Percentage Incidences of Bacteria in Mahseer (Tor putitora), Silver carp (Hypophthalmichthys molitrix) Fish Collected from Hatcheries and Local Markets of District Malakand and Peshawar of Khyber Pakhtunkhwa, Pakistan

Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.

Probiotic Bacillus safensis NPUST1 Administration Improves Growth Performance, Gut Microbiota, and Innate Immunity against Streptococcus iniae in Nile tilapia (Oreochromis niloticus)

Probiotics are considered ecofriendly alternatives to antibiotics as immunostimulants against pathogen infections in aquaculture. In the present study, protease-, amylase-, cellulase-, and xylanase-producing Bacillus safensis NPUST1 were isolated from the gut of Nile tilapia, and the beneficial effects of B. safensis NPUST1 on growth, innate immunity, disease resistance and gut microbiota in Nile tilapia were evaluated by feeding tilapia a basal diet or basal diet containing 105 and 106–107 CFU/g for 8 weeks. The results showed that the weight gain, feed efficiency and specific growth rate were significantly increased in tilapia fed a diet containing 106 CFU/g and 107 CFU/g B. safensis NPUST1. Intestinal digestive enzymes, including protease, amylase and lipase, and hepatic mRNA expression of glucose metabolism and growth-related genes, such as GK, G6Pase, GHR and IGF-1, were also significantly increased in the 106 CFU/g and 107 CFU/g B. safensis NPUST1 treated groups. Immune parameters such as phagocytic activity, respiratory burst and superoxide dismutase activity in head kidney leukocytes, serum lysozyme, and the mRNA expression of IL-1β, IL-8, TNF-α and lysozyme genes were significantly induced in the head kidney and spleen of 106 CFU/g and 107 CFU/g B. safensis NPUST1 treated fish. The cumulative survival rate was significantly increased in fish fed a diet containing 106 CFU/g and 107 CFU/g B. safensis NPUST1 after challenge with Streptococcus iniae. Dietary supplementation with B. safensis NPUST1 improves the gut microbiota of Nile tilapia, which increases the abundance of potential probiotics and reduces the abundance of pathogenic pathogens. The present study is the first to report the use of B. safensis as a potential probiotic in aquaculture, and a diet containing 106 CFU/g B. safensis NPUST1 is adequate for providing beneficial effects on growth performance and health status in tilapia.

ĐA HÌNH MICROSATELLITE LIÊN KẾT VỚI GEN HEPCIDIN/HAMP TIỀM NĂNG TRONG CHỌN GIỐNG CÁ RÔ PHI VẰN KHÁNG BỆNH DO Streptococcus iniae

Bệnh xuất huyết do vi khuẩn Streptococcus sp là mầm bệnh truyền nhiễm chính gây thiệt hại đáng kể đến sản lượng cá rô phi toàn cầu. Hepcidin/HAMP ở cá đã được báo cáo có liên quan đến miễn dịch bẩm sinh chống lại các mầm bệnh vi khuẩn. Trong nghiên cứu này, chúng tôi tiến hành phân tích mối quan hệ giữa tính đa hình microsatellites/SSRs liên kết với gen hepcidin/HAMP và khả năng kháng bệnh do Streptococcus iniae trên cá rô phi vằn dòng NT1 (Đài Loan). 17 chỉ thị SSRs và cặp mồi đặc hiệu đã được thiết kế dựa trên WebSat. Kết quả đánh giá trên 95 cá thể cho thấy 9/17 chỉ thị SSRs có tính đa hình cao và tuân theo định luật Hardy-Weinberg. Các chỉ thị này sẽ được sử dụng để đánh giá khả năng kháng vi khuẩn S. iniae. 29 cá rô phi NT1 thế hệ thứ nhất (khối lượng 23,59 ± 5,388 g/con) đã được cảm nhiễm với vi khuẩn S. iniae 89353 bằng phương pháp tiêm, với liều tiêm LD50 là 1,3x105 cfu/mL. Kết quả phân tích cho thấy, có sự sai khác có ý nghĩa thống kê về kiểu gen và tần số alen giữa nhóm sống và nhóm chết sau cảm nhiễm vi khuẩn ở 3 chỉ thị SSRs (SSR7, SSR9 và SSR16) (p<0,05). Đây là marker có tiềm năng cho chọn giống cá rô phi Đài Loan kháng bệnh do S. iniae. ABSTRACT Streptococcus has been recognized as a major infectious disease-causing significant economic loss in tilapia aquaculture in many countries. The hepatic antimicrobial peptide hepcidin/HAMP was reported to be associated with innate immunity which defends against various bacterial pathogens and viruses. In this study, we analyzed the corelation between the microsatellites/SSRs polymorphism in the hepcidin/HAMP genes and the resistance to Streptococcus iniae in the NT1strain (tilapia strain in Taiwan). Seventeen of hepcidin/HAMP-related SSRs and 17 SSR-specific PCR primer were designed using WebSat. The result showed that 9/17 hepcidin/HAMP-related SSRs were polymorphic markers and there is significant deviation from Hardy-Weinberg Equilibrium (HWE) (p<0,05). These SSRs were examined for disease resistance to S. iniae. Twenty-nine the First generation (G1) tilapia of NT1 strain (average weight of 23,59 ± 5,388g/fish) were challenged with virulent S. iniae 89353 through intraperioneal injection at dose of LD50 (1,3x105 cfu/ml). In this study, the genotype and the allele frequency in three SSRs (SSR7, SSR9 và SSR16) were significantly different between two groups (death fish with infected signals of S. iniae and alive fish infected with S. iniae) (p<0,05). Three SSRs (SSR7, SSR9 và SSR16) are considered as potential molecular markers for selective breeding of Taiwanese tilapia which resists to S. iniae.

