Diagnosis and management of atypical Mycobacterium spp. infections in established laboratory zebrafish (Brachydanio rerio) facilities.

Two established zebrafish colonies experienced increased mortality and decreased reproductive performance. Initial examination of several fish from one facility revealed hyperemic gills, petechia around the opercula, abdominal distention, and emaciation. Affected fish had congested liver with inflammation and multifocal hepatic necrosis. Large numbers of acid-fast-positive, rod-shaped bacteria were evident in multiple tissues and the blood. Mycobacterium fortuitum was subsequently isolated from several fish. Zebrafish from the second facility had skin erosions and ulceration along the flank just caudal to the pectoral fins. Large numbers of acid-fast-positive, rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas in gonads, liver, and peritoneum from affected fish. Mycobacterium abscessus and M. chelonae were isolated and identified biochemically. Definitive diagnosis in these outbreaks was obtained by culture on selective media. Because Mycobacterium spp. grow extremely slowly and positive confirmation may require 45 to 60 days, Mycobacterium species-specific polymerase chain reaction analysis was used to provide a rapid screening assay for Mycobacterium spp. as well as for verification of culture results. To our knowledge, this is the first documentation of mycobacterial infection in laboratory-maintained zebrafish and provides guidelines for diagnosis, management, and prevention of atypical mycobacteriosis in laboratory zebrafish colonies.

Correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in vascular endothelium with toxicity in early life stages of lake trout.

Edema and cardiovascular dysfunction occur in vertebrates exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development. This study examined cytochrome P4501A (CYP1A) induction in endothelium and its possible association with mortality due to the edema and vascular effects of TCDD in lake trout early life stages. Lake trout (Salvelinus namaycush) eggs were injected at 24-50 hr postfertilization with 0.2 microl of 50 mM phosphatidylcholine liposomes or liposomes containing TCDD to give seven doses ranging from 11 to 176 pg TCDD/g egg. Doses of TCDD greater than 44 pg/g egg elicited hemorrhages; yolk sac, pericardial, and meningial edema; craniofacial malformations; regional ischemia; growth retardation; and mortality at the sac fry stage of development. Expression of CYP1A was assessed at four developmental stages, by immunohistochemical analysis of serial sections of individual fish with monoclonal antibody 1-12-3 to teleost CYP1A. CYP1A staining occurred in endothelial cells of many organs of TCDD-exposed but not vehicle-exposed embryos at 1 week prehatch and sac fry at 2 weeks posthatch. Earlier developmental stages examined were negative for CYP1A expression at any dose of TCDD. The strongest response occurred in sac fry at TCDD doses greater than 88 pg TCDD/g egg but was detected at doses as low as 22 pg TCDD/g egg. CYP1A staining in endothelium appeared at lower doses and was stronger than that in other cell types, in both prehatch embryos and posthatch sac fry. Thus, the vascular system is a major initial site affected by TCDD in lake trout early life stages, and the vascular endothelium is a cell type uniquely sensitive to induction of CYP1A in these developing animals. Based on an index of immunohistochemical staining of CYP1A, endothelial CYP1A induction in sac fry by TCDD occurred with an ED50 of 64-69 pg TCDD/g egg, similar to the dose-response for mortality occurring during the sac fry stage of development (LD50 = 47 pg TCDD/g egg). The correlations seen here suggest that CYP1A or aryl hydrocarbon receptor (AhR) in the endothelium may be linked to early lesions that result in TCDD-induced vascular derangements leading to yolk sac, pericardial, and meningial edema that is associated with lake trout sac fry mortality, but the precise mechanism remains to be determined.

Experimental and Histological Studies of Four Life-History Stages of the Eastern Oyster, Crassostrea virginica, Exposed to a Cultured Strain of the Dinoflagellate Prorocentrum minimum.

Effects of the dinoflagellate Prorocentrum minimum (strain EXUV) upon four life-history stages of the eastern oyster--embryos, feeding larvae, newly set spat, and juveniles--were investigated in laboratory exposure studies. Embryonic development was not affected significantly by living, heat-killed, or sonicated cells, or by growth-medium extracts from P. minimum cultures. Feeding larvae, however, showed poor growth and poor development of the digestive system when fed P. minimum, as compared with larvae fed Isochrysis sp. (strain T-ISO). Growth of larvae fed mixed P. minimum + Isochrysis diets was intermediate. Larvae and newly set spat that had been fed a diet of 1/3 P. minimum + 2/3 Isochrysis exhibited distinctive changes in digestive-system anatomy. Spat showed an abnormal accumulation of lipid in the stomach epithelium. Absorptive cells in the digestive glands of both larvae and spat contained accumulation bodies, often with a laminated, fibrous appearance in preparations for transmission electron microscopy. These accumulation bodies were PAS (periodic acid-Schiff) positive and may correspond to autolysosomal bodies within P. minimum cells. Juvenile oysters developed the ability to digest P. minimum, but only after a refractory period of about 2 weeks, during which most P. minimum was filtered but rejected as pseudofeces. The linking of accumulation bodies within absorptive cells of oyster digestive diverticula with dinoflagellate autolysosomal bodies suggests a mechanism by which some dinoflagellates interfere with feeding in phytoplankton grazers.

