For this study blocks of tissues embedded in paraffin were provided by Northwest ZooPath and Zoo/Exotic Pathology Services. These blocks were cut for histological staining and were then returned to the providers. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: LC AF EW MC DGD LG KK JAH ERJ. Performed the experiments: LC AF EW MC DGD KK ERJ. Analyzed the data: LC AF DGD JAH ERJ. Contributed reagents/materials/analysis tools: AF MC DGD LG KK JAH ERJ. Wrote the manuscript: LC AF EW MC DGD KK JAH ERJ.
Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (
Inclusion Body Disease (IBD) is a unique and insidious worldwide disease commonly seen in captive members of snakes in the families Boidae and Pythonidae [
Immunohistochemical (IHC) staining is a well recommended method with high sensitivity and specificity for diagnosing both infectious and non-infectious diseases [
For IHC staining in veterinary medicine, there is a lack of high quality antibodies that are made to identify specific antigens among different species [
In this study, we presented a modified method for isolating IBD inclusion bodies from crude tissue homogenates. An anti-IBDP MAB was produced against the semi-purified IBD inclusion bodies isolated from a boa constrictor, and the reactivity was confirmed by western blots, enzyme-linked immunosorbent assay (ELISA), immuno-transmission electron microscopy (immuno-TEM), and IHC staining on paraffin embedded tissues. Based on the guidelines suggested by Ramos-Vara et al. [
The collection of fresh and formalin fixed tissue samples, euthanasia of snakes, and the production of monoclonal antibody was authorized under University of Florida Institutional Animal Care and Use Committee Protocol 201101156.
A total of 112 snakes were used in this study, including 94 boa constrictors, 4 annulated tree boas (
Paraffin embedded tissue blocks | 1990-2007 | Anatomic Pathology Service, College of Veterinary Medicine, UF | Boa constrictors | IBD+ | 29 |
IBD- | 17 | ||||
Ball python | IBD+ | 2 | |||
Carpet python | IBD+ | 1 | |||
Corn snakes | IBD- | 2 | |||
Emerald tree boa | IBD+ | 1 | |||
Rainbow boas | IBD+ | 4 | |||
1990-2007 | Zoological Medicine Infectious Disease Testing Laboratory, College of Veterinary Medicine, UF | Boa constrictors | IBD+ | 21 | |
IBD- | 12 | ||||
2008-2011 | Northwest ZooPath | Ball pythons | IBD+ | 2 | |
Carpet python | IBD+ | 1 | |||
Palm viper | IBD+ | 1 | |||
2008-2011 | Zoo/Exotic Pathology Services | Annulated tree boas | IBD+ | 3 | |
IBD- | 1 | ||||
Boa constrictors | IBD+ | 2 | |||
Fresh or formalin fixed tissues | 2008-2011 | Zoological Medicine Infectious Disease Testing Laboratory, College of Veterinary Medicine, UF | Boa constrictors | IBD+ |
8 |
IBD- |
5 |
a Of 8 IBD+ samples collected from boa constrictors, 5 were submitted as formalin fixed tissues and the other 3 were submitted as fresh tissues on ice. For each sample, portions of the fresh tissues were fixed in 10% NBF for processing into paraffin embedded blocks, and another portion was fixed and embedded for immuno-TEM. The remaining materials were stored at -80°C. Tissues that were submitted as formalin fixed tissues were processed into paraffin blocks for the use in this study.
b Of 5 IBD- samples collected from boa constrictors, 3 were submitted as formalin fixed tissues and 2 were submitted as fresh tissues on ice. For each sample, portions of the fresh tissues were fixed in 10% NBF for processing into paraffin embedded blocks. The remaining materials were stored at -80°C. Tissues that were submitted as formalin fixed tissues were processed into paraffin blocks for use in this study.
The purification method was modified from a published protocol for isolating human Mallory bodies [
Suspensions collected from each protein purification step were diluted 50 fold with water, and 50 µL of each diluted sample was placed in a cytocentrifuge chamber (Biomedical Polymers Inc., BMP-CYTO-S50), and centrifuged for 6 minutes at 800 rpm onto a glass microscopic slide using a Cytospin centrifuge (Shandon, Cytospin 2). The slides were air dried, fixed in 10% neutral buffered formalin (10% NBF) (Fisher Scientific, SF100-20) for 10 minutes, washed with distilled water, and stained with H&E stain.
