Early detection of Brucella canis via quantitative polymerase chain reaction analysis.

Canine brucellosis is a reportable zoonotic disease that can lead to canine reproductive losses and human infection through contact with infected urine or other genitourinary secretions. Although many locations require testing and euthanasia of positive dogs, current diagnosis is limited by the time required for seroconversion, for example, presence of B. canis-specific antibodies. The goal of this study was to determine the diagnostic ability of Brucella canis-specific quantitative polymerase chain reaction (qPCR) assay to detect B. canis in field samples prior to serological positivity for faster diagnosis and prevention of transmission within kennels or in households. Two kennels, one of which was located in the owner's home, were sampled following observation of suggestive clinical signs and positive serology of at least one dog. Specimens obtained were comparatively analysed via serology and qPCR analysis. 107 dogs were analysed for B. canis infection via qPCR: 105 via whole-blood samples, 65 via vaginal swab, six via urine and seven via genitourinary tract tissue taken at necropsy. Forty-five dogs were found to be infected with canine brucellosis via qPCR, of which 22 (48.89%) were seropositive. A statistically significant number (P = 0.0228) of qPCR-positive dogs, 5/25 (20.00%), seroconverted within a 30-day interval after initial serologic testing. As compared to serology, qPCR analysis of DNA from vaginal swabs had a sensitivity of 92.31% and specificity of 51.92%, and qPCR analysis of DNA from whole-blood samples had a sensitivity of 16.67% and specificity of 100%. B. canis outer membrane protein 25 DNA qPCR from non-invasive vaginal swab and urine samples provided early detection of B. canis infection in dogs prior to detection of antibodies. This assay provides a critical tool to decrease zoonotic spread of canine brucellosis, its associated clinical presentation(s), and emotional and economic repercussions.

SPUD qPCR assay confirms PREXCEL-Q softwares ability to avoid qPCR inhibition.

Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with about 15,500 SPUD amplicons yielded a Cq of 27.39 plus/minus 0.28 (at about 80.8% efficiency), while reactions spiked with about 7,750 SPUD plasmids yielded a Cq of 23.82 plus/minus 0.15 (at about 97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.

Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses.

Foals are particularly vulnerable to infection by Rhodococcus equi during the first 2 weeks of life whereas mature horses are not. While an innate immunodeficiency likely accounts for this clinically relevant vulnerability, the factors that contribute to infection by R. equi have not been fully elucidated. In this study, we demonstrate that cells of the monocyte lineage, including monocytes, macrophages, and dendritic cells, that have been activated with LPS and IFN-gamma, respond with a statistically significant, greater amount of cytokine mRNA production of IL-10, IL-12p35, and IL-12p40 than unstimulated control cells. Interestingly, activation of neonatal cells resulted in a twofold log increase in baseline cytokine mRNA expression of IL-10 compared with adult cells. In contrast, no significant differences in mean cytokine mRNA expression of IL-12p35 and IL-12p40 were detected, suggesting that the defect in chromosomal remodeling that prevents IL-12p35 gene transcription as a cause for decreased IL-12 synthesis in human neonates is not a likely occurrence in equine neonates. Collectively, these differences indicate that in vivo activation of equine cells of the monocyte lineage may result in different autocrine and paracrine cellular responses that vary according to age, with potential impact on regulation of adaptive and innate immune responses.

Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all-trans retinoic acid and VEGF.

Beta-defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all-trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP-A) and SP-D, and sheep beta-defensin-1 (SBD-1) were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cells had significantly increased expression of SP-D mRNA at 6 h and 24 h, decreased expression of SP-A mRNA at 12 h, and unchanged levels of SBD-1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP-A and SP-D have different transcription regulation pathways, and that expression of SBD-1 is not inducible by RA similar to its human homolog HBD-1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.

Monocytic/macrophagic pneumonitis after intrabronchial deposition of vascular endothelial growth factor in neonatal lambs.

