Search

The aim of this study was to assess the levels of heavy metals in both wild and farmed tilapia on Lake Kariba in Zambia and to evaluate the impact of intensive fish farming on wild tilapia. Three sites for wild fish (2 distant and 1 proximal to fish farms) and two fish farms were selected. One hundred fish (52 from distant sites; 20 near fish farms; 28 farmed fish) were sampled and muscle tissues excised for analysis of heavy metals (Mg, Fe, Zn, Al, Cu, Se, Co, Mo, As, Cr, V, Ni, Hg, Pb, Li, Cd, and Ag) by acid (HNO3) digestion and ICP-MS. All metals were found to be below the maximum limits (MLs) set by WHO/EU. Essential metals were higher in farmed tilapia, whereas non-essential metals were higher in wild tilapia. Significantly higher levels of essential metals were found in wild fish near the fish farms than those distant from the farms. Estimated weekly intake (EWI) for all metals were less than the provisional tolerable weekly intakes (PTWI). Target hazard quotients (THQ) and Hazard Indices (HI) were

Objectives Aeromonads cause severe diseases in farmed aquatic organisms. Herein, we examined 28 isolates causing disease in farmed aquatic organisms from India (n = 24) and Taiwan (n = 4) to gain insight of their genotypic and phenotypic properties. Results API 20NE biochemical phenotyping showed ≥ 90% similarity classifying all isolates as Aeromonas hydrophila. 16S rRNA genotyping showed ≥ 98% homology among all isolates with A. sobria (NR119044.1ATCC), A. veronii (MK990549.1), A. caviae (NR029252.1) and A. hydrophila (MG984625.1ATCC) and other reference strains. In contrast, gyrB showed a higher intraspecies diversity (≥ 96%) than 16S rRNA delineating the 28 isolates into three groups. Group-I consisted of seven Indian isolates clustered with A. sobria (MK484163.1ATCC), group-II comprised of five Indian and two Taiwanese isolates clustered with A. veronii AF417626.1ATCC while group-III had 11 Indian and three Taiwanese isolates grouped with A. hydrophila (AY987520.1 and DQ519366.1) reference strains. None of our isolates clustered with A. caviae (AJ868400.1ATCC) reference strain. These findings suggest that A. sobria, A. veronii and A. hydrophila could be the etiological agents of diseases observed in farmed fish and soft-shelled turtles (Pelodiscus sinensis) examined in this study. Overall, our findings accentuate the importance of combining phenotyping with genotyping for correct taxonomic classification of Aeromonas spp. in Aquaculture.

Tilapia lake virus (TiLV) is an emerging viral pathogen of tilapiines worldwide in wild and farmed tilapia. TiLV is an orthomyxo-like, negative sense segmented RNA virus, belonging to genus Tilapinevirus, family Amnoonviridae. Here we developed a quantitative real-time PCR (qRT-PCR) assay testing primer sets targeting the 10 segments of TiLV. Sensitivity, specificity, efficiency and reproducibility of these assays were examined. Detection sensitivity was equivalent to 2 TCID

The current study was carried out to determine occurrence and levels of persistent organic pollutants (POPs) in wild and farmed tilapia (Oreochromis niloticus) in Lake Kariba, Zambia, and possible implications for fish health. Concentrations of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyls (PBDEs), hexabromocyclododecane (HBCDD) and perfluoroalkyl substances (PFASs) were determined in liver samples of tilapia. Concentrations of POPs in wild tilapia were in general higher than in farmed tilapia, however, concentrations of DDTs and PCBs in wild tilapia foraging near the fish farms were more in the range of the farmed fish. The highest median ∑DDTs (93 and 81 ng/g lw) were found in wild tilapia from sites 1 and 2, respectively 165 km and 100 km west of the farms. DDE/DDT ratios seem to indicate recent exposure to DDT. The highest median of ∑ 17 PCBs (3.2 ng/g lw) and ∑ 10 PBDEs (8.1 ng/g lw) were found in wild tilapia from site 1 and 2, respectively. The dominating PCB congeners were PCB-118, -138, -153 and -180 and for PBDEs, BDE-47, -154, and -209. Of PFASs, only PFOS, PFDA and PFNA were detected in wild fish with highest median PFOS levels in site 1 (0.66 ng/g ww). The PCB and BDE concentrations in wild and farmed fish were above EQS biota limits set by the EU. This may suggest a risk to the fish species and threaten biodiversity.

