We have been surveying incidences in this country of drug resistant bacteria and their conjgative R plasmids isolated from cultured fish and the culturing poud water. We found out the high incidences of drug resistant bacteria and their conjugative R plasmids in various fish culturing fields.Since high amounts of chemotherapeutics have been widely used for fish diseases in this country, it is no wonder to have high incidences of drug resistant bacteria. To evaluate the effects of environmental factors on high incidences of drug resistant bacteria, we surveyed incidences of drug resistant bacteria and their conjugative R plasmids in fish culturing fields in U. S. A. We visited 74 fish culturing farms in eight fish culturing areas in the mid-summer of 1974 and sampled cultured fish and the culturing pond water. Average numbers of bacteria detected in fish intestines was 3.5 ×106/g intestinal materials and incidences of bacteria resistant to sulfonamide, streptomycin, chloramphenicol and tetracycline were 1.7%, 1.4%, 0.31% and 0.95%, respectively.Drug resistant bacteria were isolated from Salmonide fish and catfish with relatively high frequencies. Average number of bacteria detected in culturing pond water was 4.3 ×103/ml and incidences of bacteria resistant to sulfonamide, streptomycin, chloramphenicol and tetracycline were 2.8%, 8.5%, 3.5% and 3.8%, respectively. In general, incidences of drug resistant bacteria were higher in water than in intestines. All the results indicated that incidences of drug resistant bacteria in U.S.A. were far lower than those in this country. High proportion of the drug resistant bacteria isolated from the intestinal materials belonged to Enterobacteriaceae. Genera of the isolated bacteria were Escherichia, Citrobacter, Pseudomonas, Hafnia, Enterobacter, Klebsiella, Proteus and other unidentified bacteria in the order of the isolation frequencies. Genera of the drug resistant bacteria isolated from culturing pond water were the same as those from the intestinal materials except Proteus. In this surveillance, we isolated no drug resistant fish-pathogens, such as Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, and Edwardsiella tarda. This is quite different from the results which we had obtained in this country, where we had been isolated many drug resistant fish pathogenic bacteria from the intestines of healthy fish and pond water as well as from diseased fish. There are many reports on fish diseases in U.S.A. caused by bacteria. However, most of them might be caused by drug sensitive bacteria, since we did not isolate in U.S.A. any drug resistant bacteria pathogenic for fish. Thus, most of the drug resistant Enterobacteriaceae isolated from cultured fish might be selected in other fields, such as in domestic animal farms and city sewerage. Conjugative R plasmids were detected in 14 out of 532 drug resistant bacterial strains (2.6%). All the conjugative R plasmids were detected in the strains belonged to Enterobacteriaceae, and most of the R+ strains were isolated from materials from catfish farms. High water temperature and no water change might allow the growth of Enterobateria contaminated by the materials from human and domestic animals. Drug resistance markers of the detected conjugative R plasmids were combinations of sulfonamide-, streptomycin-, tetracycline- and kanamycin-resistances and no chloramphenicol- or ampicillin-resistance. These combination patterns of drug resistance were different from those detected in the fish pathogenic bacteria in Japan, but rather close to those detected in bacteria isolated from domestic animals in this country.
The preceding papers(MUROGA et al., 1976 a, b)dealt with the biochemical characteristics, taxonomical status, physiological characters and pathogenicity of Vibrio sp. isolated from a new eel vibriosis. In this paper the effect of NaCl concentrations of cultrue media on the NaCl tolerance and on the flagellation of the vibrio were examined, and these two characters of the organism were reconsidered by way of comparison with those of Vibrio fischeri. The experimental results together with considerations are given as under. 1) NaCl tolerance : The lower limit falled to 0.1% after consecutive culture in the media containing 0.3% NaCl. The upper limit, in contrast with this, raised to 6% in case of the media with 4% NaCl. The range of 0.1 to 6% is applicable to the NaCl tolerance range of this organism in view of these results. 2) flagellation: Cells having more than one flagellum at one pole occurred in cultures grown on nutrient agar plate, of which the higher the NaCl concentration (0.5-4.5%), the higher the percent occurrence (0.22-20%). Cells, however, had one polar flagellum in cultures grown in nutrient broth except in the broth with extremely high concentration (4%) of NaCl. Accordingly it may be safely said that the flagellation of this organism is monotrichous. In conclusion it is impossible to differentiate this organism provisionally classified as Vibrio anguillicida BRUUN and HEIBERG in the previous report (MUROGA et al., 1976a) from Vibrio fischeri in terms of the above-mentioned two characters.