A novel adhesion protein expressed by ciliated epithelium, hemocytes, and leukemia cells in soft-shell clams.

The ontogeny of circulating hemocytes and tumor cells in mollusks has been approached using monoclonal antibodies to normal cells. A monoclonal antibody, previously shown to identify an adhesion related protein (p130), has been used to define the reactivity of cells in tissues from normal soft-shell clams (Mya arenaria) and soft-shell clams with leukemia. Using immunoperoxidase technology, we have determined that hemocytes, connective tissue cells, and a subset of leukemia cells that are adherent share a cross-reactive epitope with cilia.

Cytochrome P4501A induction in tissues, including olfactory epithelium, of topminnows (Poeciliopsis spp.) by waterborne benzo[a]pyrene.

Topminnows of the genus Poeciliopsis are susceptible to hepatocarcinogenesis by waterborne exposure to procarcinogenic polycyclic aromatic hydrocarbons (PAH). We examined induction of cytochrome P4501A (CYP1A) in liver and other organs of the species P. monacha and P. lucida exposed to benzo[a]pyrene (B[a]P) in water (added in acetone carrier) at 1 mg/l for 48 and 90 h. Fish were fixed whole in formalin, and CYP1A was examined immunohistochemically in sagittal sections of whole animals by staining with monoclonal antibody 1-12-3, which recognizes a single cross-reacting CYP1A protein in Poeciliopsis liver microsomes. Fish exposed to B[a]P for 48 h showed moderate staining, and those exposed for 90 h showed strong specific staining in various epithelial cells in both species. These included hepatocytes, pancreatic cells, epithelial cells in gill, enterocytes of the gut, and kidney tubular epithelium. Endothelial cells in several organs, including gill pillar cells and endocardial cells in the heart, showed strong staining. Staining was stronger in P. monacha than in P. lucida. Untreated animals of both species showed mild staining of the same cells stained in B[a]P-treated fish. In P. monacha, carrier (acetone) elicited a moderate increase in staining in most cell types, including those of liver and gill; the basis for this acetone effect is not known. There was a very strong specific induction by B[a]P in olfactory epithelium and epidermal taste bud epithelium of P. monacha, the first demonstration of strong CYP1A induction in chemosensory epithelia exposed to inducer in a physiologically relevant way. This study clearly establishes that waterborne PAH can elicit induction of P4501A proteins in multiple cell types in many organs of fish, with some sites of induction (olfactory epithelium) possibly related directly to the route of exposure. The species differences in the induction response, with induction in liver and some other organs generally being greater in P. monacha than in P. lucida, could be related to previously recognized species differences in PAH toxicities in Poeciliopsis.

Pathologic Cuticular Changes of Winter Impoundment Shell Disease Preceding and During Intermolt in the American Lobster, Homarus americanus.

Cuticular lesions from twenty-four market sized lobsters (Homarus americanus) with winter impoundment shell disease were examined. Histological descriptions of cuticular lesions were correlated with the substage of molt for each lobster, because cuticle components and inflammatory mechanisms vary in each. A lesion severity grading system was developed and applied to four specific substages of the five-stage (A-E) molting cycle. Lesions present in substage C4, in which the membranous layer is deposited, and D0 (passive premolt) were divided into five grades, ranging from mild erosions (Grade I) to severe ulceration (Grade V) of the cuticle. Cuticular lesions from lobsters in C4/D0 were compared with cuticular lesions from lobsters in substages C2/C3. Defensive mechanisms exhibited by animals in all substages were epicuticle deposition, melanization, inflammatory cell infiltration, and pseudomembrane formation. In addition, animals in C4 and D0 showed proliferation of the membranous layer in affected foci. The lesion grading scheme presented in this paper can be used to describe and compare both inter- and intraspecies crustacean shell lesions.

Immunohistochemical localization of environmentally induced cytochrome P450IA1 in multiple organs of the marine teleost Stenotomus chrysops (Scup).

