Comparison between light and electron microscopy in canine and feline renal pathology: a preliminary study.

The aim of this study is to compare the accuracy and clinical use of light and transmission electron microscopy in detecting the early stages of renal pathologies in domestic animals. We examined 30 samples of renal tissue from cats and dogs referred to the Veterinary Hospital of the Department of Animal Pathology for different systemic diseases. The progressions of the kidney pathologies were classified using the scheme system proposed by the International Renal Interest Society. All samples were submitted for conventional histology and ultrastructural examination. Our study shows that electron microscopy is necessary to complete the histological examinations, especially to define early stages of kidney diseases (minimal changes disease, epithelial tubular pathologies, tubular basement membrane and glomerular basement membrane changes). Electron microscopy can be more accurate in defining the level of focal lesion, and permits discrimination between different clinical and pathological alterations such as fibrillary deposits. In conclusion, transmission electron microscopy associated with clinical, histological, histochemical and immunological examinations, is an essential method for diagnosis and prognosis of renal disease.

Krabbe's disease in two West Highland White terriers.

Two 3-month-old male West Highland White terriers were referred for progressive neurological disease. Histological examination of the central nervous system of the animals euthanized at the owner' request, revealed diffuse, bilateral and symmetrical white matter lesion consisting of varying degrees of demyelination and axonal degeneration. Accumulation of round to ovoid large mononuclear cells was especially observed along the blood vessels in the white matter. These cells were characterized by central or eccentric nuclei and highly eosinophilic, granular and PAS-positive cytoplasm. Stored material was stained with toluidine blue both at pH 4 and pH 11 and exhibited a strong PAC and no PALK activities. Staining for lectins revealed a positivity using Ricinus communis agglutinin-I, Ricinus communis agglutin-II, Triticum vulgaris and Concavalin A. Histochemical evaluation of intracellular material was performed on the kidney and on the liver, too. Ultrastructural investigations allowed to observe the cytoplasmic contents of globoid cells that is an admixture of degraded myelin membranes and different kinds of tubular aggregates. To verify if the two dogs bore the mutation at position 473, a method involving PCR amplification of genomic DNA followed by restriction-digestion was used. The diagnosis of Krabbe's disease was performed based on the clinical evaluation, morphological, histochemical and ultrastructural features.

Biochemical, ultrastructural and molecular characterization of the triphenyltin acetate (TPTA)-induced apoptosis in primary cultures of mouse thymocytes.

Triphenyltin acetate (TPTA), a triorganotin compound used in agriculture as a biocide, is immunotoxic in vivo and in vitro. The present study was undertaken to ascertain whether apoptosis might play a role in the TPTA toxicity in vitro. Mouse thymocyte primary cultures were exposed to 0, 4 and 8 micromol/L TPTA; methyl prednisolone (1 micromol/L) was used as a positive control. Cell aliquots were harvested after 0, 1, 2, 4, and 8 h and the presence of early or late apoptotic phenomena was checked by (a) morphological investigations; (b) spectrophotometric quantification of fragmented DNA and agarose gel electrophoresis; (c) cell flow cytofluorometry, using an annexin V-FITC kit; and (d) detection of in situ apoptosis by a colorimetric detection kit (Titer-Tacs). TPTA cytotoxicity was also evaluated using the trypan blue dye exclusion test. Morphological investigation indicated apoptosis and/or necrosis. After 8 h of incubation, cells exposed to 4 micromol/L TPTA showed an increase in DNA fragmentation (on electrophoresis), which was confirmed by spectrophotometry (p < 0.05). Flow cytofluorometry pointed out an early (p < 0.05) increase of annexin V-positive (apoptotic) cells in TPTA-exposed flasks, whereas at least partly contradictory, results were obtained with the Titer-Tacs kit. Overall, these results provide evidence that TPTA, at low concentrations (4 micromol/L) induces early and late apoptotic phenomena, whereas cells exposed to the highest concentrations (8 micromol/L) are likely to undergo necrosis rather than apoptosis.

Histological and immunohistochemical study of a neuroblastoma in a dog.

