Fluorescence in situ hybridization confirms clearance of visible Helicobacter spp. associated with gastritis in dogs and cats.

BACKGROUND: The results of studies examining the role of Helicobacter spp. in the pathogenesis of canine and feline gastritis are inconclusive. Furthermore, data evaluating the effectiveness of medical therapy for eradication of Helicobacter infection are limited. AIM: To detect Helicobacter spp. in mucosal biopsies of dogs and cats diagnosed with gastritis, with fluorescence in situ hybridization (FISH). ANIMALS: Three dogs and 2 cats with signs of chronic gastrointestinal disease. METHODS: Dogs and cats infected with Helicobacter spp. were treated with triple antimicrobial therapy and fed an elimination diet for 21 days. Helicobacter spp. status in endoscopic (3 dogs, 1 cat) or surgical biopsies (1 cat) of gastric mucosa was compared pre- and posttreatment in each animal by histology, FISH analysis, and polymerase chain reaction (PCR). RESULTS: Gastritis of varying severity with intraglandular spiral bacteria was observed in all animals. Pretreatment diagnostic tests confirmed the presence of mucosal Helicobacter spp. in all animals by FISH and histopathology and in 4/5 animals by PCR. Rapid resolution of vomiting episodes was observed in all animals. Gastric biopsies performed after triple therapy revealed clearance of visible Helicobacter spp. by histopathology and negative FISH analysis, as well as PCR in all animals. CONCLUSIONS AND CLINICAL IMPORTANCE: Application of FISH to routine biopsy specimens enabled rapid and specific identification of Helicobacter spp. within the gastric mucosa of dogs and cats. Although medical therapy was useful in resolution of clinical signs and clearance of visible Helicobacter spp. in gastric biopsies, gastric inflammation persisted.

Characterization of immune response of young pigs to porcine circovirus type 2 infection.

A longitudinal study was conducted to characterize the immune response of young swine to infection with porcine circovirus type 2 (PCV-2). Five 8-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally and intramuscularly with a field isolate of PCV-2 at a concentration of 10(4) TCID50/mL. Along with monitoring for clinical signs and viremia, serum samples were collected from all pigs at day 0 and thereafter every 7 days postinoculation (PI) until the termination of the study on day 35 PI. No clinical signs were observed in any of the animals during the study period. In all pigs, PCV-2 was detected by polymerase chain reaction (PCR) in serum samples collected on days 7, 14, and 21 PI. Viral DNA and antigens were detected by in situ hybridization and immunohistochemistry in tonsil, spleen, medial iliac lymph nodes, and ileum collected from each pig at the end of the study. Collectively, naïve young swine were shown to be susceptible to PCV-2. Virus-specific antibody was detected by an indirect fluorescent antibody (IFA) assay on day 14 PI, but virus-neutralizing antibody was not detected until day 28 PI. As neutralizing antibodies developed, cross-reactivity with PCV type 1 (PCV-1) also developed on the IFA test. Western immunoblot analysis revealed three PCV-2 proteins with molecular masses of 28 kd, 28.5 kd, and 35 kd. The 35-kd protein was also demonstrated in PCV-1, suggesting that this protein induced the cross-reactivity between PCV types 1 and 2. Antibody to the 28-kd protein was detected on day 14 PI and later, indicating that this protein was the most immunogenic. Because of its immunogenicity and specificity to PCV-2, and 28-kd protein might provide the antigenic basis for the development of diagnostic tests for detection of PCV-2 antibody.

Development of probes to differentiate porcine circovirus types 1 and 2 in vitro by in situ hybridization.

Porcine circovirus type 1 (PCV1), a PK-15 cell line contaminant, and porcine circovirus type 2 (PCV2), associated with post-weaning multisystemic wasting syndrome (PMWS), are genetically and antigenically related. Several techniques have been developed to detect PCV, including in situ hybridization (ISH). Previously reported probes used for ISH may hybridize with both PCV1 and PCV2 nucleic acids. We attempted to produce probes for ISH that can detect and differentiate PCV2 from PCV1 in PCV-infected cells. Riboprobes were synthesized from the sense and antisense strands of both open reading frames 1 and 2 (ORF1 and ORF2) of PCV2. At 42 and 58 degrees C, the ORF1 antisense probe hybridized with nucleic acid from both PCV1- and PCV2-infected cells. At 58 degrees C, the ORF2 antisense probe hybridized with PCV2 nucleic acid but not with PCV1 nucleic acid. The ORF1 and ORF2 sense probes bound only with PCV2 nucleic acid. Both antisense strand probes produced stronger signals than the sense strand probes. The results showed that the PCV2 ORF1 antisense probe is the most likely probe to detect both PCV types while the ORF2 antisense probe is capable of discriminating between PCV1 and PCV2.

