Development of a Reverse Line Blot Hybridization method for Detection of some Streptococcal/Lactococcal Species, the causative agents of Zoonotic Streptococosis/Lactococosis in farmed fish
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M Soltani
,
E Pirali
,
P Shayan
,
B Eckert
,
S Rouholahi
,
N Sadr-Shirazi
Abstract
Background and Objective: Streptococcosis/lactococcosis is the cause of high morbidity and mortality in aquaculture sector and to date a number of species of Streptococcus and Lactococcus genera including S. iniae, S. agalactiae, S. dysagalactiae, S. parauberis, S. feacalis, L. garvieae and L. lactis have been discriminated as the cause of disease in aquatic animals. Despite the use of diagnostic molecular methods for each of these bacterial species, no data is available on a suitable, rapid and simple simultaneous detection tool for these pathogens. This paper describes a simultaneous detection method which is PCR based on a reverse line blot (RLB) for rapid detection and differentiation of four species of genera of Streptococcus and Lactococcus genera consisting of S. iniae, S. agalactiae, S. parauberis and L. garvieae the most important agents of the disease in fish.
Materials and Methods: A reverse line blot (RLB) assay was developed for the simultaneously identification of four species of Streptococcus/lactococcusconsisting of S. iniae, S. parauberis, S. agalactiaeand Lactococcusgarvieae. The assay employs one set of primer pair for specific amplification of the 16S rRNA gene. These were designed based on the nucleotide sequences of 16S rRNA gene sharing a homology region with Streptococcus spp. and Lactococcus spp. DNA was extracted from the pure bacterial colonies and amplified. A membrane was prepared with specific oligonucleotide for each bacterial species. PCR products were then hybridized to a membrane.
Results and Conclusion: The amplification resulted in PCR product of 241 bp in length. No cross-reactions were observed between any of the tested bacterial species, and mixed DNAs from these four bacterial species were correctly identified. This RLB method is a suitable technique for a simultaneous detection of these species of bacterial fish pathogens that are some of the main causes of streptococcal/lactococcal infections in both freshwater and marine aquatic animals, and so we recommend its use for integrated epidemiological monitoring of streptococcosis/lactococcis in aquaculture industry.
Agnew W, Barnes AC. Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination. J Vet Microbiol 2007; 122: 1-15.
Austin B, Austin D A (2007). Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. Springer Praxis Publication. Chichester, pp. 58-63, 156, 238-239.
Vendrell D, Balcázar JL, Ruiz-Zarzuela I, Blas I, Olivia- Gironés O, Múzquiz JL. Lactococcusgarvieae in fish: A review. Com ImmunolMicrobiolInf Dis 2006; 29: 177-198.
Eldar A, Ghittino C.Lactococcusgarvieae and Streptococcus iniae infection in rainbow trout Oncorhynchusmykiss: similar, but different disease. Dis Aqu Org 1999; 36: 227-231.
Hassan AA, Khan IU, Lammler C. Identification of Streptococcus dysgalactiae strains of Lancefield’s group C, G and L by polymerase chain reaction. J Vet Med 2003; B50: 161-165.
Shoemaker CA, Klesius PH, Evans JJ. Prevalence of Streptococcus iniaein tilapia, hybrid striped bass and channel catfish on commercial fish farms in the United States. Am J Vet Res 2001; 62: 174-177.
Soltani M, Nikbakht-Borojeni GH, Ebrahimzadeh- Moussavi HA, Ahmadzadeh N. Epizootic outbreaks of Lactoccoccosis caused by Lactococcusgarvieae in farmed rainbow trout (Onchorhynchusmykiss) in Iran. Bull Eur Ass Fish Pathol 2008; 5: 209-214.
Mata AI, Gibello A, Casamayor A, Blanco MM, Dominguez L, Fernandez-Garayzabal JF. Multiplex PCR assay for detection of bacterial pathogens associated with warm-water streptococcosis in fish. App EnvMicrobiol 2004; 70: 3183-3187.
Meiri-Bendek I, Lipkin E, Friedmann A, Leitner G, Saran A, Friedman S, Kashi K. A PCR-Based Method for the Detection of Streptococcus agalactiae in Milk.J Dairy Sci 2002; 85: 1717–1723.
Zlotkin A, Eldar A, Ghittino C, Bercovier H.Identification of Lactococcusgarvieae by PCR. J ClinMicrobiol 1998; 36: 983-985.
Gubbels J M, De Vos AP, Van der Weide M, Viseras J, Schouls L M, De Vries E,Jongejan F. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization.J ClinMicrobiol 1999;37: 1782-9.
Soltani M, Jamshidi S, Sharifpour I. Streptococcosis caused by Streptococcus iniae in farmed rainbow trout (Onchorhynchusmykiss) in Iran: Biophysical characteristics and pathogenesis. Bull Eur Ass Fish Pathol 2005; 25: 95-106.
Yung W, Li A. Isolation and characterization of Streptococcus dysagalactiae from diseased Acipenser schrenckii. Aquaculture 2009; 294: 14-17.
14. Goh SH, Driedger D, Gillett S, Low DE, Hemmingsen SM, Amos M, Chan D, Lovgren M, Willey BM, Shaw C, Smith JA. Streptococcus iniae, a human and animal pathogen: specific identification by the chaperonin 60 gene identification method. J ClinMicrobiol 1998; 36:2164-2166.
Nomoto R, Munasinghe LI, Jin DH, Shimahara Y, Yasuda H, Nakamura A, Misawn N, Itami T, Yoshida T. Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan. J Fish Dis 2004; 27: 679-686.
HaghighiS, Soltani M,Nikbakhat-BrojeniGh , Gha M.Skall D. Molecular epidemiology of zoonotic streptococcosis/ lactococcosis in rainbow trout (Oncorhynchusmykiss) aquaculture in Iran. Iranian J Microbiol 2010; 2: 198-209.
| Files | ||
| Issue | Vol 4 No 2 (2012) | |
| Section | Articles | |
| Keywords | ||
| L. garviae Reverse line blot (RLB) bS. agalactiae S. iniae S. parauberis | ||
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