Infection Of Crabs Research Articles

Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus

Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.

A Novel Serine Protease Inhibitor Acts as an Immunomodulatory Switch while Maintaining Homeostasis

Serine protease cascades boost immune responses while maintaining homeostasis. These crucial actions are intricately regulated by cognate serine protease inhibitors. However, the mechanism underlying such a dynamic immunomodulation during acute phase infection remains obscure, particularly where the pathogen’s serine protease adds a new challenge to the host. Here, we found that infection of horseshoe crab, Carcinoscorpius rotundicauda, induced reciprocal profiles of CrSPI (serine protease inhibitor) and CrFurin (serine protease) with respect to their transcription and protein activities. Using recombinant rCrSPI, we explored its inhibitory activity against various microbial proteases and found it most efficacious against a model serine protease, subtilisin A. rCrSPI inhibited subtilisin at Ki 10^–9M with a molar ratio of 1 rCrSPI:2 subtilisin. The rCrSPI also inhibited plasma CrFurin, suppressed subtilisin-mediated activation of prophenoloxidase (PPO) and interacted with complement C3. Taken together, CrSPI acts as a key immunomodulatory ‘on-off’ switch in a 2-way regulation of serine protease microbial subtilisin and host serine proteases (CrFurin and CrC3), thereby controlling immune responses involving the complements and the PPO-mediated antimicrobial activities, while maintaining homeostasis.

Diseases of the European edible crab (Cancer pagurus): a review

AbstractStentiford, G. D. 2008. Diseases of the European edible crab (Cancer pagurus): a review. – ICES Journal of Marine Science, 65: 1578–1592. The edible crab (Cancer pagurus) supports an important fishery in European waters. The fishery is increasing in size and in relative importance as stocks of marine finfish decline. Despite its importance, though, studies on the pathogens and parasites of this crab species are relatively lacking compared with studies of commercially exploited finfish and molluscan hosts. Recent basic surveys of C. pagurus stocks from the English Channel carried out by the Cefas laboratory at Weymouth have identified a new viral infection (C. pagurus bacilliform virus, CpBV) in juvenile crabs, and several new species of protistan parasite (Hematodinium sp., Paramarteilia canceri, and Enterospora canceri) in the adult population. The histopathology and prevalence of each of these pathogens suggests that each can induce host mortality and, further, that specific pathogens are differentially prevalent in juvenile and adult cohorts from similar geographic locations and at different times of the year. In this review, these newly discovered pathogens are placed in context with previously described bacterial, fungal, protistan, and metazoan pathogens of C. pagurus, and the potential for these pathogens to impact on the health of individuals and populations within the English Channel fishery is discussed.

Detection and Discovery of Crustacean Parasites in Blue Crabs ( Callinectes sapidus ) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.

Pharmacokinetics of intravascular itraconazole in the American horseshoe crab (Limulus polyphemus)