Differences in expression of cytochrome P450 forms and their functions in different organs and cell types could determine the response of those cells and organs to xenobiotics. Recently, we described the cellular localization of cytochrome P450IA1 (P450E) induced in 10 organs or organ systems of the fish, Stenotomus chrysops (scup) treated with 3,3',4,4'-tetrachlorobiphenyl or with 2,3,7,8-tetrachlorodibenzofuran. (R.M. Smolowitz, M.E. Hahn, and J.J. Stegeman, Drug Metab. Dispos. 19, 113, 1991). Here we describe the presence and localization of P450IA1 in organs of scup sampled directly from an environment contaminated by chlorinated biphenyls and bibenzofurans, the outer New Bedford Harbor of Massachusetts. Western blot analysis of microsomes from selected organs (liver, kidney, gill, and heart), using monoclonal antibody 1-12-3, revealed induced levels of P450IA1 in each. The localization of P450IA1 in these and other organs was determined in sections prepared by standard histological methods and stained with MAb 1-12-3 in an indirect peroxidase labeling method. P450IA1 was detected in multiple cell types in liver, including hepatic, pancreatic, and vascular tissue. Kidney and gut also showed prominent P450IA1 levels in epithelial structures and in vascular endothelial cells. Specific staining was detected in endothelial cells, but not other cell types, in heart, gill, spleen, testis, ovary, nose, and brain. In heart, the staining was present in the endocardium of atrium and ventricle, and endothelium of the coronary vasculature and great vessels. The results demonstrate that P450IA proteins are induced in many organs of fish exposed to environmental chemicals in the wild, with patterns of cellular localization like those seen in fish experimentally treated with known inducers. The strong staining of P450IA1 in endothelial cells in all organs examined supports experimental results indicating that endothelium is a major site of P450IA1 induction. Our results indicate further that immunohistochemistry is a useful method for detecting P450 induction as a biomarker for exposure.

Immunohistochemical localization of cytochrome P-450IA1 induced by 3,3',4,4'-tetrachlorobiphenyl and by 2,3,7,8-tetrachlorodibenzoafuran in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup).

The regulation of different cytochrome P-450 forms and their functions in different organs and cell types could determine the susceptibility of those cells and organs to toxic effects of xenobiotics, including chemical carcinogenesis. Here we describe the cellular localization of cytochrome P-450E (P-450IA1) induced in 10 major organs or organ systems of a marine vertebrate species, the fish, Stenotomus chrysops (scup). Scup were injected ip with 3,3',4,4'-tetrachlorobiphenyl (TCB) at 1 mg/kg, or with 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 micrograms/kg. Induction was verified by Western blot analysis of microsomes from selected organs (liver, kidney, and gill) using monoclonal antibody (MAb) 1-12-3 to scup P-450IA1. The localization of P-450IA1 was subsequently determined in sections prepared by standard histological methods (10% buffered formalin fixation, paraffin embedding), and stained with MAb 1-12-3 and peroxidase-labeled second antibody. P-450IA1 was induced in epithelial and endothelial cells in liver (including pancreatic tissue), kidney, gill, gut, spleen, testis, and ovary. Induction also was detected in endothelial cells, but not other types, in heart, brain, and red muscle. In heart, the staining was present in the endocardium as well as in the endothelium of the coronary vasculature and great vessels. Although TCDF and TCB both induced P-450IA1 in various cells of all organs examined, the effect of TCB was in most cases greater than that of TCDF. This may be due to a relatively higher TCB dosage. A wider staining distribution was seen in gut, gill, kidney, and gonad of TCB-treated fish, which might be explained by a greater penetration, or by excretion of parent TCB, as opposed to TCDF. In any case, the results show that these important environmental agents induce P-450IA1 in generally similar patterns in all organs examined. The common finding of a strong induction of P-450IA1 in endothelial cells in all organs examined supports the suggestion that the endothelium may be a primary site of P-450IA1 induction.

Leukemia cell specific protein of the bivalve mollusc Mya arenaria.

Soft shell clams, Mya arenaria, develop leukemias in the hemolymph which are fatal. Tissue sections and hemolymph samples from normal and tumor-bearing clams were tested with an anti-leukemic cell specific monoclonal antibody (Mab) "IEII." Evaluation of leukemic cells and normal hemocytes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses showed that Mab IEII bound to a large protein of approximately 200 kDa from the tumor cell, but not from the normal cell preparation.

Ontogeny of leukemic cells of the soft shell clam.

A leukemic disease of the soft shell clam, Mya arenaria, has been identified along the east coast of the United States since 1977. This disease, first called hematopoietic neoplasia, is characterized by circulating tumor cells which are found in the hemolymph even before significant tissue invasion or localization of the cells can be demonstrated. The ontogeny of the leukemic cells, however, has not been resolved and remains an area of controversy. Monoclonal antibodies (Mab) developed by our laboratory were screened for specificity against the leukemic cell and normal hemocytes using an indirect immunoperoxidase technique. The method demonstrated that a new Mab "4A9" reacted both with leukemic cells and with a small subpopulation of normal circulating hemocytes (NSC). Mab 4A9 not only stained leukemic cells and NSC in peripheral hemolymph, but, more importantly, reacted specifically with another cell, the "connective tissue cell" (CTC) in clams with leukemia. The data presented in this paper show that Mab 4A9 stains a subset of normal circulating cells, leukemic cells, and the CTCs. These data lead to the hypothesis that the CTC may be the cell of origin for not only the NSC (a normal hemocyte) but also for the leukemic cell.