Preterm and young neonates are prone to inadequate surfactant production and are susceptible to respiratory distress syndrome characterized by alveolar damage and hyaline-membrane formation. Glucocorticoid therapy is commonly used in preterm and young infants to enhance lung maturation and surfactant synthesis. Recently, vascular endothelial growth factor (VEGF) was suggested to be a novel therapeutic agent for lung maturation that lacked adverse effects in mice. The purpose of this study was to assess the safety of incremental concentration (0.0005, 0.005, and 0.05 mg/ml) and duration (16, 24, and 32 hours) of recombinant human VEGF after bronchoscopic instillation (10 ml) in neonatal lambs. High-dose VEGF caused locally extensive plum-red consolidation that was microscopically characterized by interstitial and alveolar infiltrates of cells that were morphologically and phenotypically (CD68+) consistent with monocytes/macrophages. T cells (CD3+) and B cells (CD79+) were located primarily in bronchus/bronchiole-associated lymphoid tissue and were not consistently altered by treatment with VEGF. The dose of VEGF had significant effects on both gross lesions (P < .0047) and microscopic monocyte/macrophage recruitment scores (P < .0001). Thus, the VEGF dose instilled into the lung greatly influenced cellular recruitment and lesion development. The post-dosing interval of VEGF in this study had minor impact (no statistical significance) on cellular recruitment. This study showed that airway deposition of VEGF in the neonatal lamb induces monocyte/macrophage recruitment to the lung and high doses can cause severe lesions. The cellular recruitment suggests further research is needed to define dosages that are efficacious in enhancing lung maturation while minimizing potential adverse effects.

Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus.

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.

Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus.

The objective of this study was to determine whether vaccination with bacterins commonly used in the USA, when administered at a time typical of US protocol, enhances porcine circovirus type 2 (PCV2) replication and the incidence and severity of clinical signs and lesions characteristic of postweaning multisystemic wasting syndrome (PMWS) in conventional pigs. Sixty-one pigs free of PCV2 were randomly assigned to four groups. Groups 1 (n = 15) and 2 (n = 15) pigs served as sham-inoculated negative controls. Groups 3 (n = 14) and 4 (n = 17) pigs were inoculated intralymphoid with PCV2 field isolate ISU-40895. Pigs in groups 2 and 4 were vaccinated with Actinobacillus pleuropneumoniae (APP) and Mycoplasma hyopneumoniae (M. hyopneumoniae) bacterins 21 days before and again 1 day before inoculation with PCV2. Mild transient respiratory disease and diarrhea were observed from 13 to 34 days postinoculation (DPI) in pigs in groups 3 and 4. Half the pigs from each group were necropsied at 22 and 34 DPI, respectively. Moderately enlarged, tan-colored lymph nodes were observed in the majority of pigs in groups 3 and 4. There was a significantly (P < 0.05) longer length of viremia (2.14 +/- 0.26 versus 4.44 +/- 0.23 weeks), a higher copy number of the PCV2 genome in serum, a wider range of tissue distribution of PCV2 antigen, and an increased severity of lymphoid depletion in pigs vaccinated with commercial APP and M. hyopneumoniae vaccines and inoculated with PCV2 compared with PCV2-inoculated unvaccinated pigs. Swine producers and veterinarians may need to consider changes in vaccination protocols in herds with recurrent PCV2-associated PMWS.

A selectin inhibitor decreases neutrophil infiltration during acute Mannheimia haemolytica pneumonia.

The degree to which the selectin inhibitor TBC1269 reduces neutrophil infiltration in specific microscopic locations of the lung during acute pneumonia of neonates was determined. Neonatal calves were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 2 and 6 hours postinoculation (PI). One 6-hour group inoculated with M. haemolytica received TBC1269 intravenously before and after inoculation with M. haemolytica. Infiltrates of neutrophils were significantly higher in the alveolar lumen and septae but lower in the bronchial lumen and epithelium at 6 hours PI than at 2 hours PI. Significantly fewer neutrophils (P < 0.05) were present in the alveolar lumen and septae, and the bronchiolar lumen and lamina propria in the lungs of TBC1269-treated calves compared with untreated calves at 6 hours PI. TBC1269 did not alter the infiltration into bronchi and blood vessels or the expression of the selectin-independent adhesion molecule, ICAM-1. This work suggests that during acute pneumonia of neonates 1) neutrophil infiltrates progressively increase in the alveolar lumens and septae but decrease in the bronchial lumen and epithelium with time, 2) TBC1269 reduces neutrophil infiltration into specific regions of alveoli and bronchioles rather than uniformly throughout the lung, and 3) selectin inhibition does not affect the location and intensity of ICAM-1 expression.

Cellular distribution of anionic antimicrobial peptide in normal lung and during acute pulmonary inflammation.

