Fluid Thioglycollate Medium Assay Research Articles

Are Juvenile Manila Clam Ruditapes philippinarum Free from Perkinsus olseni Infection in Korean Waters?

As a suspension feeder, Manila clam Ruditapes philippinarum (A. Adams and Reeve 1850) plays a crucial role in the coastal soft bottom ecosystem in the temperate region, linking the benthic primary production to the upper trophic level. Manila clam density on tidal flats on the west coast of Korea has been declining for the past decades, and infection by the protozoan parasite Perkinsus olseni (Lester and Davis 1981) is one of the major causes for the decline. Recent studies carried out in Japan revealed that P. olseni induces mortalities of the juveniles in their natural habitats, which may lead to the recruitment failure and subsequent decline in the clam population. In this study, we surveyed P. olseni infection in juvenile Manila clam occurring on two tidal flats on the Taean coast. Ray’s fluid thioglycollate medium assay (RFTM) revealed that P. olseni infection was not limited to the adult clams, and the juvenile and small-sized clams are also infected by P. olseni. As young as four-month-old juveniles from Jugyo tidal flat were infected by P. olseni, with the prevalence (i.e., percentage of the infected individuals) of 75.0% and the intensity of 7.77 × 105 cells g−1 wet tissue weight (WT). The adult Manila clams (SL > 30 mm) from Jugyo tidal flat showed a prevalence of 96.0%, and the intensity as 5.80 × 10 cells g−1 WT. The observed infection prevalence and intensity of the juvenile are somewhat comparable to those of the adult clams, suggesting that a high level of P. olseni infection in the juveniles may lead to mortality and a long term decline in the clam population density.

First discovery of Perkinsus beihaiensis in Mediterranean mussels (Mytilus galloprovincialis) in Tokyo Bay, Japan

During analyses of the invasive Mediterranean mussel Mytilus galloprovincialis for pathologies in Tokyo Bay, infection by the protozoan parasite Perkinsus beihaiensis was found through histological examination, Ray’s Fluid Thioglycollate Medium assays, and molecular analyses. Specific PCR assays for each Perkinsus species also revealed the presence of an indigenous congeneric species, Perkinsus olseni, but P. beihaiensis was dominant in M. galloprovincialis. Sequences of the ribosomal internal transcribed spacer region I of P. beihaiensis found in Japan were genetically more similar to those found in South American countries (Panama and Brazil) than in Asian countries (China and India). Though Mediterranean mussels have become widespread in Japanese waters since their invasion in the 1930s, epidemiological surveys show that mussels collected outside Tokyo Bay are free of any Perkinsus infections. Based on these results, it was strongly suggested that P. beihaiensis invaded Tokyo Bay by transportation of bivalves originating from South America but has not yet spread to other parts of Japan. The possibility is not ruled out, however, that the parasite is indigenous in Japan but the environment in Tokyo Bay favors its transmission to Mediterranean mussels.

Survey on <i>Perkinsus</i> Species in Manila Clam <i>Ruditapes philippinarum</i> in Korean Waters Using Species-Specific PCR

We investigated possible infection with Perkinsus olseni and/or P. honshuensis in Manila clams in Korean waters using Perkinsus species-specific PCR. Ray’s fluid thioglycollate medium assay revealed that 97.3% of the clams examined were infected with Perkinsus sp., with the mean intensity ranging from 2.2 × 104 to 6.8 × 106 cells/g gill. In the Perkinsus species-specific PCR assay, all the amplified Perkinsus-positive bands were identified as P. olseni, while none of P. honshuensis-specific band was amplified. Our survey results indicate that P. olseni is the main causative agent of perkinsosis in Manila clam populations in Korean waters, while more extensive survey must be carried out.

Perkinsus beihaiensis infectando a ostra Crassostrea rhizophorae em cultivo e em estoque natural na Baía de Camamu, Bahia, Brasil

Foi investigada a infecção de Perkinsus beihaiensis (Perkinsozoa) na ostra Crassostrea rhizophorae em um sistema de cultivo do tipo espinhel e em um estoque natural de ostras no manguezal adjacente ambos localizados no estado da Bahia, Nordeste do Brasil. As colheitas foram realizadas em outubro e novembro de 2012 e em janeiro de 2013. As ostras (n = 300) foram medidas, examinadas macroscopicamente quanto a sinais da infecção e submetidas às técnicas laboratoriais: histologia, ensaio em meio de cultivo de tioglicolato de Ray (RFTM), reação em cadeia da polimerase (PCR) e sequenciamento, que confirmou a identificação do patógeno. As análises histológicas e o RFTM mostraram, respectivamente, prevalência média de 93,3% e de 69%. A infecção foi geralmente leve ou muito leve. Não houve diferença significativa (p > 0,05) entre os ambientes em termos de prevalência ou severidade da infecção. Este é o primeiro registro de P. beihaiensis no estado da Bahia e o segundo em ostras do Brasil e América do Sul.

Immunological responses of the mangrove oysters Crassostrea gasar naturally infected by Perkinsus sp. in the Mamanguape Estuary, Paraíba state (Northeastern, Brazil)

Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1–4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of hemocytes proliferation. The immunological parameters varied over the studied months, which may be attributed to the dynamics of infection by Perkinsus sp. The results of the present study demonstrate that Perkinsus sp. has a deleterious effect on C. gasar immune system, mainly in high intensities, which likely renders oysters more susceptible to other pathogens and diseases.