A 13-year-old, male German Shepherd dog was euthanasized for a frontal temporal mass revealed by the MRI. The histological examination showed a proliferation composed of small round undifferentiated cells arranged in sheets or nests and sometimes in pseudorosettes interrupted by hypocellular zones of fibrovascular stroma. Immunohistochemical studies revealed the expression of neuroblastic epitopes. The presented neoplasm has many histological and immunohistochemical features in common with the group of olfactory neuroblastomas reported in man, so it could be classified as primitive neuroectodermal tumor with neuronal differentiation.

Triphenyltin acetate-induced cytotoxicity and CD4(+) and CD8(+) depletion in mouse thymocyte primary cultures.

Organotin compounds (OTs) find application worldwide as catalysts, stabilizers and biocides. Triphenyltin derivatives (TPs), including the fungicide triphenyltin acetate (TPTA), are OTs mostly used in our country. Some OTs were proved to be immunotoxic and in this paper the cytotoxicity, the possible selective activity upon definite lymphocyte subsets as well as the antiproliferative effect of TPTA was investigated in vitro by using primary cultures of mouse thymocytes. TPTA (5, 10 and 25 microM) was cytotoxic to these cells, as demonstrated by the significant (P<0.05) reduction of the cell viability percentage (trypan blue dye exclusion test), the neutral red uptake and the reduction of tetrazolium salts to formazan products (MTT assay). These overt effects were already noticed after 4 h of exposure to TPTA. The fungicide otherwise significantly reduced, after 24 h of incubation, the percentage of mature single positive thymocytes, particularly the CD4(+)/CD8(-) one. Finally, a significative dose-dependent inhibition of the T-cell mitogen-induced cell proliferation was observed in thymocytes exposed to 1 and 8 microM TPTA. These results are indicative of the TPTA immunotoxic properties, according to previous published reports concerning the in vitro and in vivo toxicity of some di- and triorganotin compounds.

Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein as antigen.

The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. In this study, complementary DNA corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein. The resulting GST-N recombinant fusion protein was purified by affinity chromatography and used as antigen for serological testing by indirect enzyme-linked immunosorbent assay (ELISA). Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), obtained following fusion experiments with spleen cells of BAlb/c mice that were immunized with the purified virus, were used in a competitive assay to increase the specificity of the ELISA. Both MAbs were found to be directed against highly conserved conformational epitopes of North American isolates of PRRSV. Optimal concentration of GST-N protein was determined by checkerboard titration, using hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a range of 0.1-0.5 microg protein per well. When tested on 95 sera from pigs that were experimentally infected with the IAF-Klop strain, the competitive ELISA (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); Idexx), with specificity values of 100 and 96.2%, respectively. When tested on clinical samples (542 sera) from 28 positive and 28 negative pig herds, the K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) tests as shown by kappa values of 0.762 and 0.803. The sensitivity and specificity of K8-ELISA were 100% on a herd basis, whereas sensitivity values of 80 and 82% with a specificity of 98.7% were determined on an individual basis in comparison with HerdCheck(R) and IF tests.

Histopathological and immunohistochemical studies on cetaceans found stranded on the coast of Italy between 1990 and 1997.

Detailed histopathological and immunohistochemical studies were carried out on 24 cetaceans, 17 of which were striped dolphins (Stenella coeruleoalba), four bottlenose dolphins (Tursiops truncatus) and three Risso's dolphins (Grampus griseus), all found stranded on the Italian coast between 1990 and 1997. The most frequently detected lesions were chronic pneumonia (73.7% of the examined lungs), focal chronic pancreatitis (71.4%), non-purulent encephalitis (50%), chronic hepatitis (42.1%), and chronic focal interstitial nephritis (31.2%). The skin and the subcutaneous panniculus were often (33.3%) affected by parasitic diseases (Phyllobothrium delphini and Pennella sp.). An appreciable percentage of animals showed lymphoid depletion in their lymphatic organs (47.2%), as well as a high rate of parasitic infestations in their alimentary tracts (25%).

Effects of the ionophore antibiotic monensin on hepatic biotransformations and target organ morphology in rats.

