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This paper reports the identification of Streptococcus iniae from a large collection of isolates previously identified as Streptococcus sp., Lactococcus lactis subsp. lactis or Leuconostoc sp. A total of 204 bacterial isolates recovered either from the brain, eye, or kidney of red tilapias in previous disease outbreaks and disease monitoring in Malaysia from 2006 to 2008 were used. PCR identification revealed that 34 (16.7%) of the isolates were confirmed as S. iniae. Our records showed that S. iniae-infected fish exhibited lethargy, exophthalmia, and erratic swimming patterns. Pathological lesions including generalised congestion of the internal organs, splenic infarction with soft and oedematous brain. Histopathological examination revealed multifocal encephalitis as one of the major findings. However, 44% and 26.5% of the tilapias from which S. iniae was isolated did not manifest any clinical sign and pathological lesion, respectively. This study revealed that S. iniae was responsible for streptococcosis in cultured red tilapia in Malaysia since 2006.

Pangasius hypophthalmus or famously known by local Malaysians as Patin Hitam is one of the most important sources of food in Malaysia. It is widely cultured in the Peninsular Malaysia especially in Pahang as Patin and is a popularly consumed freshwater fish. Global economic interest in the fish has increased its demand in the USA and Europe. However, high mortality due to bacterial and viral infections is the main problem that needs to be solved. Therefore, bacteria in P. hypophthalmus in Pahang is being focused with the factors connected to the prevalence of bacteria and virus in P. hypophthalmus. This research was conducted for two cycles (February-September 2016 and January-August 2017) in different farms in Temerloh, Pahang. Bacteria and virus samples were taken from three organs of Patin Hitam which are kidney, liver and spleen. Physical parameters for water quality were measured using a multiparameter probe sensor (YSI, USA) and chemical parameters were analysed with DR900 colorimeter (Hach, USA). Bacteria samples were identified using biochemical test kits, API 20NE and 20E, followed by confirmation of the bacteria using Polymerase Chain Reaction (PCR). Virus samples were identified using conventional PCR. There are several bacteria isolated throughout the culture period. The highest prevalence of Aeromonas hydrophila in Temerloh in the first cycle was in May 2016 (40%), however, was equally evident in four out of the seven months of the second cycle, which was in April, May, June and July 2017 (20%). There was a relationship between the prevalence of A. hydrophila and iron, nitrite and pH in the first cycle in Temerloh. However, there was no relationship in the second cycle. Significantly, these results could contribute to better treatment of fish disease and development of standard operating procedure of future fish culture for early disease prevention.

The study determines the presence of Vibrionaceae, Betanodavirus and Iridovirus in marine cage-cultured fish, while identifying the roles of fish size, water physicochemical parameters and relationships among the pathogens itself. Cultured grouper and snapper were randomly sampled from a commercial fish farm between February and December 2014. The total body weight and length of individual fish were measured. The kidney, liver and spleen were sampled for bacteria isolation, while for viral identification, the sample of brain, eye, kidney and spleen were used. Water physicochemical parameters during the sampling activities were also determined. Laboratory results revealed isolations of multiple pathogens including Vibrio alginolyticus, V. vulnificus, Photobacterium damselae subsp. damselae (PD), Nervous Necrosis Virus (NNV) and Iridovirus (IV) at low to high prevalence throughout the study period. The weight of affected groupers ranged between 98 g and 719 g, while snappers between 67 g and 982 g. There was a weak and moderate negative correlation between the grouper’s weight and the presence of NNV (R = −0.3684; P < 0.05) and V. vulnificus (R = −0.6451; P < 0.05), respectively. No significant (P > 0.05) difference was noted in the rate of isolated pathogens between groupers and snappers, and between the pathogens affecting snappers. However, detection of IV in groupers was significantly (P < 0.05) higher than V. alginolyticus, V. vulnificus and PD. Isolations of V. vulnificus in groupers showed strong and moderate positive correlations with isolations of PD (R = 0.7069; P < 0.05) and IV (R = 0.6665; P < 0.05), respectively. In snappers, there was strong positive correlation between isolation of V. alginolyticus and NNV (R = 0.7526; P < 0.05). Multivariate analyses showed that water temperature, dissolved oxygen, ammonia, iron and nitrite were the most significant water physicochemical parameters associated with presence of these pathogens.