Aliquots of IB preps or crude tissue homogenates were reduced by addition of equal volume of 2X loading sample buffer (25 mM Tris, 4% SDS, 100mM DTT, and 30% glycerol). For western blots, the proteins were reduced by addition of 4X NuPAGE LDS sample buffer (Invitrogen, NP0008) and 10X NuPAGE reducing agent (Invitrogen, NP0004) with 500 mM DTT. Following addition of the reducing buffer, the samples were heated to 95-100 °C for 10 minutes. Next, the total protein concentration of a 1:10 dilution of each reduced and unreduced sample was estimated by standard Bradford Protein Assay (Bio-Rad Laboratories, Inc.), subsequently, the total protein concentration of the IB preps were estimated. The reduced proteins were resolved on a 10% or 4%~12% NuPAGE Bis-Tris gel (Invitrogen) with MES or MOPS buffer (Novex) at 200V constant voltage. For evaluation of protein quality, 5 µg of protein were loaded in each well, and the resolved protein was visualized by SimplyBlue (Novex) stain, using the microwave protocol provided by the manufacturer (Novex).
The resolved 68 KDa IBDP bands were cut from the gels, minced, and packed inside glass tubes of the Model 422 Electro-eluter (Bio-Rad, 165-2976). The protein was electro-eluted in Tris-Glycine buffer without SDS, following the protocol provided by the manufacturer (Bio-Rad).
Several solubilizing reagents were used alone or in combination in an attempt to solubilize the isolated insoluble inclusion bodies (IB preps). The tested solubilizing reagents included, 8-12 M urea, 6 M guanidine hydrochloride (Gu-HCl), 1% Triton-100, 2% octyl beta-glucoside (OBG), 1% dodecyl maltoside (DDM), 2-4% SDS, 20% lithium dodecyl-sulfate (LDS), 1 M DTT, dimethyl sulfoxide (DMSO), bicarbonate buffer and 1% acetic acid. Additionally, a combination of 1 M DTT with 8 M urea, or 6 M Gu-HCl, or 1% Triton-100, or 2% OBG, or 1% DDM, or 20% LDS, or 4% SDS were tested. Thirty microliters of IB prep was placed in a 1.5 mL tube, and centrifuged at 12,000 rpm (15,294 x g) for 20 minutes at 4°C using an Eppendorf 15 Amp Centrifuge Model 5810R (Eppendorf North America, Hauppauge, New York). After centrifugation, the supernatant was removed, and the pellet was resuspended in 30 µL of a solubilizing reagent. For the use of combined reagents, 30 µL of DTT was added into the tube. After thorough vortexing, the tube was maintained at room temperature (RT) for 30 minutes, followed by centrifugation again at 12,000 rpm for 20 minutes. Solubilization was monitored by visually comparing the size of the pellet in the treated sample with that in an untreated sample. If the inclusion bodies were partially solubilized by the reagent, the pellet would appear smaller than the pellet of the untreated sample. If the inclusion bodies were completely solubilized, no pellet would be observed after centrifugation.
Mouse monoclonal antibody against IBDP was produced using the standard protocol of the Hybridoma and Protein Core Laboratories, UF Interdisciplinary Center for Biotechnology Research (ICBR) [
The resolved protein gel was transblotted onto a nitrocellulose membrane using standard protocol of the iBlot dry blotting system (Invitrogen). The membrane was blocked, washed, incubated, and developed following previously described method [
Flat bottom 96 well assay plates were coated with IB prep that was diluted in optimal concentrations (10, 20, 30, 40 µg/mL) with bicarbonate buffer. The IB preps isolated from liver and kidney of 2 IBD positive boa constrictors (#08-76, #08-122) were used as coating antigens on separate plates. The general ELISA procedure was the standard protocol used in Hybridoma and Protein Core Laboratories, UF ICBR [
Fresh liver and kidney tissues collected from three IBD+ boa constrictors were submitted for examination using TEM. The tissues were fixed with electron microscopy grade 4% paraformaldehyde (4% PF), 1% glutaraldehyde in 1X PBS, pH 7.24. The samples were washed in PBS pH 7.24, subsequently water washed and dehydrated in a graded ethanol series (25%, 50%, 75%, 95%, 100%, 100%), infiltrated in Lowicryl HM20 acrylic resin (Electron Microscopy Sciences, Hatfield, PA, USA) and UV cured at -10°C for 48 hours. Cured resin blocks were trimmed, thin sectioned and collected on Formvar coated Ni 400 mesh grids (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were immuno-labeled at RT as follows; the grids were treated with 200 mM NH4Cl in high salt Tween-20 (HST) for 20 minutes, rinsed in HST, incubated 1 hour with blocking solution (1.5% BSA, 0.5% cold water fish skin gelatin, 0.01% Tween-20 in HST, pH 7.2), and incubated with immunized mouse 1 serum in 1:100 dilution, or the purified anti-IBDP MAB in 1:10 dilution overnight at 4°C. For negative controls, the grids were incubated with non-related mouse IgG antibodies (ICBR-EMBL private stock). The following day, the grids were washed three times in PBS, and incubated for 1 hour at 21°C on 18 nm colloidal gold affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:30 in PBS. Subsequent washes in PBS and distilled water, post-stained with 2% aq. uranyl acetate and Reynold’s lead citrate. Sections were examined with a Hitachi H-7000 TEM (Hitachi High Technologies America, Inc. Schaumburg, IL, USA) and 2k x 2k digital images acquired with a Veleta camera and iTEM software (Olympus Soft-Imaging Solutions Corp, Lakewood, CO, USA).