As a preliminary in vivo approach in order to study the mechanism of toxicity of the veterinary anticoccidial monensin, male Wistar rats were orally administered 0, 2 and 12 mg kg-1 body wt. day-1 of monensin for 7 days. At the end of the experiment, effects of the ionophore on serum creatine kinase, lactic dehydrogenase and selected drug metabolising enzyme activities were investigated. Furthermore, liver, heart and quadriceps femoris muscle samples were submitted to morphological investigations. Clinical signs or increasing levels of enzymic markers of muscle injury attributable to monensin toxicosis have never been observed in treated animals. As a matter of fact all drug metabolising enzymes activities checked have not shown significant changes, except for a significant decrease of ethoxyresorufin O-deethylase (up to 31%) and aminopyrine N-demethylase (17%) activities. Morphologically, mitochondrial cristae fragmentation and initial formation of 'myelinic sheaths-like' structures have been noticed in heart and muscle fibres. As far as rat study is concerned, these results confirm heart and muscle as target organs of monensin toxicity. In addition, these findings suggest that the inhibition of hepatic biotransformation processes following the i.p. administration of the ionophore, as reported previously by other authors, might reflect unspecific cellular toxic effects rather than a specific enzyme damage.

Ultrastructural morphology of sarcosporidiosis in alpine ibex (Capra ibex).

The authors describe the ultrastructural morphology of the cyst wall of sarcocysts in the muscles from Ibex, found dead and sent to this department to be examined. Several samples of the diaphragm were taken from 52 Ibex of different age and sex, one Hispanic and the others from the Gran Paradiso National Park (GPNP). The samples were prepared for electron microscopy after testing for sarcocystiosis through extemporaneous microscopic examination. Eighty-six percent were positive. The morphology of the cyst wall led to the identification of three types of sarcocysts in the Ibex of the GPNP and a further type in the Hispanic one. The morphology of the sarcocysts wall is similar to the wall of the species described in the domestic ruminants from several authors.

Antigenic variation among bovine viral diarrhea virus (BVDV) strains and the role of different cell fixation methods in immunoassays.

Antigenic variation among 13 Quebec isolates of bovine viral diarrhea virus (BVDV), 4 reference strains and 2 American isolates were studied by peroxidase-linked antibody assay (PLA assay) and neutralization test (NT). The Quebec strains consisted of 3 isolates before 1993 and 10 isolates from 1993. In the PLA assay, we compared 2 different fixatives, acetone and formalin. Acetone-fixation allowed us to identify 6 groups from amongst the viruses tested. All the Quebec isolates were different from the reference strains. In addition, antigenic variation was detected between Quebec isolates obtained before and during 1993. However, PLA assays performed after formalin fixation did not detect these antigenic variations. Neutralization tests were carried out with 2 polyclonal antibodies (PAb) and 6 monoclonal antibodies (MAb). They were used to classify BVDV strains and isolates into 4 groups and 7 subgroups respectively. In conclusion, we demonstrated that the BVDV isolates from the 1993 outbreak in Quebec are antigenically different from reference strains and from isolates existing in Quebec before 1993. In addition, we have shown that 2 internationally used fixation-methods in PLA assay give different results. The usefulness of each method is discussed.

Evaluation of a protective immunity induced by an inactivated influenza H3N2 vaccine after an intratracheal challenge of pigs.

A challenge study was conducted to evaluate the safety and efficacy of an inactivated influenza H3N2 virus vaccine combined with Quil A/Alhydrogel mixture under controlled conditions in piglets. Twenty-four piglets from 12 sows were allocated to 2 groups; injected intramuscularly with 2 doses of the tested vaccine or with PBS at 2 wk intervals and challenged intratracheally with 105TCID50 of the H3N2 swine influenza virus 6 d after the 2nd immunization. Clinical and virological parameters were recorded for 4 d after the challenge. The use of the tested vaccine produced high serum hemagglutination-inhibition titers against the swine H3N2 strain virus. This strong immune response suppressed all clinical signs and viral shedding and reduced pulmonary lesions due to the challenge in the vaccinated group, without causing any secondary effects. Our results suggest that the serum HI titers correlated with the degree of protection induced by an inactivated swine influenza H3N2 vaccine.

Comparison of polymerase chain reaction and monoclonal antibodies for G-typing of group A bovine rotavirus directly from fecal material.

