewartii subsp. stewartii strain DC283 (NCBI accession no. CP017581.1) was used as a reference genome, where the query hit 72% coverage and average sequencing depth of 68. In total, 28,717 nucleotide polymorphisms, 520 small insertion/deletions and 142 structure variants were identified. The complete genome was deposited at the European Nucleotide Archive under the sample accession number ERP119356 and study accession number PRJEB36196.

Live vaccines against bacterial fish diseases: A review.

Fish diseases are often caused either by bacteria, viruses, fungi, parasites, or a combination of these pathogens. Of these, bacterial fish diseases are considered to be a major problem in the aquaculture industry. Hence, the prevention of such diseases by proper vaccination is one of the integral strategies in fish health management, aimed at reducing the fish mortality rate in the aquaculture farms. Vaccination offers an effective yet low-cost solution to combat the risk of disease in fish farming. An appropriate vaccination regime to prevent bacterial diseases offers a solution against the harmful effects of antibiotic applications. This review discusses the role of live-attenuated vaccine in controlling bacterial diseases and the development of such vaccines and their vaccination strategy. The current achievements and potential applications of live-attenuated and combined vaccines are also highlighted. Vaccine development is concluded to be a demanding process, as it must satisfy the requirements of the aquaculture industry.