Comparison between specific immunoperoxidase staining and bacteriological culture in the diagnosis of renal leptospirosis of pigs

Seventy-two pigs were examined for the presence of leptospires in the kidney by both bacteriological culture and an immunoperoxidase procedure performed on formalin-fixed, paraffin-embedded sections of tissue with a primary antibody raised in rabbits against serovar pomona. The methods were in accordance in 62 of 70 (89 per cent) of the specimens. Compared with culture the sensitivity of the immunoperoxidase procedure was 30 of 38 (78 per cent) and its specificity 100 per cent; the predictive value of a positive result was 100 per cent, of a negative result, 80 per cent. The major advantages of the immunoperoxidase procedure are specificity, speed of execution and the possibility of simultaneous visualisation of leptospiral antigen and microscopic lesion.     

Immunohistochemical detection of 3 viral infections in paraffin-embedded tissue from mink (Mustela vison): a tissue-microarray-based study

Immunohistochemical (IHC) assays were developed and tested for the detection of 3 viral infections in archived paraffin-embedded mink tissue. Specimens had been obtained from mink with diagnoses of acute Aleutian disease (AD), mink parvoviral enteritis (MVE), or canine distemper (CD) made by means of routine diagnostic procedures. To improve the efficiency and reduce the costs of IHC analyses, tissue microarray (TMA) technology was used. Representative cores 2 mm in diameter from each tissue specimen and from positive- and negative-control specimens were collected in a TMA block. Immunohistochemical reactions to viral antigens were assessed and graded. Positive reactions were found in 91% of the 32 specimens from mink with AD, 53% to 80% of the 60 specimens from mink with MVE, and all 66 of the specimens from mink with CD. To validate the use of TMAs, the IHC methods were applied to whole-mount paraffin-embedded sections of 10 of the positive specimens for each disease, together with whole-mount sections of small intestine and lung tissue from 2 healthy mink. The IHC grading of the TMA cores and the whole-mount sections from the same animal corresponded completely. These results suggest that IHC demonstration of viral antigen allows rapid and reliable diagnosis of the 3 viral infections in mink and is a potential supplement to histologic diagnostic procedures. The TMA technique proved useful for screening large numbers of samples for expression of specific viral antigens, while reducing overall costs.     

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.     

Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors.

OBJECTIVE: To determine the estrogen receptor (ER) content of canine mammary gland tumors by use of immunohistochemical (IHC) examination of formalin-fixed sections. SAMPLE POPULATION: 21 mammary gland tumors from 20 adult dogs. PROCEDURE: ER were detected in formalin-fixed tissues, using an avidin-biotin alkaline phosphatase IHC assay and were quantified on fresh-frozen tumor samples, using a modified dextran-coated charcoal (DCC) assay. RESULTS: 7 of 21 tumors had visually detectable nuclear ER by use of IHC staining, whereas 8 of 21 tumors were positive for ER by use of the DCC assay. The ER-positive cells in 5 IHC-positive tumors were epithelial cells with histologic criteria of early malignancy. The remaining 2 ER-positive tumors detected by use of IHC had ER-positive mast cells within areas of connective tissue around the tumor. CONCLUSIONS: Immunohistochemistry is an additional method for detection of ER in canine mammary tumors. The major advantage of this type of assay is that it may be performed on formalin-fixed tissues, and individual ER-positive cells may be identified. Discovery of ER-positive mast cells by use of IHC is of concern, particularly if the ER status of a tumor is based on DCC results alone. CLINICAL RELEVANCE: Because most canine mammary tumors are fixed in formalin prior to histologic evaluation, an IHC assay that identifies ER-positive cells is desirable. Adjunctive antiestrogen therapy could be administered to dogs with ER-positive tumors.     

A novel method for the detection of porcine circovirus type 2 replicative double stranded viral DNA and nonreplicative single stranded viral DNA in tissue sections.

Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.     

