Optimization of in situ hybridization protocols for detection of feline herpesvirus 1

In situ hybridization (ISH) protocol including microwaving pre-treatment regimes was developed and compared with protease digestion as a pre-treatment regime for its effects on detecting feline herpesvirus 1 (FHV-1) in formalin fixed, paraffin embedded tissue. We found that optimum results were obtained using microwave pre-treatment. The results showed that the use of microwave irradiation would be recommended as a means of supplementing ISH methods, especially when using long-term formalin fixed, paraffin-embedded tissue.     

Treadmill exercise is not an effective methodology for producing the dark-cutting condition in young cattle

Holstein steer calves (n = 25) were used to evaluate the effects of treadmill exercise (TME) on blood metabolite status and formation of dark-cutting beef. Calves were blocked by BW (156 +/- 33.2 kg) and assigned randomly within blocks to 1 of 5 TME treatments arranged in a 2 x 2 factorial design (4 or 8 km/h for a duration of 10 or 15 min) with a nonexercised control. Venous blood was collected via indwelling jugular catheters at 10, 2, and 0 min before TME and at 2-min intervals during exercise. Nonexercised steers were placed on the treadmill but stood still for 15 min. Serum cortisol levels, as well as plasma concentrations of glucose, lactate, and NEFA, were similar (P > 0.05) before TME. Serum cortisol concentrations were unaffected (P > 0.05) during the first 6 min of TME, but between 8 and 15 min of TME, cortisol concentrations were greater (P < 0.05) in steers exercised at 8 km/h than those exercised at 4 km/h or controls (speed x time, P < 0.001). Although TME did not affect (P > 0.05) plasma glucose levels, plasma lactate concentrations in steers exercised at 8 km/h increased (P < 0.05) sharply with the onset of the TME treatment and remained elevated compared with steers exercised at 4 km/h or unexercised controls (speed x time, P < 0.001). Exercised steers had the lowest (P < 0.05) plasma NEFA concentrations during the first 6 min of TME compared with unexercised steers; however, NEFA concentrations were similar after 10 and 12 min of TME, and by the end of TME, steers exercised at 8 km/h had greater (P < 0.05) NEFA levels than nonexercised controls or steers exercised at 4 km/h (speed x time, P < 0.001). Even though muscle glycogen levels and pH decreased (P < 0.001) and muscle lactate concentrations increased (P < 0.001) with increasing time postmortem, neither treadmill speed nor TME duration altered postmortem LM metabolism. Consequently, there were no (P > 0.05) differences in the color, water-holding capacity, shear force, or incidences of dark-cutting carcasses associated with preslaughter TME. It is apparent that preslaughter TME, at the speeds and durations employed in this study, failed to alter antemortem or postmortem muscle metabolism and would not be a suitable animal model for studying the formation of the dark-cutting condition in ruminants.     

半套式PCR检测石蜡包埋组织中猪圆环病毒2型方法的探讨

半套式 PCR是采用一对半特异性引物对目的基因进行扩增的一种方法 ,该方法较常规 PCR的更敏感和更特异。本试验即采用该种方法 ,从石蜡包埋组织中提取圆环病毒 DNA进行 PCR扩增 ,来检测猪圆环病毒。结果表明组织石蜡切片经二甲苯脱蜡 ,蛋白酶 K消化 ,利用半套式 PCR方法 ,得到 43 2 bp的特异性扩增产物 ,从而建立了一种新的检测圆环病毒的方法 ,并为从存档石蜡研究该病提供了很好的前景     

Transmission of transmissible mink encephalopathy to raccoons (Procyon lotor) by intracerebral inoculation.

To determine the transmissibility of transmissible mink encephalopathy (TME) agent to raccoons and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of transmissible spongiform encephalopathies (TSEs) in raccoons, 4 raccoon kits were inoculated intracerebrally with a brain suspension from mink experimentally infected with TME. One uninoculated raccoon kit served as a control. All 4 animals in the TME-inoculated group showed clinical signs of neurologic disorder and were euthanized between 21 and 23 weeks postinoculation (PI). Necropsy examinations revealed no gross lesions. Spongiform encephalopathy was observed by light microscopy, and the presence of protease-resistant prion protein (PrPres) was detected by immunohistochemistry and Western blot techniques. Scrapie-associated fibrils were observed by negative-stain electron microscopy in the brains of 3 of the 4 inoculated raccoons. These findings confirm that TME is experimentally transmissible to raccoons and that diagnostic techniques currently used for TSE in livestock detect prion protein in raccoon tissue. According to previously published data, the incubation period of sheep scrapie in raccoons is 2 years, whereas chronic wasting disease (CWD) had not shown transmission after 3 years of observation. Because incubation periods for the 3 US TSEs (scrapie, TME, and CWD) in raccoons appear to be markedly different, it may be possible to use raccoons for differentiating unknown TSE agents. Retrospective genotyping of raccoons using frozen spleens showed that the raccoon PrP gene is identical to the mink gene at codons 179 and 224. Further studies, such as the incubation periods of bovine spongiform encephalopathy and other isolates of scrapie, CWD, and TME in raccoons, are needed before the model can be further characterized for differentiation of TSE agents.     