A reverse transcriptase and polymerase chain reaction (RT-PCR)-based assay for G-typing of bovine rotaviruses (BRV) was compared with a monoclonal antibody-based immunoassay (MAbs-ELISA) in the characterization of BRV field strains obtained from calves in different regions of Quebec between 1992 and 1994. The strains were analysed for two G types (G6 and G10) which are the most predominate BRV field strains. Fecal samples positive for BRV by polyacrylamide gel electrophoresis (n = 74) were typed by both methods revealing 77% correlation. RT-PCR detected 10 more G6 and 2 more G10 serotypes than MAbs-ELISA. Nine of the 12 discrepant samples could be cultivated and were confirmed as G6 (8) or G10 (1) by both methods. RT-PCR was able to efficiently detect artificial mixes of G6 and G10 and detected two mixed field infections. Four additional infections considered as mixed by MAbs-ELISA and as only G6 by RT-PCR were possibly MAbs-ELISA cross-reactions. RT-PCR provided a very sensitive method for typing BRV field isolates.

Triphenyltin acetate toxicity: a biochemical and ultrastructural study on mouse thymocytes.

1. Triphenyltin acetate (TPTA) has been shown to exert in vivo a selective toxic effect on the immune system. To assess in vitro possible alterations induced by TPTA exposure, primary cultures of mouse thymocytes were incubated up to 24 h with graded amounts (1-12 microM) of the organotin. 2. The cytotoxic activity has been evaluated with the MTT colorimetric assay, the neutral red (NR) assay and the lactic dehydrogenase (LDH) cellular release. Cell pellets were fixed with 2.5% glutaraldehyde, resin-embedded and ultrathin sections were observed through transmission electron microscopy. 3. After 2 h of incubation, dose-dependent increases of cytotoxicity were observed in thymocytes submitted to MTT and NR tests (up to 41.43% and 18.9%, respectively), while 22 h later this overt effect on cell viability was noticed merely in cells exposed to 12 microM TPTA. Dose-dependent increases of LDH leakage in the culture medium were observed all throughout the study. 4. Morphological investigations revealed features (chromatin condensation, cell membranes fragmentation and formation of membrane bound apoptotic bodies) suggestive of apoptosis. 5. This study indicates that TPTA is cytotoxic to mouse thymocytes: morphologically, the rising of apoptosis is likely to be recognized, as previously reported in different in vitro studies with other immunosuppressive agents as dioxin and corticosteroids.

Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec.

Fecal samples from diarrheic and nondiarrheic dairy calves (1 to 3 weeks old) from 12 regions of Quebec, collected between 1992 and 1994, were screened for group A bovine rotavirus (BRV) using a combination of 2 VP6-specific monoclonal antibodies (MAbs) in an enzyme-linked immunosorbent assay (ELISA). The overall prevalence of BRV infection was 26.4% (107/405). In diarrheic calves, BRV infection reached 74.3% (55/74), but only 15.7% (52/331) in nondiarrheic calves. BRV-positive samples were serotyped by enzyme-linked immunosorbent assay (ELISA) using G6 and G10 specific MAbs. The analysis of 107 field samples revealed that, in diarrheic calves, 34.5% (19/55) were G6, 27.2% (15/55) were G10, 9% (5/55) were G6 and G10 positive, and 29.9% (16/55) were G6 and G10 negative. In nondiarrheic calves, 19.2% (10/52) were G6, 19.2% (10/52) were G10, 7.6% (4/52) were G6 and G10 positive, and 53.6% (28/52) were G6 and G10 negative. Rotavirus dsRNA was extracted from BRV-positive samples and examined by polyacrilamide gel electrophoresis (PAGE). Of 107 samples tested, 74 (69.1%) were positive, and all the samples demonstrated a typical group A rotavirus migration pattern.

Experimental model of cirrhosis in rabbits exposed to carbon tetrachloride by inhalation.

