Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages

Viable Haemophilus pleuropneumoniae bacteria were toxic for porcine alveolar macrophages in vitro. This cytotoxic effect proved to be dose-related. A cell-free extract of H. pleuropneumoniae, heat-killed bacteria, and a Pasteurella multocida field strain were nontoxic. When macrophages were cultured with H. pleuropneumoniae bacteria in a ratio of 100 macrophages to six bacteria, ultrastructural signs of cellular degeneration were observed within 1 h. This degeneration was observed in macrophages with or without phagosomes containing H. pleuropneumoniae. A cytotoxic substance was filtered from a H. pleuropneumoniae culture in Eagle's minimal essential medium supplemented with Earle's salts (EMEM) and 10% foetal calf serum that was incubated for 10 h at 37 degrees C. This substance was destroyed by heating at 65 degrees C for 30 min. Macrophages were less susceptible to the toxic effect of H. pleuropneumoniae when serum of convalescent pigs was added.     

Activated porcine alveolar macrophages: are biological response modifiers the answer?

We report that 75% of conventionally housed 3- to 4-week-old swine already have detectable activated alveolar macrophages as measured by cytotoxicity of tumor cells. These macrophages can not be further activated by the biological response modifier N-acetylmuramyl-L-alanyl-Disoglutamine-2H2O (MDP). These macrophages lose cytotoxic activity when cultured for 24 h and this loss of activity can not be reversed by MDP. We suggest that MDP biological response modifier therapy of swine alveolar macrophages may not be a useful technique in preventing respiratory disease in swine.     

Effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, Bordetella bronchiseptica, or a combination of both organisms in pigs

OBJECTIVE: To examine effects of co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and Bordetella bronchiseptica in pigs. ANIMALS: Forty 3-week-old pigs. Procedure-30 pigs (10 pigs/group) were inoculated with PRRSV, B bronchiseptica, or both. Ten noninoculated pigs were control animals. RESULTS: Clinical signs, febrile response, and decreased weight gain were most severe in the group inoculated with both organisms. The PRRSV was isolated from all pigs in both groups inoculated with virus. All pigs in both groups that received PRRSV had gross and microscopic lesions consistent with interstitial pneumonia. Bordetella bronchiseptica was cultured from all pigs in both groups inoculated with that bacterium. Colonization of anatomic sites by B bronchiseptica was comparable between both groups. Pigs in the group that received only B bronchiseptica lacked gross or microscopic lung lesions, and B bronchiseptica was not isolated from lung tissue. In the group inoculated with B bronchiseptica and PRRSV, 3 of 5 pigs 10 days after inoculation and 5 of 5 pigs 21 days after inoculation had gross and microscopic lesions consistent with bacterial bronchopneumonia, and B bronchiseptica was isolated from the lungs of 7 of those 10 pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical disease was exacerbated in co-infected pigs, including an increased febrile response, decreased weight gain, and B bronchiseptica-induced pneumonia. Bordetella bronchiseptica and PRRSV may circulate in a herd and cause subclinical infections. Therefore, co-infection with these organisms may cause clinical respiratory tract disease and leave pigs more susceptible to subsequent infection with opportunistic bacteria.     

Organic barn dust extract exposure impairs porcine macrophage function in vitro: Implications for respiratory health

Respiratory diseases are responsible for a significant amount of animal morbidity and mortality in the swine industry, including the majority of nursery and grower/finisher deaths. Innate immunity, including the maintenance of lung macrophage health and function, is an important defense mechanism against respiratory pathogens and their associated losses. Chronic exposure of swine industry workers to airborne barn dust results in significant predisposition to airway diseases and impairment of alveolar macrophage (AM?) function. Because of their importance in maintaining normal respiratory function, this study was designed to evaluate the impact of barn dust on swine macrophages. As measures of macrophage function, we evaluated the activation of NF-κB, cytokine production, cell surface marker expression and the phagocytic and antibacterial capabilities of porcine macrophages after in vitro exposure to an organic swine barn dust extract (ODE). ODE treatment induced AM? secretion of both pro- and anti-inflammatory cytokines, suggesting a complex activation profile. Additionally, ODE induced expression of genes (TLR2, NOD2) involved in sensing Gram-positive bacteria, a major component of barn dust. ODE exposure also enhanced the expression of several cell surface markers of activation, including a receptor for the porcine reproductive and respiratory syndrome virus. Moreover, two key functions of AM?, phagocytosis and bacterial killing, were impaired after exposure to ODE. Treatment with ODE for the first 72 h of differentiation also inhibited the ability of monocyte-derived macrophages to translocate NF-κB to the nucleus following endotoxin stimulation. Taken together, these results demonstrate, for the first time, that organic dust extract exposure negatively affects pig macrophage activation and function, potentially enhancing host susceptibility to a variety of respiratory infections.     