Anionic peptides (APs) are small antimicrobial peptides present in human and ovine lung. In this study APs were also detected in bovine lung, and production of APs in lungs with acute inflammation induced by various stimuli was determined. The distribution and intensity of APs were determined by immunohistochemistry in lungs of 1) neonatal calves (1-3 days of age) inoculated with Mannheimia (Pasteurella) haemolytica, a known inducer of the bovine beta-defensin lingual antimicrobial peptide (LAP) or pyrogen-free saline (PFS), and 2) growing calves (3 months of age) similarly inoculated with M. haemolytica, a lipopolysaccharide (LPS) from M. haemolytica, an LPS-associated protein from M. haemolytica, or PFS. APs were also detected by western blots with the same antibody in lungs of the calves above, as well as in calves inoculated with Pseudomonas aeruginosa, and an adult cow. Anionic peptide (AP) immunoreactivity was detected in bands (approximate weights) in the western blots of lung at 28-30 (strongest signal), 31, 45, and 52-60 kd regardless of inoculum. The adult cow lacked bands at 45 kd, but it had additional bands at 64 (inconsistently) and 35-38 kd. All these band sizes are consistent with those of the western blots of human and ovine lung. The cellular distribution of APs in lung of neonatal and growing cattle was similar to that in lung of human and sheep. In lungs with acute inflammation induced by live bacteria, LPS, or protein, AP distribution and intensity were similar to those in control (PFS-inoculated) lungs and slightly decreased in bronchioles. This work demonstrates that AP is present in lung of cattle and is thereby conserved among two ruminant species and man. Distribution and intensity of AP production are not enhanced by infection or acute inflammation and are decreased in bronchioles, which suggests that AP is not induced like beta-defensins such as LAP, but, instead, is produced constitutively.

T cell-dependent inducible nitric oxide synthase production and ultrastructural morphology in BALB/c mice infected with Mycobacterium avium subspecies paratuberculosis.

Euthymic BALB/c and athymic nude BALB/c mice aged 3-8 days were infected intraperitoneally with Mycobacterium avium subspecies paratuberculosis (ATCC strain 19698). After euthanasia at 5 months post-inoculation, hepatic granulomas were evaluated by morphometric analysis of digital images captured from light microscopy sections, by electron microscopy and by immunohistochemical methods. Euthymic mice differed from athymic mice in that (1) their hepatic granulomas were smaller, contained fewer bacteria, and produced more inducible nitric oxide synthase, and (2) their hepatic macrophages contained fewer bacteria, a higher percentage of degraded bacteria, and increased numbers of primary lysosomes. The study showed that macrophage activation was markedly less in the T cell-deficient athymic mice than in the euthymic mice.

Mast cell density and substance P-like immunoreactivity during the initiation and progression of lung lesions in ovine Mannheimia (Pasteurella) haemolytica pneumonia.

To determine the density of mast cells (MCs) and the extent of substance P (SP) immunoreactivity during initiation and progression of pneumonic pasteurellosis (PP), 18 lambs were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 1, 15 and 45 days post-inoculation (n=3, each group). Additionally, the left (non-inoculated) contralateral lungs in bacteria-inoculated animals were collected as controls. At 1 day after bacterial inoculation the lungs had typical M. haemolytica lesions. These pneumonic lesions had fewer numbers of MCs and reduced histamine content. Macrophages infiltrating some of the inflamed areas were strongly immunoreactive for SP. At 15 days, MCs remained scarce at sites where lung damage persisted, i.e. pyogranulomatous foci, but were increased in number in areas of interstitial damage. Pulmonary ganglion neurons were strongly immunoreactive for SP. By 45 days the fibrosing changes became more defined as pleural fibrosis, fibrosing alveolitis, alveolar epithelial hyperplasia and bronchiolitis obliterans. These lungs had increased numbers of MCs, but histamine content was not different from saline- and non-inoculated left lungs. Substance P immunoreactivity occurred only in nerves and was scarce and mild. This work demonstrates that MC density decreases initially with PP, but increases with progression of PP. SP fibres tend to be decreased during the initiation and at 45 days of PP, but other cells, such as macrophages and neuronal ganglion cells, produce substance P during progression of PP and thereby constitute an additional source of substance P.

Dihydrocapsaicin treatment depletes peptidergic nerve fibers of substance P and alters mast cell density in the respiratory tract of neonatal sheep.

In the present study we administered dihydrocapsaicin (DHC) to neonatal lambs to deplete C-fibers of neuropeptides. We measured the density of substance P (SP)-fibers in nasal septum to assess the effectiveness of the treatment at 3, 9, and 21 days. The numbers of mast cells in the upper and lower respiratory tract were determined at the same time points and histamine content was determined from lung tissue. DHC treatment depleted SP-fibers for up to the 21 day time point. This depletion was estimated as 85% in comparison with controls. In vehicle-treated lambs, the density of SP-fibers decreased progressively with age, but not to the degree of DHC-treated lambs whose SP-fibers were depleted from the initial 3-day measurement. In both, vehicle- and DHC-treated lambs, numbers of mast cells increased progressively with time; however, the density of mast cells was augmented in the entire respiratory tract of DHC-treated animals. Apparently, DHC treatment exerts a single and initial effect in increasing mast cells whereas time maintains a continuous influence; both factors exert their influence independently. Despite large numbers of mast cells in DHC-treated animals, histamine content in the lung had similar levels as controls. Our study provides fundamental data for a better understanding of conditions that may influence defense mechanisms dependent on the mast cell-nerve axis in the respiratory tract.