Interstitial pneumonia in feedlot cattle: concurrent lesions and lack of immunohistochemical evidence for bovine respiratory syncytial virus infection.

The objectives of this study were to describe the nature and distribution of microscopic lung lesions in feedlot cattle with interstitial pneumonia and to determine whether bovine respiratory syncytial virus (BRSV) antigen was present in affected lungs. Lungs with macroscopic lesions compatible with interstitial pneumonia were collected from cattle from 5 west-central Saskatchewan feedlots that had been on feed for greater than 60 days at the time of death. Interstitial pneumonia was most consistently present in dorsal portions of caudal lung lobes and in 21/28 cases (75%) had a multifocal to coalescing distribution. All 28 lungs exhibited hyaline membrane formation and some degree of type II alveolar epithelial cell hyperplasia, consistent with an acute to subacute duration. Twenty-one of 28 cases (75%) had concurrent bronchopneumonia in at least 1 lung lobe; bronchopneumonia was grossly evident in 9/28 cases (32%). Chronic bronchitis or bronchiolitis was present in at least 1 section in 12/28 (43%) of the lungs, and 25/28 (89%) had at least 1 focus of bronchiolitis fibrosa obliterans. Bronchopneumonia and bronchiolitis fibrosa obliterans were markedly less common in 10 sets of bovine lungs obtained from an abattoir. Bovine respiratory syncytial virus antigen was demonstrated using immunohistochemistry in 2/28 cases and was associated with bronchiolar epithelial necrosis that was more severe than the bronchiolar lesions in the BRSV antigen-negative cases. Interstitial pneumonia in feedlot cattle in this study was more frequently associated with suppurative bronchopneumonia and bronchiolitis fibrosa obliterans than with BRSV infection.

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.

Fatal pulmonary edema in white-tailed deer (Odocoileus virginianus) associated with adenovirus infection.

Sporadic sudden deaths in adult white-tailed deer occurred from November 1997 through August 1998 on an Iowa game farm. Three of the 4 deer necropsied had severe pulmonary edema, widespread mild lymphocytic vasculitis, and amphophilic intranuclear inclusion bodies in scattered endothelial cells in blood vessels in the lung and abdominal viscera. Immunohistochemistry with bovine adenovirus 5 antisera and transmission electron microscopy demonstrated adenoviral antigen and nucleocapsids, respectively, within endothelial cells. Adenovirus was isolated in cell culture from 1 of the affected deer. The isolate was neutralized by California black-tailed deer adenovirus antiserum. These findings indicate that adenovirus should be considered in the differential diagnosis of both black-tailed and white-tailed deer with pulmonary edema and/or hemorrhagic enteropathy.

Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome.

A rapid in situ hybridization (ISH) technique for the detection of porcine circovirus (PCV) nucleic acid in cell culture and formalin-fixed paraffin-embedded tissues was developed. A fluorescein-labeled RNA probe was transcribed from a plasmid containing 530 bp of the ORF1 of a PCV isolated from a pig with postweaning multisystemic wasting syndrome (PMWS). Hybridization using standard hybridization buffer was performed at 42 C for 16 hours and was compared to hybridization using rate enhancement hybridization (REH) buffer at 67 C for 2 hours. Hybridization was detected with an alkaline phosphatase-conjugated antifluorescein antibody. In both cultured cells and tissues from pigs with PMWS, the signal intensity and number of labeled cells in sections hybridized with REH buffer were equal to those of sections hybridized with standard hybridization buffer. The total time required for ISH using the REH buffer is 7-8 hours, thus making this protocol suitable for application in routine PCV diagnosis.

Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue.

An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.

Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS).

