Pneumonia In Pigs Research Articles

Benefits of Antibiotics During Viral Infections: Immuno‐Modulating Properties of Tulathromycin in Porcine Reproductive and Respiratory Syndrome

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive‐strand RNA virus that grows in primary alveolar macrophages and causes acute pneumonia in pigs. PRRSV is a major concern in the swine industry with a total cost of productivity losses estimated at $600 million annually in the U.S. alone. However, due to its high antigenic variability, and poorly understood immunopathogenesis, there is currently no treatment to control PRRSV infection. The common occurrence of PRRSV infection with bacterial infections, including Actinobacillus pleuropneumoniae, begs the question of the value of antibiotics for the treatment of the disease it causes. Tulathromycin, a macrolide used for the treatment and prevention of respiratory disease in pigs and cattle, has been shown to exhibit potent immuno‐modulating properties [1]. We hypothesize that tulathromycin attenuates the detrimental effects of PRRSV in porcine macrophages. This may help characterize novel mechanism through which an anti‐microbial agent may deliver clinical benefits in the context of a viral infection.Aims1) Determine if Tulathromycin has direct anti‐viral effects in PRRSV‐infected porcine macrophages. 2) Identify new immuno‐modulating effects of Tulathromycin in PRRSV‐infected macrophages.MethodsPorcine monocytes were isolated from peripheral blood of healthy piglets. Seven days‐old monocyte‐derived macrophages were treated with Tulathromycin (1 mg/ml) or untreated (control) and incubated for 1h at 37°C. Macrophages were then cultured in media alone, or with PRRSV (MOI of 1) for 2h to 24h. (1) Extracellular and intracellular Viral titers were measured at 2h, 24h, and 48h post‐infection. (2) Macrophage activation was monitored by morphological changes and confirmed with IL‐8 ELISA and ROS production assays at 2h, 6h, 12h or 24h. (3) Phagocytosis properties were assessed by measuring zymosan particles (1 μg/mL) or 3μM opsonized latex beads (ratio beads: cells, 10:1) engulfment assay (4) Cell death ELISA, Annexin V immunostaining and lactate dehydrogenase assay were performed to evaluate the effects of Tulathromycin on PRRSV‐induced necrosis and apoptosis.Results(1) Tulathromycin did not change PRRSV particle forming unit (pfu) in macrophages at any time of infection (2.1×104 PFU/mL with tulathromycin versus 1.9×104 PFU/mL for control) (2). Exposure to PRRSV increased macrophage‐induced production of IL‐8 and ROS by more than four times compared to control. Pre‐treatment with tulathromycin significantly attenuated PRRSV‐induced macrophage activation (i.e. 1.85 times compared to untreated macrophages). (3) Macrophage phagocytosis of both zymosan and latex beads was impaired by PRRSV. Tulathromycin was able to restore phagocytic functions in PRRSV‐infected macrophages to control values. (4) Finally, Tulathromycin reduced viral induced necrosis but synergized with the virus to induce apoptosis.ConclusionOur results demonstrate that Tulathromycin attenuates macrophage‐driven inflammatory response induced by PRRSV, restores PRRSV‐induced phagocytic impairment, and inhibits cell necrosis. These effects occur in the absence of a direct anti‐viral activity. Together, these data demonstrate the potential clinical benefits of Tulathromycin in the context of PRRSV‐induced pneumonia through the modulation of a viral‐induced macrophage‐driven inflammation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Update on syndromes and clinical problems associated with porcine circovirus type 2 infection