Comparison of bacterial cultivation, PCR, in situ hybridization and immunohistochemistry as tools for diagnosis of Haemophilus somnus pneumonia in cattle

The aim of the present study was to compare the potential of bacterial cultivation (BC), PCR, in situ hybridisation (ISH), and immunohistochemistry (IHC) in the diagnosis of Haemophilus somnus, when applied to pneumonic bovine tissue. Lungs from 65 field cases submitted for bacteriological examination were included in the study. The PCR-detection was performed on three different samples: plate-PCR (detection on plate washes after incubation of lung tissue on agar plates); swab-PCR (direct detection on a swab from the cut surface); and, whenever possible, a bronchus-PCR (direct detection on a swab from the main bronchus of the right cranial lung lobe). In order to examine the pathological significance of the findings, a histopathological examination of the cases was performed. H. somnus was detected by one or more techniques in 33 cases in total. By BC the bacterium was isolated from 10 cases, IHC and ISH were positive in 17 and 19 cases, and plate- and swab-PCR were positive in 21 and 29 cases, respectively. The bronchus-PCR was positive in 30 out of 61 cases examined. The PCR-technique was the most sensitive method, and as this technique is fast and relatively inexpensive, it should be considered as a supplementary tool in the diagnosis of H. somnus induced calf pneumonia.     

Comparison of different methods for diagnosis of porcine proliferative enteropathy

The objectives of this study were: (1) to compare 2 methods of serology; (2) to compare 3 histologic techniques; and (3) to compare 2 methods of detecting shedding in pigs experimentally challenged with Lawsonia intracellularis. The sensitivities of these tests were determined by the detection of infection. Forty 5-week-old pigs were inoculated on day 0 with intestinal homogenate from pigs with proliferative enteropathy (PE). Clinical evaluation was done on day 7 and daily from day 14 to 28 postinoculation. Fecal shedding of L. intracellularis was monitored by use of polymerase chain reaction (PCR) analysis and immunoperoxidase staining at 7-day intervals. Serum was obtained on days 0 and 28 for serologic testing by glass slide and tissue culture indirect fluorescent antibody tests. At euthanasia on day 28, gross intestinal lesions were evaluated and ileum samples collected for histologic analyses. Ileal histologic sections from each animal were stained by hematoxylin and eosin, Warthin-Starry silver stain, and immunohistochemistry (IHC). Of the 40 pigs, 36 had gross lesions typical of PE at necropsy. The percentage of agreement between the 2 serologic methods was 94.4%. Immunoperoxidase stain of fecal smears was more sensitive than PCR for detecting fecal shedding, especially on day 21 (89.5% and 60.5%, respectively) and day 28 (59.4% and 37.5%, respectively) post-inoculation. The IHC stain was much more sensitive for detecting infection than the routinely used hematoxylin and eosin and Warthin-Starry silver stains. In conclusion, in experimentally infected pigs, both serologic methods were appropriate techniques for detecting infection. For fecal samples, PCR has low sensitivity. Immunohistochemistry is the best diagnostic tool for formalin-fixed samples.     

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.     

Comparison of direct culture versus PCR for the detection of Brucella in aborted fetuses of cattle and sheep in Turkey

The aim of this study was to detect Brucella in samples from aborted fetuses of sheep and cattle in Turkey using PCR and bacteriological analysis, and to determine the sensitivity and specificity of the PCR. Organ homogenates from 38 aborted fetuses of cattle and 56 aborted fetuses of sheep were tested. All organ homogenates were cultured for bacteriological analysis, and all of the homogenates and the Brucella isolates obtained by culture were examined with a commercial PCR kit. On bacteriological analysis, Brucella species were found in 30 (31.9 per cent) of the 94 organ homogenates, eight (21.1 per cent) of which were from cattle and 22 (39.3 per cent) from sheep. Using PCR, a total of 29 (30.9 per cent) homogenates were positive for Brucella species, eight (21.1 per cent) of which were from cattle and 21 (37.5 per cent) from sheep. Compared with the bacteriological method, the diagnostic sensitivity and specificity of the PCR kit used in this study were 83 per cent and 94 per cent, respectively.     

Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens

Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.     

Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalin-fixed skin biopsy specimens.