Adherence of Bordetella bronchiseptica and Pasteurella multocida to porcine nasal and tracheal epithelial cells.

The ability of 19 different Bordetella bronchiseptica isolates and 25 Pasteurella multocida isolates to adhere in vitro to porcine nasal and tracheal epithelial cells was examined. It was found that B. bronchiseptica adhered well to upper respiratory tract cells. In contrast the number of P. multocida organisms which adhered was four to six times less than the number of B. bronchiseptica adherent organisms. This difference was statistically significant (p less than 0.0001). Both microorganisms adhered in greater numbers to nasal cells than to tracheal cells (p less than 0.005). The data indicated that B. bronchiseptica possesses a greater ability than P. multocida to attach to porcine upper respiratory tract cells.     

Effects of intranasal inoculation with Bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with Pasteurella multocida in pigs

OBJECTIVE: To determine effects of intranasal inoculation with porcine reproductive and respiratory syndrome virus (PRRSV) or Bordetella bronchiseptica on challenge with nontoxigenic Pasteurella multocida in pigs. ANIMALS: Seventy 3-week-old pigs. PROCEDURE: In experiment 1, pigs were not inoculated (n= 10) or were inoculated with PRRSV (10), P. multocida (10), or PRRSV followed by challenge with P. multocida (10). In experiment 2, pigs were not inoculated (n = 10) or were inoculated with B. bronchiseptica (10) or PRRSV and B. bronchiseptica (10); all pigs were challenged with P. multocida. Five pigs from each group were necropsied 14 and 21 days after initial inoculations. RESULTS: Pasteurella multocida was not isolated from tissue specimens of pigs challenged with P. multocida alone or after inoculation with PRRSV. However, in pigs challenged after inoculation with B. bronchiseptica, P. multocida was isolated from specimens of the nasal cavity and tonsil of the soft palate. Number of bacteria isolated increased in pigs challenged after coinoculation with PRRSV and B. bronchiseptica, and all 3 agents were isolated from pneumonic lesions in these pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Infection of pigs with B. bronchiseptica but not PRRSV prior to challenge with P. multocida resulted in colonization of the upper respiratory tract and tonsil of the soft palate with P. multocida. Coinfection with PRRSV and B. bronchiseptica predisposed pigs to infection of the upper respiratory tract and lung with P. multocida. Porcine reproductive and respiratory syndrome virus and B. bronchiseptica may interact to adversely affect respiratory tract defense mechanisms, leaving pigs especially vulnerable to infection with secondary agents such as P. multocida.     

Detection of respiratory pathogens in air samples from acutely infected pigs

Pathogens causing significant respiratory disease in growing pigs include Porcine reproductive and respiratory syndrome virus, Porcine circovirus 2, swine influenza virus, porcine respiratory coronavirus, Mycoplasma hyopneumoniae, and Bordetella bronchiseptica. The objective of this research was to characterize the respiratory excretion of these pathogens by acutely infected pigs. Pigs were inoculated under experimental conditions with 1 pathogen. Samples were collected from the upper respiratory tract and exhaled air. All pathogens were detected in swabs of the upper respiratory tract, but only M. hyopneumoniae and B. bronchiseptica were detected in expired air from individually sampled, acutely infected pigs. These findings suggest either that the acutely infected pigs did not aerosolize the viruses or that the quantity of virus excreted was below the detection threshold of current sampling or assay systems, or both, at the individual-pig level.     

Experimental respiratory disease in dogs due to Bordetella bronchiseptica.

Young dogs of two age groups, six weeks and 12 weeks respectively, were infected by aerosol with a strain of Bordetella bronchiseptica which had been isolated from a dog with pneumonia. Clinical respiratory disease characterised by coughing and in some cases purulent nasal discharge was induced in both groups of infected dogs and also in dogs kept in contact. B bronchiseptica was recovered from the nasal cavity, trachea, bronchi and lung parenchyma of infected and contact animals. At necropsy, masses of Gram-negative bacteria were found trapped in the cilia of the respiratory epithelia and there was an exudate containing neutrophils in the mucosae of the respiratory tract at all levels. A close similarity was noted between the lesions produced in the dog and those described in pertussis infection in man. Experimental respiratory disease in the dog due to B bronchiseptica may offer a model system for the study of the human disease.     