The prevalence of different pathogens detected in combination with porcine circovirus type 2 (PCV-2) was studied retrospectively in field cases of postweaning multisystemic wasting syndrome (PMWS) diagnosed at the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, between January 2000, and September 2001. The presence of PCV-2 antigen in lymphoid tissues and/or lung, demonstrated by immunohistochemistry, together with moderate to severe lymphoid depletion and/or granulomatous lymphadenitis, was used as the criteria for the diagnosis of PMWS. A total of 484 cases fulfilled these criteria. Most of the cases (294/369) of PMWS occurred in pigs between the ages of 8 and 18 weeks, with a peak at 10 weeks of age. Porcine reproductive and respiratory syndrome virus was detected in 51.9% of the cases, Mycoplasma hyopneumoniae in 35.5%, bacterial septicemia in 14.0%, bacterial pneumonia in 7.6%, swine influenza virus in 5.4%, and PCV-2 alone in 1.9%. In cases with bacterial septicemia the most frequently isolated pathogen was Streptococcus suis. In cases with bacterial pneumonia, Pasteurella multocida was the most prevalent.

Detection and isolation of coronavirus from feces of three herds of feedlot cattle during outbreaks of winter dysentery-like disease.

Clinical signs of a winter dysentery-like syndrome in 6- to 9-month-old cattle in 3 feedlots included acute onset of diarrhea with high morbidity and low mortality, respiratory tract problems that included dyspnea, coughing, and nasal discharge, and high rectal temperatures. Bovine coronavirus was detected by use of an ELISA and immune electron microscopy in fecal and nasal swab samples and by immunohistochemical analysis of intestinal sections collected from calves during necropsy. Bovine coronavirus should be considered in the differential diagnoses for diseases that cause acute onset of bloody diarrhea in feedlot cattle.

Brown Norway rats are high responders to bronchiolitis, pneumonia, and bronchiolar mastocytosis induced by parainfluenza virus.

The pathogenesis of parainfluenza 1 (Sendai) virus infection was compared among 25-day-old BN, F344, and LEW rats to identify a sensitive as well as a resistant inbred rat strain to Sendai virus-induced lung injury during early life. At 7 days after inoculation, BN rats had 65-fold higher (P less than .001) pulmonary viral titers and threefold higher (P less than .002) numbers of neutrophils in bronchoalveolar lavage fluid than did F344 rats. At 14 days after inoculation, when most virus-induced inflammation had been resolved, BN rats had a threefold greater (P less than .01) incidence of bronchioles with aggregates of lymphocytes and macrophages than did F344 rats. Control BN rats had higher numbers of bronchiolar eosinophils than did F344 or LEW rats. Although viral inoculation resulted in increased numbers of bronchiolar mast cells in all three strains at 14 days, bronchiolar mast cell density was greater (P less than .005) in virus-inoculated BN and LEW rats than in F344 rats. We conclude that BN rats are high responders and F344 rats are low responders to Sendai virus-induced bronchiolitis, pneumonia, and airway mastocytosis. These strain differences may be useful in elucidating important pathogenetic mechanisms in virus-induced airway injury and mastocytosis.

Virus-induced increases in bronchiolar mast cells in Brown Norway rats are associated with both local mast cell proliferation and increases in blood mast cell precursors.

BACKGROUND: Parainfluenza type 1 (Sendai) virus-induced bronchiolitis during early life in rats induces increases in bronchiolar mast cells that persist for months after infection and are associated with airway obstruction and airway hyperresponsiveness. Brown Norway (BN) rats are highly susceptible, and Fischer 344 (F344) rats are relatively resistant to, Sendai virus-induced increases in airway responsiveness and mast cell density. EXPERIMENTAL DESIGN: To identify mechanisms responsible for the virus-induced mast cell increases, BN rats were studied using in vivo bromodeoxyuridine labeling, in vitro culture of bone marrow, blood, and lung mast cell progenitors (colony-forming unit-mast cell (CFU-MC)), and in vivo treatment with the rodent mast cell stabilizers disodium cromoglycate and nedocromil sodium. Bone marrow, blood, and lung CFU-MC were also quantitated in F344 rats. RESULTS: At 10 days after inoculation, there was a fivefold increase (p = 0.001) in the bromodeoxyuridine labeling index of bronchiolar mast cells in virus-inoculated BN rats. Viral inoculation increased CFU-MC/ml blood greater than fivefold (p < 0.05) in BN rats at 10 days after inoculation, and BN rats had greater numbers of both blood and lung CFU-MC than did F344 rats. Treatment with inhibitors of mast cell degranulation had no effect on Sendai virus-induced bronchiolar mast cell increases in BN rats. CONCLUSIONS: Virus-induced increases in bronchiolar mast cells result from proliferation of preexisting mast cells and may be augmented by recruitment of circulating mast cell precursors.

