Effects of ketoprofen and flunixin in pigs experimentally infected with Actinobacillus pleuropneumoniae

The antipyretic effect of the non-steroidal anti-inflammatory drugs (NSAIDs) ketoprofen (3 mg/kg) and flunixin (2 mg/kg) were studied in pigs. The drugs were administered intramuscularly at 8 and 32 h following endobronchial challenge with Actinobucillus pleuropneumoniae. Infected (non-medicated) and non-infected (non-medicated) controls were used. Endobronchial challenge with Actinobucillus pleuropneumoniae induced laboured breathing, coughing, fever, reduced food and water consumption and increased white blood cell counts. At autopsy, pleuropneumonia was evident. Ketoprofen showed a highly significant antipyretic effect but flunixin did not. The decrease in food consumption of ketoprofen-treated pigs was significantly less than that of the infected (non-medicated) controls. Blood parameters were not significantly influenced by either NSAID and, at necropsy, gastric and renal side-effects were not observed for either drug.     

Effect of oral enrofloxacin and florfenicol on pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 1

This study was carried out to compare the efficacy of two oral anti-microbials as metaphylactic medication to pigs inoculated with Actinobacillus pleuropneumoniae serotype 1. Forty-two pigs with an average weight of 22.64 kg were randomly assigned to three treatment groups: group F was given doses of 40 ppm of florfenicol, group E received 150 ppm of enrofloxacin and group C received no medication. Groups F and E received medicated feed 12 h before being inoculated and for 7 days after inoculation. All the pigs were inoculated by aerosol, with 2 x 10(7) CFU/ml of A. pleuropneumoniae serotype 1 each. The average body temperature was higher in group C than in groups E and F, between 12 and 96 h after inoculation (P < 0.05). No differences were found between groups F and E in respiration pattern, nasal secretion and general condition (P > 0.05): however, differences were found in group C for respiration pattern and general condition (P < 0.05), 12 h after inoculation. There was no mortality in groups F and E, whereas a 50% mortality was recorded in group C during the first 48 h after inoculation (P < 0.05). Necropsies and bacterial cultures were performed 12 days after inoculation. Lesions were observed in five pigs of group F (35.71%) with an average damage of 1.16%; in four pigs of group E (28.57%) with 1.24%; and in 13 animals in group C (92.85%) with 34.5% of affected lung tissue (P < 0.05). The infective agent was cultured from various organs of animals in groups F and C, but not from those in group E.     

Effect of giving enrofloxacin in the diet to pigs experimentally infected with Actinobacillus pleuropneumoniae

Three groups of 16 pigs were exposed individually when four weeks old to intranasal infection with 10(8.9) viable Actinobacillus pleuropneumoniae (serovar 3, strain 2/(10P2); a fourth group was kept in isolation from the others as uninfected controls. Seven days later the 62 surviving animals were killed and necropsied. The organism had caused typical, mainly subacute disease in 12 of the 16 unmedicated animals but in only two of the 16 which had had continuous access to a diet containing 150ppm of enrofloxacin from four hours before exposure to infection, and in six of the 16 given 32 ppm enrofloxacin. However, only 150 ppm enrofloxacin produced marked control of the infection in terms of reduced average severity of thoracic lesions and much reduced prevalence of the organism in the lung at necropsy, and the mean weight gain (1.55 kg) and feed conversion efficiency (2.08) of this infected group over seven days were similar to those of the unmedicated, uninfected controls (1.67 kg and 2.25). The infected but untreated group on average produced detectable antibody seven days after infection whereas in the infected and medicated groups a specific response against serovar 3 was absent.     

Evaluation of Actinobacillus pleuropneumoniae diagnostic tests using samples derived from experimentally infected pigs

