Histopathological and immunological changes in green mussel, Perna viridis, challenged with Vibrio alginolyticus.

Understanding of pathogenicity and immunity is crucial in producing disease-resistant cultured mollusk varieties. This study aimed to isolate pathogenic Vibrio alginolyticus from naturally infected Perna viridis, and to determine histopathological and immunological changes after challenge test with the same bacteria. Biochemical tests and 16S rDNA identified the pathogen as V. alginolyticus (99%). Antibiotic susceptibility test showed ampicillin resistance of the pathogen. Pathogenicity assay was conducted by immersing P. viridis in 1.5 × 106 CFU mL-1V. alginolyticus for 60 min and observed for 5 days. Clinical signs, histopathological and immunological alterations were observed and monitored. Infected groups showed 60% mortality and decreased immunity factors, including total hemocyte count and lysozymes activity. Histopathological examination revealed pathological lesions in the hepatopancreas at 24 h post-challenge and hemocyte proliferation as part of a severe inflammatory reaction. Karyomegaly in the hepatopancreas tissue, concomitant with necrosis demolition of tubules cells, was also observed. V. alginolyticus was determined to be pathogenic to P. viridis, causing mortality as a result of multiple organ lesions and dysfunction in digestive gland and immune organs. This study demonstrated the role of histopathological and immunological parameters as potential biomarkers in assessing vibriosis caused by Vibrio species in green mussel, P. viridis.

Efficacy of live attenuated vaccine derived from the Streptococcus agalactiae on the immune responses of Oreochromis niloticus.

Streptococcus agalactiae species have been recognized as the main pathogen causing high mortality in fish leading to significant worldwide economical losses to the aquaculture industries. Vaccine development has become a priority in combating multidrug resistance in bacteria; however, there is a lack of commercial live attenuated vaccine (LAV) against S. agalactiae in Malaysia. The aim of this study is to compare two methods using attenuated bacteria as live vaccine and to evaluate the efficacy of selected LAV on the immune responses and resistance of Oreochromis niloticus (tilapia) against S. agalactiae. The LAV derived from S. agalactiae had been weakened using the chemical agent Acriflavine dye (LAV1), whereas the second vaccine was weakened using serial passages of bacteria on broth media (LAV2). Initial immunization was carried out only on day one, given twice-in the morning and evening, for the 42 day period. Serum samples were collected to determine the systemic antibody (IgM) responses and lysozymal (LSZ) activity using ELISA. On day 43 after immunization, the fish were injected intraperitoneally (i.p) with 0.1 mL of S. agalactiae at LD50 = 1.5 × 105 (CFU)/fish. Fish were monitored daily for 10 days. Clinical signs, mortality and the relative percent of survival (RPS) were recorded. Trial 1 results showed a significant increased (P < 0.05) in serum IgM titers and LSZ activity as compared to LAV2 and the control group (unvaccinated fish). The efficacy of LAV1 was proven effective as determined by the RPS values, LAV1 at 81.58% as compared to LAV2 at 65.79%. Trial 2 of LAV1 and control group were further determined by administering primary and booster doses revealed a RPS value for LAV1 of 82.05%, with the significant enhancement on the immune responses of tilapia as compared to control group. In conclusion, LAV revealed to elevate antibody IgM levels, LSZ activity and provide long-term protection when added to feed. LAV is a low-cost vaccine shown to rapidly increase the immune response of fish and increase survival rates of fish against S. agalactiae infection.

Effect of Excoecaria agallocha on non-specific immune responses and disease resistance of Oreochromis niloticus against Streptococcus agalactiae.

