Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus.

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (>99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

Efficacy of heat-killed and formalin-killed vaccines against Tilapia tilapinevirus in juvenile Nile tilapia (Oreochromis niloticus).

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture-based heat-killed (HKV) and formalin-killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 µl of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 × 106 TCID50- inactivated virus. A booster vaccination was carried out at 21-day post-vaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV-specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival of 71.3% and 79.6% for HKV and FKV, respectively. Significant up-regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantly increased in the spleen of fish vaccinated with the HKV, but not with FKV. Both vaccines induced a specific IgM response in both serum and mucus. In summary, this study showed that both HKV and FKV are promising injectable vaccines for the prevention of disease caused by TiLV in Nile tilapia.

Immunization of Nile Tilapia (Oreochromis niloticus) Broodstock with Tilapia Lake Virus (TiLV) Inactivated Vaccines Elicits Protective Antibody and Passive Maternal Antibody Transfer.

Tilapia lake virus (TiLV), a major pathogen of farmed tilapia, is known to be vertically transmitted. Here, we hypothesize that Nile tilapia (Oreochromis niloticus) broodstock immunized with a TiLV inactivated vaccine can mount a protective antibody response and passively transfer maternal antibodies to their fertilized eggs and larvae. To test this hypothesis, three groups of tilapia broodstock, each containing four males and eight females, were immunized with either a heat-killed TiLV vaccine (HKV), a formalin-killed TiLV vaccine (FKV) (both administered at 3.6 × 106 TCID50 per fish), or with L15 medium. Booster vaccination with the same vaccines was given 3 weeks later, and mating took place 1 week thereafter. Broodstock blood sera, fertilized eggs and larvae were collected from 6-14 weeks post-primary vaccination for measurement of TiLV-specific antibody (anti-TiLV IgM) levels. In parallel, passive immunization using sera from the immunized female broodstock was administered to naïve tilapia juveniles to assess if antibodies induced in immunized broodstock were protective. The results showed that anti-TiLV IgM was produced in the majority of both male and female broodstock vaccinated with either the HKV or FKV and that these antibodies could be detected in the fertilized eggs and larvae from vaccinated broodstock. Higher levels of maternal antibody were observed in fertilized eggs from broodstock vaccinated with HKV than those vaccinated with FKV. Low levels of TiLV-IgM were detected in some of the 1-3 day old larvae but were undetectable in 7-14 day old larvae from the vaccinated broodstock, indicating a short persistence of TiLV-IgM in larvae. Moreover, passive immunization proved that antibodies elicited by TiLV vaccination were able to confer 85% to 90% protection against TiLV challenge in naïve juvenile tilapia. In conclusion, immunization of tilapia broodstock with TiLV vaccines could be a potential strategy for the prevention of TiLV in tilapia fertilized eggs and larvae, with HKV appearing to be more promising than FKV for maternal vaccination.