The aim of this study was to assess the levels of heavy metals in both wild and farmed tilapia on Lake Kariba in Zambia and to evaluate the impact of intensive fish farming on wild tilapia. Three sites for wild fish (2 distant and 1 proximal to fish farms) and two fish farms were selected. One hundred fish (52 from distant sites; 20 near fish farms; 28 farmed fish) were sampled and muscle tissues excised for analysis of heavy metals (Mg, Fe, Zn, Al, Cu, Se, Co, Mo, As, Cr, V, Ni, Hg, Pb, Li, Cd, and Ag) by acid (HNO

The pathogenesis of Lactococcus garvieae (L. garvieae) was assessed in Nile tilapia (Oreochromis niloticus) following administration by two different routes of infection (intraperitoneal versus immersion), using 180 fish divided into three groups. The first group of fish was injected intraperitoneally (IP) with 3 × 105 colony-forming units (cfu) of L. garvieae; the second group was infected by immersion (IMM) into water containing 9.6 × 105 cfu/ml L. garvieae, and in group 3 (Control), the fish were injected IP with sterile normal saline. Mortalities were recorded daily, and on 3, 5, 7, and 13 days post-infection (dpi), liver, kidney, spleen, brain and eyes were sampled. The level of infection between groups was assessed by number of mortalities that occurred, pathology/histopathology of internal organs, bacterial re-isolation and presence of bacteria in situ determined using immunohistochemistry. A significant difference (p

In this study, fish farmers’ management practices, occurrence, and knowledge of fish diseases in Nyeri County, Kenya, were evaluated. Fish farming management practices for small-scale farmers in Kenya have numerous challenges which have led to disease occurrence and reduced production. Moreover, the impact and association of these challenges to farmers’ knowledge of fish diseases and their burden has not been fully studied. A semistructured questionnaire was used to capture farmers’ biodata, fish species farmed, and farmers’ management practices such as handling of nets, pond fertilization, and disposal of fish waste. Farmers’ knowledge of fish diseases was based on their ability to identify independent and dependent variable indicators. Independent variables included clinical signs, decreased feeding, bulging eyes, floating on water, abdominal swelling, bulging eyes, abnormal skin color, reduced growth, and abnormal swimming with fish death as were the dependent variable. A total of 208 farmers were interviewed and included those of tilapia (134), mixed tilapia and catfish (40), catfish (22), rainbow trout, and five dams under cooperative management. Tilapia was the most kept fish species (66.8%) followed by polyculture of tilapia and catfish (20%) and rainbow trout (2%). Most respondents were male (78.5%) over 51 years of age (50%). Fifty percent of the respondents had secondary school education. There was a significant association between deaths and sharing of nets in Kieni East subcounty ( p = 0.0049 , chi-square), while on-farm fish waste disposing appeared to cause higher deaths compared to burning of the waste although not statistically significant ( p = 0.13 ). Few respondents observed decreased feed uptake (

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis, a disease of salmonid responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in Kenya. In this study, rainbow trout fry and growers from two farms in Nyeri County were screened for IHNV. Whole fry (n = 4 from each farm) and kidney samples from growers (n = 15 and n = 6 from the two farms, respectively) were collected and preserved for cell culture examination or PCR analysis. Screening of samples was done by PCR followed by sequencing of the glycoprotein gene of the virus. Demonstration of the virus was done by propagation in EPC cells followed by the indirect fluorescence antibody test (IFAT). The results revealed the presence of IHNV at low prevalence of 0.1 and 0.4 for the two farms. The virus was confirmed both by IFAT and by partial sequencing of the G gene. Phylogenetic analysis revealed that the Kenyan isolates were identical to those of the J genogroup found mostly in Asia. The findings have implications for biosecurity measures and import regulations for the Kenyan rainbow trout industry.