Spontaneous cardiomyopathy and exophthalmos in cotton rats (Sigmodon hispidus).

Exophthalmos and clinical signs of heart failure occurred sporadically in 3- to 12-month-old cotton rats (Sigmodon hispidus) in a colony originally derived from three male and four female littermates. Macroscopic lesions in severely affected animals included subcutaneous edema, hydrothorax, right ventricular dilatation, unilateral or bilateral atrial thrombosis, and exophthalmos. Hearts from 17 cotton rats that were found dead or were euthanatized because of exophthalmos or dyspnea and 33 control cotton rats were examined microscopically. Myocardial lesions were present in 46 of 46 cotton rats > or = 1 month of age and consisted of multifocal cardiac myocyte necrosis, mineralization, and mononuclear inflammatory cell infiltration. Cotton rats > 5 months of age also had foci of interstitial fibrosis and myocyte atrophy. Twelve of 24 (50%) necropsied cotton rats had chronic pulmonary congestion, and livers from eight of 24 (33%) had chronic periacinar congestion and atrophy. Thrombi were present in one or both cardiac atria in nine of 50 (18%) hearts, and in at least one orbital venous sinus in 14 of 24 (58%) necropsied cotton rats and in 12 of 14 (86%) with exophthalmos. Exophthalmos in this colony of cotton rats appears to have resulted predominantly from orbital venous sinus thrombosis caused by stasis of venous blood secondary to right heart failure associated with a heritable cardiomyopathy.

Proliferative vasculopathy and cutaneous hemorrhages in porcine neonates infected with the porcine reproductive and respiratory syndrome virus.

Severe cutaneous hemorrhages with dermal and subcutaneous capillary angioplasia were seen in aborted and stillborn piglets, concurrently with an acute outbreak of porcine reproductive and respiratory syndrome virus (PRRSV) abortions. Histologically, the lesions consisted of angioblastic endothelial cells and immature capillary vascular structures coursing through the edematous myxomatous dermis and subcutis. Proliferating capillaries often were surrounded by large and foamy macrophages that stained positively for PRRSV by immunohistochemistry. The sudden appearance of these vascular lesions during the PRRSV outbreak and their abrupt disappearance after the abortion storm, along with the immunohistochemical localization of PRRSV-positive macrophages adjacent to the proliferating capillaries, suggest that PRRSV likely played a role in the development of these unusual lesions.

Virus-induced increases in airway mast cells in brown Norway rats are associated with enhanced pulmonary viral replication and persisting lymphocytic infiltration.

Brown Norway (BN) rats are more susceptible than Fischer 344 (F344) rats to parainfluenza virus-induced lung injury and to bronchiolar mast cell increases that are associated with persistent airway hyperresponsiveness. In this study, pulmonary viral replication as well as immune, inflammatory, and airway mast cell responses to Sendai virus infection were compared between neonatal BN and F344 rats. BN rats supported prolonged viral replication, and viral titers in BN rats were 5-fold higher (p < .05) than in F344 rats at 7 days after inoculation. F344 rats had 18-fold higher (p < .06) numbers of lymphocytes in bronchoalveolar lavage fluid at 7 days after inoculation than did BN rats. Persisting bronchiolar aggregates of lymphocytes, plasma cells, and macrophages were more common, and increases in bronchiolar mast cells were greater in BN rats than in F344 rats. No strain differences were detected in bronchiolar intramural infiltrates of CD4 + or CD8 + cells. The greater susceptibility of BN rats to virus-induced increases in bronchiolar mast cells and airway responsiveness may be the result of their less efficient viral clearance mechanisms and more persistent bronchiole-centered inflammatory response.

Pulmonary eosinophilia and granulomatous pulmonary arteritis induced in rats by intravenous Sephadex.