Fresh tissues including liver, kidney, and pancreas obtained from five euthanized boa constrictors were dissected into approximately 5 mm thick sections, placed in cassettes, fixed in 10% NBF or 4% PF for 48 hours, and finally embedded into paraffin. In order to evaluate the effects of formalin fixation time, freshly obtained liver, kidney, and pancreas of an IBD+ boa constrictor were each cut into 10 sections that were approximately 5 mm thick. One piece of each sectioned liver, kidney, and pancreas were placed in a cassette (ten sets of tissues), followed by fixation in ten identical containers filled with 10% NBF. On Day 2 (48 hours after initial fixation), Day 7, Day 8, Day 9, Day 15, Day 23, Day 32, Day 39, Day 50, and Day 58, one cassette was removed and the tissues were embedded in paraffin. For tissue set of Day 58, only kidney and pancreas were embedded.
Paraffin embedded tissues obtained from the case repositories were embedded in the laboratory of Anatomic Pathology Service in the UF Veterinary Hospital (Lab 1). Using an automated processor (Thermo Electric Corporation, Shandon Excelsior), the fixed tissues were dehydrated in graded ethanol, followed by infiltration of xylene and paraffin. The processed tissues were manually mounted in paraffin blocks. In order to evaluate the IHC staining condition, liver and pancreas of one boa constrictor were fixed, processed, and embedded separately in two laboratories, Lab 1 and the laboratory of Molecular Pathology Core (Lab 2), College of Medicine, UF. The embedding procedures of the two labs can be found in
All H&E staining were done by an automatic slide stainer (Gemini Varistain, Thermo Shandon, Illinois, IL). For paraffin embedded tissues, the slides were deparaffinized with xylene, and the tissue sections were rehydrated in a graded series of ethanol solutions. The rehydrated tissues were stained with hemotoxylin (Richard-Allan Scientific, 7212) for 2 minutes, incubated with clarifier 2 (Richard-Allan Scientific, 7402) for 30 seconds, followed by incubating with bluing reagent (Richard-Allan Scientific, 7301) for 1 minute, then incubated one minute in 80% ethanol before staining with eosin (Richard-Allan Scientific, 71311) for 1 minute. In between the application of each reagent, the slides were washed with running water. For the cytospin prepared IB preps, the fixed microscopic slides were stained with the above protocol with minor modifications as follow. After staining with hemotoxylin, the slides were incubated with clarifier 2 for 15 seconds. After bluing, the slides were stained with eosin without incubating with 80% ethanol. Finally, the H&E stained slides were dehydrated in a graded ethanol series, dipped in xylene, and coverslipped.
Immunohistochemical staining was used for confirming the reactivity of the antibodies to the IBD inclusion bodies within tissue sections. The staining was performed in Lab 2 using the methods provided below.
For paraffin embedded tissues, the sections were deparaffinized in xylene, followed by rehydrating in graded ethanol, and finally rinsed with water. The deparaffined tissues were either treated with AR reagents or were not treated with AR reagents. The following AR reagents were evaluated: trypsin (Invitrogen, Digest-All2), Trilogy (Cell Marque), Citra (Biogenex), Dako Target Retrieval Solution, pH 6.0 (DAKO), Dako Target Retrieval Solution, pH 9.0 (DAKO). The trypsin AR was done by incubating the slides for 5 minutes at 37°C. The AR treatments with other reagents were done by incubating the slides for 30 minutes at 95°C. For double AR treatment, the slides were incubated with Trilogy for 30 minutes at 95°C, followed by additional 5 minutes of incubation with trypsin at 37°C.
