Nopadon Pirarat

Modulation of intestinal morphology and immunity in nile tilapia (Oreochromis niloticus) by Lactobacillus rhamnosus GG.

The use of lactic acid bacteria from human origins as a potential probiotic supplementation in aquaculture feed is now widely accepted. Here, we examined some of the properties and mechanisms of the action of Lactobacillus rhamnosus GG, originating from humans, on growth performance, gut mucosal immunity and humoral and cellular immune response in tilapia (Oreochromis niloticus). The results suggested that supplementation of L. rhamnosus gave an advantage in promoting the intestinal structure and the mucosal immunity of tilapia. Probiotic fish had a greater villous height in all parts of the intestines and, significantly, in the proximal and middle part. The population of intraepithelial lymphocytes was significantly higher in the probiotic group than in the control group in all parts of the intestines. The population of acidophilic granulocyte in the probiotic group was significantly higher at the proximal and distal parts when compared with the control group. The higher serum complement activity as well as the enhanced phagocytosis and killing ability of the head kidney leukocytes in the probiotic supplemented fish corresponded with the higher level of TNF alpha and IL-1 gene expression, suggesting that the induction of IL-1 and TNF alpha cytokines by L. rhamnosus served as an important regulator of gut associated immune systems.

Increasing of temperature induces pathogenicity of Streptococcus agalactiae and the up-regulation of inflammatory related genes in infected Nile tilapia (Oreochromis niloticus).

Temperature strongly affects the health of aquatic poikilotherms. In Nile tilapia (Oreochromis niloticus), elevated water temperatures increase the severity of streptococcosis. Here we investigated the effects of temperature on the vulnerability and inflammatory response of Nile tilapia to Streptococcus agalactiae (Group B streptococci; GBS). At 35 and 28 °C, GBS took 4 and 7h, respectively to reach the log-phase and, when incubated with tilapia whole blood, experienced survival rates of 97% and 2%, respectively. The hemolysis activity of GBS grown at 35 °C was five times higher than that of GBS grown at 28 °C. GBS expressed cylE (β-hemolysin/cytolysin), cfb (CAMP factor) and PI-2b (pili-backbone) much more strongly at 35 °C than at 28 °C. Challenging Nile tilapia reared at 35 and 28 °C with GBS resulted in accumulated mortalities of about 85% and 45%, respectively. At 35 °C, infected tilapia exhibited tremendous inflammatory responses due to a dramatic up-regulation (30-40-fold) of inflammatory-related genes (cyclooxygenase-2, IL-1β and TNF-α) between 6 and 96 h-post infection. These results suggest that the increase of GBS pathogenicity to Nile tilapia induced by elevated temperature is associated with massive inflammatory responses, which may lead to acute mortality.

Molecular characterization and virulence gene profiling of pathogenic Streptococcus agalactiae populations from tilapia (Oreochromis sp.) farms in Thailand

Streptococcus spp. were recovered from diseased tilapia in Thailand during 2009–2010 (n = 33), and were also continually collected from environmental samples (sediment and water) from tilapia farms for 9 months in 2011 (n = 25). The relative percent recovery of streptococci from environmental samples was 13–67%. All streptococcal isolates were identified as S. agalactiae (group B streptococci [GBS]) by a species-specific polymerase chain reaction. In molecular characterization assays, 4 genotypic categories comprised of 1) molecular serotypes, 2) the infB allele, 3) virulence gene profiling patterns (cylE, hylB, scpB, lmb, cspA, dltA, fbsA, fbsB, bibA, gap, and pili backbone–encoded genes), and 4) randomly amplified polymorphic DNA (RAPD) fingerprinting patterns, were used to describe the genotypic diversity of the GBS isolates. There was only 1 isolate identified as molecular serotype III, while the others were serotype Ia. Most GBS serotype Ia isolates had an identical infB allele and virulence gene profiling patterns, but a large diversity was established by RAPD analysis with diversity tending to be geographically dependent. Experimental infection of Nile tilapia (Oreochromis niloticus) revealed that the GBS serotype III isolate was nonpathogenic in the fish, while all 5 serotype Ia isolates (3 fish and 2 environmental isolates) were pathogenic, with a median lethal dose of 6.25–7.56 log10 colony-forming units. In conclusion, GBS isolates from tilapia farms in Thailand showed a large genetic diversity, which was associated with the geographical origins of the bacteria.

Genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm‐raised tilapia Oreochromis niloticus (L.) in Thailand

Betanodavirus infection was diagnosed in larvae of farm-raised tilapia Oreochromis niloticus (L.), in central Thailand. Extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. Molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. The virus strain was highly homologous (93.07-93.88%) and closely related to red-spotted grouper nervous necrosis virus (RGNNV).

The Pathological Effects of Melamine and Cyanuric Acid in the Diet of Walking Catfish (Clarius batrachus)

The toxicity of melamine and its analogue in man and animals has been reported widely. The aim of the present study was to examine the pathological effects of feeding melamine and cyanuric acid, separately or in combination, to walking catfish (Clarius batrachus). The catfish developed darkening of the skin as early as 3 days post feeding. Melamine-related crystals were distributed multifocally throughout the liver, kidney, heart, spleen and corpuscle of Stannius of fish fed melamine and cyanuric acid in combination. Oil red O staining and electron microscopy revealed that the melamine-related crystals had structure resembling that of plastic polymer crystals. Elevations in the serum concentrations of alanine transaminase, aspartate transaminase, creatinine and uric acid were related to the crystal-associated granulomatous inflammation in the liver and kidney of affected fish. None of the catfish died during the 2-week experiment. Melamine and cyanuric acid are therefore systemically toxic to fish in addition to causing renal crystal formation and renal damage as seen in man and animals. The finding of extrarenal crystals implies that the metabolism and biotransformation of these toxic compounds should be further investigated in aquatic animals.

Short-term Exposure of Nile Tilapia (Oreochromis niloticus) to Mercury Histopathological Changes, Mercury Bioaccumulation, and Protective Role of Metallothioneins in Different Exposure Routes

To investigate effects of short-term mercury (Hg) exposure in tilapia (Oreochromis niloticus) including histopathological changes, Hg bioaccumulation, and protective role of metallothionein (MT) in different exposure routes, adult tilapias were intraperitoneally injected, orally intubated, or semistatically exposed to 0.5, 1, 2, 5 µg/g mercuric chloride. Histopathology, autometallography (AMG), inductive coupled plasma–atomic emission spectrometry (ICP-AES), and MT immunohistochemistry were determined at 0, 3, 6, 9, 12, and 15 days postexposure. Microscopic lesions were observed in the kidney, hepatopancreas, spleen, and intestine. AMG positive grains were found in renal tubule epithelium, melanomacrophage centers (MMCs), and intestinal epithelium of treated tilapias. Hg concentrations measured by ICP-AES in abdominal visceral organs were significantly higher than in other organs. All exposure routes caused lesions of increasing severity and Hg accumulations in a dose-dependent manner. Semistatic groups produced the highest intensity of lesions, AMG positive staining, as well as total Hg concentrations. Positive MT expression in renal tubule epithelium, pancreatic acini, and splenic MMCs was observed only in semistatic groups. The semistatic exposure route demonstrated the most significant microscopic lesions, Hg bioaccumulation, and MT expression.

Systemic Neosporosis in a White Rhinoceros

Abstract Neosporosis was diagnosed in a 16- year-old female white rhinoceros (Ceratotherium simum) that died suddenly without clinical signs. Histopathology revealed disseminated protozoan tachyzoites in liver, adrenal cortex, kidney, and intestine, with morphology compatible with either Toxoplasma or Neospora. The organism was identified as Neospora caninum with the use of primary rabbit anti–N. caninum antibody immunohistochemistry and polymerase chain reaction. The exact source of infection remains unknown, but it is suspected that N. caninum oocysts were ingested from the soil.