The American horseshoe crab (Limulus polyphemus), a marine member of the Phylum Arthropoda is a popular hands-on exhibit animal in aquariums and zoological parks. At Ripley s Aquarium of the Smokies, white, proliferative, irregularly expansile lesions were observed on the soft tissue of the gills and the joint spaces of the legs and tails of crabs kept in a hands-on tank. Additionally, 1–2 mm diameter, circular, discolored red-brown, ulcerated lesions of the ventral carapace were recognized. A fungal etiology of the lesions was determined by cytology and histopathology. Itraconazole is a triazole anti-fungal agent and is widely used in human and veterinary medicine for the treatment of fungal infections. Pharmacokinetic studies are scarce for invertebrates (Gore et al., 2005), and those involving anti-fungal agents are lacking. This study was designed to determine the pharmacokinetics of itraconazole for the treatment of susceptible fungal infections in American horseshoe crabs. The study group consisted of 10 horseshoe crabs (six males and four females) equally divided into two 150 gallon recirculating tanks. Water quality parameters were assessed regularly, including temperature (23–25 C), pH (7.77–8.31), salinity (33–38 ppt), total ammonia ⁄NH3 ⁄NH4 -N (0.0 mg ⁄L), nitrite (0.02–0.05 ppm), nitrate (98–104 ppm), dissolved oxygen saturation (71–97.8%), dissolved oxygen (4.0–6.2 ppm), copper (0.003–0.009 ppm), and alkalinity ⁄CaCO3 (290–400 ppm). Sample collection and drug administration were performed through the cardiac sinus with the animal restrained in ventral flexion (Smith, 2006). A pilot study was conducted in one animal to establish a dose and sampling interval for itraconazole. The animal was given 5 mg ⁄kg i.v. itraconazole (Sporanox; Ortho Biotech, L.P., Raritan, NJ, USA) diluted with 0.9% sodium chloride to a concentration of 3.33 mg ⁄mL per manufacturer s instructions. This mixture was further diluted to 2 mg ⁄mL with 0.9% sodium chloride (Hospira, Lake Forest, IL, USA) to attain a pH of 6.60 as determined by Beckman F pH meter (Beckman Instruments, Inc., Irvine, CA, USA). Hemolymph was collected in a 3-mL syringe with a 22-guage needle at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h following itraconazole administration. The sample volume was 0.3 mL for a total of 3.3 mL hemolymph collected over the sampling period. Hemolymph was placed immediately in a lithium heparin microtainer (Becton Dickinson, Franklin Lakes, NJ, USA), centrifuged, and the hemolymph supernatant was placed in a cryovial and stored at )80 C until analysis. Based on the results from the pilot study, a dose of 10 mg ⁄kg was established for the full study. Procedures for itraconazole preparation, sample collection and handling were identical to those used in the pilot study. One untreated animal in the tank served as a negative control to assess the redistribution of itraconazole circulating in the water. Analyses for itraconazole, and its active metabolite, hydroxyitraconazole were performed using a modified HPLC method as previously described (Cox et al., 1997). Hemolymph samples were analyzed by reverse phase HPLC with fluorescence detection. The HPLC system consisted of a 2996 separations module, a 2475 fluorescence detector and a computer equipped with EMPOWER software (Waters, Milford, MA, USA). Standard curves for hemolymph analysis were prepared by fortifying untreated hemolymph with itraconazole and hydroxyitraconazole to produce a linear concentration range of 10– 5000 ng ⁄mL. The intra-assay variability for itraconazole ranged from 0.6% to 1.8% and 0.9% to 4.2% for the hydroxy metabolite. Inter-assay variability ranged from 0.5% to 10% for itraconazole and 1.8% to 9.2% for the hydroxy metabolite. Recovery ranged from 86% to 93% for itraconazole and 79% to 90% for hydroxyitraconazole. Analysis of concentration–time data and estimation of pharmacokinetic parameter values for itraconazole and its metabolite J. vet. Pharmacol. Therap. 31, 83–86, doi: 10.1111/j.1365-2885.2007.00924.x. SHORT COMMUNICATION

Molecular Basis for Invertebrate Innate Immune Recognition of (1→3)-β- D-Glucan as A Pathogen-Associated Molecular Pattern

Innate immunity responds to various pathogen-associated molecular patterns (PAMPs) to evaluate the biological nature of foreign materials by using limited numbers of receptors. Analyses of interactions between PAMPs and its receptors are essential to understand the molecular basis regarding how we discriminate self and non-self materials. Upon infection of horseshoe crabs, an arthropod species, rapid hemolymph coagulation is induced to engulf invading microorganisms by a cascade-type reaction. The reaction is very sensitive to lipopolysaccharide and (1-->3)-beta-D-glucans on Gram-negative bacteria and fungi, respectively, and hence is utilized as assay reagents that detect and quantitate these PAMPs with a name of "limulus test." In this mini-review, recognition of (1-->3)-beta-D-glucans by a unique serine protease zymogen factor G of horseshoe crab is described. Molecular dissection and detailed kinetic analyses have revealed that multivalent binding to polymers of a simple target structure is one of the principles that allows stable and specific recognition of PAMPs by pattern recognition receptors in innate immunity.

Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda

Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the co-regulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained down regulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immuno-modulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.