The prepared slides were washed with tris-buffered saline (TBS), and blocked with Sniper blocking reagent (Biocare Medical, BS966) for 15 minutes at RT. After washing again with TBS, the blocked tissues were covered with diluted mouse serum in antibody diluent (Invitrogen, 00-3218) or with medium collected from the growing hybridoma clones, and incubated overnight at 4°C. For antibody validation, the slides were covered by anti-IBDP MAB in a specific dilution (1:1,000, 1:2,000, 1:5,000, 1:10,000, 1:20,000), and incubated for 1 hour at RT, or overnight at 4°C.
After washing, the paraffin embedded tissues were incubated with 3% peroxide in methanol for 10 minutes. The slides were rinsed with water and washed by TBS before covering the tissue with HRP conjugated goat-anti-mouse antibody (Biocare Medical, MHRP520) for 30 minutes at RT. After washing, the HRP-conjugated secondary antibody was visualized by development with diaminobenzidine (DAB; Vector Laboratory, SK-4100) or VECTOR NovaRED (Vector Laboratory, SK-4800) according to the manufacturer’s protocol. For the standardized IHC staining, all slides were stained using NovaRED HRP chromagen. The tissues were counterstained with hematoxylin, dehydrated in graded ethanol, placed in xylene, and cover slipped by Cytoseal XYL (Thermo Scientific).
For the tissues to be stained automatically, after AR and blocking, the slides were applied onto the automated staining machine (Autostainer Plus, DAKO), in which the washes and incubation of the primary and secondary antibodies were performed. Subsequently, the slides were developed manually using the procedures described above.
Using light microscopy, the intensity of the IHC stain was given one of the four scores: 0 (no staining), 1 (faint, barely visible), 2 (moderate), 3 (strong). For each IHC staining run, a positive control slide (IHC score 3) and negative control slides (IHC score 0) were stained parallel to the slides to be scored. The negative control of each sample was a duplicated slide that stained with a commercial non-specific mouse IgG instead of anti-IBDP antibody.
For evaluating the effect of storage time in paraffin and cross reactivity of the anti-IBDP MAB, the IHC staining results were interpreted as either IHC stain positive or negative. A positive IHC stain (IHC+) was defined by a visible staining pattern (IHC score 1 to 3) compared to the negative control (IHC score 0). A negative IHC stain (IHC-) was defined by no visible staining pattern (IHC score 0) compared to the negative control (IHC score 0).
In this study, samples with a positive or negative diagnosis for IBD by examination of the H&E stain (current gold standard) were classified as IBD+ or IBD-, respectively. The sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of the IHC test, compared to H&E, were calculated following standard procedures [
In order to assess the effect of storage time in paraffin, the paraffin embedded tissues were divided into two groups: Group 1. tissue samples embedded within the time period 1990-2000, and Group 2. tissue samples embedded within the time period 2001-2011. The sensitivity and specificity of the IHC test in Group 1 and Group 2 were compared using the Fisher Exact X2 test, following standard procedures [
Inclusion body preps and total liver homogenates from 3 IBD+ and 2 IBD- boa constrictors were obtained. The IBD- liver did not result in any solid pellet after the incubation with sarkosyl, whereas a tightly bound pellet was obtained from all three IBD+ samples (
The arrows showing pelleted inclusion bodies in 1% Sarkosyl after centrifugation.
The semi-purified inclusion bodies were used to immunize mice for antibody production. Bar = 20 µm.
Ten microliters of protein were loaded on each lane. Lane 1 to 3 are three different IB preps obtained from three IBD positive boa constrictors. Lane 4 is the liver homogenate from the same boa as lane 3. Lane 5 and 6 are liver homogenates from two IBD negative boas. Lane 7 is the IB prep derived from an IBD negative boa, which no pellet were left after incubating with 1% sarkosyl. Lane 8 is HB served as a blank control. The IB prep from #08-76 showed a major intense band approximately at 68 KDa (arrow), showing that the IBDP is concentrated in the IB prep compared to the liver homogenate (Lane 4). M. Molecular weight marker (Invitrogen, Mark 12).
All attempts to completely solubilize the IB prep using the common reducing and solubilizing agents were unsuccessful. Following centrifugation, the semi-purified inclusion bodies formed a tightly bound pellet that was difficult to resuspend. Small amounts of inclusion bodies were lost as a consequence of binding to the inner surface of plastic tubes and pipets. This was more prominent when placed in aqueous solutions (such as water, HB or PBS), than in solutions containing urea, Gu-HCl, OBG, and sarkosyl. Combination of 4% SDS or 12 M urea with 500 mM DTT could not completely solubilize IBDP. Prior to protein electrophoresis, the IB preps were reduced by sample buffers containing high concentration of solubilizing reagents (2% SDS or 2% LDS with 50-500 mM DTT) that were heated to 95-100 °C for 10 minutes. Still, a portion of inclusion bodies remained insoluble. The reduced IBDP that was resolved by electrophoresis became insoluble again when electro-eluted. The eluted IBDP coated the membrane of the gel eluter collecting chamber, and could not be retrieved. The solution collected from the collecting chamber had a protein concentration that was too low for antibody production. Due to the insolubility of the IB preps, further protein purification methods such as 2D-electrophresis and liquid chromatography were not feasible. Nevertheless, the IBDP (IB prep) from snake #08-76 was considered to be of sufficient purity to serve as the best immunogen available for anti-IBD MAB production.
