Virulence properties and pathogenicity of multidrug-resistant Vibrio harveyi associated with luminescent vibriosis in Pacific white shrimp, Penaeus vannamei.

Global high demand for Pacific white shrimp Penaeus vannamei has led to intensified cultivation and a wide range of disease problems, including bacterial diseases due to vibrios. Three presumptive luminescent Vibrio harveyi strains (Vh5, Vh8 and Vh10) were isolated from the hepatopancreas (Vh5) and haemolymph (Vh8 and Vh10) of diseased growout Pacific white shrimp from a farm in Setiu, Terengganu, Malaysia, using Vibrio harveyi agar (VHA) differential medium. All three strains were identified as V. harveyi by biochemical characteristics. 16S rRNA gene-based phylogenetic analyses by neighbour-joining, maximum likelihood and maximum parsimony methods showed all three strains in the V. harveyi cluster. All three strains were ß-haemolytic and positive for motility, biofilm formation and extracellular products (caseinase, gelatinase, lipase, DNase, amylase and chitinase). Vh10 was subjected to pathogenicity test in Pacific white shrimp by immersion challenge and determined to have a LC50 of 6.0 × 108 CFU mL-1 after 168 h of exposure. Antibiotic susceptibility tests showed that all strains were resistant to oxytetracycline (OXT30), oleandomycin (OL15), amoxicillin (AML25), ampicillin (AMP10) and colistin sulphate (CT25) but sensitive to doxycycline (DO30), flumequine (UB30), oxolinic acid (OA2), chloramphenicol (C30), florfenicol (FFC30), nitrofurantoin (F5) and fosfomycin (FOS50). Each strain was also resistant to a slightly different combination of eight other antibiotics, with an overall multiple antibiotic resistance (MAR) index of 0.40, suggesting prior history of heavy exposure to the antibiotics. Vh10 infection resulted in pale or discoloured hepatopancreas, empty guts, reddening, necrosis and luminescence of uropods, as well as melanised lesions in tail muscle. Histopathological examination showed necrosis of intertubular connective tissue and tubule, sloughing of epithelial cells in hepatopancreatic tubule, haemocytic infiltration, massive vacuolation and loss of hepatopancreatic tubule structure.

First detection of Shiga toxin producing Escherichia coli O157:H7 (HlyA gene) and enumeration of most probable number (MPN) of fecal coliforms and Escherichia coli in cage cultured oysters (Crassostrea iredalei) and water from southern Malaysia

Aims@#Escherichia coli O157:H7 is known to be transmitted via fecal-oral route, where water plays a role in the transmission process. Oysters as bivalves, bio accumulate pathogens from the water through filter feeding and are suspected to play a role as disease transmission vector. In Malaysia, the data on oyster’s microbiological quality are limited. Hence, it was vital to conduct oyster related studies in Malaysia. The main objectives of this study include the enumeration of most probable number (MPN) of fecal coliforms and E. coli and isolation of E. coli from oyster (Crassostrea iredalei) and water sample for the detection of 16S rRNA and HlyA (Hemolysin A) genes of E. coli O157:H7. @*Methodology and results@#A total of 120 oysters and water samples (n=6) were collected from a fisherman village located in southern Malaysia. Total fecal coliforms and E. coli were determined using the MPN procedure. Colonies of E. coli were identified based on Gram staining, biochemical test, and PCR detection for the presence of 16S rRNA and HlyA gene of E. coli O157:H7. The enumeration results showed that the MPN of the fecal coliforms and E. coli found in the collected oyster samples do not meet the standard to be directed for human consumption (0.72 ± 0.19 × 104 MPN/100 g and 0.13 ± 0.03 × 10 4 MPN/100 g, respectively). The PCR assays showed that 16 out of the 104 (15.38%) of E. coli isolated from water and oysters showed the presence of HlyA gene. The phylogenetic tree analysis showed there were genetic relationships between the HlyA gene of the E. coli isolated in this study with the ones isolated from calf and human faeces.@*Conclusion, significance and impact of study@#The detection of Shiga toxin producing E. coli O157:H7 (HlyA gene) in cage cultured oysters (C. iredalei) and water from southern Malaysia was first time reported here. In the future, more study can be conducted to study the expression of the HlyA gene and confirm of its identity as E. coli O157:H7 using different target genes such as eaeA (encodes a 94 kD outer membrane protein called intimin) and Stx1 (Shiga toxin, Shigella dysenteriae type 1).

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.