Epidemiology of bitter crab disease (Hematodinium sp.) in snow crabs Chionoecetes opilio from Newfoundland, Canada

The parasitic dinoflagellate Hematodinium sp. causes a condition known as bitter crab disease (BCD) in snow crabs Chionoecetes opilio and Tanner crabs C. bairdi. As the name of the condition implies, crabs infected with BCD are unmarketable due to their bitter flavor. We surveyed the distribution of BCD in 3 regions within the snow crab fishery of Newfoundland from 1997 to 2003. Over time, the disease has become firmly established in Conception and Bonavista Bays and persists at low levels on the Avalon fishing grounds. An epizootic occurred within Bonavista and Conception Bays in 1999 and persisted in Conception Bay in 2000, reaching prevalences of over 2% to 9% in trapped and trawled male crabs and from 19 to 26% in trawled and trapped female crabs, respectively. Infections were highest in females and small males, i.e. the unfished and pre-recruit portions of the fishery. In a mortality study, all of the naturally infected crabs died and 50% of the experimentally inoculated crabs died. Patterns in the molting cycle and prevalence of infection indicate that transmission occurs during the post-molt condition, and that overt infections probably develop 2 to 4 mo later with mortalities occurring at least 3 to 4 mo thereafter. The hydrography of this bay may have contributed to the epizootic as infections were centered within the deeper confines of the bay. Analysis of various abiotic factors uncovered a significant positive association between prevalence, depth and mud/sand substrates; the nature of this relationship was not apparent but may be related to diet or alternate hosts. Lastly, given the increase in BCD in snow crabs in Newfoundland, we recommend that fishery management programs for Chionoecetes fisheries employ non-selective gear to monitor for Hematodinium infections in female and juvenile crabs because these under-sampled members of the population may forewarn of impending recruitment declines that might otherwise remain unexplained.

18-kDa protein as a marker to detect WSSV infection in shrimps

White spot syndrome virus (WSSV) was purified from infected shrimp tissues by ultracentrifugation and run on SDS-PAGE. Nine major proteins were observed when the profiles of uninfected and infected shrimp tissues were compared. A prominent 18-kDa (kilodalton) protein was purified from the gel and a specific antiserum was produced that detected WSSV infection of both shrimps and crabs. A time course study with different shrimp tissues using Western blot analysis and ELISA indicated that tail, head tissue and eyestalk had high viral load compared to other organs. Fluoresceinated microspheres and latex beads coated with 18-kDa antiserum agglutinated upon mixing with infected shrimp tissue and failed to agglutinate with normal shrimp tissue. An easy cost-effective diagnostic assay has been developed using 18-kDa antiserum, and it detected the virus as early as 24 h postinfection in shrimps.

Co-infection by a yeast-like organism in Hematodinium-infected European edible crabs Cancer pagurus and velvet swimming crabs Necora puber from the English Channel.

During the winter months, edible crabs Cancer pagurus and velvet swimming crabs Necora puber from the English Channel can harbour infections by a Hematodinium sp. dinoflagellate. This parasite is responsible for a highly pathological condition known as 'Pink Crab Disease' (PCD) in the edible crab. In the current study, a high proportion (between 25 and 100%) of Hematodinium-infected edible and velvet swimming crabs captured from 2 sites in the English Channel also harboured a systemic infection by a yeast-like organism. This is the first report of such an infection in crabs. Budding yeast-like cells were observed intracellularly in circulating haemocytes and free in the host plasma. These cells stained positively with silver and periodic acid-Schiff (PAS) reactions. Despite an apparent haemocytopoenia in Hematodinium-infected crabs, haemocytic encapsulation of yeast-like cells was evident, while no such response was observed against Hematodinium sp. plasmodia. It is hypothesised that Hematodinium infection in these crabs may either increase the likelihood of secondary infections via an indirect suppression of the host immune system, or alternatively, decrease the likelihood of competitive growth inhibition by stimulating the host immune system to encapsulate and destroy secondary pathogens. Results are discussed with regard to the likely identity of the yeast-like organism, and the role of secondary pathogens in the eventual mortality of Hematodinium-infected hosts.

Monitoring the prevalence of the parasitic dinoflagellate Hematodinium sp. in snow crabs Chionoecetes opilio from Conception Bay, Newfoundland.