Due to the insolubility of the IB preps, the IBDP could not be detected in the Bradford Protein Assay without reduction by heating up to 95-100°C for 10 minutes in 2% SDS and 50 mM DTT. However, with 10 fold dilution of the reduced sample, the concentration of the SDS (0.2%) still exceeded the tolerance of SDS level (0.025%) in Bradford Protein Assay. Thus, it was suspected that there was interference with the estimated protein concentration. Therefore, for all assays (western blots and ELISA) used in antibody selection, IB preps were quantified by the volume of the IB prep needed for a sufficient reaction in the assay determined by a test run prior to the actual assay, and the amount of protein equivalent to the volume was documented.
Polyclonal antibody that was reactive to the 68 KDa protein band was detectable in the mouse serum by western blots and IHC on day 57 post-immunization. Cultured media of hybridoma mass cultures derived from splenic lymphocytes of mouse 1 were screened for reactivity to the IB preps by ELISA. Four different IB preps were used as coating antigens, including liver and kidney isolates from two IBD+ boa constrictors (#08-76 and #08-122). Each hybridoma mass culture was tested for the reactivity against 2 to 3 IB preps. Of 303 hybridoma mass cultures screened, only 1 culture (5B3) showed a low positive reactivity (OD reading approximately 5 folds higher than the baseline) and cross-reacted with 3 different IB preps. Additionally, 32 mass cultures that had significantly higher OD readings compared to the background were also selected. Collected cultured media of the 33 selected mass cultures were further tested for the reactivity to IB preps by western blots. Only antibodies produced by mass culture 5B3 showed reactivity to the 68 KDa protein band, and reacted to all 4 IB preps.
The mass culture 5B3 was further cloned by limiting dilution and seeded at a single cell per well density. Of 72 single colony wells that were screened for reactivity to IB prep by ELISA, 10 wells that showed low positive reactivity (OD read approximately 5 fold of the baseline) were selected, and further tested for their reactivity to all four IB preps. The monoclonal antibodies were isotyped and determined to be IgG subtype by ELISA. Seven out of the 10 selected clones were further tested for reactivity to the 68 KDa band of 4 IB preps by western blots, and for their IHC reactivity to the inclusion bodies in liver and pancreas of boa #08-76 and #08-122. All of the tested clones showed positive reactivity to the 68 KDa and inclusion bodies by western blot and IHC staining respectively. The clone 5B3-3D9 that showed less background in IHC staining was selected and grown to high density for subsequent purification. From 120 mL of cultured medium that was harvested, and purified, a total yield of 16.9 mg anti-IBDP monoclonal antibody at a concentration of 10.37 mg/mL was obtained. This antibody was isotyped as IgG1 with kappa light chains by IsoStrip.
Under TEM, the inclusion bodies within liver and kidney of 3 IBD+ boa constrictors were detected by mouse 1 serum or anti-IBDP MAB, which were labeled with conjugated gold particles (
A. gold particles conjugated with purified anti-IBDP MAB labeling an inclusion body within liver of a boa constrictor. Bar = 2 µm. B. gold particles conjugated with polyclonal antibody (Mouse 1 serum) labeling an inclusion body within kidney of a boa constrictor. Bar = 1 µm. C. gold particles conjugated with polyclonal antibody (Mouse 1 serum) labeling an inclusion bodies within kidney of a boa constrictor. Bar = 2 µm.
In order to establish a consistent staining performance, we initially used the paraffin embedded tissues of two boa constrictors for IHC staining to determine the ultimate staining condition for our system using the anti-IBDP MAB. The criteria for standardization were based on the suggested guidelines by Ramos-Vara et al. [
We found that tissues fixed in 4% PF stained adequately (IHC score 3) with only mild AR treatment or no treatment at all. Tissues that were fixed in 10% NBF sometimes required AR treatment for adequate IHC staining intensity (IHC score greater than 0 or 1). However, considering that 10% NBF was most commonly used in routine histopathological evaluation, we selected it as the standard fixation method.