鸡包涵体肝炎过程中IFN-γ的mRNA原位杂交

FAV-Ⅷ型禽腺病毒口服接种感染2日龄SPF雏鸡,攻毒后取不同日龄的肝脏于福尔马林溶液中固定,并进行石蜡包埋,石蜡切片进行原位杂交染色.结果表明,着染部位主要出现在坏死灶或血管周围的成团或散在的淋巴细胞的胞浆内,在病情严重时的5 dpi开始增多,至20 dpi表达量也很高,以后开始减少,直到30 dpi基本消失.说明鸡包涵体肝炎过程中肝脏IFN-γ的mRNA大量上调,可能与T淋巴细胞的增多有关;而且IFN-γ作为免疫增强剂与抗病毒感染和病症恢复也有关.     

Demonstration of bovine virus diarrhoea virus antigen in formalin fixed, paraffin embedded tissue using a streptavidin/biotin technique

The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining.     

Cyclin D1 immunohistochemical expression and somatic mutations in canine oral melanoma

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non‐UV induced melanomas. Twenty‐eight cases of COM were retrieved from paraffin‐blocks archives. A total of 4 μm thick sections were immunostained with an antibody against human Cyclin D1 and Ki‐67. Cyclin D1 and Ki‐67 expressions were scored through two counting methods. DNA was extracted from 20 μm thick sections of formalin‐fixed paraffin‐embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki‐67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki‐67 (r = 0.56; P = .003). The correlation found between Ki‐67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single‐nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.     

Microsporidial keratopathy in two dogs

A microsporidial keratopathy is described in two dogs. Both dogs presented with a unilateral stromal keratopathy characterized by multifocal coalescing opacities, and the diagnosis was made on histopathologic examination of keratectomy specimens. Transmission electron microscopy (TEM) on formalin‐fixed, paraffin‐embedded corneal tissue was performed in one dog, and the morphologic features were consistent with Nosema species infection. Both dogs were initially diagnosed and treated by superficial keratectomy. One dog received additional antifungal medication and underwent a penetrating keratoplasty following local recurrence two years later. No other systemic lesions attributable to the microsporidial infection were identified clinically. The clinical and diagnostic pathology findings, treatment, and follow‐up are discussed.     

A comparison of the polymerase chain reaction with standard laboratory methods for the detection of EHV-1 and EHV-4 in archival tissue samples

A detection system incorporating the polymerase chain reaction was compared with the use of histopathology and virus isolation to determine the presence of equid herpesvirus type 1 or equid herpesvirus type 4 in equine tissues submitted to a diagnostic laboratory. When the polymerase chain reaction was performed, these tissues had been stored for up to 3 years. Thirty-eight tissues representing 14 cases had been stored embedded in paraffin wax. Analysis of these tissues using the PCR gave predictive values of 1.0 and 0.91 for a positive and negative result respectively, and sensitivity and specificity values of 75% and 100% respectively. Fifty-three tissues representing 28 cases had been stored immersed in 10% formalin. Analysis of these tissues gave predictive values of 0.44 and 0.42 for a positive and negative result respectively, and sensitivity and specificity values of 28% and 57% respectively. The poor results obtained with this group of tissues was attributed to contamination of the samples during wax embedding. Viral DNA could not be amplified from older tissues. These results indicate that under appropriate conditions the polymerase chain reaction is reliable when applied to tissues collected for routine diagnosis. However, it is less reliable when samples for analysis are handled collectively. Also, storage of tissues in wax blocks for 14 or more years inhibits later amplification of viral DNA from these tissues. The implications of these results to the application of the polymerase chain reaction to routine laboratory diagnosis are discussed.     