Bitter crab disease (BCD) of snow crabs Chionoecetes opilio is caused by a parasitic dinoflagellate, Hematodinium sp. In Newfoundland's commercial fishery, infected snow crabs are identified using visual, macroscopic signs of disease for separation prior to processing. We estimated the sensitivity and specificity of gross, macroscopic diagnosis of Hematodinium sp. by comparing these results with microscopic examination of prepared hemolymph smears. The sensitivity of a diagnostic test is the probability that the test will yield a positive result given that the animal has the disease. The specificity is the probability of a negative result given the animal is not diseased. In October 1998, we conducted a design-based survey using cluster sampling in 2 strata. Over 10 000 snow crabs from pot and trawl surveys were examined macroscopically for BCD. In addition, over 350 crabs were randomly examined microscopically for disease. The double sampling resulted in an estimated sensitivity of 52.7% and an estimated specificity of 100%. That is, a positive result from macroscopic examination is definitive, if the observer is well trained, but macroscopic examination will fail to detect infections in crabs with borderline clinical signs of disease. The prevalence estimated from macroscopic observations (p(st) = 2.24%) was corrected for misclassification by dividing p(st) by the estimated sensitivity (0.527), giving a corrected estimate of 4.25%. The use of double sampling provides for efficient estimation of prevalence in that large numbers of crabs can be quickly examined for gross signs of infection and the results corrected for misclassification based on a limited number of observations with a better, but time-consuming test. In addition, the prevalence of macroscopically infected male crabs was lower in a trap survey (0.57%) compared to a trawl survey (1.59%). In the trawl survey, female crabs had a significantly higher prevalence of macroscopically diagnosed infections than males (6.34%). The prevalence of BCD has shown an alarming increase since it was first detected in Newfoundland during the early 1990s. Transmission and mortality studies are warranted to better understand the effect of the disease on its commercially important host.

Experimental host range and histopathology of white spot syndrome virus (WSSV) infection in shrimp, prawns, crabs and lobsters from India

Experimental studies were conducted by injecting or feeding white spot syndrome virus (WSSV) derived from infected shrimp, Penaeus monodon (Fabricius), collected from the south‐east coast of India, to five species of shrimp, two species of freshwater prawns, four species of crabs and three species of lobsters. All species examined were susceptible to the virus. Experimental infections in the shrimp had the same clinical symptoms and histopathological characteristics as in naturally infected P. monodon. A cumulative mortality of 100% was observed within 5–7 days in shrimp injected with WSSV and 7–9 days in shrimp fed with infected tissue. Two species of mud crab, Scylla sp., survived the infection for 30 days without any clinical symptoms. All three species of lobsters, Panulirus sp., and the freshwater prawn, Macrobrachium rosenbergii (De Man), survived the infection for 70 days without clinical symptoms. However, bioassay and histology using healthy P. monodon revealed that crabs, prawns and lobsters may act as asymptomatic carriers/reservoir hosts of WSSV. This is the first report to suggest the carrier/reservoir capacity of these hosts through histological and bioassay evidences. Ultrastructural details of the virus in experimentally infected shrimp, P. vannamei, (Boone), were also studied.

Hematodinium perezi infections in adult arid juvenile blue crabs Callinectes sapidus from coastal bays of Maryland and Virginia, USA

In coastal bays of Maryland and Virginia, USA, adult and juvenile blue crabs Callinectes sapidus were severely infected with the parasitic dinoflagellate I-lematodinium perezi. Dinoflagellates were observed in the hemocoel of all infected crabs; associated histopathological changes were evident in some tissues. Dinoflagellates could be observed with an inverted microscope through the 5th pleopod of heavily infected juvenile crabs (5 to 29 mm) without invasion. This note documents a high prevalence of H. perezj infections in juvenile blue crabs from coastal bays in Maryland and Virginia. The seasonal infection prevalence cycle reported by previous authors is consistent with observations made during this study.

Parasitological survey of the first intermediate host of Paragonimus westermani in Iga area of Mie Prefecture, Japan.

