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- | ====== Diagnostic | + | ====== Diagnostic |
- | Content coming | + | ===== Histopathology ===== |
+ | {{ : | ||
+ | Fixed fish are processed for histological sectioning and staining with hematoxylin | ||
+ | and eosin. Additional special stains may be used to identify infectious organisms. | ||
+ | Histopathology allows for evaluation of all tissues for normal organ development, | ||
+ | presence of infectious agents, responses to suboptimal water quality, and a range of | ||
+ | tissue changes and pathologies. We, therefore, recommend histopathology for | ||
+ | routine health screening of sentinel and sick fish, and additional diagnostic tests are | ||
+ | provided as appropriate based on histological findings. | ||
+ | |||
+ | ===== Bacteriology ===== | ||
+ | {{ : | ||
+ | We perform aseptic cultures of the kidney and swim bladder for diagnosis of | ||
+ | systemic infections and bacterial aerocystitis, | ||
+ | blood agar plates. Fish are shipped live for bacteriology, | ||
+ | taken from frozen specimens if | ||
+ | necessary. We require advanced | ||
+ | notification prior to shipment of live | ||
+ | fish to ensure appropriate personnel | ||
+ | and lab supplies are available. | ||
+ | Bacterial infections in zebrafish are | ||
+ | often sporadic and opportunistic. We | ||
+ | recommend bacteriology as a follow-up | ||
+ | to histopathology if bacterial infections | ||
+ | are observed in tissue sections and in | ||
+ | conjunction with histopathology | ||
+ | during an incidence of increased | ||
+ | morbidity or mortality. | ||
+ | |||
+ | |||
+ | ===== Bacteriology + Histopathology ===== | ||
+ | {{ : | ||
+ | Bacterial culture and histopathology can be performed on the same fish. We use a | ||
+ | dorsal approach to perform aseptic cultures of the kidney and swim bladder. After | ||
+ | samples are taken for cultures, the carcasses are fixed in Dietrich’s fixative and | ||
+ | processed for histological sectioning and H&E staining. Interpretation of culture | ||
+ | results is greatly enhanced by the addition of histological evaluation on the same | ||
+ | fish. By also examining tissue sections for evidence of bacterial infection, we | ||
+ | mitigate the risks of false culture results. False negative culture results may occur | ||
+ | with improper sampling of focal infections and with slow-growing and difficult to | ||
+ | culture bacteria. False positive | ||
+ | cultures may occur with skin or gut | ||
+ | contamination of samples, which is | ||
+ | common with small fish, and in | ||
+ | mixed bacterial infections where | ||
+ | the easily cultured bacteria are not | ||
+ | responsible for the observed fish | ||
+ | morbidity and mortality. By | ||
+ | adding histopathology we also have | ||
+ | the opportunity to diagnose | ||
+ | numerous other pathologies not | ||
+ | associated with bacterial infection. | ||
+ | |||
+ | |||
+ | ===== Necropsy ===== | ||
+ | |||
+ | Fish should be shipped live for necropsy examination. Fish are euthanized, | ||
+ | dissected, and organs examined for gross changes. Fin clips, gill clips, and skin | ||
+ | scrapes are also performed. Tissues may be fixed and processed for histological | ||
+ | sectioning and staining. | ||
+ | {{ : | ||
+ | ===== Molecular Assays ===== | ||
+ | |||
+ | Real-time PCR assays for diagnosis of Pseudoloma neurophilia, | ||
+ | chelonae, M. haemophilum, | ||
+ | embedded tissue at the Oregon Veterinary Diagnostic Laboratory. PCR assays for | ||
+ | additional zebrafish pathogens, such as Pseudocapillaria tomentosa, will be available | ||
+ | soon. Samples are received by ZIRC and transferred to OVDL for appropriate | ||
+ | testing. | ||
+ | |||
+ | |||
+ | ===== Mycobacteriosis ===== | ||
+ | |||
+ | Diagnosis of mycobacteriosis and species identification of Mycobacterium spp. | ||
+ | require different approaches compared to Gram negative bacteria. Diagnosis of | ||
+ | mycobacteriosis to the genus levels only requires observation of acid-fast bacteria in | ||
+ | infected tissues. Species identification usually requires DNA sequence evaluation | ||
+ | (e.g., 16S rDNA or hsp65) or even HPLC comparisons of mycolic acids from cultured | ||
+ | bacteria. We use the former. For severe outbreaks, we often compare the DNA | ||
