Persistent organic pollutants (POPs) and per- and polyfluoroalkyl substances (PFASs) in liver from wild and farmed tilapia (Oreochromis niloticus) from Lake Kariba, Zambia: Levels and geographic trends and considerations in relation to environmental quality standards (EQSs).

The current study was carried out to investigate a wide variety of persistent organic pollutants (POPs) in wild and farmed tilapia (Oreochromis niloticus) in Lake Kariba, Zambia, and assess levels of POPs in relation to Environmental Quality Standards (EQSs). Concentrations of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), polybrominated diphenyls (PBDEs), and perfluoroalkyl substances (PFASs) were determined in liver samples of tilapia. PFASs compounds PFOS, PFDA and PFNA were only detected in wild fish, with the highest median PFOS levels in site 1 (0.66 ng/g ww). Concentrations of POPs were in general highest in wild tilapia. 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 fish farms. Lower DDE/DDT ratios in sites 1 and 3 may indicate relatively recent exposure to DDT. The highest median of ∑17PCBs (3.2 ng/g lw) and ∑10PBDEs (8.1 ng/g lw) were found in wild tilapia from sites 1 and 2, respectively. The dominating PCB congeners were PCB-118, -138, -153 and -180 and for PBDEs, BDE-47, -154, and -209. In 78% of wild fish and 8% of farmed fish ∑6PBDE concentrations were above EQSbiota limits set by the EU. This warrants further studies.

Lactococcus garvieae isolated from Lake Kariba (Zambia) has low invasive potential in Nile tilapia (Oreochromis niloticus).

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 < .0001) was observed between L. garvieae re-isolation from tilapia following administration by IP injection and IMM. Similarly, more clinical signs and mortalities (p < .001) were observed in the IP group compared to the IMM group where no mortalities were observed. These findings suggest that L. garvieae has a low invasive potential in Nile tilapia with intact skin/external barriers and highlights the importance of maintaining fish without cuts or abrasions under field conditions.

First detection and isolation of infectious haematopoietic necrosis virus from farmed rainbow trout in Nyeri County, Kenya.

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.

Genotypic and phenotypic characterization of Edwardsiella isolates from different fish species and geographical areas in Asia: Implications for vaccine development.

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.

Isolation and Characterization of Flavobacteriaceae from Farmed and Wild Nile Tilapia in Tanzania.

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.

Multilocus sequence analysis revealed a high genotypic diversity of Aeromonas hydrophila infecting fish in Uganda.

A multilocus sequence analysis (MLSA) was carried out to delineate Aeromonas hydrophila from fish in Uganda. Five housekeeping genes including recA, gyrB, metG, gltA and pps; and the 16S rRNA gene were amplified and sequenced from a total of nine A. hydrophila isolates. The obtained sequences were edited, and consensus sequences generated for each gene locus. The housekeeping gene sequences were concatenated and phylogenetic analysis performed in MEGA version 7.0.2. Pairwise distances ranged from 0.000 to 0.118, highest within the gltA gene locus and lowest within the 16S rRNA gene. The average evolutionary diversity within isolates from the same source ranged between 0.002 and 0.037, and it was 0.033 between the different sources. Similar tree topologies were obtained from the different gene loci with recA, metG and gyrB being more consistent in discriminating isolates according to sources while the 16S rRNA gene had the lowest resolution. The concatenated tree had the highest discriminatory power. This study revealed that A. hydrophila strains infecting fish in Uganda are of diverse genotypes suggesting different sources of infection in a given outbreak. Efforts to minimize spread of the bacteria across sources should be emphasized to control infections of mixed genotypes.

Gamma irradiation during gametogenesis in young adult zebrafish causes persistent genotoxicity and adverse reproductive effects.

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.

Parental gamma irradiation induces reprotoxic effects accompanied by genomic instability in zebrafish (Danio rerio) embryos.

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.

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L).

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 107.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon.

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.

Assessment of heavy metals in wild and farmed tilapia (Oreochromis niloticus) on Lake Kariba, Zambia: implications for human and fish health.