Detection of leptospires in tissue using an immunoperoxidase staining procedure

An immunoperoxidase technique for the localisation of leptospires in sections of formalin fixed paraffin embedded kidney tissue is described. The procedure utilises a two-layered antibody sandwich with rabbit anti-leptospiral immunoglobin. Using antiserum to specific leptospiral serovars the presence and distribution of specific serovar in the tissue could be determined. The technique was also used to detect leptospires of given serovars in smears made from infected tissues and fluids. There was good correlation between culture results and results of the immunoperoxidase staining method on kidneys infected with leptospires. The diagnostic possibilities of the technique on formalin fixed tissue specimens are discussed.     

脊髓等柔软组织石蜡组织切片制作技术探讨

以家兔脊髓组织为实验材料,探讨制作大批量脊髓组织教学标本简便、可行的方法。结果表明,用常规方法制作优质的脊髓组织切片时,在10%的中性福尔马林固定液中固定7 d,进行H.E染色时,苏木精染色的时间为30 min,伊红染色时间6 min,制作的切片细胞核嗜碱性、呈蓝紫色,细胞质和细胞间质嗜酸性、呈粉红色,胞质内尼氏体为蓝紫色颗粒,两者间颜色对比度好,容易区分,是制作大批量教学标本的可选方法。     

Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues

Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases.     

A modified immunoperoxidase assay for detection of antibody porcine reproductive and respiratory syndrome (PRRS) virus in swine sera

MARC-145 cell monolayers infected with PRRS virus were fixed in 3% neutral buffered formalin and embedded in paraffin. The sections were stained by avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The positive reactions were detected by ABC method and indirect immunofluorescent assay (IFA). There was good correlation between ABC and IFA, and the titers in ABC were higher than those in IFA. The present results indicate that the immunohistochemical staining is a useful test for the detection and quantitation of PRRS virus antibody in swine sera as well as IFA.     

The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.     

Modified assay for true and apparent metabolisable energy based on tube feeding

1. To improve the accuracy with which true metabolisable energy (TME) values of feedingstuffs are determined, a modification to the assay based on tube-feeding is proposed. 2. To ensure that the gastrointestinal tracts of the experimental birds are as empty as possible at the start of the assay it is recommended that the normal food is withdrawn 48 h before tube-feeding. 3. In order to partly alleviate the effects of starvation, all birds are given two doses of 25 g glucose (as an aqueous solution) about 40 and 16 h before tube-feeding. Birds, from which endogenous energy losses are to be derived, are fed 50 g glucose rather than given no food. 4. All birds are given 50 ml water by tube about 32 h after feeding to overcome any effects induced by low water intake. 5. A comparison of the two procedures with 8 feedingstuffs showed that the mean coefficient of variation was reduced from 5.5% to 1.5% for TME and from 4.7% to 1.8% for TME.     

Detection of antibodies against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7 using the immunohistochemical staining.

Whole cells of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) serotype 1, 2, 5 or 7 attached to fibrins were fixed in 10% neutral buffered formalin and embedded in paraffin. The sections on a slide glass were stained by the avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The serum antibodies against A.pleuropneumoniae (serotypes 1, 2, 5 and 7) were measured by the ABC method and complement fixation (CF) test. There was good correlation between the ABC and CF tests. The present results indicate that the immunohistochemical staining is as useful as the CF test for the detection and quantification of antibody in swine sera.     

Effect of a hydrocolloid dressing on first intention healing surgical wounds in the dog: a pilot study

OBJECTIVES: To evaluate the efficacy of a hydrocolloid dressing for the treatment of surgical wounds in dogs. METHODS: Six healthy young female dogs of medium size and different breed underwent ovariohysterectomy. Histological evaluation was performed on biopsies taken from the edges of the wounds at day 7. The dressing was applied on one half of the wound according to manufacturer's instructions; the second half served as control. Biopsy specimens were fixed in a 10% formalin buffered solution pH 7.4, paraffin embedded and stained with haematoxylin and eosin. For clinical assessment, the presence and quality of exudate, erythema of the surrounding area, swelling and correct apposition of the wound margins were evaluated. RESULTS: The hydrocolloid dressing was easy to use. The clinical quality of the treated skin wounds was superior to the non-treated ones. Comparison of histological features between treated and untreated wounds showed a more regular organisation of the granulation tissue in the treated wounds, with fibroblasts being aligned parallel to the overlying epidermis. The number of inflammatory cells and the extension of granulation tissue were less prominent and less widespread in treated compared to untreated wounds. CONCLUSION: The dressing performed very well in terms of adhesiveness and flexibility. It was useful in the management of surgical wounds to avoid contamination and ameliorate the epithelialisation rate and granulation tissue morphology of the surgical scar.