Intracellular accumulation,subcellular distribution and efflux of tilmicosin in swine phagocytes

Tilmicosin is a semi-synthetic macrolide antibiotic, currently approved for veterinary use in cattle and swine respiratory disease. As the concentrations of tilmicosin are generally low in swine lung tissue, the interaction of tilmicosin with three types of swine phagocytes (monocyte-macrophages, alveolar macrophages, and neutrophils) was evaluated to provide an understanding of clinical efficacy. After incubation with radiolabelled tilmicosin, uptake was determined and expressed as the ratio of the intracellular (Ci) to the extracellular (Ce) drug concentration (Ci/Ce). Tilmicosin was avidly accumulated by the swine phagocytes (Ci/Ce 48–69 at 4 h incubation) with 51 to 85% localized in the lysosomes. Uptake was dependent on cell viability, temperature and pH, but was not influenced by the metabolic inhibitors, sodium cyanide or potassium fluoride. However, lipopolysaccharide (LPS) exposure increased tilmicosin uptake by the swine phagocytes. In neutrophils, upon removal of extracellular tilmicosin, 60% of the intracellular tilmicosin was effluxed within the first 30 min, but after 4 h of incubation in drug-free medium, 25% remained cell-associated. In contrast, after 4 h of incubation in drug-free medium, 60% and 45% of tilmicosin remained cell-associated, within alveolar macrophages and monocyte-derived macrophages, respectively. Tilmicosin uptake was observed to increase lysosomal enzyme (acid phosphatase, lysozyme and β-glucuronidase) production. Finally, neutrophils were shown to transport and efflux bioactive tilmicosin in a test system measuring both neutrophil chemotaxis under agarose and a bioassay measuring inhibition of bacterial growth in the presence of antibiotic in agar. These in vitro interactions of tilmicosin with swine phagocytes suggest an integral role in effecting clinical efficacy.     

Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung

The potential synergistic effect of bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica in the production of pneumonia after aerosol/intranasal infection of conventionally reared lambs was evaluated. A mild clinical response was observed in lambs given virus and/or bacteria. Gross pulmonary lesions were seen in 3 of 6 lambs given RSV and then P haemolytica 3 or 6 days later, respectively (groups D and E), and in 1 lamb of 5 given virus and bacteria simultaneously (group G). Gross lesions were not seen in control sheep (group A), in lambs given virus or bacteria alone (groups B and C), or in lambs exposed to bacteria and then virus 3 days later (group F). Bovine RSV and P haemolytica were recovered from the lungs of 5 of 7 lambs with macroscopic lesions. Gross pulmonary lesions were cranioventral firm areas of red consolidation. Microscopically, the predominant lesion was a suppurative bronchopneumonia. Bovine RSV was recovered from the nasal cavity of 8 of 27 (30%) lambs given RSV during days 3 to 6 after viral inoculation, including 1 lamb in group B, 2 in groups D, E, and F, and 1 in group G. Pasteurella haemolytica was recovered from the nasal cavity of 9 of 28 (32%) inoculated lambs, including 2 lambs from groups C and E, 3 in group D, and 1 in groups F and G. Viral antigen, as determined by immunofluorescence, was concentrated mainly in individual cells in alveolar walls, some alveolar macrophages, and a few bronchiolar epithelial cells. In vitro alveolar macrophage assays indicated decreased numbers of Fc receptors on those macrophages collected from lambs given RSV 6 days before P haemolytica infection, as compared with that in the other groups. These cellular defects disappeared after 24 hours of culture. Seemingly, bovine RSV does facilitate P haemolytica pulmonary infection in conventional, immuno-competent lambs and provides evidence for decreased Fc receptors on alveolar macrophages.     

In vitro phagocytosis and killing of Corynebacterium equi by alveolar macrophages of foals

Bronchoalveolar lavage was performed 5 times, sequentially, on 3 healthy foals while each foal was 6 to 63 days of age. Phagocytosis and bactericidal assays were performed on recovered alveolar macrophages. Corynebacterium equi and alveolar macrophages at a ratio of 10:1 were incubated for 1 hour in medium containing 1% heat-inactivated rabbit anti-C equi serum. After incubation, greater than 90% of the alveolar macrophages contained at least 1 ingested bacterium and each alveolar macrophage contained 9.4 +/- 1.0 bacteria (mean +/- SE). After alveolar macrophages and C equi were incubated for 1 hour in medium containing heat-inactivated pooled normal horse serum, approximately 24% of the alveolar macrophages contained at least 1 bacterium and each alveolar macrophage contained 0.8 +/- 0.7 bacteria. From 6 to 61 days of age, each foal had significantly (P less than 0.05) decreased phagocytic activity by alveolar macrophages, but a significant change in killing of C equi by alveolar macrophages was not found in the foals from 21 to 61 days of age. After incubating alveolar macrophages and C equi for 4 hours in vitro, approximately 75% of ingested C equi remained viable.     