The porcine circoviruses (PCV) are members of the genus Circovirus, family Circoviridae. Two types of PCV have been characterized and were named Porcine Circovirus type 1/PCVl and Porcine Circovirus type 2/PCV2. PCV1 is considered to be avirulent, since it has never been associated with the occurrence of swine disease. On the contrary, PCV2 is identified as virulent porcine pathogen and the virus is considered to have a potential pathogenic role to Post Weaning Multisystemic Wasting Syndrome (PMWS), Porcine Dermatitis and Nephropathy Syndrome (PDNS) and other porcine diseases, although the pathogenesis of this emerging viral infection has not been yet determined. The association of PCV2 and PMWS is now well established. However, it is believed that co-infection with the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is necessary for the clinical expression of the syndrome, while Mycoplasma hyopneumoniae, a common finding in PMWS cases, increases the incidence and the severity of PMWS in pigs. The results of experimental trials indicate that PCV2 may need co-factors and/or a long period of incubation to fully reproduce PMWS, while the stimulation of the immune system after vaccination might be a pivotal event in the production of PMWS. The diagnosis is based on clinical signs, necropsy, on the presence of the characteristic histopathological lesions in lymphoid tissues and the presence of PCV2 (antigens, nucleic acid) within the lesions. PDNS has been reported in several pig-producing countries and is characterized by the presence of hemorrhagic skin lesions, oedema of the limbs, necrotizing vasculitis and glomerulonephritis, affecting pigs of 1.4-4 months old. The demonstration of PCV2 antigens and nucleic acid, closely associated with skin and renal lesions, has led to the speculation that PCV2 is an aetiologic agent of this syndrome. Although PCV2 has been consistendy detected in PDNS, lesions consistent with this syndrome have yet to be reproduced experimentally. It is likely that many factors, such as overcrowding, poor ventilation, commingling of different agegroups, co-infection of viruses and bacteria, and other stressors, may act in a synergistic fashion to trigger as yet unidentified mechanisms to induce PDNS. Porcine Respiratory Disease Complex (PRDC) is a serious health problem in growing and finishing pigs aged around 16-22 weeks. Pneumonia in pigs with PRDC is due to a combination of both viral and bacterial agents, such as PRRSV,swine influenza virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae and Pasteurella multocida. Some authors suggest that PCV2 could be an important contributor to PRDC, probably by interaction or synergism with other respiratory pathogens, such as PRRSV. PCV2-associated PRDC should be differentiated from PMWS clinically and histopathologically. There have been several experimental and field reports of PCV2-associated reproductive failure (elevated abortion, stillbirths and fetal mummification). Furthermore, it is suggested that porcine proliferative and necrotising pneumonia is a result of a PRRSV and PCV2 coinfection. Moreover, it has been demonstrated that PCV2 and porcine parvovirus may act as cofactors in the experimental reproduction of exudative epidermitis caused by Staphylococcus hyicus. Thus, it is likely that these viruses are involved in lesion development and/or progression of the disease. Respectively, the results of a recent survey indicate that both viruses are highly prevalent in pigs with exudative epidermitis. Additionally, PCV2 is associated with granulomatous enteritis, congenital tremor and lately with necrotizing lemphadenitis, which might be also manifestations of PCV2 infection. It has recendy been hypothesized that different types of PCV2 may be responsible for different disease manifestations. Most of the relevant studies have found minor differences in the respective PCV2 genomes, but at this time remains unclear what significance these minor differences may have. It has been suggested that they may account for different in tropism with respect to the host organism. In addition, other host factors, such as age or route of infection, may affect the pathogenicity and clinical manifestation of PCV2 infection. However, the pathogenical role of PCV2 remains unclear and further studies are therefore needed to define it. The presence of PDNS, PMWS and for the first time the occurrence of PMWS after vaccination against enzootic pneumonia, and the relationship between PMWS-PRRS and PMWS-PRDC as well, have already been described and diagnosed in Greece. Additionally, the genetic analysis of the first PCV2 isolates from pigs presenting various clinical conditions in Greece has been performed.

Anatomopathological pneumonic aspects associated with highly pathogenic Pasteurella multocida in finishing pigs

ABSTRACT: The bacterium Pasteurella multocida is a frequent cause of porcine respiratory disease complex in finishing pigs. Historically, the bacterium is recognized as an opportunistic agent, causing secondary bacterial pneumonia in pigs. Several Brazilian reports have suggested the ability of P. multocida to cause primary pulmonary infection that leads to the death of finishing pigs prior to slaughter. The aim of this study was to evaluate anatomopathological pulmonary findings associated with P. multocida infection that were obtained from animals with clinical respiratory disease and from animals at slaughter. Twenty-five lung samples from 14 herds of finishing pigs with acute clinical respiratory disease and 19 lungs collected at slaughter from a different set of 14 herds were studied. In all lung samples, bacterial isolation was performed, and only samples with pure P. multocida growth were included in the study. Gross and histopathological lesions were evaluated, as well as Influenza A, porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae co-infections. Pleuritis and pericarditis were more often observed in clinical samples (P<0.05). Moreover, there was a numerical trend indicating that pericarditis, lymphadenomegaly and cavity exudates were more often present in clinical samples. Thirteen lung samples were negative to M. hyopneumoniae, Influenza A and PCV2 by immunohistochemistry (IHC), with only P. multocida identified. In these cases, gross lesions such as pleuritis, pericarditis and lymphadenomegaly were always present, and no histologic lesions indicative of other agents such as Actinobacillus pleuropneumoniae, Actinobacillus suis or Haemophilus parasuis were observed. These findings suggest the ability of some P. multocida isolates to cause primary respiratory and systemic infection. However, in this study, it was not possible to determine specific virulence markers to explain these findings.