The biological effects of gamma radiation may exert damage beyond that of the individual through its deleterious effects on reproductive function. Impaired reproductive performance can result in reduced population size over consecutive generations. In a continued effort to investigate reproductive and heritable effects of ionizing radiation, we recently demonstrated adverse effects and genomic instability in progeny of parents exposed to gamma radiation. In the present study, genotoxicity and effects on the reproduction following subchronic exposure during a gametogenesis cycle to 60Co gamma radiation (27 days, 8.7 and 53 mGy/h, total doses 5.2 and 31 Gy) were investigated in the adult wild-type zebrafish (Danio rerio). A significant reduction in embryo production was observed one month after exposure in the 53 mGy/h exposure group compared to control and 8.7 mGy/h. One year later, embryo production was significantly lower in the 53 mGy/h group compared only to control, with observed sterility, accompanied by a regression of reproductive organs in 100% of the fish 1.5 years after exposure. Histopathological examinations revealed no significant changes in the testis in the 8.7 mGy/h group, while in 62.5% of females exposed to this dose rate the oogenesis was found to be only at the early previtellogenic stage. The DNA damage determined in whole blood, 1.5 years after irradiation, using a high throughput Comet assay, was significantly higher in the exposed groups (1.2 and 3-fold increase in 8.7 and 53 mGy/h females respectively; 3-fold and 2-fold increase in 8.7 and 53 mGy/h males respectively) compared to controls. A significantly higher number of micronuclei (4–5%) was found in erythrocytes of both the 8.7 and 53 mGy/h fish compared to controls. This study shows that gamma radiation at a dose rate of ≥ 8.7 mGy/h during gametogenesis causes adverse reproductive effects and persistent genotoxicity (DNA damage and increased micronuclei) in adult zebrafish. Gamma irradiation during gametogenesis in young adult zebrafish causes persistent genotoxicity and adverse reproductive effects

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120-2,343 bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.

Gamma radiation represents a potential health risk to aquatic and terrestrial biota, due to its ability to ionize atoms and molecules in living tissues. The effects of exposure to 60Co gamma radiation in zebrafish (Danio rerio) were studied during two sensitive life stages: gametogenesis (F0: 53 and 8.7mGy/h for 27 days, total doses 31 and 5.2Gy) and embryogenesis (9.6mGy/h for 65h; total dose 0.62Gy). Progeny of F0 exposed to 53mGy/h showed 100% mortality occurring at the gastrulation stage corresponding to 8h post fertilization (hpf). Control and F0 fish exposed to 8.7mGy/h were used to create four lines in the first filial generation (F1): control, G line (irradiated during parental gametogenesis), E line (irradiated during embryogenesis) and GE line (irradiated during parental gametogenesis and embryogenesis). A statistically significant cumulative mortality of GE larva (9.3%) compared to controls was found at 96 hpf. E line embryos hatched significantly earlier compared to controls, G and GE (48-72 hpf). The deformity frequency was higher in G and GE, but not E line compared to controls at 72 hpf. One month after parental irradiation, the formation of reactive oxygen species (ROS) was increased in the G line, but did not significantly differ from controls one year after parental irradiation, while at the same time point it was significantly increased in the directly exposed E and GE lines from 60 to 120 hpf. Lipid peroxidation (LPO) was significantly increased in the G line one year after parental irradiation, while significant increase in DNA damage was detected in both the G and GE compared to controls and E line at 72 hpf. Radiation-induced bystander effects, triggered by culture media from tissue explants and observed as influx of Ca2+ ions through the cellular membrane of the reporter cells, were significantly increased in 72 hpf G line progeny one month after irradiation of the parents. One year after parental irradiation, the bystander effects were increased in the E line compared to controls, but not in progeny of irradiated parents (G and GE lines). Overall, this study showed that irradiation of parents can result in multigenerational oxidative stress and genomic instability in irradiated (GE) and non-irradiated (G) progeny of irradiated parents, including increases in ROS formation, LPO, DNA damage and bystander effects. The results therefore highlight the necessity for multi- and transgenerational studies to assess the environmental impact of gamma radiation.

Tilapia (Oreochromis niloticus) farming on Lake Kariba, Zambia has been practiced since early 2000s. In recent years, the industry has experienced disease outbreaks with clinical signs consistent with streptococcus-like infections. Affected fish were growers weighing approximately 200 g or above from farms designated A and B, reared in cages on the lake. The objective of this study was to identify and characterize the causative bacteria of these outbreaks. A total of eighty-six diseased fish were sampled between 2014 and 2016. Bacteria were cultured from internal organs and were characterized by biochemical tests, PCR and sequencing of the 16S rRNA gene or 16S 23S intergenic spacer region. The results showed the prevalence of Lactococcus garvieae at farm A in 2015 and 2016 outbreaks (15/20 and 23/40, respectively) and at farm B (6/20) in 2016. Infection of naive fish with one isolate of L. garvieae recovered from the outbreaks resulted in clinical signs and mortalities akin of what had been observed during the outbreaks, confirming Koch's postulates. In the 2014 outbreaks, the low prevalence of Streptococcus agalactiae (4/15 and 2/11 respectively at farms A and B) and S. iniae (2/15) at farm A suggests that other pathogens were likely involved in the disease outbreak observed.

