Differential detection of vibrios pathogenic to shrimp by multiplex PCR

Abstract

IntroductionVibrio infestations in the mid-1990s led to the nearcollapse of the black tiger shrimp Penaeus monodon industry in the Philippines (Lavilla-Pitogo et al., 1998) with V. harveyi (Karunasagar et al., 1994; LavillaPitogo et al., 1990) and V. campbellii (De la Peña et al., 2001) as main causative agents in shrimp disease or vibriosis. In addition to these two species, Vibrio isolates from infected shrimps in different parts of the Philippines, were observed to cause high mortalities of shrimp postlarvae (De la Peña et al., 2001). These isolates were identified on the basis of biochemical tests to belong to either V. harveyi or V. campbellii but were observed to exhibit characteristics distinct from type strains of both species (Cortado et al., 2004;Rañoa and Hedreyda, 2005 The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65°C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65°C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.

Development of a haemolysin gene-based multiplex PCR for simultaneous detection ofVibrio campbellii,Vibrio harveyiandVibrio parahaemolyticus

“…Simultaneous detection of the three target species was neglected in previous studies. A number of PCR‐based methods have been developed previously for individual identification of V. harveyi and V. parahaemolyticus targeting various genes e.g ., ornithine decarboxylase (Hedreyda and Ranoa 2007), 16S rRNA (Fukui and Sawabe 2007), hly (Castroverde et al. 2006), toxR (Lin et al.…”

Rapid diagnosis of vibriosis and white spot syndrome (WSS) in the culture of shrimp, Penaeus monodon in Philippines

“…Several PCR protocols have been developed to detect certain gene sequences of pathogenic Vibrio spp. infecting shrimp in the Philippines Hedreyda 2003, 2004;Rañoa and Hedreyda 2005;Castroverde et al 2006;Franco and Hedreyda 2006). It has been reported that the Vibrio strains, which are pathogenic to shrimps in the Philippines belong to either V. harveyi or V. campbellii (de la Peña et al 2001).…”

“…These have been routinely used in various shrimp health laboratories all over the world. On the other hand, PCR protocols to detect pathogenic Vibrios have also been developed (Maeda et al 2002;Hedreyda 2003, 2004;Rañoa and Hedreyda 2005;Castroverde et al 2006;Franco and Hedreyda 2006) and Thailand (Thaithongnum et al 2006). Most of the PCR assays for the detection of WSSV have been tested in field assays, but the protocols for detecting pathogenic Vibrios have been developed using established strains of Vibrio spp.…”

Bacteriological and Histopathological Studies on Vibrio Species Isolated from Naturally Infected Freshwater Fish in Delta Region, Egypt

“…The pathogenic V. harveyi is known to harbor many virulence encoding genes, including hemolysins (vhh) (Parvathi et al, 2009), and lots of literature of works established the use of vhh genes targeted PCR as a proof of pathogenic V. harveyi (Castroverde et al, 2006;Conejero and Hedreyda, 2004). Collagenase virulence encoding genes have been shown to be produced by various species of bacteria, including Vibrioalginolyticus for more details about the precise role of collagenase in the pathogenesis of bacterial diseases including Vibriosis see (Harrington, 1996).…”