The objective of this study was to evaluate the efficacy of immunohistochemical (IHC) staining for diagnosis of persistent bovine viral diarrhea virus (BVDV) infection using formalin-fixed, paraffin-embedded skin biopsy specimens. Skin from 41 of 42 calves shown to be persistently infected (PI) with BVDV by repeated virus isolation more than 3 weeks apart were immunohistochemically positive for BVDV antigen. Positive IHC staining was most pronounced in the keratinocytes and in hair follicle epithelium, hair matrix cells of the hair bulb, and the dermal papilla. All of the skin sections from 10 calves experimentally infected postnatally with BVDV (10(5) median tissue culture infective doses [TCID50]) and biopsied on days 0, 5, 7, and 9 postinfection were negative for viral antigen. Ten calves from a second group experimentally infected with a higher dose of BVDV (10(8) TCID50) were biopsied when viremic between 10 and 14 days postinfection and 4 calves exhibited positive IHC staining for BVDV; however, staining in these skin biopsies was confined to small foci in the nonfollicular epidermis and follicular ostia. This staining was distinct from that observed in skin obtained from PI cattle. Skin biopsy represents an effective method for identifying animals PI with BVDV.     

An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues

Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.     

Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues.

An immunohistochemical staining technique (IHC) was developed to detect avian pneumovirus (APV) antigen in formalin-fixed, paraffin-embedded tissue sections using streptavidin-biotin immunoperoxidase staining. Samples of nasal turbinates and infraorbital sinuses were collected from 4-week-old poults experimentally inoculated with APV and from older turkeys infected during naturally occurring outbreaks of avian pneumovirus. Tissue was fixed in 10% buffered neutral formalin, embedded in paraffin, sectioned and stained. Inflammatory changes were observed microscopically in the mucosa and submucosa of the nasal turbinates and infraorbital sinuses of both experimentally inoculated poults and naturally infected birds. Viral antigen was detected by IHC in the ciliated epithelial cells of nasal turbinates and infraorbital sinuses.     

A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity.     

Use of in situ hybridization with a biotinylated probe for the detection of bovine herpesvirus-1 in aborted fetal tissue

Forty-five cases of bovine abortion were examined using in situ hybridization (ISH) with a biotinylated DNA probe specific for bovine herpesvirus-1 (BHV-1). Of the 45 cases, 16 were diagnosed as due to BHV-1, 15 were determined to be due to other causes, and 14 were of undetermined etiology. Direct comparisons between ISH and an immunoperoxidase (IP) test specific for BHV-1 were performed on formalin-fixed, paraffin-embedded tissue sections of lung, liver, kidney, spleen, thymus, and placenta; fluorescent antibody tests for BHV-1 and virus isolation were performed on fresh lung and liver. In comparison to these routine BHV-1 detection techniques, ISH had an overall sensitivity of 88.2% and a specificity of 89.3% in detecting BHV-1 in aborted fetuses. Immunoperoxidase was more sensitive than ISH with tissue sections from lung (87.5% vs. 69%), liver (92% vs. 17%), spleen, and placenta; results of the tests on tissue sections from kidney were concordant. Liver sections presented special problems in that nonspecific reactions were frequently observed with hybridization. With thymus sections, the rate of detection was higher by hybridization than by IP, but the specificity of some of these reactions could not be confirmed.     

Progesterone receptors in normal,dysplastic and tumourous feline mammary glands. Comparison with oestrogen receptors status

The aims of the study were to standardise an immunohistochemical (IHC) method for the detection of progesterone receptors (PR) on formalin-fixed, paraffin wax-embedded tissue sections of feline mammary gland tumours and dysplasias, comparing the results with those obtained using the radiolabelled ligand dextran coated charcoal (DCC) assay applied to frozen tissue samples from the same cases. Also, to define the immunohistochemical distribution of PR in the different cellular compartments of the lesions and to compare the oestrogen receptor (ER) and PR status of the feline mammary lesions. Proliferative mammary lesions collected from 34 cats were studied; 25 malignant tumours and 9 benign tumours and dysplasias. PR protein was present at a concentration of 5 fmol mg(-1) (positivity threshold) in 37.5 per cent of malignant tumours and 66.7 per cent of benign tumours and dysplasias while immunoreative products to PR antibody were found in the nuclei of tumour cells in 38.5 per cent and 66.7 per cent of the cases, respectively. Concordance between DCC-PR and IHC-PR was 88.5 per cent (P<0.001). The specificity (true negatives) and sensitivity (true positives) of the IHC method were 89.4 per cent and 87.5 per cent respectively. The presence of PR was linked to the absence of ovariectomy (P<0.02). Oestrogen receptors, detected by either method, were also detected in half the cases in which PR had been detected. In malignant tumours, the most prevalent groups were the ER + PR + and ER-PR + groups.     