The autovaccine was produced in-house using a bacterial isolate from a diseased fish from the target farm. Three groups of 150 fish each were injected with either 1) an oil-adjuvanted, inactivated whole cell autovaccine, 2) adjuvant only or 3) PBS (negative control). Approximately 660 degree days post vaccination, the fish were challenged with 9x105 cfu bacteria/fish by intraperitoneal injection and monitored for a further 28 days. Protection against infections was measured by lack of/reduced bacterial loads both by bacterial re-isolation and immunohistochemistry as well as absence of clinical signs/pathology. Significantly less L. garvieae (p

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH4Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH4Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH4Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

This study aimed to assess antimicrobial susceptibility of members of the family Flavobacteriaceae isolated from Nile tilapia (Oreochromis niloticus). Antimicrobial susceptibility of 67 Flavobacteriaceae isolates originating mainly from ponds and Lake Victoria against 19 antimicrobial agents was determined by the broth micro dilution method. Overall, most isolates were susceptible to enrofloxacin (97%; MIC90 2μg/ml) followed by novobiocin (85%, MIC90, 4μg/ml) and the aminoglycoside streptomycin (85%; MIC90, 128μg/ml). Some isolates were also susceptible towards trimethoprim/sulfamethoxazole (77.6%), neomycin and florfenicol both at 62.7%. Susceptibility levels were low for tylosin tartrate (32.8%), clindamycin and sulphathiazole both at (23.9%), ceftiofur (6%), spectinomycin (6%) and tetracyclines/ oxtetracyclines (4.5%). In contrast, β-Lactams (amoxicillin, penicillin), gentamycin and erythromycin exhibited very poor activity against Flavobacteriaceae isolates. The extent of antimicrobial susceptibility did not vary significantly among isolates from farmed and wild fish isolates (P > 0.01). The highest Multiple Antimicrobial Resistance (MAR) index was observed in Chryseobacterium indologenes (0.89) and the lowest in C. indicum isolates (0.32). Our results indicate that most of Flavobacteriaceae isolates are multidrug resistance, and this may be associated with intrinsic resistance mechanisms to a broad range of antimicrobial agents. However, the need remains to carryout in-depth study to understand better the underlying genetic mechanisms given that the magnitude and trend for susceptibility was comparable between isolates from aquaculture and fisheries. The findings from this study give us insight into appropriate choice of antimicrobial agents for effective treatment of infections caused by these isolates.

Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 feed manufacturing plants in Nyeri, Kenya for aflatoxin analysis using competitive enzyme-linked immunosorbent assay. Fish were sampled from 12 farms for gross and microscopic pathological examination. Eighty-four percent of feeds sampled tested positive for aflatoxins, ranging from 1.8 to 39.7 &micro, g/kg with a mean of 7.0 &plusmn, 8.3 &micro, g/kg and the median of 3.6 &micro, g/kg. Fifteen feeds (18.5%) had aflatoxins above the maximum allowable level in Kenya of 10 &micro, g/kg. Homemade and tilapia feeds had significantly higher aflatoxin levels than commercial and trout feeds. Feeds containing maize bran and fish meal had significantly higher aflatoxin levels than those without these ingredients. Five trout farms (41.7%) had fish with swollen abdomens, and enlarged livers with white or yellow nodules, which microscopically had large dark basophilic hepatic cells with hyperchromatic nuclei in irregular cords. In conclusion, aflatoxin contamination of fish feeds is prevalent in Nyeri, and may be the cause of adverse health effects in fish in this region.

The genus Edwardsiella is one of the major causes of fish diseases globally. Herein, we examined 37 isolates from ten different fish species from India, South Korea and Taiwan to gain insight into their phenotypic and genotypic properties, of which 30 were characterized as E. tarda with phenotypic homology estimated at 85.71% based on API-20E biochemical tests. Genotyping using 16S rRNA put all isolates together with E. anguillarum, E. hoshinae, E. tarda, E. piscicida and E. ictaluri reference strains in a monophyletic group. In contrast, the gyrB phylogenetic tree clearly separated E. ictaluri, E. tarda and E. hoshinae reference strains from our isolates and put our isolates into two groups with group I being homologous with the E. anguillarum reference strain while group II was homologous with the E. piscicida reference strain. Hence, our findings point to E. piscicida and E. anguillarum as species infecting different fish species in Asia. Homology of the ompW protein suggested that strains with broad protective coverage could be identified as vaccine candidates. This study underscores the importance of combining genotyping with phenotyping for valid species classification. In addition, it accentuates the importance of phylogenetic comparison of bacterial antigens for identification of potential vaccine candidates.