A variety of AR reagents including, trypsin, Trilogy, Citra, Dako Target Retrieval solution pH 6.0, and Dako Target Retrieval solution pH 9.0 were tested to determine which reagent resulted in the best staining intensity in IHC staining on the selected tissues fixed with 10% NBF. When trypsin was used, the blocks made in Lab 2 stained strongly with MAB (IHC score 3), but the blocks made in Lab1 stained faintly (IHC score 1) or no staining (IHC score 0). Using harsher AR treatment such as, Trilogy, Citra, Dako Target Retrieval solution (pH 6.0 and pH 9.0), the staining intensity was improved to medium or high (IHC score 2 to 3) (
The standard dilution for the primary antibody was determined by testing anti-IBDP MAB in a series of dilutions until the IHC staining intensity started to decrease. The staining intensity remained high with very minimal background in a dilution of 1:10,000, but the staining intensity decreased in a dilution of 1:20,000. Thus, the dilution of 1:10,000 was determined as the standard dilution for the anti-IBDP MAB. The incubation time of the primary antibody showed no significant differences in the staining intensity between 1 hour incubation at RT and overnight incubation at 4°C. Therefore 1 hr was used as the standard incubation time. Since there was no significant difference in the IHC staining intensity between manually and automated staining methods, the later method was selected as the standard protocol. This would enhance ‘run to run’ and ‘inter-run’ consistency.
The chromogenic peroxidase detection system was used throughout all IHC staining, with HRP conjugated goat-anti-mouse antibody detected by DAB or NovaRED. The substrate DAB stained reactive sites with a brown color that sometimes could be confused with the brown pigments of the hepatic melanomacrophages in liver tissues. The substrate NovaRED stained the reactive sites with a reddish-purple color that contrasted better than DAB in the bluish hemotoxylin stained background (
The tissue was fixed and stained under standardized IHC staining conditions using NovaRED as substrate. The negative control was stained with non-specific mouse antibody. The cell nucleus stained dark blue with hematoxylin, and the inclusion bodies are indicated by arrows. A. The inclusion bodies were not stained in the negative control slide. Bar = 20 µm.. B. The inclusion bodies stained dark red by anti-IBDP MAB. Bar = 20 µm. C. The inclusion bodies were not stained in the negative control slide. Bar = 100 µm. D. The positive stained pancreas by anti-IBDP MAB. Bar = 100 µm.
Finally, the IHC staining conditions for the use of anti-IBDP MAB were standardized and summarized in
Species | |
Tissue type | Liver, kidney, pancreas |
Fixative | 10% NBF |
Fixation time | 48 hours |
Antigen retrieval | Triology (95°C 30 minutes) |
Primary antibody |
Anti-IBDP MAB (1:10,000 dilution) |
Incubation time |
1 hour RT |
Secondary antibody |
HRP conjugated goat-anti-mouse antibody (MACH 2 Mouse HRP-Polymer, Biocare Medical) |
Incubation time |
30 minutes RT |
Detection system | Chromogenic peroxidase with NovaRed substrate |
Counter staining | Hematoxylin (blue) |
* For standardized IHC staining of this study, these steps were performed in automated staining machine.
According to the IHC
Ten blocks (Block 1 to 10), each containing liver, kidney, and pancreas that were fixed for a specific length of time (48 hours to 58 days), were used in this study. Three slides were made from each block, two stained with anti-IBDP MAB, and one stained with non-specific mouse antibody as negative control. The inclusion bodies within the embedded tissue remained detectable with the anti-IBDP MAB up to 58 days following initial fixation (
Slide 1 | Liver | 2 | 2 | 2,3 | 3 | 2,3 | 3 | 2,3 | 1,2 | 1,2 | N/A |
Kidney | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 2,3 | 2,3* | 3 | |
Pancreas | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
Slide 2 | Liver | 2 | 3 | 2,3 | 2 | 2,3 | 3 | 3 | 1,2 | 2 | N/A |
Kidney | 3 | 3 | 3 | 2,3 | 3 | 3 | 3 | 2,3 | 3 | 3 | |
Pancreas | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
Slide 3 | Liver | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | N/A |
Kidney | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
Pancreas | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
The IHC score was not uniformed in some samples, in which two scores were given.
* In some areas the inclusion bodies were not stained by anti-IBDP MAB. N/A: sample not available.