Airway eosinophilia has been proposed as a major pathogenetic event in bronchial asthma and airway hyperresponsiveness. Intravenous injection of Sephadex G200 in rats induces pulmonary and blood eosinophilia and alters pulmonary responsiveness to 5-hydroxytryptamine. To characterize the early pulmonary inflammatory responses following Sephadex administration, and to determine the timing of the onset of pulmonary eosinophilia relative to blood eosinophilia, Sprague-Dawley rats were injected intravenously with Sephadex G200 beads. Lungs and other tissues were examined by light and transmission electron microscopy at 4, 12, 24, 48, 72, and 96 hours after injection. Blood eosinophil counts were determined at 0, 24, and 96 hours after injection. Sephadex beads were trapped initially in small caliber muscular pulmonary arteries associated with terminal bronchioles and in intra-acinar locations. There was marked infiltration of eosinophils and macrophages around the beads and into arterial walls and edematous periarterial and peribronchiolar connective tissue as early as 4 hours after injection. Periarterial-peribronchiolar eosinophil aggregates peaked in density at 24 and 48 hours. Macrophages and multinucleated cells dominated the inflammatory cell responses in arteries immediately surrounding partially degraded Sephadex beads from 24 to 96 hours. Bone marrow eosinophilopoiesis and blood eosinophilia were not detected until 96 hours. We conclude that Sephadex induces pulmonary eosinophilia prior to blood eosinophilia and suggest that Sephadex may induce pulmonary release of one or more eosinophil chemotactic substance(s). This model may prove useful in the study of factors that influence eosinophil migration into the lung in various disease states.

Expression of the neutrophil chemoattractant interleukin-8 in the lesions of bovine pneumonic pasteurellosis.

We investigated the expression of interleukin-8 (IL-8) in pneumonic pasteurellosis of cattle because neutrophils are important mediators of tissue injury in this disease and because IL-8 is a major neutrophil chemoattractant in other species. We also compared IL-8 expression in bacterial and viral pneumonia, since the latter lacks the severe neutrophil exudation typical of pneumonic pasteurellosis. IL-8 expression was assessed by northern analysis, in situ hybridization, enzyme-linked immunosorbent assay, and in vivo bioassay. IL-8 mRNA expression was elevated dramatically in lesions of pneumonic pasteurellosis compared to unaffected lung from the same calves. In situ hybridization revealed intense expression of IL-8 mRNA in alveolar macrophages and neutrophils and milder expression in bronchiolar and alveolar epithelium, interstitial cells, and pleural mesothelium. Bronchoalveolar lavage fluid from lesional lung contained 16.06+/-4.00 ng/ml IL-8, whereas those from nonlesional and normal lung contained 0.34+/-0.11 and 0.01+/-0.002 ng/ml, respectively. We detected IL-8 expression at only minimal levels in bovine respiratory syncytial viral pneumonia. Lung extracts from lesions of pneumonic pasteurellosis induced vigorous neutrophil infiltration following injection into bovine skin, and 89% depletion of IL-8 from the extract reduced this neutrophil influx by 60%. These results demonstrate consistent upregulation of IL-8 expression in lesions of pneumonic pasteurellosis, implying a role for IL-8 in the ongoing recruitment of neutrophils to established lesions of pneumonic pasteurellosis. Because neutrophil-mediated tissue injury is critical to the pathogenesis of pneumonic pasteurellosis, these data suggest that neutralization of IL-8 activity could ameliorate the severe clinical signs and lesions of this disease.

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.

Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome.

Swine infectious agents, especially viruses, are potential public health risks associated with the use of pig organs for xenotransplantation in humans. Therefore, there is a need for better characterization of swine viruses and for the development of diagnostic tests for their detection. We report here isolation of a novel strain of porcine circovirus (PCV) from pigs with postweaning multisystemic wasting syndrome (PMWS). Affected pigs exhibited severe interstitial pneumonia and lymphoid depletion. The complete nucleotide sequence (1,768 nucleotides) of the genome of the PCV isolate was determined and compared with the sequence of the PCV strain isolated from PK-15 cells. Sequence comparison revealed significant differences between the two PCV strains, with an overall DNA homology of 76%. Two major open reading frames (ORFs) were identified. ORF1 was more conserved between the two strains, with 83% nucleotide homology and 86% amino acid homology. ORF2 was more variable, with nucleotide homology of 67% and amino acid homology of 65%. PCR and in situ hybridization demonstrated abundant viral DNA in various organs of pigs with PMWS. In situ hybridization demonstrated that this strain of PCV targets multiple organs and infects macrophages, lymphocytes, endothelial cells, and epithelial cells.