One impediment to the successful oral vaccination in fish is the hostile stomach environment that antigens must cross. Furthermore, uptake of antigens from the gut to systemic distribution is required for induction of systemic immunity, the dynamics of which are poorly understood. In the present study, groups of Atlantic salmon parr were intubated with live or inactivated infectious pancreatic necrosis virus (IPNV), either orally or anally. At 1, 24 and 72 h post infection (p.i.), the fish were sacrificed. Serum was used for assessing IPNV by ELISA, while formalin-fixed head-kidney, spleen, liver and intestine tissues were used for the demonstration of antigens by immunohistochemistry. Both live and inactivated IPNV antigens were observed in enterocytes of the intestines and in immune cells of the head-kidneys and spleens of all groups. In the liver, no antigens were observed in any of the groups. Significantly higher serum antigen OD values (p < 0.04) were observed in orally- compared to anally-intubated fish. By contrast, no difference (p = 0.05) was observed in tissue antigens between these groups by immunohistochemistry. No significant difference (p = 0.05) in serum antigens was observed between groups intubated with live and inactivated IPNV, while in tissues, significantly more antigens (p < 0.03) were observe in the latter compared to the former. These findings demonstrate that both live and inactivated IPNV are taken up by enterocytes in the intestines of Atlantic salmon, likely by receptor-mediated mechanisms. Higher IPNV uptake by the oral compared to anal route suggests that both the anterior and posterior intestines are important for the uptake of the virus and that IPNV is resistant to gastric degradation of the Atlantic salmon stomach.

The present study was conducted to explore the occurrence of Flavobacteriaceae in wild Nile Tilapia Oreochromis niloticus (n = 108) collected from Lake Victoria and farmed Nile Tilapia (n = 187) collected from 12 ponds in the Morogoro region of Tanzania. The size of the ponds surveyed ranged from 130 to 150 m2 . Pond parameters and fish morphometric data were recorded during sampling. In total, 67 Flavobacterium-like isolates (n = 44 from farmed fish; n = 23 from wild fish) were identified on the basis of colony morphology and biochemical tests. Sequences from the 16S ribosomal RNA (rRNA) gene revealed that all 67 isolates belonged to the genera Flavobacterium and Chryseobacterium. Based on 16S rRNA nucleotide identity, 26 isolates showed high similarity with C. indologenes (99-100% identity), 16 showed similarity to C. joostei (98-99.9%), and 17 were similar to diverse species of Chryseobacterium (97-99%). Three isolates were similar to F. aquatile and three were similar to F. indicum, with 99-100% nucleotide identity in both cases, and two isolates were similar to F. oryzae (99-100% identity). The findings obtained in this study provide a baseline for future studies and contribute to an understanding of the threats presented by the aquatic Flavobacteriaceae reservoir toward the development of healthy fish farming in Tanzania. Such knowledge is vital for the development of a sustainable aquaculture industry in Tanzania that will contribute to increased food security.

The global expansion of the aquaculture industry has brought with it a corresponding increase of novel viruses infecting different aquatic organisms. These emerging viral pathogens have proved to be a challenge to the use of traditional cell-cultures and immunoassays for identification of new viruses especially in situations where the novel viruses are unculturable and no antibodies exist for their identification. Viral metagenomics has the potential to identify novel viruses without prior knowledge of their genomic sequence data and may provide a solution for the study of unculturable viruses. This review provides a synopsis on the contribution of viral metagenomics to the discovery of viruses infecting different aquatic organisms as well as its potential role in viral diagnostics. High throughput Next Generation sequencing (NGS) and library construction used in metagenomic projects have simplified the task of generating complete viral genomes unlike the challenge faced in traditional methods that use multiple primers targeted at different segments and VPs to generate the entire genome of a novel virus. In terms of diagnostics, studies carried out this far show that viral metagenomics has the potential to serve as a multifaceted tool able to study and identify etiological agents of single infections, co-infections, tissue tropism, profiling viral infections of different aquatic organisms, epidemiological monitoring of disease prevalence, evolutionary phylogenetic analyses, and the study of genomic diversity in quasispecies viruses. With sequencing technologies and bioinformatics analytical tools becoming cheaper and easier, we anticipate that metagenomics will soon become a routine tool for the discovery, study, and identification of novel pathogens including viruses to enable timely disease control for emerging diseases in aquaculture.