Temporal Variation ofPerkinsus olseniInfection Intensity in the Manila ClamRuditapes philippinarumin Gomso Bay, Off the West Coast of Korea

Interannual variation of Perkinsus olseni infection intensity in the adult Manila clam Ruditapes philippinarum in Gomso Bay, off the west coast of Korea, was monitored from 1999 to 2000. Infection intensity of P. olseni (i.e., total number of P. olseni cells in unit tissue weight) was determined using Ray's fluid thioglycollate medium assay and Choi's 2-M NaOH digestion. In Gomso Bay, P. olseni monthly infection prevalence ranged from 83 (April 2000)–100%. It was remarkable that of the 18 mo of sampling, the prevalence remained at 100 % in 12 sampling months. Infection intensity of P. olseni in Manila clam ranged 366,001 (July 1999)–2,235,325 cells/g wet tissue (October 1999). The infection intensity recorded in 1999 was significantly higher than the level measured in 2000, suggesting an interannual variation in the intensity (P < 0.05). A very high level of infection intensity observed during the fall (September, October, and November) coincided with a relatively low condition index and mass morality of clams in the bay. Our data suggest that the mass mortality of clams observed during late summer to mid fall in Gomso Bay could be, in part, explained by the high level of Perkinsus infection coupled with the poor physiological condition of clams during the postspawning season.

Pathologies in commercial bivalve species from Santa Catarina State, southern Brazil

The State of Santa Catarina in southern Brazil is the most important Brazilian producer region. The Pacific oysters Crassostrea gigas and the brown mussels Perna perna are the main species currently produced. The occurrence and impact of parasites on bivalves from natural or culture populations from Brazil are still poorly studied. This work describes several diseases, parasites and commensals from four edible bivalve species, from cultured and exploited stocks, from five sites at Santa Catarina State. Unspecific histopathological symptoms of stress, parasites and commensals were found, including viral gametocytic hypertrophy and rickettsia-like organisms with very low prevalences; the protozoan Nematopsis sp. with high prevalence and, ovarian parasites with low prevalences; and, diverse metazoans, such as turbellarian, sporocysts and metacercariae of trematodes, metacestoda larvae and spionid polychaete. Ray's fluid thioglycollate medium assays for detection of Perkinsus spp. did not detect any infected bivalves. Polymerase chain reaction assays for Marteiliodies chungmuensis in oysters with ovarian lesions produced negative results.

Pathology survey of the short-neck clam Ruditapes philippinarum occurring on sandy tidal flats along the coast of Ariake Bay, Kyushu, Japan

The pathological condition of the short-neck clam Ruditapes philippinarum was surveyed along the coast of Kumamoto, Japan, in June 2004. DNA sequences of the non-transcribed spacer region and internal transcribed spacer region flanking 5.8S rRNA identified Perkinsus olseni among the clams. Ray’s fluid thioglycollate medium assay indicated that 96.7% of the clams surveyed from the Kiguchi River tidal flat (native clams, Stn KR-N) and 96.7% of the clams surveyed from the Midori River tidal flat (Stn MR) were infected with P. olseni with an infection intensity of 464,278 and 199,937 Perkinsus cells/gram tissue wet weight (gWW), respectively. In contrast, 66.7% of the clams imported from China and stored along the Kiguchi River tidal flat (Stn KR-I) and 20.2% of clams from the Arao tidal flat (Stn AT) were infected with P. olseni with an infection intensity of 37,547 and 3382 Perkinsus cells/gWW, respectively. Brown ring disease was detected in the clam population from Stn KR-I at a prevalence of 90.0%. Polymerase chain reaction and the 16S rRNA sequence suggested that the agents of brown ring disease observed at Stn KR-I were Vibrio tapetis-like bacteria. Sporocysts and metacercariae of unidentified trematodes were also observed in the gonads and mantle of the clams from Stn KR-I, Stn MR, and Stn AT, at prevalences of 7.1–42.9%. Metacestodes (larval tapeworms) were found in the foot and digestive gland at a prevalence of 52.5%, 30.0%, and 14.3% in clams from Stns MR, AT, and KR-N, respectively. Histology also showed massive hemocyte infiltration and inflammation among clams heavily infected with P. olseni. Castration of the follicle was typical among clams infected with the trematode. The data indicate that most of the clams along the coast of Kumamoto are infected with various pathogens at various rates of infection, and these pathogens could have negative effects on the clam population in the long term.

Occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis in Korean waters

This is the first report of the occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis off the western and southern coasts of South Korea. Histological observations revealed Perkinsus-like organisms in the mantle, gills, digestive tubules, and gonad. Haemocytic infiltration and tissue necrosis were also observed in heavily infected clams. Hypnospore formation of the Perkinsus-like organism was confirmed with Ray’s fluid thioglycollate medium assay (RFTM). When incubated in filtered and aerated seawater, the hypnospore gave rise to cell division and subsequently discharged hundreds of motile zoospores. Genus- and species-specific polymerase chain reaction (PCR) assays and the DNA sequences of the internal transcribed spacer region (ITS) of the Perkinsus sp. isolated from the Venus clam were identical to those of P. olseni reported from the Manila clam Venerupis (= Ruditapes) philippinarum. Based on the DNA sequences and microscopic data, the Perkinsus-like pathogen isolated from P. jedoensis was identified as P. olseni, which parasitizes the Manila clam in European and Asian waters and Haliotis rubra (abalone) in Australian waters. The prevalence and infection intensity of a clam population collected from Yosu, Korea, was determined using RFTM and Choi’s 2 M NaOH digestion technique. The intensities averaged 10,768 and 7438 Perkinsus cells per gram tissue in 2003 and 2004, and the prevalence ranged from 37.0 to 53.9%, respectively.