Vibrio sp. RT group infection had broken out among the salmonids in autumn 1974. Histopathological observation was made on 15 diseased rainbow trout yearlings and 5 diseased amago salmon yearlings. Diseased fish showed a small boil or erosive lesions in the body surface or/and in the head in early stages but advanced cases showed large ulcerative lesions in the body surface or/and in the head, exophthalmos and enlarged spleen. Histopathologically an early lesion was formed in the skin in which considerable bacterial multiplication, edematous dissociation and infiltration of inflammatory cells were observed in the dermal loose connective tissue. In the extended ulcerative lesions bacteria spread extensively through the lateral musculatures and made many large colonies in the interstitial tissues. In these lesions dermal necrosis associated with epithelial slough and considerable edema, slight hemorrhages, infiltration of inflammatory cells, degeneration or necrosis of muscle fibers in the affected musculature were observed. In the lesion formed in the head bacteria diffusely spread through subcutaneous connective tissue and adipose tissue and edema, slight hemorrhage, infiltration of inflammatory cells and tissues necrosis were caused. In an eye ball showing exophthalmos, which was usually associated with lesions formed in the head, marked dilatation of coroid capillaries, detachment of the retina accompanied by necrosis of the pigmented layer, iris hemorrhage and degeneration of the conea were observed. In cases of systemic infection metastatic lesions appeared in the heart and spleen, and bacterial emboli were caused in sinusoids of the liver and renal hematopoietic tissue and in lamellae capillaries.
From May in 1975 to October in 1976, field investigations were made on dactylogyrids infestation on carp cultured in ponds at Hiroshima Prefectural Freshwater Fish Experiment Station. The results obtained are summarized as follows: 1) Dactylogyrus extensus and D. minutns were observed on fish of all ages almost throughout the year. 2) Apparent seasonal changes in abundance of both the dactylogyrids were recognized, and the changes seem to depend mainly on the water temperature. 3) D. extensus and D. minutus increased in abundance in the spring and the fall. The incresse of the former species was remarkable in the spring, and that of the latter in the fall. Both the species decreased conspicuously at the mid summer. 4) The same tendency of the above-mentioned changes in D. extensus was confirmed by experiments. D. extensus apparently increased in number with artificial raise of water temperature from 11°C to 20°C, and decreased sharply when the water temperature was over 20°C and reached about 25°C.
An epizootic of fungus disease occurred among kokanee salmon, Oncorhynchus nerka var. adonis in Lake Shikotsu of Hokkaido Island in 1974 to 1975. Fungi infected mainly on the peduncle and caudal fin of fish. One kind of fungus was isolated from diseased fish by inoculating materials from the caudal fin onto Sabouraud dextrose agar and incubating at 15°C. Inoculation experiments using moon fish, Xyphorus maculatus showed that all the isolates were virulent (Table 2). The fungus belonged to the genus Saprolegnia according to DICK (1973) from its characteristics of the asexual reproductive stages. Characteristics of the sexual reproductive structures of diclinous antheridia and subcentric oospores placed the fungus close to S. parasitica and S. australis according to SEYMOUR (1970). Comparison in biological characteristics between the present fungus, and S. parasitica and S. australis described by SEYMOUR (1970) are summarized as follows. The zoospores of the fungus were larger in size than those of S. parasitica and S. australis. The oogonia were sparsely formed only after prolonged period of time, and in this point it resembles to S. parasitica rather than S. australis. The fungus can be distinguished from S. parasitica and S. australis by the characteristic of catenulate oogonia. The fungus has smaller oogonia than those of S. parasitica and S. australis. As regards characteristics of pits of the oogonial wall the fungus lies between S. parasitica and S. australis. Oospores per oogonium of the fungus were less in number than those of S. parasitica and S. australis. Antheridial barnches are usually present in S. parasitica and S. australis, but were often lacking in the fungus. From the above mentioned the fungus was regarded as a new species of the Saprolegnia.