Envision免疫组化技术在诊断猪繁殖与呼吸综合征中的应用

为探讨免疫组织化学Envision法在诊断猪繁殖与呼吸综合征(PRRS)中应用的可行性,采用免疫组织化学Envision二步法对30例疑似PRRS病猪肺组织的病毒抗原进行定位、半定量检测。结果表明,Envision法可原位检测病猪肺组织PRRSV抗原的分布;其抗原的阳性表达主要出现在肺巨噬细胞胞浆内,其次为肺泡上皮细胞和细支气管黏膜上皮细胞。依据阳性细胞百分率,进行染色结果判断,检出阳性病猪23例,阳性检出率为76.7%。试验证明,Envision法具有高敏感、低背景、快速简便的特点,可在PRRS诊断中推广应用。     

Serum haptoglobin concentration in growing swine after intranasal challenge with Bordetella bronchiseptica and toxigenic Pasteurella multocida type D.

The acute phase reaction, in association with progressive atrophic rhinitis (AR), was monitored for 3 wk using serum haptoglobin (HPT) quantification in thirty-six, 15 kg swine after intranasal challenge with varying doses of Pasteurella multocida type D (toxigenic strain) and Bordetella bronchiseptica. The challenge doses were administered alone or in combination with pigs divided into 9 isolated treatment groups. Increasing doses of B. bronchiseptica were associated with lower serum HPT (P < 0.05), whereas increasing doses of P. multocida tended to increase serum HPT (0.05 < P < 0.10). Significant and positive correlation of mean HPT and AR score was found in these pigs; increased AR scores were associated with elevated mean HPT concentration (r = 0.41, P < 0.01). A significant interaction between P. multocida and B. bronchiseptica dose indicated that increasing the dose of B. bronchiseptica, for a fixed P. multocida dose, was associated with less AR (P < 0.05). The AR scores were greater in pigs given P. multocida, than B. bronchiseptica alone. These results indicate that a complex interaction between Pasteurella multocida and Bordetella bronchiseptica causes progressive atrophic rhinitis and alters serum HPT concentration in swine.     

Bovine leukocyte phagocytosis and bacteria killing monitored by intracellular acridine orange fluorescence and extracellular fluorescence quenching

The time course of phagocytosis and intracellular killing of serum-opsonized Escherichia coli K12 and Staphylococcus aureus SG511 by glass-adherent bovine peripheral blood polymorphonuclear leukocytes (PMNLs) and cultured monocytes (macrophages) was monitored by fluorescence microscopy of single cells using the acridine orange (AO)/crystal violet (CV) technique. After interaction of glass-adherent leukocytes (20, 40, 60 min, 37 degrees C) with opsonized bacteria, cells were stained with the fluorescent dye AO. Living bacteria stained green, dead bacteria stained orange. The addition of CV to AO-stained bacteria quenched the fluorescence of extracellular bacteria only. CV does not penetrate living bovine PMNLs which allows the discrimination of ingested (fluorescent) and extracellular (nonfluorescent) bacteria during attachment and phagocytosis of bacteria by adherent PMNLs. We investigated quantitatively phagocytosis and intracellular killing of serum-opsonized bacteria by bovine PMNLs from 22 bulls of 4 different Swiss dairy breeds. Within 60 min maximum uptake (approximately 12 bacteria/PMNL) and killing (approximately 80%) of serum-opsonized Escherichia coli K12 and Staphylococcus aureus SG511 were achieved. The AO/CV technique was also used to quantify the uptake and intracellular killing of serum-opsonized Escherichia coli K12 by cultured monocytes (macrophages). Within 60 min maximum uptake of bacteria (approximately 16/MO) was achieved; approximately 83% of bacteria were killed.     