Inactivated whole virus vaccines represent the majority of commercial preparations used to prevent infectious pancreatic necrosis (IPN) in salmonids today. The production of these vaccines requires high virus concentrations that are resource-demanding. In this study, we describe the cultivation of high yields of IPN virus in Asian Grouper strain K (AGK) cells. The mechanism by which this is achieved was investigated by comparison with commonly used salmonid cell lines (RTG-2 and CHSE-214 cells). The cells were counted before and sequentially after infection. Thereafter, protein shut down, virus yields and apoptosis were assessed. The effects of poly(I:C) pre-treatment and Mx expression on IPNV concentrations were examined and the results show that high virus yields were associated with high cell numbers per unit volume, delayed cell death and apoptosis in AGK cells while the opposite was observed in RTG-2 cells. Poly(I:C) treatment and Mx expression resulted in a dose-dependent inhibition of virus multiplication. The production capacity of AGK and CHSE-214 cells were compared and higher split ratio and shorter split interval of AGK cells documents dramatic differences in virus antigen production capacity. Collectively, the results suggest that high cell numbers and prolonged survival of AGK cells are responsible for the superior virus yields over RTG-2 and higher split ratio/shorter split interval makes AGK superior over CHSE cells.

Infectious pancreatic necrosis virus (IPNV) is the causative agent of IPN, an important disease of salmonids. IPNV infections result in either sub-clinical or overt disease and the basis of this difference is not well-understood. The objective of the present study was to determine the VP2 gene of the virus associated with the different forms of clinical manifestation. Groups of Atlantic salmon (Salmo salar L.) reared in farms located in different IPN disease pressures were monitored from brood stock until grow-out over a 3 year period. Hatcheries A1 and B1 as well as cooperating seawater farms were located in a low disease risk area while hatcheries A2 and B2 as well as their cooperating seawater farms were in high IPN risk areas. Samples including eggs, milt, whole fry, kidney depending on the stage of production were collected during outbreaks or in apparently healthy populations where no outbreaks occurred. The virus was re-isolated in CHSE cells and the VP2 gene amplified by RT-PCR followed by sequencing. During the freshwater stage, there were no disease outbreaks at hatcheries A1, A2, and B1 (except in one fish group that originated from hatchery B2), although IPNV was isolated from some of the fish groups at all 3 hatcheries. By contrast, all fish groups at hatchery B2 suffered IPN outbreaks. In seawater, only groups of fish originating from hatchery A1 had no IPN outbreaks albeit virus being isolated from the fish. On the other hand, fish originating from hatcheries A2, B1, and B2 experienced outbreaks in seawater. The VP2 amino acid fingerprint of the virus associated with subclinical infections from A1 and co-operating seawater sites was V64A137P217T221A247N252S281D282E319. By contrast, all virus isolates associated with clinical infections had the motif I64T137T217A221T247V252T281N282A319, where underlined amino acids represent the avirulent and highly virulent motif, respectively. Phylogenetic analysis of amino acid sequences showed 2 clades, one of isolates associated with subclinical infections (from A1 and cooperating seawater farms) and the other of isolates from fish with overt disease (all other sites). Furthermore, the clustering pattern of isolates suggests more circulation of virus within fish groups rather than between them.

An anthrax outbreak occurred in November 2010 in five villages of Sesheke district in Western Zambia. Control measures and data collection was carried out immediately the outbreak was reported. The prevalence of the disease in cattle was estimated at 7.4% (45/609) while the average herd size of infected cattle in affected villages was estimated at 121.8 (95% CI 48.8-194.8). Individual mortality per herd varied between 1.70% (3/179) and 20.25% (6/79). The relative risk of infection of cattle in the five affected villages varied between 0.18 (95% CI 0.4-5.7) and 3.7 (95% CI 1.99-6.68). In humans, the disease only affected three people and was characterized by cutaneous carbuncles. The ratio of infected persons per number of infected carcasses varied between 1:37 and 1:49 in affected villages while the overall ratio of people at risk to the number of carcasses was 42:1 indicating that despite availability of a large number of carcasses, human contact with infected carcasses was low. The findings of this study underline the importance of timely disease control measures in reducing the risk of human infections to anthrax in the face of an outbreak.

Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime–boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log 10 reduction of virus titer in heart for the IWV group. Prime–boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime–boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines.

Two strains of IPNV made by reverse genetics on the Norwegian Sp strain NVI-015 (GenBank AY379740) backbone encoding the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs were used to prepare inactivated whole virus (IWV), nanoparticle vaccines with whole virus, Escherichia coli subunit encoding truncated VP2-TA and VP2-PT, VP2-TA and VP2-PT fusion antigens with putative translocating domains of Pseudomonas aeruginosa exotoxin, and plasmid DNA encoding segment A of the TA strain. Post challenge survival percentages (PCSP) showed that IWV vaccines conferred highest protection (PCSP=42-53) while nanoparticle, sub-unit recombinant and DNA vaccines fell short of the IWV vaccines in Atlantic salmon (Salmo salar L.) postsmolts challenged with the highly virulent Sp strain NVI-015 (TA strain) of IPNV after 560 degree days post vaccination. Antibody levels induced by these vaccines did not show antigenic differences between the virulent and avirulent motifs for vaccines made with the same antigen dose and delivery system after 8 weeks post vaccination. Our findings show that fish vaccinated with less potent vaccines comprising of nanoparticle, DNA and recombinant vaccines got infected much earlier and yielded to higher infection rates than fish vaccinated with IWV vaccines that were highly potent. Ability of the virulent (T(217)A(221)) and avirulent (P(217)T(221)) motifs to limit establishment of infection showed equal protection for vaccines made of the same antigen dose and delivery systems. Prevention of tissue damage linked to viral infection was eminent in the more potent vaccines than the less protective ones. Hence, there still remains the challenge of developing highly efficacious vaccines with the ability to eliminate the post challenge carrier state in IPNV vaccinology.

A serolocigical survey was conducted for the detection of Theileria parva antibodies in 176 African buffaloes (Syncerus caffer) sampled between 1996 and 2005 in livestock-wildlife interface areas of Zambia. Rhipicephalus appendiculatus, Rhipicephalus species, and Amblyomma variegatum were the most abundant tick species identified on buffaloes. T. parva sero-positives were reported in buffaloes sampled from game management areas at Mlanga and Nanzhila bordering the Kafue National Parks and in the Lochnivar National Park while buffaloes sampled from Lower Zambezi National Park were sero-negative. Given that Game Management Areas serve as interface areas that permit the co-existence of livestock and wildlife in similar ecological habitats our findings suggest that buffaloes could play a significant role in the epidemiology of theileriosis in livestock-wildlife interface areas. Thus far, the disease has only been reported in livestock and is herein being reported in the African buffalo for the first time in Zambia.

The inflammatory response of Atlantic cod ( Gadus morhua L.) following vaccination with oil-based vaccines has not been previously characterized in any detail. In this study, groups of Atlantic cod were intraperitoneally injected with commercial oil-adjuvanted vaccines ALPHA JECT 3000 (AJ 3000) and AJ 6-2. A water-based vaccine ALPHA MARINE™ Vibrio (AVM), an experimental liposome vaccine and physiological saline (placebo) were also included for comparison. Histopathological changes at the injection sites were evaluated semi-quantitatively at 1, 2, 4, 8, 12, 16, 20 and 25 weeks post-vaccination (p.v.), parallel with the examination of vaccine antigen retention. Gross intra-abdominal lesions were only examined at 12 and 25 weeks. The results show that the onset of inflammation in all vaccinated groups was rapid to develop, with intense cellular infiltrations predominated by mononuclear cells especially in groups injected by oil-based vaccines. Inflammation induced by AVM and liposome vaccines resolved within 12 weeks. In contrast, oil-adjuvanted vaccines produced mild, persistent but ultimately decreasing reactions. Persistent antigens were observed in oil-based and liposome vaccines. The results show that the cod inflammatory response is similar to other bony fish species. The findings also suggest that cod has an efficient innate immune system that is able to rapidly remove or sequester antigens from the injection site leading to the down-regulation of inflammation. Oil-adjuvanted vaccines appear to be well-tolerated by this species and show promise as a possible approach for disease control.