Molecular Investigation of Recurrent Streptococcus iniae Epizootics Affecting Coral Reef Fish on an Oceanic Island Suggests at Least Two Distinct Emergence Events

Streptococcus iniae is an emerging zoonotic pathogen of increasing concern for aquaculture and has caused several epizootics in reef fishes from the Caribbean, the Red Sea and the Indian Ocean. To study the population structure, introduction pathways and evolution of S. iniae over recurring epizootics on Reunion Island, we developed and validated a Multi Locus Sequence Typing (MLST) panel using genomic data obtained from 89 isolates sampled during epizootics occurring over the past 40years in Australia, Asia, the United States, Israel and Reunion Island. We selected eight housekeeping loci, which resulted in the greatest variation across the main S. iniae phylogenetic clades highlighted by the whole genomic dataset. We then applied the developed MLST to investigate the origin of S. iniae responsible for four epizootics on Reunion Island, first in inland aquaculture and then on the reefs from 1996 to 2014. Results suggest at least two independent S. iniae emergence events occurred on the island. Molecular data support that the first epizootic resulted from an introduction, with inland freshwater aquaculture facilities acting as a stepping-stone. Such an event may have been facilitated by the ecological flexibility of S. iniae, able to survive in both fresh and marine waters and the ability of the pathogen to infect multiple host species. By contrast, the second epizootic was associated with a distinct ST of cosmopolitan distribution that may have emerged as a result of environment disturbance. This novel tool will be effective at investigating recurrent epizootics occurring within a given environment or country that is despite the fact that S. iniae appears to have low genetic diversity within its lineage.

Fatal Streptococcus iniae Infection in a Juvenile Free-Ranging Short-Beaked Common Dolphin (Delphinus delphis)

Streptococcus iniae (S. iniae) is a significant aquatic pathogen of farmed fish species, important zoonotic pathogen, and reported cause of disease in captive Amazon River dolphins (Inia geoffrensis) and a bottlenose dolphin (Tursiops truncatus). Here we report S. iniae as the cause of subcutaneous abscesses, sepsis and mortality in a juvenile free-ranging short-beaked common dolphin (Delphinus delphis) found deceased on a metropolitan Australian beach. Body surfaces were covered by multifocal, depressed, deep, irregular cutaneous ulcerations, which microscopically were characterised by ruptured subcutaneous abscesses with intralesional cocci. Routine microbiological investigations revealed a heavy growth of beta-haemolytic Streptococcus sp. identified as Streptococcus iniae in skin lesions as well as from heart blood, the latter supportive of sepsis. Tissues were negative for cetacean morbillivirus and no other disease processes were identified. S. iniae has not been reported in free-ranging marine mammals, nor in Australian delphinids, previously. More notably a pathogen of captive animals, this case report identifies S. iniae as a pathogen of wild dolphins also. In addition to expanding the host reservoir of a significant zoonotic pathogen, determining the source of infection as well as possible consequences for other marine mammals and wild and intensive fish stocks warrants further investigations.