+ | finger prints of various isolates using molecular techniques (Ostland et al. 2008; | ||
+ | Whipps et al. 2008). | ||
+ | Several methods can be employed to identify mycobacteria in zebrafish. | ||
+ | As histopathology is our primary diagnostic method, mycobacteriosis is usually first | ||
+ | identified by observing lesions consistent with the disease (e.g., granulomas) in | ||
+ | tissue sections. For confirmation, | ||
+ | additional sections from the same fish. Diagnosis to the genus level can also be | ||
+ | accomplished by staining and identification of acid fast bacilli in tissue imprints. | ||
+ | Following diagnosis, different options are available for species identification. | ||
+ | |||
+ | ==== a. PCR Identification from culture. ==== | ||
+ | |||
+ | Whereas Mycobacterium spp. generally grow | ||
+ | slower in culture than Gram negative bacteria, their resistance to many antibiotics | ||
+ | and disinfectants such as cetyl-pyridinium chloride (CPC) allows for selective | ||
+ | culture on specific media. Here organs such as liver, spleen and kidney are | ||
+ | aseptically cultured, mycobacteria are isolated on media, and then identified using | ||
+ | molecular techniques. | ||
+ | |||
+ | ==== b. PCR identification from tissues. ==== | ||
+ | |||
+ | Some mycobacteria, | ||
+ | are extremely fastidious and slow growers. Therefore, we often find it more | ||
+ | appropriate to conduct PCR analysis directly on infected tissues (Whipps et al. | ||
+ | 2007). When Mycobacterium infection is suspected, spleen can be removed and | ||
+ | frozen back before the fish carcass is preserved in fixative and processed for | ||
+ | histopathology. PCR analysis can then be performed on the spleen if acid fast bacilli | ||
+ | are observed in histological sections. | ||
+ | |||
+ | ==== c. PCR identification from paraffin blocks. ==== | ||
+ | |||
+ | Molecular diagnosis of mycobacteria | ||
+ | in zebrafish in which the fish have been preserved in formalin-based fixatives and | ||
+ | processed into paraffin can be obtained with reasonable success (Peterson et al. | ||
+ | 2013). Paraffin blocks containing fish identified as positive by histopathology are | ||
+ | sent to the Veterinary Diagnostic Laboratory (VDL) at Oregon State University | ||
+ | where infected fishes are cored from blocks and evaluated with specific PCR tests | ||
+ | for M. marinum, M. chelonae and M. haemophilum, | ||
+ | causing mycobacteriosis in zebrafish (http:// | ||
+ | The above methods (a-c) are often employed with multiple fish during an outbreak | ||
+ | – e.g., some fish preserved in Dietrich’s processed for histology and other fish frozen | ||
+ | or preserved in ethanol for PCR analysis. However, we often use an alternative | ||
+ | approach in which use a single fish is processed for both histology and other tests | ||
+ | (e.g., culture/ | ||
+ | cavity, 2) aseptically obtain biospies of spleen, liver, mesenteries, | ||
+ | Dietirich’s for histopathlogy, | ||
+ | acid fast stains, 5a) freeze biopsy specimens for future PCR analysis or 5b) setup | ||
+ | bacterial cultures for mycobacteria using CPC disinfection followed by culture on | ||
+ | mycobacteria-specific media. With this approach, we can correlate bacteriology | ||
+ | results with specific severities of infection. Alternatively, | ||
+ | fish for histology, and then select a subset for further bacterial identification based | ||
+ | on histology results. | ||
+ | |||
+ | Ostland, V.E., Watral, V., Whipps, C.M., Austin, F.W., St-Hilaire, S., Westerman, M.E., | ||
+ | Kent, M.L. 2008. Biochemical, | ||
+ | select Mycobacterium marinum isolates in hybrid striped bass (Morone chrysops x | ||
+ | Morone saxatilis) and zebrafish (Danio rerio). Dis. Aquat. Org. 79: 107-118. | ||
+ | Peterson, T.S, Kent, M.L. Ferguson, J.A., Watral, V.G., Whipps, C.M. 2013. Comparison | ||
+ | of fixatives and fixation time on PCR detection of Mycobacterium in zebrafish Danio | ||
+ | rerio. Dis. Aquat. Org. 104: | ||
+ | Whipps, C.M. Dougan, S.T., Kent, M.L. 2007. Mycobacterium haemophilum infections | ||
+ | of zebrafish (Danio rerio) in research facilities. FEMS Microbiol Lett. 270: 21–26. | ||
+ | Whipps, C.M., Matthews, J.L., Kent, M.L. 2008. Distribution and genetic | ||
+ | characterization of Mycobacterium chelonae in laboratory zebrafish (Danio | ||
+ | rerio). Dis. Aquat. Org. 82: 45-54. |