Pasteurella haemolytica A1 and Bovine Respiratory Disease: Pathogenesis

The severe fibrinonecrotic pneumonia associated with pneumonic pasteurellosis usually results from colonization of the lower respiratory tract by Pasteurella haemolytica biotype A, serotype 1(A1). Despite recent research efforts, the authors lack a detailed understanding of the interactions and host response to P. haemolytica in the respiratory tract. The authors hypothesize that management and environmental stress factors or viral infection alters the upper respiratory tract (URT) epithelium allowing P. haemolytica to colonize the epithelium. Once the URT is colonized, large numbers of organisms enter the lung where they interact with alveolar macrophages. Endotoxin, released from the bacteria, crosses the alveolar wall where it activates pulmonary intravascular macrophages, endothelium, neutrophils, lymphocytes, platelets, complement, and Hageman factor leading to complex interactions of cells and mediators. It is the progression of this inflammatory response with neutrophil influx that is ultimately responsible for the pulmonary injury. Leukotoxin is a major virulence factor of P. haemolytica that allows it to survive by destroying phagocytic cells. At subcytolytic concentrations it may also enhance the inflammatory response by activating cells to produce mediators and release reactive oxygen metabolites and proteases.     

Enhanced adherence of Pasteurella multocida to porcine tracheal rings preinfected with Bordetella bronchiseptica.

Adherence of 25 isolates of Pasteurella multocida to porcine tracheal rings was evaluated. Results indicated that adherence was not related to the isolate's origin, capsular or somatic types, dermonecrotoxin production or hemagglutination activity. The effect of a preinfection with Bordetella bronchiseptica on the colonization by P. multocida was then studied. On rings infected with P. multocida alone, bacteria initially adhered to the epithelium, but within a few hours, the level of colonization decreased progressively. On rings preinfected with B. bronchiseptica, or pretreated with a cell-free B. bronchiseptica culture supernate (or filtrate), a high level of P. multocida colonization was maintained for at least 24 hours. Results indicate that B. bronchiseptica appears to facilitate upper respiratory tract colonization by P. multocida by a process which involves a low molecular weight (less than or equal to 1000) heat-stable substance, possibly the tracheal cytotoxin.     

Expression of a truncated Pasteurella multocida toxin antigen in Bordetella bronchiseptica

Mild or subclinical respiratory infections caused by Bordetella bronchiseptica are widespread in pigs despite multiple control efforts. Infection with virulent B. bronchiseptica strains is a common risk factor in the establishment of toxin-producing strains of Pasteurella multocida in the nasal cavity of pigs leading to the disease, atrophic rhinitis (AR). This study was designed to explore the possibility of expressing a protective epitope of P. multocida toxin (PMT) in B. bronchiseptica to create single-component mucosal vaccine to control atrophic rhinitis in pigs. To achieve this, a P. multocida toxin fragment (PMTCE), that was non-toxic and protective against lethal challenge in mice, was cloned into a broad-host-range plasmid, PBBR1MCS2, and introduced into B. bronchiseptica by electroporation. The Pasteurella gene construct was placed under the regulatory control of a promoter region that was separately isolated from B. bronchiseptica and appears to be part of the heat shock protein gene family. B. bronchiseptica harboring the plasmid under antibiotic selection expressed the 80kDa PMTCE as determined by PAGE and Western blot with a PMT-specific monoclonal antibody. When introduced into the respiratory tracts of mice, B. bronchiseptica harboring the plasmid construct was reisolated in declining numbers for 72h post-inoculation. Antibody responses (IgM, IgA and IgG) to B. bronchiseptica were detected in serum and respiratory lavage, but PMTCE-specific antibodies were not detected. While further refinements of PMT expression in B. bronchiseptica are necessary, this study provides a basis for the development of a single-component, live-attenuated vaccine against atrophic rhinitis.     

The tenacity of Bordetella bronchiseptica in the air]

The survival time of Bordetella bronchiseptica (phase I and III) has been determined at a temperature of 21 degrees C and 27 degrees C and at a relative humidity of 32%, 58% and 76% in a rotating aerosol chamber. The highest survival time (118.8 min half-life time) has been found at 21 degrees C and 76% relative humidity in B. bronchiseptica (phase I). An increasing temperature (from 21 degrees C to 27 degrees C) causes a lower survival time. The phase I strains react more sensitive to this changing of environment than phase III strains. An increase of relative humidity leads to a higher survival time concerning phase I strains. In contrast phase III strains of B. bronchiseptica react with an increasing survival time. On the basis of the detected half-life time one must suppose an aerogenous distribution of the germ in all stages of animals, especially in intensive husbandry.