The study on the candidate probiotic properties of encapsulated yeast, Saccharomyces cerevisiae JCM 7255, in Nile Tilapia (Oreochromis niloticus)

Saccharomyces cerevisiae JCM 7255 was tested as a probiotic candidate in tilapia after encapsulating and freeze drying. Viability and morphology during storage and during transit through simulated gut and bile conditions were determined. Growth performance, anti-streptococcal activity and gut mucosal immune parameters were also tested. The viability of encapsulated yeasts was significantly high in simulated gastric and bile conditions and remained high after storage at room temperature for 14 days. The morphology of free S. cerevisiae revealed rough, bumpy, ruptured surface during incubation in gut and bile conditions. Agar spot anti-streptococcal activity showed inhibition of 20 out of 30 strains of Streptococcus agalactiae. Supplementation improved the intestinal structure and growth performance in tilapias. Intraepithelial lymphocytes in the proximal intestine were significantly observed. Lower cumulative mortality after the oral streptococcal challenge was also seen. The results suggest that encapsulated S. cerevisiae JCM 2755 could be a potential probiotic strain in tilapia culture.

Genomic comparison between pathogenic Streptococcus agalactiae isolated from Nile tilapia in Thailand and fish-derived ST7 strains

Streptococcus agalactiae, or Group B streptococcus (GBS), is a highly virulent pathogen in aquatic animals, causing huge mortalities worldwide. In Thailand, the serotype Ia, β-hemolytic GBS, belonging to sequence type (ST) 7 of clonal complex (CC) 7, was found to be the major cause of streptococcosis outbreaks in fish farms. In this study, we performed an in silico genomic comparison, aiming to investigate the phylogenetic relationship between the pathogenic fish strains of Thai ST7 and other ST7 from different hosts and geographical origins. In general, the genomes of Thai ST7 strains are closely related to other fish ST7s, as the core genome is shared by 92-95% of any individual fish ST7 genome. Among the fish ST7 genomes, we observed only small dissimilarities, based on the analysis of clustered regularly interspaced short palindromic repeats (CRISPRs), surface protein markers, insertions sequence (IS) elements and putative virulence genes. The phylogenetic tree based on single nucleotide polymorphisms (SNPs) of the core genome sequences clearly categorized the ST7 strains according to their geographical and host origins, with the human ST7 being genetically distant from other fish ST7 strains. A pan-genome analysis of ST7 strains detected a 48-kb gene island specifically in the Thai ST7 isolates. The orientations and predicted amino acid sequences of the genes in the island closely matched those of Tn5252, a streptococcal conjugative transposon, in GBS 2603V/R serotype V, Streptococcus pneumoniae and Streptococcus suis. Thus, it was presumed that Thai ST7 acquired this Tn5252 homologue from related streptococci. The close phylogenetic relationship between the fish ST7 strains suggests that these strains were derived from a common ancestor and have diverged in different geographical regions and in different hosts.

Expression of canine distemper virus receptor nectin-4 in the central nervous system of dogs

Canine distemper virus (CDV) exhibits lymphotropic, epitheliotropic, and neurotropic nature, and causes a severe systemic infection in susceptible animals. Initially, signaling lymphocyte activation molecule (SLAM) expressed on immune cells has been identified as a crucial cellular receptor for CDV. Currently, nectin-4 expressed in epithelia has been shown to be another receptor for CDV. Our previous study demonstrated that neurons express nectin-4 and are infected with CDV. In this study, we investigated the distribution pattern of nectin-4 in various cell types in the canine central nervous system and showed its relation to CDV infection to further clarify the pathology of disease. Histopathological, immunohistochemical and immunofluorescent analyses were done using formalin-fixed paraffin-embedded tissues of CDV-infected dogs. Dual staining of nectin-4 and CDV antigen or nectin-4 and brain cell markers was performed. Nectin-4 was detected in ependymal cells, epithelia of choroid plexus, meningeal cells, neurons, granular cells, and Purkinje’s cells. CDV antigens were detected in these nectin-4-positive cells, further suggesting contribution of nectin-4 for the CDV neurovirulence. On the other hand, astrocytes did not express nectin-4, although they were frequently infected with CDV. Since astrocytes are negative for SLAM expression, they must express an unidentified CDV receptor, which also contributes to CDV neurovirulence.