During the period from September 1985 to March 1988, the freshwater snails, Semisulcospira libertina, were collected from 4 mountain rivers in Ayama County of Mie Prefecture, which is known as a heavily infected locality with Paragonimus westermani (Kerbert, 1878) Braun, 1899, and were examined for cercariae and rediae of this lung fluke. Of 3,000 snails studied, 80 (2.67%) harbored Paragonimus larvae. The infected snails were found at 3 sites in the Nishitani (A), Nenobi(B), and Higashitani (C) rivers. The infection rate of Paragonimus cercariae at these sites varied from 0.13 to 6.08%. The highest incidence (6.08%) of cercarial infection occurred at site A (Nishitani river), where the prevalence and intensity of infection with P. westermani metacercariae were considerably greater in the 2nd intermediate host crabs. No Paragonimus was observed in 776 snails collected at site D (Taki river), where the incidence and degree of metacercarial infection in crabs were quite small. There was a positive correlation between the infection incidence of P. westermani and the size of S. libertina; the percentage of infected snails increased in larger size classes, reaching 24.14% at a shell length of over 40 mm. In the 80 positive snails there were 51 mixed infections with P. westermani and 1 or 2 other species of trematode larvae. Of these, 43 were double infections and 8 triple. The morphological features of P. westermani cercaria and redia are described.

Susceptibility and resistance of the rock crab, Cancer irroratus, to natural and experimental bacterial infection

The incidence of naturally occuring bacterial infection in a Connecticut population of rock crab, Cancer irroratus, varied between 10 and 60% of the population. The fluctuation in incidence was correlated significantly with hemocyte concentrations. Twelve percent of the crabs sampled during Jenuary were found to be infected with a previously undescribed Vibrio. The Vibrio was demonstrated as pathogenic over a wide range of temperatures. High mortalities in experimentally infected crabs appear to result from hemocyte clumping and extensive intravascular clotting triggered by an endotoxin. The lobster pathogen, Aerococcus viridans var. homari, was found to be pathogenic at 25°C to C. irroratus, a prey species of the lobster. Results suggest that this species can act as a reservior host for A. viridans var. homari.

A ciliate infection ( Paranophrys sp.) in laboratory-held Dungeness crabs, Cancer magister

A ciliate infection ascribed to the genus Paranophrys was found in early adult Dungeness crabs, Cancer magister, held in laboratory bioassay facilities, and in outdoor flow-through tanks in Newport, Oregon. Infections proved lethal as ciliates multiplied to high densities in the blood and tissues causing degeneration and necrosis of musculature. All infected crabs had recent wounds through the exoskeleton which probably provided a portal of entry, and the elapsed time between infliction of a wound and gross symptoms of stress was estimated at 9 days although animals died as late as 26 days. The nature of Paranophrys infections in C. magister, i.e., as an opportunistic pathogen of cultured animals and/or a disease in wild populations of this crab is discussed.

Ecological observations on the commercial sand crab, Portunus pelagicus (L.), and its parasite, Sacculina granifera Boschma, 1973 (Cirripedia: Rhizocephala)

Abstract. Sacculina granifera was found in 12% of commercial sandcrabs, Portunus pelagicus, in Moreton Bay irrespective of sex. Although male and female crabs were randomly distributed about Moreton Bay, egg bearing females were most common at the seaward station. This and the distribution of epizoic barnacles on gills and carapace suggest the sexes school separately. The increased prevalence of barnacles on the carapace of infected crabs indicates Sacculina inhibits moulting. The distribution of interna infections in small crabs towards south and west and externa infections in large crabs in the easterly, seaward stations suggests Sacculina preferentially attacks young crabs as they move inshore and then induces crabs to behave like ‘berried’ (i.e. egg bearing) females by moving seaward as they grow. Morphologically female crabs are little changed by Sacculina, but the males show considerable modification which is reflected most accurately in the shortening of the chelar propodus to proportions similar to normal females. Infected crabs are sterile and internally the hepatopancreas becomes green rather than tan. Parasitized crabs were seen to groom their externae as ‘berried’ females groom their egg masses.