New serological tests have recently been introduced for Actinobacillus pleuropneumoniae diagnosis. No information is currently available on how these tests compare regarding the detection of antibodies from subclinically infected pigs. To answer this question, 80 pigs were randomly assigned to experimental groups infected with A. pleuropneumoniae serovars 1, 3, 5, 7, 10, 12, 15 and a non-inoculated control group. Blood samples and oropharyngeal swabs were collected prior to infection and for 7 consecutive weeks thereafter. Serum samples were tested using the Swinecheck(?) APP ELISA and the Multi-APP ELISA (University of Montreal). All pigs were euthanized at 49 days post-inoculation. Tonsil and lung samples were cultured for isolation and tested by PCR. The Multi-APP ELISA detected seroconversion 1 week earlier than the Swinecheck(?) APP ELISA with the earliest seroconversion detected at 1 week post-infection (serovar 10) and the latest at 3 weeks post-infection (serovar 1). Seroconversion at day 49 was serovar-dependent and varied from 4 (44%) positives detected in the serovar 10 group to 9 positives (100%) detected in the serovar 15 group. Thirty-one pigs were serologically positive for A. pleuropneumoniae at 49 days post-infection and only 15 still carried A. pleuropneumoniae on their tonsils based on PCR results. No cross-reactions were observed when serum samples were cross-tested using the Swinecheck(?) APP ELISA. A. pleuropneumoniae was successfully isolated from the lung of 2 pigs that developed pleuropneumonia, but was not isolated from tonsils due to heavy contamination by the resident flora. This study offers a comprehensive evaluation of the diagnostic tools currently available for detection of A. pleuropneumoniae subclinical infection.     

Serum haptoglobin concentration in swine naturally or experimentally infected with Actinobacillus pleuropneumoniae.

Serum haptoglobin (Hp) concentrations were measured in swine that were naturally or experimentally infected with Actinobacillus pleuropneumoniae. In swine from a specific-pathogen-free herd, mean serum concentration of Hp (+/- SD) was 5.79 +/- 1.06 mg of cyanmethemoglobin-binding capacity (CHBC)/dl. Serum Hp concentrations in paired samples were measured at 7-day intervals in 40 swine randomly selected from a conventional herd that was experiencing an acute episode of pneumonia and deaths caused by A pleuropneumoniae serotype-5 infection. Day-0 and -7 serum Hp concentrations were 24.58 +/- 1.38 and 23.10 +/- 1.12 mg of CHBC/dl, respectively, with no significant difference between these measurements. In a second conventional herd with a history of chronic infection with A pleuropneumoniae serotype 5, serum concentrations of Hp measured in paired samples obtained 6 days apart were 12.36 +/- 0.81 and 18.63 +/- 0.76 mg of CHBC/dl, respectively, and were significantly (P < 0.05) different from each other. Twenty-nine 12-week-old conventional swine were challenged intranasally with A pleuropneumoniae serotype 1 (n = 19) and serotype 5 (n = 10). Serum Hp concentration increased from prechallenge concentrations of 7.49 +/- 1.38 and 15.10 +/- 1.22 mg of CHBC/dl, respectively, to 41.01 +/- 1.35 and 22.37 +/- 1.78 mg of CHBC/dl, respectively, 72 hours after challenge. For these 29 swine, serum Hp concentration was positively correlated with rectal temperature (r = 0.34; P < 0.001) during the immediate postchallenge period.     

Treatment of pigs experimentally infected with Mycoplasma hyopneumoniae, Pasteurella multocida, and Actinobacillus pleuropneumoniae with various antibiotics.

The authors have performed a comparative study of the efficacy of various in-feed medications for the treatment of 5- to 6-week-old specific pathogen-free (SPF) piglets experimentally infected on day 1 with Mycoplasma hyopneumoniae, on day 8 with Pasteurella multocida (serotype A), and on day 15 with Actinobacillus pleuropneumoniae (serotype 2). The treatment started on day 9 and continued for 12 consecutive days, then the piglets were euthanized for examination of macroscopic, histologic, and pathologic lesions and for the presence of mycoplasmas and bacteria in the lungs. Based on the results of clinical observations (respiratory signs, rectal temperature, body weight gain, and feed conversion efficiency), macroscopic and histologic lesions of the lungs, and microbiologic findings, the best results were obtained by treatment of pigs with Econor + chlortetracycline, followed by Tetramutin, Pulmotil, Cyfac, and lincomycin + chlortetracycline.     

Porcine pleuropneumonia in Australian pigs due to Actinobacillus pleuropneumoniae serovar 5

Book reviewed in this article: Guide to the Dissection of Domestic Ruminants . RE Habel Biology and Medicine of Rabbits and Rodents . JE Harkness and JF Wagner     

Interleukin 6, serum amyloid A and haptoglobin as markers of treatment efficacy in pigs experimentally infected with Actinobacillus pleuropneumoniae