Orthotopic hepatic transplantation is the most diffused surgical treatment used when attempting to substitute an irreversibly damaged liver. However, this practice faces two main problems. Firstly, surgeons need to explant the organ from the donor who is a just deceased human being, with all the implications involved. Secondly, the host patient must be rendered immunologically tolerant to the graft, through pharmacological suppression. Some hepatic alterations allow a structural and functional compensation by means of an autotransplant, but liver cirrhosis, which is widely diffused in humans and easily produced in experimental animals, has not yet been treated with this technique. This research was undertaken with the aim of procuring hepatic cirrhosis in a medium-sized animal--the rabbit--exposed for some months to CCl4 vapours. Preliminary experiments were performed in order to shorten the challenge period by means of liver induction. This approach was unsuccessful because of the natural susceptibility of this species, which was heightened by the barbiturate administration (0.05% sodium phenobarbital in drinking water, for one week). However, induction, rendered impossible the survival after carbon tetrachloride given orally (125 microliters/kg body weight) or by inhalation (1000 ppm for 2 hours). Finally, CCl4 was administered to normal rabbits by inhalation at the initial concentration of 100 ppm, for 2 hours twice a week. The level of the hepatotoxin was subsequently raised stepwise, according to the body weight curve, up to the maximum of 600 ppm by week 23. At the fourth week of intoxication, the metabolic activity of liver microsomes appeared severely depressed, as shown by the 300% increase of hexobarbital sleeping time. The observation of the surface of liver in situ, performed through an explorative laparatomy, showed initial but clear signs of hepatic fibrosis. At sacrifice, after six or ten months of cirrhogenic treatment, the histopathology examination of the liver demonstrated severe and massive cirrhosis, respectively for the two different steps. This experimental schedule appears to be suitable for producing hepatic cirrhosis in a medium-sized animal, which could be used as a model for pioneering attempts of liver autotransplantation. Furthermore, we point out two important aspects of these results. First, the poison has been inhaled by the experimental animals as it generally happens for humans in the environment. Second, the ratio between the length of exposure versus the life span of rabbits--6 months and 8 years, respectively--parallels that reported for human cirrhosis due to halogenated hydrocarbons--5 or 10 years versus 60 or 80 years.

The 5'-untranslated region sequence of a potential new genotype of bovine viral diarrhea virus.

The 5' untranslated region (UTR) of several bovine viral diarrhea virus (BVDV) isolates from the severe Quebec outbreak was amplified by polymerase chain reaction (PCR) and sequenced. Sequences revealed the loss, for the BVDV type II isolates, of an internal PstI restriction site, which is present in all known BVDV type 1 5' UTR sequences. A single restriction enzyme digestion (PstI) of an aliquot of PCR product allowed us to differentiate BVDV type I and BVDV type II.

Detection of porcine respiratory coronavirus and transmissible gastroenteritis virus by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) for the detection of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was developed. A bovine TGEV-specific polyclonal antibody was purified by affinity chromatography with the TRIO Bioprocessing System and was used as the capture antibody, at a concentration of 1.5 micrograms/well. The F5.39 monoclonal antibody was obtained by the fusion of spleen lymphocytes from TGEV immunized mice with NS-1 myeloma cells. This mAb was used as a second antibody for the ELISA. The ELISA detected 40 ng of TGEV and 407 ng of PRCV. To study the ability of ELISA to detect TGEV in field cases, 53 intestinal samples were taken from pigs exhibiting clinical signs of transmissible gastroenteritis. All the positive samples detected by the ELISA were confirmed as positive by immunofluorescence or cell culture immunofluorescence. To study the ability of this ELISA to detect PRCV in nasal swabs and lung samples, 20 seven-day-old piglets were inoculated with a Quebec strain of PRCV. The ELISA was able to detect PRCV in both kinds of samples.

Antigenic characterization of an H3N2 swine influenza virus isolated from pigs with proliferative and necrotizing pneumonia in Quebec.

A new strain of swine influenza A virus, designated A/Swine/Saint-Hyacinthe/150/90 has been isolated from pigs with severe proliferative and necrotizing pneumonia in Quebec. The antigenic characterization of the hemagglutinin was performed by hemagglutination inhibition test, immunoblot and indirect immunoprecipitation using polyclonal antisera. Only the last test was able to detect an antigenic relationship between the hemagglutinin of this isolate and an H3 subtype influenza virus. The immunoprecipitation test was a useful alternative for determining the hemagglutinin of influenza A virus subtypes. The neuraminidase inhibition test demonstrated a reactivity between the A/Swine/Saint-Hyacinthe/150/90 and antiserum against a N2 subtype influenza virus. Our results indicate that this new strain isolated for the first time in the porcine population of Canada is related to A/Sw/Hong Kong/76 H3N2 swine influenza virus.