Bacterial infection in the blue crab, Callinectes sapidus: course of infection and histopathology

During the summer, groups of blue crabs, Callinectes sapidus, collected in commercial crab traps in Chincoteague Bay, Virginia, often undergo heavy mortalities during the first week to 10 days in the laboratory. Gram-negative bacteria are seen in hemolymph and tissues of many of the sick and dying crabs. The bacterial infections appear to be acquired during capture and transport, suggesting that potentially pathogenic bacteria in water or on the exoskeleton may be introduced into tissues by wounding or other means during the stressful conditions suffered at that time. The pathology caused by bacterial infection includes diminution in numbers of hemocytes, reduced clotting ability of the hemolymph, and progressive formation of hemocyte aggregations with necrotic centers in the heart, arteries, and hemal sinuses and spaces. By the third day, aggregations, often with many bacteria visible in the centers, occur especially in the gills, antennal gland, and Y organ. There are large premortem plasma clots in some animals. The focal and massive necroses that occur may be due to hypoxia resulting from obstruction of hemolymph flow by cellular aggregations and plasma clots and to toxic products of necrotic cells and/or bacteria.

A Follow-Up Study To Evaluate The Efficacy Of Mass Chemotherapy For Control Of Paragonimiasis

In the survey of l1,005 subjects in l3 villages of Che Ju lsland, 1,450 persons were egg-positive by repeated sputum examinations. The response rate to intradermal test ranged 50-80% of the population and to sputum examination for skin positive 60-70% in the first survey done in l964. 93% of all egg-positive was treated with 40 mg/kg body weight of bithionol on alternate days for 10-15 doses in l965. Follow-up studies were carried out in one year, three-year, five-year, and eight year after the mass treatment. The one year follow-up study covered 86.5% of all treated cases in all villages but only a part of the population of a few villages in further follow-up studies. In this paper only the result obtained from the age group of 7-18 years, the common population studied in every follow-up for the purpose of comparison, is presented. Intermediate hosts of P. westermani were examined at the same time. In one year follow-up it was found that 91% of egg-positive treated became negative. Three parameters, skin reaction, sputum examination, and infection status of intermediate hosts by area and year were used for comparison. Skin positive rate showed gradual decrease with more marked drop in Kang Chung where more thorough survey and mass treatment (about 43% of all infected persons estimated among whole population) were carried out than other areas (only 20-30%). The proportion of intermediate skin reation (60-100 mm(3) wheal size) increased markedly indicating decreased dose of infection which corresponds to the dropped infection rate and average number of metacercaria in crabs. Egg-positive rate in sputum examination done for skin positive reactors also presented similar pattern to skin reaction although it should be taken account that the result of 1964 was from repeated sputum examinations but that of other years from a single examination. Increased proportion of intermediate skin reaction which has much lower egg-positive rate probably played major role in decreased egg-positive rate. The infection rate of snail started to drop about one year earlier than crab; crab infection rate and intensity started to drop about four year after the mass treatment. The duration needed to show the change of infection status in intermediate hosts after the mass chemotherapy may correspond to the duration of survival of the parasite in the intermediate hosts. With the data available presently, it is difficult to determine on what point of the curve the present status is situated (on decreasing phase or increasing phase?), how far the rate will go down if it is on decreasing phase, and how long would it take in returning to the original status without further management. The result of follow-up study, however, strongly indicate that continous and intense chemotherapy may be able to put the paragonimiasis under control. Further follow-up study on total population including survey on changes of environment and eating habits is planned.

Transmissible Disease, Probably Viral in Origin, Affecting the Amebocytes of the European Shore Crab, Carcinus maenas

A transmissible infection of the shore crab ("crabe enragée"), Carcinus maenas, was discovered in one of about 700 of this species collected by the Station Biologique, Roscoff, France. The infection has been transferred by subinoculation through four serial passages in crabs collected at Roscoff and through six more passages in C. maenas collected at Woods Hole, Mass. The agent causes abnormal cellular clotting, a drop in the peripheral amebocyte count, clumping of amebocytes in the peripheral tissue and blood, and abnormal behavior of the amebocytes of the infected animal on glass. Mortality over a period of some weeks is negligible. Electron microscopy shows characteristic virus particles, about 55 to 125 nm in size, in the infected amebocytes. The agent is present in large amounts in the whole blood and serum, is filterable, heat labile, and has been preserved in the frozen state. Final identification will depend upon purification, growth in tissue culture, and further electron microscopy.