health:diagnostic_testing:start

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health:diagnostic_testing:start [2016/06/23 14:25]
rubin
health:diagnostic_testing:start [2016/07/08 14:21] (current)
rubin [Bacteriology]
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-====== Diagnostic ​Tests ======+====== Diagnostic ​Testing ​======
  
-Content coming ​soon+===== Histopathology ===== 
 +{{ :​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. 
 + 
 +===== Bacteriology ===== 
 +{{ :​health:​diagnostic_testing:​bacteriology.jpg?​nolink&​200|}} 
 +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 + 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. 
 + 
 + 
 +===== 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. 
 +{{ :​health:​diagnostic_testing:​necropsy.jpg?​nolink&​800 |}} 
 +===== Molecular Assays ===== 
 + 
 +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. 
 + 
 + 
 +===== 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. 
 +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,​ 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.
health/diagnostic_testing/start.1466717145.txt.gz · Last modified: 2016/06/23 14:25 by rubin