The use of oral vaccination in finfish has lagged behind injectable vaccines for a long time as oral vaccines fall short of injection vaccines in conferring protective immunity. Biodegradable polymeric nanoparticles (NPs) have shown potential to serve as antigen delivery systems for oral vaccines. In this study the recombinant outer membrane protein A (rOmpA) of Edwardsiella tarda was encapsulated in chitosan NPs (NP-rOmpA) and used for oral vaccination of Labeo fimbriatus. The rOmpA purity was 85%, nanodiameter

Birnaviruses of aquatic organisms constitute small, nonenveloped icosahedral viruses with bisegmented, double-stranded RNA (dsRNA) genomes, and currently, two genera infect aquatic organisms; Aquabirnavirus and Blosnarvirus. Among aquabirnaviruses, the infectious pancreatic necrosis virus (IPNV) is the type species and causes severe diseases in salmonids, while there are a number of other aquabirnaviruses that infect other aquatic organisms. Blotched snakehead virus is the type strain of the Blosnarvirus genus, infecting blotched snakehead (Channa maculata). The genome of birnaviruses is organized into two segments, A and B. Segment A encodes the structural proteins VP2 and VP2 plus the virus protease (VP4) and a nonstructural protein, VP5. Segment B encodes the viral polymerase. The virulence traits of IPNV have been well defined, and for strains infecting Atlantic salmon, highly virulent strains carry defined amino acids in defined residues of the VP2 protein. Vaccination and selective breeding in Atlantic salmon have contributed to reducing the impact of IPNV infection in salmonid aquaculture worldwide.

Aquaculture is the fastest growing food-producing sector globally and, with intensification of the production, viral diseases have emerged. representing a challenge to sustainable development. Thus disease control and prevention is an imperative. This will depend on viral properties, the environment, reservoir hosts, susceptibility of farmed species, transmission dynamics and viral pathogenicity. Most of these parameters are not known for aquatic viruses, which preclude deployment of optimal control strategies. Current prevention and control targets host species utilized in aquaculture, and is likely to change as additional species are being exploited. With this come new challenges because pathogen exchange between aquatic species is difficult or impossible to predict in principle. Here we introduce current preventive and control measures, including vaccination principles that have been and are explored to reduce the prevalence and impact of viral infections in aquaculture.

The oral route offers the most attractive approach of immunization of fish for a number of reasons: the ease of administration of antigens, it is less stressful than parenteral delivery and in principle, it is applicable to small and large sized fish; it also provides a procedure for oral boosting during grow-out periods in cages or ponds. There are, however, not many commercial vaccines available at the moment due to lack of efficacy and challenges associated with production of large quantities of antigens. These are required to stimulate an effective immune response locally and systemically, and need to be protected against degradation before they reach the sites where immune induction occurs. The hostile stomach environment is believed to be particularly important with regard to degradation of antigens in certain species. There is also a poor understanding about the requirements for proper immune induction following oral administration on one side, and the potential for induction of tolerance on the other. To what extent primary immunization via the oral route will elicit both local and systemic responses is not understood in detail. Furthermore, to what extent parenteral delivery will protect mucosal/gut surfaces and vice-versa is also not fully understood. We review the work that has been done on the subject and discuss it in light of recent advances that include mass production of antigens, including the use of plant systems. Different encapsulation techniques that have been developed in the quest to protect antigens against digestive degradation, as well as to target them for appropriate immune induction are also highlighted.

Mucosal organs are principle portals of entry for microbial invasion and as such developing protective vaccines against these pathogens can serve as a first line of defense against infections. In general, all mucosal organs in finfish are covered by a layer of mucus whose main function is not only to prevent pathogen attachment by being continuously secreted and sloughing-off but it serves as a vehicle for antimicrobial compounds, complement, and immunoglobulins that degrade, opsonize, and neutralize invading pathogens on mucosal surfaces. In addition, all mucosal organs in finfish possess antigen-presenting cells (APCs) that activate cells of the adaptive immune system to generate long-lasting protective immune responses. The functional activities of APCs are orchestrated by a vast array of proinflammatory cytokines and chemokines found in all mucosal organs. The adaptive immune system in mucosal organs is made of humoral immune responses that are able to neutralize invading pathogens as well as cellular-mediated immune responses whose kinetics are comparable to those induced by parenteral vaccines. In general, finfish mucosal immune system has the capacity to serve as the first-line defense mechanism against microbial invasion as well as being responsive to vaccination.