The double AR treatment restored the reactivity of anti-IBDP MAB in liver up to 50 days of fixation in 10% NBF (Block 9), and the uneven staining pattern was not observed. When double AR treatment was used in staining liver in Block 8 and 9 (
Tissue fixed up to 50 days (Block 9) in 10% NBF was stained with standardized Trilogy AR treatment (left) and double AR treatment (right). The inclusion bodies are indicated by arrows. Bar = 40 µm. A. In pancreas, the staining of inclusion bodies decreased when double AR was used. B. In kidney, the staining of inclusion bodies decreased when double AR was used. C. In liver, the staining of inclusion bodies improved when double AR was used.
Paraffin embedded tissues of 60 IBD+ and 34 IBD- boa constrictors collected from 1990-2011were tested using IHC staining. Paraffin embedded tissues were classified into one of two groups based on the time period they were collected (Group1:1990-2000 versus Group 2: 2001-2011). The sensitivity of the IHC test was similar between Group 1 (24/29 or 83%) and Group 2 (26/31 or 84%) (p = 0.99) (
24 (83%) | 26 (84%) | |
5 (17%) | 5 (16%) | |
0 (0%) | 1 (5%) | |
15 (100%) | 18 (95%) | |
The reactivity of the anti-IBDP MAB was tested on paraffin embedded tissues of the following 15 IBD+ non-boa constrictors: annulated tree boas (n=3), ball pythons (n=4), carpet pythons (n=2), emerald tree boa (n=1), palm viper (n=1), and rainbow boas (n=4) (
3 | 0 | 3 | |
4 | 1 | 3 | |
2 | 2 | 0 | |
1 | 0 | 1 | |
1 | 0 | 1 | |
4 | 0 | 4 |
a Sample of IBD cases reported by Stenglein et al [
b Sample of IBD case reported by Raymond et al [
The reactivity of the anti-IBDP MAB to inclusion bodies of other non-IBD diseases was tested with IHC staining on two previously reported corn snakes [
Overall, 60 samples from boa constrictors classified as IBD+ and 34 as IBD- based on H&E staining were tested with IHC staining (
H&E + | H&E - | Total IHC Test results | Sensitivity | 83.3 | 75.8 | 90.9 | |
---|---|---|---|---|---|---|---|
IHC + | 50 | 1 | 51 | Specificity | 97.1 | 93.6 | 100 |
IHC - | 10 | 33 | 43 | Positive predicted value | 98.0 | 95.2 | 100 |
Total H&E test results | 60 | 34 | 94 | Negative predicted value | 76.7 | 68.2 | 85.3 |
False positive | 2.9 | ||||||
False negative | 16.7 |
The positive and negative predictive values were calculated based on the prevalence (64%) of the sample set.
5% | 57% | 99% | 100% | 99% |
15% | 79% | 96% | 100% | 96% |
64% | 98% | 77% | 100% | 77% |
80% | 98% | 58% | 100% | 59% |
PPV: Positive predicted value; NPV: Negative predicted value.
The most challenging aspect of this study was the solubilization of IBDP. Insoluble semi-purified inclusion bodies (IB preps), could not be solubilized completely by a wide range of methods that are commonly used for solubilizing protein. This feature is shared by other protein accumulating disorders such as aggregates of Huntingtin protein, a prion-like protein that is seen in Huntington’s disease [
Another problem caused by the insolubility of IBDP was the difficulty in estimating the protein concentration by Bradford Protein Assay for the use in ELISA and western blots. Bradford Protein Assay is commonly used to estimate protein concentration in solutions by comparing the coloration of protein binding dyes in an unknown sample to a set of standard proteins with known concentrations [
To ensure that the best IBDP reactive MAB with the highest specificity was selected, IB preps derived from different tissues (liver and kidney) and also from different boa constrictors were used as antigen for antibody screening. When screening the mass cultures, only those that reacted with all IB preps were selected to avoid choosing an antibody that would non-specifically react to contaminants in liver or kidney. The specificity of the clones was confirmed by ELISA, western blots, immuno-TEM and IHC staining, which ensured the antibody selected reacted to the 68 KDa protein and the inclusion bodies in situ with minimal background staining. Based on these findings, the IB preps were of sufficient purity to achieve the objectives of this study.
This study was designed to validate an anti-IBDP MAB for use in IHC staining to diagnose IBD in boa constrictors. Boa constrictors were considered the standard species, for the following reasons: 1. The anti-IBDP MAB was produced using liver tissue of a boa constrictor; 2. Within the sample repository, the majority of the samples were from boa constrictors; 3. IBD in boa constrictors was more frequently diagnosed than in other species. The outcome of our antibody validation studies designed to evaluate factors that could potentially affect the IHC staining are discussed below.