The possibility to use acute phase proteins to monitor the elimination of a bacterial infection in pigs would facilitate an objective assessment of treatment with various antimicrobial substances. To examine this possibility, the acute phase response (IL-6, serum amyloid A (SAA), and haptoglobin) elicited by Actinobacillus pleuropneumoniae and its reduction on treatment with various antibiotics was studied in serum from specific pathogen free (SPF) pigs. Pigs were infected intranasally with A. pleuropneumoniae serotype 2, and either left as non-treated control pigs or treated with different antibiotics intramuscularly at onset of respiratory disease (20h post-infection). Pigs responded to the infection with prominent increases in activity and concentrations of IL-6, SAA, and haptoglobin. These responses were to a certain extent overlapping and covered the time span from a few hours after infection until development of detectable levels of specific antibodies (7-10 days post-infection in untreated pigs). The haptoglobin response lasted until the end of the study on day 17 and thereby partly coincided with the antibody response. Treatment with antimicrobials that effectively reduced establishment of the infection with A. pleuropneumoniae also reduced the duration of all three acute phase responses, and reduced the concentration of serum haptoglobin. In contrast, less efficacious treatments did not reduce these acute phase responses. Thus, acute phase reactants can be applied to monitor therapeutic effects of antimicrobial drugs in the pig and measurements of IL-6, SAA and haptoglobin could add valuable information about the stage of infection during a disease outbreak.     

In situ hybridization for the detection of the apxIV gene in the lungs of pigs experimentally infected with twelve Actinobacillus pleuropneumoniae serotypes

The detection of the apxlV gene in lung tissues from pigs experimentally infected with the 12 major A. pleuropneumoniae serotype (1 to 12) reference strains was studied by in situ hybridization using a non-radioactive digoxigenin-labeled DNA probe. In situ hybridization produced a distinct positive signal in all pigs inoculated with the 12 A. pleuropneumoniae serotypes. Positive hybridization typically exhibited a dark-brown to black reaction product in intracellular and extracellular locations, without background staining. A strong hybridization signal was seen in degenerated alveolar leukocytes ("oat cells") adjacent to the foci of coagulative necrosis and in the alveolar spaces. The in situ hybridization methodology developed for the detection of the apxIV gene is a valuable tool for the diagnosis of porcine pleuropneumonia caused by A. pleuropneumoniae when only formalin-fixed tissues are submitted for diagnosis.     

Expression of nitric oxide synthase 2 and cyclooxygenase-2 in swine experimentally infected with Actinobacillus pleuropneumoniae

The expression of inflammatory mediators was examined in pigs experimentally infected with Actinobacillus pleuropneumoniae. The activity of nitric oxide synthase 2 (NOS2) and cyclooxygenase-2 (COX-2) was determined by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) in bronchoalveolar lavage fluid in response to A. pleuropneumoniae in vivo. By in situ hybridization and immunohistochemistry, both NOS2 and COX-2 enzymes were detected in neutrophils and macrophages that had infiltrated into alveolar spaces. The sharp increase in PGE2 concentration preceded the increase in the concentrations of NO. NO levels were highly correlated with PGE2 level (rs=0.7218, P <0.05). The NO levels were positively correlated with lung lesion scores (rs=0.9087, P <0.05) until 24 hours postinoculation (hpi) as were the lung lesion scores and PGE2 levels (rs=0.925, P <0.01). High levels of PGE2 produced by COX-2 are generated in early infection (6 hpi). However, in later stages of infection (12-36 hpi), there is participation of NO and PGE2 accompanied by coinduction of both NOS2 and COX-2.     

Effects of oral administration of tilmicosin on pulmonary inflammation in piglets experimentally infected with Actinobacillus pleuropneumoniae

OBJECTIVES: To determine the effects of oral administration of tilmicosin in piglets experimentally infected with Actinobacillus pleuropneumoniae. ANIMALS: Forty 3-week-old specific-pathogen free piglets. PROCEDURES: Piglets were assigned to 1 of 4 groups as follows: 1) uninfected sham-treated control piglets; 2) infected untreated piglets that were intratracheally inoculated with 10(7) CFUs of A pleuropneumoniae; 3) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received tilmicosin in feed (400 ppm [microg/g]) for 7 days prior to inoculation; or 4) infected treated piglets that were intratracheally inoculated with A pleuropneumoniae and received chlortetracycline (CTC) in feed (1100 ppm [microg/gl) for 7 days prior to inoculation. Bronchoalveolar lavage (BAL) fluid and lung tissue specimens of piglets for each group were evaluated at 3 or 24 hours after inoculation. For each time point, 4 to 6 piglets/group were studied. RESULTS: Feeding of CTC and tilmicosin decreased bacterial load in lungs of infected piglets. Tilmicosin delivered in feed, but not CTC, enhanced apoptosis in porcine BAL fluid leukocytes. This was associated with a decrease in LTB4 concentrations in BAL fluid of tilmicosin-treated piglets, compared with untreated and CTC-treated piglets, and also with a significant decrease in the number of pulmonary lesions. Tilmicosin inhibited infection-induced increases in rectal temperatures, as measured in untreated and CTC-treated piglets. Pulmonary neutrophil infiltration and prostaglandin E2 concentrations in the BAL fluid were not significantly different among groups at any time. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of tilmicosin to infected piglets induces apoptosis in BAL fluid leukocytes and decreases BAL fluid LTB4 concentrations and inflammatory lung lesions.     