ISHII(1931) described a nematode from the caudal fin of Japanese crucian carp, Carassius auratus (LINNé), under the name of Filaria carassii and stated that the gravid female of the nematode had three minute sharp protuberances around the mouth. Later, YAMAGUTI (1961) referred this species to the genus Philometra, but FURUYAMA (1934) and IVASHKIN et al. (1971) regarded it as the synonym of Philometroides sanguinea (RUDOLPHI, 1819) RASHEED, 1963. We found the eleven gravid females of ISHII's species in the caudal fin of nine of the 76 crucian carp which were obtained from a game fishing pond at Ichikawa city in Chiba prefecture in March, 1975, and examined for the nematode. The infected nine fish were 18-25 cm in body length. Five specimens of the eleven gravid females were examined morphologically. Possessing numerous bosess on the cuticular surface of their bodies, ISHII's species had to be certainly combined with the genus Philometroides and had to be distinguished from P. sanguinea in which the presence of the pharyngeal teeth was never described by any writer, though the sizes of gravid females, eggs and larvae were the same each other. We suggested from the combination of this species that the two characters of the genus Philometroides, that was, “Head with papillae” and “Oesophageal teeth absent”, had to be eliminated from the generic diagnosis proposed by RASHEED (1963). Philometroides carassii (ISHII, 1931) n. comb. (Figs. 1-4): Body filiform, tapered anteriorly and posteriorly with bluntly rounded end, 40-45 mm long by 0.8-1.1 mm wide when fixed in 5% formalin. Cuticle with numerous bosses irregularly scattered. Cephalic and caudal papillae absent. Three cuticularrized pharyngeal teeth present around the mouth. Pharynx swollen just under the mouth, 3.0-3.5 mm long by 0.11-0.15 mm wide at the swelling. Nerve ring 0.25-0.28 mm from the head end. Intestine 0.24-0.37 mm wide, ended blindly near the tail end. Anus and vulva absent. Uterus opposed. Anterior ovary 1.7-2.5 mm long by 0.05-0.08 mm wide and posterior ovary 1.2-2.0 mm long by 0.03-0.04 mm wide. Eggs spherical, thin-shelled, 26-40μ in diameter. Larvae filiform with rounded head and shrply pointed tail, 0.34-0.41mm long by 13-18μ wide. Types: Deposited in the National Science Museum, Tokyo. Cat. no. NSMT-As-1428.
Gravid females of Philometroides carassii (ISHII, 1931) NARAJIMA et EGUSA, 1977 were found in the caudal fin of the crucian carp, Carassius auratus (LINNé) in the early spring. Their body color originated from the red-colored fluid circulating intermittently in their body cavities and changed from clear red to light pink as eggs developed in the uterus, because the layer of the body fluid became thinner with expansion of the uterus.The pinkish gravid females penetrated the skin with their heads at the base or the posterior end of the caudal fin and moved out slowly into water for themselves. The exposed part of their bodies soon discolored, straightened out and hardened, however, yielding of larvae never occurred so far as their tails stayed in the fin. The process of delivery was observed with three gravid females taken out from hosts. When the worms dipped in water, a kind of pulse possibly due to a contraction of the uterus wall started within a minute from the posterior end to the anterior. At that instant the anterior part of the uterus broke the body wall longitudinally and rushed out into water with the other organs. Almost all larvae were released from the split uterus for several minutes. From the observations that larvae were never lereased so far as the tail of the worm stayed in the fin but released immediately when dipping the whole body in water, we supposed that gravid females might have a receptor of water at their tails.