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 <1, indicating no health risks from a lifetime of fish consumption. Selenium Health Benefit Value (HBVSe) was positive for all locations, indicating protective effects of selenium against mercury in fish. Total cancer risk (CR) due to As, Cr, Cd, Ni and Pb was less than 1 × 10-4, indicating less than 1 in 10,000 carcinogenic risk from a lifetime consumption of tilapia from Lake Kariba. Hg levels (0.021 mg/kg) in wild tilapia at site 1 were higher than the Environmental quality standard (EQS = 0.020 mg/kg) set by EU, indicating possible risk of adverse effects to fish. Except for Hg, levels of metals in fish were safe for human consumption and had no adverse effects on fish.

Efficiency, sensitivity and specificity of a quantitative real-time PCR assay for Tilapia Lake virus (TiLV).

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 TCID50/ml when tested on supernatants from cell culture-grown TiLV. Specificity tests showed that all primer sets amplified their respective TiLV segments, and standard curves showed linear correlation of R2 > 0.998 and amplification efficiencies between 93 % and 98 %. Intra- and inter-assay coefficients of variation (CV %) were in the range of 0.0 %- 2.6 % and 0.0 %- 5.9 %, respectively. Sensitivity tests showed that primer sets targeting segments 1, 2, 3 and 4 had the highest detection sensitivities (100.301 TCID50/ml). The qRT-PCR used for detection of viral genome in TiLV infected organs gave virus titers equivalent to 3.80 log10, 3.94 log10 and 3.52 log10 TCID50/ml for brain, kidney and liver tissues, respectively as calculated on the basis of Ct values. These findings suggest that primer optimization for qPCR should not only focus on attaining high amplification efficiency but also sensitivity comparison of primer sets targeting different viral segments in order to develop a method with the highest sensitivity.

Sequence similarities of the capsid gene of Chilean and European isolates of infectious pancreatic necrosis virus point towards a common origin.

The Chilean salmonid industry was developed by importing breeding materials, a practice still in effect due to deficits in the national supply of roe. Importation of breeding materials is often associated with the transmission of pathogens. The objectives of this study were to compare the infectious pancreatic necrosis virus (IPNV) isolates from Chile to those of European origin and to determine the diversity of the Chilean IPNV. The VP2 genes of IPNV from Chilean fish (whose eggs originated from Scotland, Iceland and Norway) were compared to isolates from fish in Norway and Ireland. The results show that the isolates are identical (97-99%) and cluster into one genogroup. Our findings support previous reports of association between the trade-in breeding materials and transmission of pathogens. Furthermore, our results demonstrate the genotypic diversity of Chilean IPNV isolates. These findings have important implications for IPNV disease diagnosis and control in Chile.

Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

Challenges and Solutions to Viral Diseases of Finfish in Marine Aquaculture.

Aquaculture is the fastest food-producing sector in the world, accounting for one-third of global food production. As is the case with all intensive farming systems, increase in infectious diseases has adversely impacted the growth of marine fish farming worldwide. Viral diseases cause high economic losses in marine aquaculture. We provide an overview of the major challenges limiting the control and prevention of viral diseases in marine fish farming, as well as highlight potential solutions. The major challenges include increase in the number of emerging viral diseases, wild reservoirs, migratory species, anthropogenic activities, limitations in diagnostic tools and expertise, transportation of virus contaminated ballast water, and international trade. The proposed solutions to these problems include developing biosecurity policies at global and national levels, implementation of biosecurity measures, vaccine development, use of antiviral drugs and probiotics to combat viral infections, selective breeding of disease-resistant fish, use of improved diagnostic tools, disease surveillance, as well as promoting the use of good husbandry and management practices. A multifaceted approach combining several control strategies would provide more effective long-lasting solutions to reduction in viral infections in marine aquaculture than using a single disease control approach like vaccination alone.

Management Practices, Farmers' Knowledge of Diseased Fish, and Their Occurrence in Fish Farms in Nyeri County, Kenya.