Reduction of pulmonary mast cells in areas of acute inflammation in calves with Mannheimia (Pasteurella) haemolytica pneumonia.

Mast cells in the left cranial pulmonary lobe of colostrum-deprived neonatal calves were quantified 2 and 6 h after intrabronchial inoculation with Mannheimia (Pasteurella) haemolytica A1. The mast cells were detected (1) immunohistochemically with a mouse anti-human mast cell tryptase monoclonal antibody, and (2) by metachromatic staining with low pH toluidine blue. A greater number of mast cells was demonstrated by the second method than by the first. At 6 h after inoculation, but not at 2 h, the number of mast cells was significantly reduced at the site of the main lesions. Treatment of calves with a sialyl Lewis mimetic (TBC1269) did not appreciably affect the results at 6 h.

Influence of beta(2)-integrin adhesion molecule expression and pulmonary infection with Pasteurella haemolytica on cytokine gene expression in cattle.

beta(2)-Integrins are leukocyte adhesion molecules composed of alpha (CD11a, -b, -c, or -d) and beta (CD18) subunit heterodimers. Genetic CD18 deficiency results in impaired neutrophil egress into tissues that varies between conducting airways and alveoli of the lung. In this study, we investigated whether CD18 deficiency in cattle affects proinflammatory cytokine (PIC) expression in pulmonary tissue after respiratory infection with Pasteurella haemolytica. Cattle were infected with P. haemolytica via fiberoptic deposition of organisms into the posterior part of the right cranial lung lobe. Animals were euthanized at 2 or 4 h postinoculation (p.i.), and tissues were collected to assess PIC gene expression using antisense RNA probes specific for bovine interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) along with the beta-actin (beta-Act) housekeeping gene. Expression of PIC was induced at 2 h p.i. in P. haemolytica-infected cattle and continued to 4 h p.i. At 2 h p.i., induction of gene expression and increase of cells that expressed PIC were observed both in CD18(+) and CD18(-) cattle after inoculation of P. haemolytica. The induction of gene expression with P. haemolytica inoculation was more prominent in CD18(-) cattle than in CD18(+) cattle by comparison to pyrogen-free saline (PFS)-inoculated control animals. At 4 h p.i., however, the induction of PIC, especially IL-1alpha, IL-6, and IFN-gamma, in the lungs of CD18(+) cattle inoculated with P. haemolytica was greater than that in lungs of the CD18(-) cattle. IFN-gamma and TNF-alpha genes were not increased in P. haemolytica-inoculated CD18(-) cattle lungs compared to the PFS-inoculated control lungs at 4 h p.i. In PFS-inoculated lungs, we generally observed a higher percentage of cells and higher level of gene expression in the lungs of CD18(-) cattle than in the lungs of CD18(+) cattle, especially at 4 h p.i. The rate of neutrophil infiltration into the lungs of CD18(-) cattle at 2 h p.i. was significantly higher than that of CD18(+) cattle; at 4 h p.i., there was no difference between the two groups. These data suggest that beta(2)-integrins may contribute to the induction of expression of some PIC genes, as a consequence of P. haemolytica infection.

In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non-BLAD calves.

Effects of retinoid beta-glucuronides and N-retinoyl amines on the differentiation of HL-60 cells in vitro.

Retinoyl beta-glucuronide and retinyl beta-glucuronide, which are naturally occurring water-soluble metabolites of vitamin A, induce the granulocytic differentiation of HL-60 cells in vitro, as evidenced by an increased reduction of nitroblue tetrazolium. The relative effectiveness of various retinoids in differentiation is retinoic acid greater than retinoyl beta-glucuronide greater than retinyl beta-glucuronide. Under the selected assay conditions, retinol, hydroxyphenyl-retinamide, retinamide, and N-retinoyl-phenylalanine are essentially inactive in differentiation. At concentrations of retinoids from 10(-9) to 10(-5) M, cell viability was best with the retinoid beta-glucuronides and retinamide, less with retinoic acid and retinol, and poorest with the N-retinoyl aromatic amines. Cellular growth was depressed only slightly by retinyl beta-glucuronide and retinamide, but to a greater degree by the other derivatives. Retinoyl beta-glucuronide was hydrolyzed in part to retinoic acid, whereas retinyl beta-glucuronide was cleaved to retinol, if at all, at a very slow rate. Under the selected assay conditions, retinoic acid and the retinoid beta-glucuronides primarily induce the differentiation of HL-60 cells, whereas the N-retinoyl aromatic amines show cytotoxicity.