For immuno-detection purposes, although there is no agreement on the exact fixation time, it is generally recommended that tissues should be fixed for a minimum of 12-24 hours [
In the standardized staining condition using Trilogy AR treatment, the staining of liver was assessed to be less intense compared to the staining of pancreas and kidney regardless of the fixation time. In liver, the inclusion bodies located at the margin of the tissue stained more intensively then the inclusion bodies located near the center of the tissue section. Similar staining patterns were observed in the study of Webster et al., which was attributed to differences in the rate of formalin penetration [
Antigen retrieval for IHC staining was to improve the binding of the antibodies to the antigen by breaking down the cross-linked proteins formed through formalin fixation. This can be a critical step, especially when tissues are fixed or remain in fixatives for prolonged periods of time [
During the process of standardization, we found that the same tissue sample processed and embedded in two laboratories (Lab 1 and Lab 2) had different requirements for AR (
In this study, the diagnostic performance of the IHC test was considered acceptable with the sensitivity of 83% and specificity of 97%. The higher false negative value (10/60 or 16.7%) of the IHC test estimated by this study was affected by 10 IHC- cases that were previously diagnosed as IBD+. When the H&E stained sections of the 10 cases were re-examined, the characteristic eosinophilic intracytoplasmic inclusion bodies were not found in 5 of these cases. Thus, we judged the diagnosis of these 5 cases as ‘questionable’. Four of the questionable cases were diagnosed during 1990-2000 (3 were reported in 1995), which was approximately when IBD was being diagnosed more commonly in boa constrictors in the US. This also suggested that a false positive diagnosis of IBD can likely be made by a pathologist having limited experience diagnosing IBD. The limitation of using H&E stained sections for diagnosing IBD is the inability of this method to accurately distinguish IBD inclusion bodies from other types of eosinophilic intracytoplasmic inclusion bodies. Similar problems were described in another IBD study using H&E staining [
Although the estimated specificity of the IHC test was very high (97%), the calculated false positive value (1/34 or 2.9%) may have been overestimated. The one case judged to be IBD- with H&E staining, was clearly IHC+ in liver and brain. Apparently, due to the small size of inclusions they were missed in the original examination of H&E stained tissue sections. Previous studies have shown that IBD inclusion bodies start as small and inconspicuous intracytoplasmic bodies that increase in size over time [
Ideally, in order to use the IHC test for diagnosis and control of IBD in populations of boa constrictors, the disease burden (prevalence) in the populations of interest should be known since prevalence will affect PPV and NPV of the test (
Of the total number of cases available for this project, the number of IBD+ non-boa constrictors was limited. Throughout 1990 to 2011, only 15 (6 species) cases were found in UF and collaborating laboratories, with, only 2 carpet pythons and 1 ball python staining IHC+. Even when double AR was used, the other 12 cases remained unstained. As with boa constrictors, different species of snakes may be infected with different ‘strains’ of the causative agent of IBD. Interestingly, the two species in our study, in which the anti-IBDP MAB cross-reacted were more distantly related to boa constrictors than other more closely related species tested, such as, rainbow boas, annulated tree boas, and emerald tree boas. Nevertheless, the use of anti-IBDP MAB in diagnosing IBD in boa constrictors is validated by this study and will serve as a powerful tool for diagnosing this insidious disease.
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Portions of this article and figures were from PhD dissertation of Li-Wen Chang: Chang, Li-Wen. Development of Molecular Diagnostic Tests for Inclusion Body Disease in Boid Snakes. 2012. ProQuest (Ann Arbor, MI, USA) through University of Florida, Gainesville, FL, USA.
We are grateful for significant help from the following laboratories within College of Veterinary Medicine, University of Florida: Patrick Knisley, Micaela Barter and Jeffery Abbott, of the Anatomic Pathology Service, and April Childress of Zoological Medicine Infectious Disease Testing Laboratory. Kind support from Francesco C. Origgi, Centre for Fish and Wildlife Health, Institute of Animal Pathology, College of Veterinary Medicine, University of Bern, Switzerland. And material kindly provided by following veterinarians: Mike Garner (Northwest ZooPath, Monroe, WA, USA), Freeland Dunker (California Academy of Sciences, San Francisco, California, USA), Drury Reavill (Zoo/Exotic Pathology Services, West Sacramento, CA, USA), Thomas Boyer (Pet Hospital of Penasquitos in San Diego, San Diego, CA, USA), Amy Wells (Avian and Exotic Clinic of Monterey, Del Rey Oaks, CA, USA), Lauren Powers (Carolina Veterinary Specialists, Huntersville, NC, USA).