The current study was designed to evaluate the effects of Excoecaria agallocha leaf extracts on immune mechanisms and resistance of tilapia, Oreochromis niloticus, after challenge with Streptococcus agalactiae. Fish were divided into 6 groups; groups 1-5 fed with E. agallocha leaf extracts at 10, 20, 30, 40 and 50mgkg-1 level, respectively. Group 6 were fed without extract addition and acted as control. E. agallocha extracts were administered as feed supplement in fish diet for 28days and the hematological, immunological, and growth performance studies were conducted. Fish were infected with S. agalactiae at a dose of 15×105CFUmL-1 and the total white blood cell (WBC), phagocytosis and respiratory burst activities of leukocytes, serum bactericidal activity, lysozyme, total protein, albumin, and globulin levels were monitored and mortalities recorded for 15days post infection. Results revealed that feeding O. niloticus with 50mgkg-1 of E. agallocha enhanced WBC, phagocytic, respiratory burst, serum bactericidal and lysozyme activities on day 28 pre-challenge and on 3rd, 6th, 9th, 12th and 15th day post-challenge as compared to control. Total protein and albumin were not enhanced by E. agallocha diet. E. agallocha increased the survival of fish after challenge with S. agalactiae. The highest mortality rate (97%) was observed in control fish and the lowest mortality (27%) was observed with group fed with 50mgkg-1 extract. The results indicate that dietary intake of E. agallocha methanolic leaf extract in O. niloticus enhances the non-specific immunity and disease resistance against S. agalactiae pathogen.

Molecular identification and histopathological study of natural Streptococcus agalactiae infection in hybrid tilapia (Oreochromis niloticus).

AIM: The main objective of this study was to emphasize on histopathological examinations and molecular identification of Streptococcus agalactiae isolated from natural infections in hybrid tilapia (Oreochromis niloticus) in Temerloh Pahang, Malaysia, as well as to determine the susceptibility of the pathogen strains to various currently available antimicrobial agents. MATERIALS AND METHODS: The diseased fishes were observed for variable clinical signs including fin hemorrhages, alterations in behavior associated with erratic swimming, exophthalmia, and mortality. Tissue samples from the eyes, brain, kidney, liver, and spleen were taken for bacterial isolation. Identification of S. agalactiae was screened by biochemical methods and confirmed by VITEK 2 and 16S rRNA gene sequencing. The antibiogram profiling of the isolate was tested against 18 standard antibiotics included nitrofurantoin, flumequine, florfenicol, amoxylin, doxycycline, oleandomycin, tetracycline, ampicillin, lincomycin, colistin sulfate, oxolinic acid, novobiocin, spiramycin, erythromycin, fosfomycin, neomycin, gentamycin, and polymyxin B. The histopathological analysis of eyes, brain, liver, kidney, and spleen was observed for abnormalities related to S. agalactiae infection. RESULTS: The suspected colonies of S. agalactiae identified by biochemical methods was observed as Gram-positive chained cocci, ß-hemolytic, and non-motile. The isolate was confirmed as S. agalactiae by VITEK 2 (99% similarity), reconfirmed by 16S rRNA gene sequencing (99% similarity) and deposited in GenBank with accession no. KT869025. The isolate was observed to be resistance to neomycin and gentamicin. The most consistent gross findings were marked hemorrhages, erosions of caudal fin, and exophthalmos. Microscopic examination confirmed the presence of marked congestion and infiltration of inflammatory cell in the eye, brain, kidney, liver, and spleen. Eye samples showed damage of the lens capsule, hyperemic and hemorrhagic choroid tissue, and retina hyperplasia accompanied with edema. Brain samples showed perivascular and pericellular edema and hemorrhages of the meninges. Kidney samples showed hemorrhage and thrombosis in the glomeruli and tubules along with atrophy in hematopoietic tissue. Liver samples showed congestion of the sinusoids and blood vessel, thrombosis of portal blood vessel, and vacuolar (fatty) degeneration of hepatocytes. Spleen samples showed large thrombus in the splenic blood vessel, multifocal hemosiderin deposition, congestion of blood vessels, and multifocal infiltration of macrophages. CONCLUSION: Therefore, it can be concluded that pathological changes in tissues and organs of fish occur proportionally to the pathogen invasion, and because of their high resistance, neomycin and gentamicin utilization in the prophylaxis or treatment of S. agalactiae infection should be avoided.