양식 바지락, Ruditapes philippinarum에 대한 Vibrio tapetis의 병원성과 PCR법에 의한 진단
Pathogenicity and PCR detection of Vibrio tapetis in Manila clams, Ruditapes philippinarum
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"Brown ring disease (BRD)"의 원인균인 Vibrio tapetis (NCIMB 13622)의 바지락(Ruditapes philippinarum)에서의 병원성을 조사하고, 바지락 조직 내의 균의 존재를 PCR법으로 진단하였다. 실온에 12시간 노출시킨 바지락 개체당 1.0×107 cells과 1.0×104 cells의 V. tapetis를 멸균주사기를 사용하여 인위감염 시켰을 때의 누적폐사율은 각각 67.5%와 7.5%로 나타났다. 폐사개체에서 BRD의 특징적인 증상인 패각내면의 chonchiolin 침착에 의해 형성되는 갈색반점은 관찰되지 않았으나, PCR법에 의해 균의 존재를 확인할 수 있었다.10종의 Vibrio균을 대상으로 PCR법의 검출 특이성을 조사한 결과 V. tapetis에 대해서만 414 bp의 특이밴드가 검출되었다. 또 V. tapetis를 개체당 1.0×104 cells과 1.0×108 cells 접종시킨 다음 사육하면서 경시적으로 균의 동태를 조사한 결과 개체당 1.0×104 cells을 접종하였을 때는 1일 후에는 아가미에서만 특이밴드가 검출되었고, 3일 후에는 모든 조직에서 검출되지 않았다. 한편 개체당 1.0×108 cells을 접종하였을 때 1일 후에는 아가미와 소화관, 3일 후에는 모든 조직에서 검출되었으며, 5일과 7일 후에는 외투막과 발, 9일 후에는 아가미와 발에서 검출되어, 검출장기로는 아가미가 가장 효과적인 것으로 판명되었다. PCR법으로 태안과 고창 지역의 바지락, 동죽, 굴 및 피뿔고둥의 조직내 V. tapetis의 존재를 검사한 결과 바지락과 피뿔고둥에서는 전혀 검출되지 않은 반면 굴과 동죽에서 각각 23.1%, 와 9.4% 검출되어 바지락 이외의 다른 패류의 진단에도 유용한 방법으로 판단되었다.
Abstract
Pathogenicity of Vibrio tapetis, the causative bacterium of "brown ring disease (BRD)" was evaluated in Manila clams (Ruditapes philippinarum) by artificially 0.1 ㎖ infection of 1.0×105 cells and 1.0×108 cells at 20℃. A PCR assay based on 16S rRNA to detect the bacteria in clam tissues was established. Accumulative mortality of clams infected with 1.0×107 cells and 1.0×104 cells per an individual of the bacteria was 67.5% and 7.5%, respectively. However, the deposit of brown pigment in the inner shells by accumulation of chonchiolin was not found. The bacteria were not be able to re-isolate from the infected clams by the conventional agar plate method but were easily detected by PCR assay established in this experiment. In clams artificially infected with 10 species of Vibrio, a 414bp for V. tapetis was detected in PCR assay. The specific band in the clams infected with 1.0×104 cells per an individual of V. tapetis was detected only in gills one day after the infection but never be found in any tissues including gills three days after the infection. In the case of clams infected with 1.0×108 cells per an individual of V. tapetis the specific band was detected in gills and intestine one day after the infection, in all tissues three days after the infection, and then in gills and adductor muscle nine days after the infection. The PCR assay was applied to detect V. tapetis in manila clam, surf clam (Mactra veneriformis), oyster (Crassostrea gigas) and Thomas' rapa whelk (Rapana venosa) taken from Taean and Gochang from April to July 2004. The infection rates were detected to 23.1% and 9.4% in the oyster and surf clam, while manila clam and Thomas' rapa whelk were not found.
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