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--- health:diagnostic_testing:start [2016/04/19 09:14]
rubin created
+++ health:diagnostic_testing:start [2021/05/14 09:10] (current)
katy
@@ Line 1: / Line 1: @@
-====== Diagnostic Testing Services ======
+====== Diagnostic Testing ======
-  * Consultation
+===== Histopathology =====
-    * Zebrafish Husbandry and Health
+{{ :health:diagnostic_testing:histopathology.gif?nolink&600 |}}
+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.
-  * Histopathology
+===== Bacteriology =====
-    * Disease Investigation
+{{ :health:diagnostic_testing:bacteriology.jpg?nolink&200|}}
-    * Sentinel Testing
+We perform aseptic cultures of the kidney and swim bladder for diagnosis of
+systemic infections and bacterial aerocystitis, respectively. Samples are streaked on
+blood agar plates. Fish are shipped live for bacteriology, although samples can be
+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
-  * Necropsy Exam
+===== Bacteriology + Histopathology =====
+{{ :health:diagnostic_testing:bacteriology_histopathology.jpg?nolink&300|}}
+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.
-[[https://zebrafish.org:4442/zirc/health/pdf/submission.pdf|Download Submission Form]]
-[[:health:submission:start|Sample Submission Information]]
+===== Necropsy =====
-[[health:submission:preperation|Submission Preperation]]
+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.
+{{ :health:diagnostic_testing:necropsy.jpg?nolink&800 |}}
+===== Molecular Assays =====
-[[health:submission:shipping|Shipping Information]]
+Real-time PCR assays for diagnosis of Pseudoloma neurophilia, Mycobacterium
+chelonae, M. haemophilum, and M. marinum are performed on frozen and paraffin-
+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.
-[[https://zebrafish.org:4442/zirc/documents/fees.php#pathology|Pricing]]
+===== 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, we then follow with acid fast staining of
+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, such as M. haemophilum,
+are extremely fastidious and slow growers. Therefore, we often find it more
+appropriate to conduct PCR analysis directly on infected tissues (Whipps et al.
+). 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.
+). 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, the three most common species
+causing mycobacteriosis in zebrafish (http://vetmed.oregonstate.edu/diagnostic).
+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/imprints/direct PCR) as follows: 1) Aseptically expose the coelomic
+cavity, 2) aseptically obtain biospies of spleen, liver, mesenteries, 3) preserve fish in
+Dietirich’s for histopathlogy, 4) prepare imprints of biopsied tissues and perform
+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, we can process several
+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, molecular, and virulence characteristics of
+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. 104:113-120.
+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.

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