Circulating cortisol, tumor necrosis factor-alpha interleukin-1beta, and interferon-gamma in pigs infected with Actinobacillus pleuropneumoniae

This study evaluated the time course of systemic cytokine concentrations in an acute model of pneumonia in pigs challenged intranasally with Actinobacillus pleuropneumoniae. Feed intake and serum cortisol were measured as overt clinical and systemic markers of disease onset, respectively, and serum tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma as representative systemic inflammatory markers. Crossbred barrows (n = 15), approximately 5 wk of age, were used in the study. Pigs were housed in an environmentally controlled facility at 25 degrees C and under continuous illumination in pens measuring approximately 1.5 m2. Pigs had free access to water and an unmedicated diet. Approximately 1 wk prior to disease challenge, pigs were fitted nonsurgically with venous catheters. At challenge, pigs were given 5 x 10(8) CFU Actinobacillus pleuropneumoniae intranasally (n = 8) or a similar volume of sterile growth media intranasally (Control; n = 7). Feed intake was estimated by the change in feeder weight at 12-h intervals from -12 to 72 h relative to the time of disease challenge. Blood sampling began 12 h prior to challenge and continued until 72 h after challenge. Pigs were sampled at -12, -6, and 0 h, then at 90-min intervals until 12-h post-challenge, continuing at 3-h intervals until 24-h post-challenge, then again at 6-h intervals until 72 h after challenge. Serum was harvested and frozen until assayed for cortisol, tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Feed intake was reduced in Actinobacillus pleuropneumoniae pigs during the intervals 0 to 12 h (P < 0.001), 24 to 36 h (P < 0.001), 48 to 60 h (P <0.05), and 60 to 72 h (P < 0.05). TheActnobacillus pleuropneumoniae-challenged pigs had elevated serum cortisol from 180-min to 18-h post-challenge (P < 0.001) and also at 36 (P < 0.05), 42 (P < 0.001), and 60 (P < 0.05) h following infection. Circulating cytokines were not affected by disease challenge. Thus, in this experimental model of pneumonia, weaned pigs demonstrated expected behavioral and endocrine characteristics of disease in the absence of significant changes in circulating inflammatory cytokines.     

Early in vivo interactions of Actinobacillus pleuropneumoniae with tonsils of pigs.

Twenty gnotobiotic piglets were inoculated with 5 x 10(8) colony forming units of an Actinobacillus pleuropneumoniae biotype 1-serotype 9 strain onto their tonsils. Five other piglets (controls) were inoculated with phosphate-buffered saline solution. Pigs were euthanized at 30 min, 90 min, 180 min, 6 h, 9 h, 12 h or 24 h after inoculation. At necropsy, samples were taken from the tonsils for bacteriological, histological, immuno-histochemical and electron microscopical examination. A. pleuropneumoniae was isolated from tonsils of all the infected pigs, but not from tonsils of the control pigs. Early after inoculation bacteria were mainly associated with the stratified squamous epithelium and detached epithelial cells. Vacuolization and desquamation of the epithelium was observed and many transmigrating neutrophils were present. At later times after inoculation, bacteria were found closely associated with the crypt-walls and with detached cells present in the crypts. A strong neutrophil migration was observed mainly in the deeper parts of the crypts. It is concluded that attachment of A. pleuropneumoniae to tonsillar epithelial cells probably constitutes a first step in establishing bacteria at this body site.     

