Susan M. Bower

Cryptobia catostomi: Incubation in plasma of susceptible and refractory fishes

Abstract The mechanism of resistance of rainbow trout to Cryptobia catostomi was studied in laboratory raised fish using the in vitro plasma incubation test. In this test a suspension of washed C. catostomi was incubated in freshly collected plasma for 3 hr at 4 C; after that, the mixture was examined for living C. catostomi . The plasma of six refractive fishes (goldfish, creek chub, hornyhead chub, rainbow trout, pike, and brown bullhead) had cryptobiacidal titers ranging from 1:2 to greater than 1:16. Heat inactivation (56 C for 30 min) of the trout plasma reduced the cryptobiacidal titer from 1:16 to 1:4. Selected removal of Mg 2+ and Ca 2+ ions from the plasma using EGTA and EDTA reduced the titer to 1:2 and 1:4; however, the titer was restored to 1:16 when the chelated plasmas were resupplemented with the ions. Zymosan treatment of the plasma also reduced the titer to 1:4. Even partial coagulation of the blood reduced the titer; the titer for serum was only 1:4 as compared to 1:16 for plasma. It is proposed that rainbow trout is refractive to C. catostomi because its blood contains a natural properdin system that is activated by the parasite. Thus the alternate pathway of complement activation is suggested as one of the mechanisms of “natural immunity” by vertebrates that are related to the susceptible host.

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location.

Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

Immunological comparison of four Trypanosoma spp. (sub-genus Schizotrypanum) from bats.

The antigenic differences and similarities between culture forms of Trypanosoma (Schizotrypanum) hedricki, Trypanosoma (Schizotrypanum) myoti, Trypanosoma (Schizotrypanum) vespertilionis and Trypanosoma (Schizotrypanum) dionisii, all isolated from bats, were compared using the double-diffusion technique. Antiserum to each trypanosome species was produced in rabbits by inoculating them with sonicated culture forms. Two isolates of T. hedricki were antigenically identical to each other, as were 2 isolates of T. myoti. There were several common antigens between the species. However, at least 1 antigen of each species was distinct from those of the other species.

An in vitro comparison of Trypanosoma spp. (subgenus Schizotrypanum) from bats

SummaryEpimastigotes of Trypanosoma vespertilionis from diphasic blood agar cultures were on the average longer and the distance between the nucleus and kinetoplast greater than epimastigotes of T. hedricki, T. myoti and T. dionisii. Also, no yellow granules were seen in the epimastigotes of T. vespertilionis whereas they were obvious in the other three species. Long thin trypomastigotes which are characteristic of T. hedricki, T. myoti and T. dionisii cultures were not seen in T. vespertilionis. T. dionisii was much less infective to fibroblasts from mice and did not develop in fibroblasts from chicken, as did T. hedricki and T. myoti. Blood trypomastigotes were seen in chicken embryos inoculated with blood agar cultures of T. hedricki and T. myoti, but none was seen in embryos infected with T. dionisii.The cultural characteristics examined could not be used to differentiate T. hedricki from T. myoti. ac]19810317

Detection of a parasitic amoeba (Order Dactylopodida) in the female gonads of oysters in Brazil

The impacts of oocyte parasites on the reproductive success of molluscs are largely unknown. In this study, we evaluated the presence of gonad parasites in 6 species of marine bivalve molluscs native to southern Brazil. Cultured bivalves included the mangrove oyster Crassostrea gasar (sometimes called C. brasiliana), the brown mussel Perna perna, the lions paw scallop Nodipecten nodosus and the wing pearl oyster Pteria hirundo. Another species of mangrove oyster, C. rhizophorae, and the carib pointed venus clam Anomalocardia brasiliana (syn. A. flexuosa) were collected from the wild. Molluscs were collected in winter 2009 and summer 2010 for histopathological and molecular evaluation. An unknown ovarian parasite (UOP) was observed in histopathological sections of female gonads of C. gasar and C. rhizophorae. The UOP possessed features suggestive of amoebae, including an irregular outer membrane, frothy cytoplasm, a nucleus with a prominent central nucleolus and a closely associated basophilic parasome. PCR analysis was negative for Marteilioides chungmuensis, Perkinsus spp. and Paramoeba perurans. However, real-time PCR successfully amplified DNA from oyster gonads when using universal Paramoeba spp. primers. Also, conventional PCR amplified DNA using primers specific for Perkinsela amoebae-like organisms (syn. Perkinsiella), which are considered as endosymbionts of Parameoba spp., previously thought to be the parasome. Our results suggest that this UOP is a species of amoeba belonging to 1 of the 2 families of the order Dactylopodida, possibly related to Paramoeba spp. This study represents the first report of this type of organism in oysters. We found that C. gasar and C. rhizophorae were the most susceptible molluscs to these UOPs.

Disease and mortality among Yesso scallops Patinopecten yessoensis putatively caused by infection with Francisella halioticida

During the fall of 2015, up to 40% mortality occurred in juvenile Yesso scallops Patinopecten yessoensis at an aquaculture site in Baynes Sound, British Columbia, Canada. Macroscopic lesions were present in 11% of the scallops, and histopathology consisting of multifocal and diffuse haemocyte infiltration was observed in 44% of the specimens examined. Histologically, small Gram-negative intracellular bacteria-like particles were observed within necrotic haemocytes of the lesions, suggesting a bacterial aetiology. DNA was extracted from adductor muscle lesions of diseased scallops, and the 16s rDNA gene as well as the DNA-directed RNA polymerase beta subunit (rpoB) were amplified by PCR. Sequence analyses of the resulting 413 and 925 bp fragments were a 100% match to the reference sequence for Francisella halioticida, originally described as the cause of mortality in abalone from Japan. Isolation and culture of the bacteria was not possible at the time, as no further diseased specimens were available. Results from in situ hybridization assays as well as examination by transmission electron microscopy provide further evidence supporting the hypothesis that F. halioticida was the most probable causative agent of the lesions and mortality.

Rare occurrence of heart lesions in Pacific oysters Crassostrea gigas caused by an unknown bacterial infection

On rare occasions, small cream-coloured cysts have been observed in the heart and pericardial cavity of Pacific oysters Crassostrea gigas from British Columbia, Canada. Histopathology revealed the presence of large colonies of bacteria (up to 800 µm in diameter) causing significant host response and hypertrophy of the heart epithelium. The causative bacteria were characterized as follows: Gram-negative, coccoid to small rod-shaped, typically <1.5 µm in size, cell walls highly endowed with surface fimbriae and division via binary fission. Although these bacteria shared some morphological characteristics with the order Rickettsiales, they did not require an intracellular existence for multiplication. Unfortunately, a cultured isolate was not available, and a retrospective attempt to further characterize the bacteria using DNA sequence analysis of a fragment from the 16S rDNA region proved to be uninformative.