Aerobic Bacteria Occurring in the Vagina of Bitches with Reproductive Disorders

A retrospective survey was performed of aerobic bacterial species found in the vagina of 203 bitches with genital disorders, e.g. infertility, vaginitis, pyometra and puppy death. Escherichia coli, beta-hemolytic streptococci, Staphylococcus intermedius and Pasteurella multocida were the species most often isolated. From bitches with pyometra E. coli in pure culture was the most frequent isolate. In contrast, the majority of infertile bitches gave rise to mixed cultures, and no specific bacterial species was consistently associated with infertility. Thus, bacterial sampling from infertile bitches was concluded to be of low diagnostic value. Bacterial species isolated from the bitches having vaginitis were present in pure culture in 26.9% of the samples while nonspecific mixed cultures were obtained from 34.6% of the samples from these bitches. E. coli was the most frequently isolated bacterial species from bitches with dead puppies. However, in such cases it is important to relate the vaginal bacterial findings to autopsy findings and the results of bacteriological cultures of the pups.     

A survey of equine abortion and perinatal foal losses in Hungary during a three-year period (1998-2000)

Cases of equine abortion and perinatal foal losses were investigated in Hungary during a three-year period (1998-2000). Samples from aborted equine fetuses and newborn foals (total n = 96) were examined using bacteriological, virological, pathological, immunohistochemical (IHC), molecular biological and serological methods. The cause of abortion and perinatal foal loss was identified in 67/96 cases (70%); viral infection was found in 22 (23%), viral and bacterial coinfection in 1 (1%), bacterial infection in 23 (24%), protozoan infection in 1 (1%) and fungal infection in 2 cases (2%). Morphological lesions suggestive of infection were recorded in 2 (2%) and non-infectious causes in 16 cases (17%).     

Pathogenesis of six pigeon-origin isolates of Newcastle disease virus for domestic chickens

The pathogenesis of six pigeon-origin isolates of Newcastle disease virus (NDV) was investigated in chickens. Four isolates were previously defined as the variant pigeon paramyxovirus 1 (PPMV-1), and two isolates were classified as avian paramyxovirus 1 (APMV-1). Birds inoculated with PPMV-1 isolates were euthanatized, and tissue samples were collected at 2, 5, and 10 days postinoculation (DPI). Birds inoculated with APMV-1 isolates died or were euthanatized, and tissue samples were collected at 2, 4, and 5 DPI. Tissues were examined by histopathology, immunohistochemistry (IHC) for the presence of NDV nucleoprotein, and in situ hybridization (ISH) for the presence of viral mRNA for the matrix gene. Spleen sections were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and by IHC using an anti-active caspase-3 antibody (IHC-Casp) to detect apoptotic cells. Brain sections of PPMV-1-infected birds were examined by IHC to detect T and B lymphocytes and glial fibrillary acidic protein (GFAP). Histologically, birds inoculated with PPMV-1 isolates had marked lesions in the heart and brain. Presence of viral nucleoprotein and viral mRNA in the affected tissues was confirmed by IHC and ISH, respectively. Numerous reactive astrocytes were observed in brain sections stained for GFAP Among all the isolates, the IHC-Casp demonstrated that apoptosis was very prominent in the ellipsoid-associated cells of the spleen at 2 DPI. Results of the TUNEL assay indicated that apoptotic cells were prominent at 5 DPI and were more randomly distributed. The clinical signs and gross and histopathologic changes observed in the APMV-1-infected birds were characteristic of an extensive infection with highly virulent NDV evident by IHC.