Polycystic lesions have been reported in many different fish species. This paper reports for the first time a case of polycystic lesions in the liver of tilapia. A group of 700 tilapia juveniles of approximately 50 g was obtained from a hatchery in Negeri Sembilan, Malaysia. When they were between 130 and 250 g, they started to show distended abdomen but appetite remained normal. Post-mortem examination on 30 randomly selected tilapia revealed numerous large masses containing jelly-like material in the peritoneal cavity of 33% of the fish. Histopathology revealed typical cystic lesion of liver tissue. This is the first report in hybrid tilapia and is presumed to be the result of a genetic anomaly.

Tilapia is one of the most common cultured fish in many countries. However,productions of tilapia might decrease due to various diseases, including streptococcosis that can kill 100% of the fish. In Malaysia, outbreaks of streptococcosis are frequently observed during the dry months, particularly between April and August when the water temperature is high. Streptococcosis in fish is caused by either Streptococcus agalactiae or S. iniae. Although vaccination is practiced to control streptococcosis, the protection remains unclear. Thus, this project attempted to develop and evaluate recombinant cell for vaccine preparation to improve vaccine efficacy against streptococcosis. The hypothesis of this study are major outer surface protein of Streptococcus agalactiae is suitable as vaccine candidate and can be cloned and expressed in the Escherichia coli prokaryotic system to produce the recombinant vaccine. The newly develop feed based recombinant vaccine can elicit certain level of systemic and mucosal antibody and gave protection against Streptococcosis in red hybrid tilapia. The outer surface proteins (OSPs) of S. agalactiae isolated earlier from fish that died of streptococcosis were extracted, purified and characterized. The 48kDa protein band was found to be the most antigenic following SDS-PAGE and Western immunoblotting methods. Therefore, the 48kDa protein band was selected for further studies as a vaccine candidate. The 48kDa band was processed for N-terminal sequencing, and the results revealed that the protein was the cell wall surface anchor family protein of the outer surface protein (OSP) of S. agalactiae, which was encoded by a gene of approximately 1263bp in size. The DNA gene was amplified by the polymerase chain reaction (PCR) method, purified and cloned in pET-32 Ek/Lic vector. The successful clones were then transformed into Novablue Escherichia coli strain before the positive clones were screened for the end product of 1335bp; the vector contributed

The present invention relates to a vaccine for treating streptococcosis disease that occurs in fish. The vaccine comprises of a recombinant microorganism expressing an antigenic protein of Streptococcus agalactiae, wherein the antigenic protein is cell wall surface anchor family protein. The vaccine is administered orally and gives longer period of protection against the disease. The present invention also relates to a method for producing the vaccine.

One hundred and sixty fingerlings and 80 adult tilapias were experimentally infected with Streptococcus agalactiae and Stapylococcus aureus to determine their LDso. Four concentrations of Streptococcus agalactiae (109, 108,107, 106 CFU/mL) were used in this experimental infection. These tilapias were divided into 4 groups of 40 fingerlings and 20 adults per group. Groups 1, 2, 3 and 4 of the fingerlings were exposed to 109, 10,107, 106 CFU/mL of S. agalactiae by immersion in 2 L inoculum solution for 20 min. Similarly, the adult groups were exposed to the same concentrations of S. agalactiae but by intraperitoneal injection at the rate of 1 mL of the inoculum per gram. Similar procedures were repeated using exposure to Staphylococcus aureus alone or a combination of S. agalactiae and S. aureus. All test groups were observed for signs of infections. On Day 7 post-infection (pi), all fish that were still alive were humanely killed. The LDso of the adult tilapia that were exposed to S. agalactiae, S. aureus or mixed infection was 2.3884 x 107,2.8151 X 108 , and 4.2409 x io', respectively. For the fingerling groups, the LDso for S. agalactiae, S. aureus, and mixed infection was 2.9242 x 1020,2.8665 x 1017 , and 4.9748 x IO!', respectively. Experimental infection in adults could be established within 12 h post-injection to 6.3 x 109 CFU per mL and 9.7 x 109 CFU per mL of S. agalactiae and S. aureus, respectively. For fingerlings, infection could be established within 72 h following bath immersion to 6.3 x 109 CFU per mL and 9.7 x 109 CFU per mL of S. agalactiae and S. aureus, respectively.