In trout hatcheries, most of loss of brood stock had been caused after egg-stripping. If the mortality of brood stock is lowered more economical and stable egg-supply could be expected. Under these circumstances, pathological studies have been conducted to clarify the cause of loss after eggstripping. In the present study mortality and symptoms of brood rainbow trout after egg-stripping were studied from 1972 to 1977 at Koide Branch, Niigata Prefectural Freshwater Fisheries Experimental Station. The mortality during 7 months from September to March, 1972-1976, ranged between 15.9 and 25.0% for 2-year-old and between 13.0 and 52.7% for 3-year-old trout. The mortality of 3-year-old was higher than that of 2-year-old trout except 1974. The mortality increased one week after egg-stripping and ceased after about 3 weeks. Macroscopical examinations of the fish died after egg-stripping were carried out during 2 seasons, from December 1975 to January 1976 (Exp.1) and from November 1976 to December 1976 (Exp. 2). The remarkable symptoms were fungus affection on skin and gills, inflammation of intestine, redness of pyloric coeca, and atrophy of spleen. Boils due to furunculosis were observed in some instances. Incidence of infestation of external parasites were examined for the samples obtained in Exp. 2. Trichodina were observed on gill of 25.5% of 2-year-old fish and 30.0% of 3-year-old.
An epizootic of mycotic granulomatosis occurred among pond-cultured ayu, Plecoglossus altivelis at a fish farm in Shiga Prefecture. A fungus was isolated by inoculating materials taken from lesions of diseased fish onto FME agar and incubating at 25°C (Table 1). Inoculation experiments using ayu and gold fish, Carassius auratus showed that the fungus was the causative agent of mycotic granulomatosis. The experiments were done by injecting a mass of hyphae cultured on the FME agar into intramuscularly each fish. Granulomatosis was histopathologically demonstrated in all the artificially infected fishes, from which the fungus was reisolated by means of inoculating materials from lesions on FME agar. Spore formation was not observed in the fungus growing in the lesion of naturally infected ayu, and the source of infection of mycotic granulomatosis was thought to be hyphae themselves. The present authors suggest the temporary designation, MG-fungus (Mycotic Granulomatosisfungus), until the fungus is named.
In the process of artificial production of Ayu fry (Plecoglossus altivelis), considerable mortalities of the fry (aged one to two months) were frequently found. Each of these dead fish had a white girdle along the spinal cord, apparently showing the characteristic of the disease. Fourty one of the diseased fish were histopathologically investigated by the present authors. All the diseased fish erroneously swallowed the diets through the pneumatic duct into the swim bladder, without transferring into the alimentary canal. In 39 fish, the lumen of the swim bladder showed hyperplasia or necrosis due to bacteria, which probably multiplied abruptly in the organ. In 2 fish, a great number of fungi were observed in the diet found in the swim bladder; some organs (kidney and body musculature) near the swim bladder were remarkably affected by the fungi. From the present work, it is considered that the erroneous-swall-owing of diet causes the mortalities of Ayu fry.
Ceroidosis occured in snapping turtle(Trionyx sinensis)fed on rotten silk-warm pupae and fish meat continuously in Oita in summer, 1975. They showed the dark-yellowish coloration of the adipose tissue all over the body.Six diseased turtles were examined histopathologically and histochemically. In the adipose tissue of the mild cases, angitis and necrosis of a part of fat cells around the inflammed capillaries were observed.Lipopigment, indentified as ceroid, was deposited inside each necrotized fat cells. In the adipose tissue of the advanced cases, angitis and necrosis of fat cells around the inflammed vessels, ceroid accumulation inside and among each necrotized fat cells were found in more extensive areas and in larger quantities. A great number of macrophages were present around the inflammed vessels and most of them devoured ceroid.It seems that the dark-yellowish coloration of the adipose tissue is due to above-mentioned changes.A small number of ceroid-laden macrophages appeared in all the visceral organs. Degenerating striated muscle fibers appeared in the extensive muscula tures. The authors consider that above-mentioned changes closely resemble the yellow fat disease in mammals and aves.