Expression of cyclooxygenase-2 in swine naturally infected with Actinobacillus pleuropneumoniae

Cyclooxygenase-2 (COX-2) was detected and localized in 15 pigs with naturally occurring pleuropneumonia using a 437-base pair digoxigenin-labeled cDNA probe in an in situ hybridization protocol. Histopathologic changes in the acute stage were characterized by coagulative necrosis of lung parenchyma, hemorrhage, vascular thrombosis, edema, fibrin deposition, and infiltration of lung parenchyma by neutrophils and alveolar macrophages in nine pigs. In chronic lesions, a thick layer of granulation tissue surrounded foci of pulmonary necrosis in six pigs. All 15 pigs infected with Actinobacillus pleuropneumoniae, confirmed by bacterial isolation, had distinct positive hybridization signals for COX-2 in bronchial, bronchiolar epithelial cells, alveolar macrophages, neutrophils, and type I pneumocytes. COX-2 expression was detected primarily in neutrophils from pigs with acute lesions and primarily in alveolar macrophages from pigs with chronic lesions. The results suggest that a prostanoid product of COX-2 is an important component of the inflammatory response to acute and chronic A. pleuropneumoniae infection.     

Blood lymphocyte subsets in pigs vaccinated and challenged with Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae bacterins do not induce protection in pigs while infection with low doses of the CM5 strain of A. pleuropneumoniae given by aerosol induces complete protection. To evaluate possible correlates of protection in blood lymphocyte subset phenotypes, pigs were treated with a commercial bacterin given intramuscularly, low dose (10(5)cfu/ml) aerosol infection with CM5 or control treatments of the bacterin adjuvant or phosphate buffered saline. All pigs were challenged with a high dose (10(7)cfu/ml) of A. pleuropneumoniae. Lymphocytes and sera were collected prior to and following primary and secondary immunizations and challenge, for evaluation of B- and T-cell markers and antibody to four A. pleuropneumoniae antigens. IgM(micro)+ B-cells were increased following primary exposure to antigen in the bacterin-vaccinated group only. An increase in CD4+ cells in the LD aerosol-infected group was apparent following secondary exposure to antigen. These early changes suggest little difference in lymphocyte populations between treatment groups, however, greater differences were observed following high-dose challenge; CD4+ lymphocytes were increased significantly in both bacterin and LD-challenged groups (p<0.05) while CD8+ cells decreased in the LD-group at this time period. Consequently, there were significant differences (p<0.05) in the CD4:CD8 ratio after high-dose challenge compared to earlier time points and control groups. Variation in cellular expression of SLA-DR and DQ was observed but trends correlating to treatment group were not evident. Complete protection or lack of protection associated with LD challenge or immunisation resulted in significant differences in B-cell frequencies and CD4:CD8 ratio phenotypes in pigs, but only changes in CD4:CD8 ratios appeared relevant to protection.     

Actinobacillus pleuropneumoniae osteomyelitis in pigs demonstrated by fluorescent in situ hybridization.

Necrotizing osteomyelitis and fibrinopurulent arthritis with isolation of Actinobacillus pleuropneumoniae serotype 2 is reported in two pigs from a herd with lameness and mild coughing problems among 8 to 12-week-old pigs. Application of fluorescent in situ hybridization targeting 16S ribosomal RNA of A. pleuropneumoniae in formalin-fixed tissue was performed to verify the association of A. pleuropneumoniae with the bone and joint lesions. By in situ hybridization A. pleuropneumoniae was demonstrated as multiple microcolonies or single cells dispersed in focal fibrinonecrotizing pleuropneumonia, in joints with arthritis, and in bone necroses including lysis of growth plate and suppurative inflammation in the adjacent trabecular metaphysis, thus demonstrating that well-known infections manifest new, unusual lesions.     

Pulmonary lesions in lambs experimentally infected with ovine adenovirus 5 strain RTS-42

Twelve lambs were inoculated transtracheally and intranasally with Mastadenovirus ovi 5 strain RTS-42 and killed sequentially. Pulmonary lesions were studied by light and electron microscopy. Four lambs served as sham inoculated controls. Pulmonary lesions consisted of multifocal areas of bronchiolitis and alveolitis associated with necrosis and sloughing of isolated type I and type II alveolar epithelial cells and nonciliated bronchiolar epithelial cells. This was followed rapidly by hyperplasia of the remaining epithelium and repair of the damage. A cellular infiltrate of neutrophils and macrophages began at 2 days after inoculation, peaked at 4 days after inoculation, gradually diminished until minimal at 12 days after inoculation, and was resolved at 21 days after inoculation. Surfactant was abundant and, along with debris, was removed from the alveoli by macrophages. Clinical disease was not seen, but lesions were believed to be sufficient to allow bacteria to colonize the lungs and cause severe disease.