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 (<20%) and poor growth. Fifty-seven percent of farmers reported mortalities. Fish poor growth, floating in water, and management practices in subcounties had significant effect on fish deaths. The farmers had knowledge of signs of diseased fish, but there was paucity of knowing the specific causes of disease. Farmers need to be empowered on best aquaculture husbandry to avoid disease transmission and specific fish disease signs to enhance proper reporting of disease for subsequent mitigation measures.

Aeromonas species obtained from different farmed aquatic species in India and Taiwan show high phenotypic relatedness despite species diversity.

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.

High gene expression of inflammatory markers and IL-17A correlates with severity of injection site reactions of Atlantic salmon vaccinated with oil-adjuvanted vaccines.

BACKGROUND: Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but the underlying profile of inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices.Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscosa antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response. RESULTS: Granulomatous lesions were observed in all vaccinated fish. The presence of severe granulomas was associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, TGF-beta (p = 0.001), IL-17A (p = 0.007) and its receptor (IL-17AR) (p = 0.009) representing TH17 were significantly up-regulated in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of T(H)1 T cell lineage (IFN-gamma, CD4) or T(H)2 (GATA-3) responses were differentially expressed. CONCLUSIONS: Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon have the profile of strong expression of genes related to innate immune responses. The expression of TGF-beta, IL-17A and its receptor suggests an involvement of T(H)17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating also a T(H)2-profile. To what extent the expression of IL-17A and its receptor reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon when vaccinated with oil-based vaccines.

Trypanosoma brucei Infection in asymptomatic greater Kudus (Tragelaphus strepsiceros) on a game ranch in Zambia.

Trypomastogotes of Trypanosoma brucei were detected from 4 asymptomatic kudus (Tragelaphus strepsiceros) on a game ranch located approximately 45 km north east of Lusaka, Zambia. Blood smears examined from 14 wildlife species comprising of the impala (Aepyceros melampus), Kafue lechwe (kobus leche kafuensis), sable antelope (Hippotragus niger), tsessebe (Damaliscus lunatus), warthog (Phacochoerus aethiopicus), puku (Kobus vardoni), zebra (Equus burchelli), waterbuck (Kobus ellipsiprymnus), bushbuck (Tragelaphus scriptus), reedbuck (Redunca arundinum), wilderbeest (Connochaetes taurinus), hartebeest (Alcephelus lichtensteini), African buffalo (Syncerus caffer), and kudu (Tragelaphus strepsiceros) showed that only the kudu had T. brucei. Although game ranching has emerged to be a successful ex-situ conservation strategy aimed at saving the declining wildlife population in the National Parks, our findings suggest that it has the potential of aiding the re-distribution of animal diseases. Hence, there is a need for augmenting wildlife conservation with disease control strategies aimed at reducing the risk of disease transmission between wildlife and domestic animals.

Tilapia lake virus downplays innate immune responses during early stage of infection in Nile tilapia (Oreochromis niloticus).

Tilapia lake virus (TiLV) causes high mortality and high economic losses in tilapines. We describe an experimental challenge study focusing on early post challenge innate immune responses. Nile tilapia (Oreochromis niloticus) were infected with 105 TCID50/mL TiLV intraperitoneally, followed by virus quantification, histopathology and gene expression analysis in target (brain/liver) and lymphoid (spleen/headkidney) organs at 3, 7, 12, 17, and 34 days post challenge (dpc). Onset of mortality was from 21 dpc, and cumulative mortality was 38.5% by 34 dpc. Liver and kidney histopathology developed over the period 3-17 dpc, characterized by anisocytosis, anisokaryocytosis, and formation of multinucleated hepatocytes. Viral loads were highest at early time (3 dpc) in liver, spleen and kidney, declining towards 34 dpc. In brain, viral titer peaked 17 dpc. Innate sensors, TLRs 3/7 were inversely correlated with virus titer in brain and headkidney, and IFN-ß and Mx showed a similar pattern. All organs showed increased mRNA IgM expression over the course of infection. Overall, high virus titers downplay innate responses, and an increase is seen when viral titers decline. In silico modeling found that TiLV segments 4, 5 and 10 carry nucleolar localization signals. Anti-viral effects of TiLV facilitate production of virus at early stage of infection.