Efficacy of one dose vaccination against experimental infection with two Mycoplasma hyopneumoniae strains

BackgroundMycoplasma hyopneumoniae (M. hyopneumoniae) is the primary agent of enzootic pneumonia in pigs. Pigs are often infected with different M. hyopneumoniae strains. This study assessed the efficacy of vaccination against experimental infection with two genetically different M. hyopneumoniae strains in weaned piglets. At 33 days of age (D0), 45 M. hyopneumoniae-free piglets were randomly assigned to three different groups: 1) negative control group (NCG; n = 5): not vaccinated, not infected, 2) positive control group (PCG; n = 20): not vaccinated, infected, and 3) vaccination group (VG; n = 20): single vaccination with an inactivated whole-cell M. hyopneumoniae vaccine (Hyogen®, Ceva) (D1), infected. The PCG and VG were endotracheally inoculated with 7 × 107 CCU in 7 ml of the highly virulent M. hyopneumoniae strain F7.2C (D24) and 7 × 107 CCU in 7 ml low virulent strain F1.12A (D25). A respiratory disease score (RDS) was assessed from D24 until D53. At D53 (euthanasia), macroscopic lung lesions (MLL) were scored, log copies of M. hyopneumoniae DNA (qPCR) and IL-1 and IL-6-concentrations (ELISA) on bronchoalveolar lavage fluid were determined.ResultsThe RDS and MLL at euthanasia were respectively 0, 1.20 and 0.55 (P < 0.001) and 0, 7.56 and 0.68 (P < 0.001) for NCG, PCG and VG, respectively. The qPCR results for PCG and VG were 3.99 and 1.78 log copies (P < 0.001), respectively, with a significant difference between PCG and VG. The IL-1 and IL-6 results at euthanasia for NCG, PCG and VG were 17.61, 1283.39 and 53.04 pg/ml (P < 0.001) and 148.10, 493.35 and 259.80 pg/ml (P = 0.004), respectively with a significant difference between PCG and VG.ConclusionsVaccination with Hyogen® in pigs was efficacious against an experimental challenge with both a low and highly virulent M. hyopneumoniae strain as the vaccinated pigs coughed significantly less, and showed significantly less lung lesions compared to the non-vaccinated challenged pigs: the vaccinated animals showed a 52.9% lower RDS and 91.0% lower MLL compared to the PCG. In the bronchoalveolar lavage fluid collected at the necropsy of the vaccinated pigs, a significantly lower amount of M. hyopneumoniae-DNA and a significantly lower IL-1 and IL-6 concentration was found compared to the pigs of the PCG.

Prediction of marbofloxacin dosage for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida by pharmacokinetic/pharmacodynamic modelling

BackgroundBacterial pneumonia in pigs occurs widely and requires antimicrobial therapy. It is commonly caused by the pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida. Marbofloxacin is an antimicrobial drug of the fluoroquinolone class, licensed for use against these organisms in the pig. In recent years there have been major developments in dosage schedule design, based on integration and modelling of pharmacokinetic (PK) and pharmacodynamic (PD) data, with the objective of optimising efficacy and minimising the emergence of resistance. From in vitro time-kill curves in pig serum, PK/PD breakpoint Area under the curve (AUC) 24h /minimum inhibitory concentration (MIC) values were determined and used in conjunction with published PK, serum protein binding data and MIC distributions to predict dosages based on Monte Carlo simulation (MCS).ResultsFor three levels of inhibition of growth, bacteriostasis and 3 and 4log10 reductions in bacterial count, mean AUC24h/MIC values were 20.9, 45.2 and 71.7 h, respectively, for P. multocida and 32.4, 48.7 and 55.5 h for A. pleuropneumoniae. Based on these breakpoint values, doses for each pathogen were predicted for several clinical scenarios: (1) bacteriostatic and bactericidal levels of kill; (2) 50 and 90% target attainment rates (TAR); and (3) single dosing and daily dosing at steady state. MCS for 90% TAR predicted single doses to achieve bacteriostatic and bactericidal actions over 48 h of 0.44 and 0.95 mg/kg (P. multocida) and 0.28 and 0.66 mg/kg (A. pleuropneumoniae). For daily doses at steady state, and 90% TAR bacteriostatic and bactericidal actions, dosages of 0.28 and 0.59 mg/kg (P. multocida) and 0.22 and 0.39 mg/kg (A. pleuropneumoniae) were required for pigs aged 12 weeks. Doses were also predicted for pigs aged 16 and 27 weeks.ConclusionsPK/PD modelling with MCS approaches to dose determination demonstrates the possibility of tailoring clinical dose rates to a range of bacterial kill end-points.

Persistence of Mycoplasma hyopneumoniae sequence types in spite of a control program for enzootic pneumonia in pigs

Enzootic pneumonia (EP) in pigs caused by Mycoplasma (M.) hyopneumoniae has successfully been combatted in Switzerland. A control program was fully implemented in 2004 which is based on total depopulation strategies of affected fattening farms as well as partial depopulation on breeding farms. Thereby, the number of cases has dropped drastically from more than 200 in 2003 to two cases in 2013. Currently monitoring is done based on clinical observation and subsequent diagnostic of coughing pigs. Moreover, in case of more than 10% gross pathological lesions per slaughter batch laboratory confirmation for EP is compulsory. Despite these strict measures it was not possible to eliminate M. hyopneumoniae from Swiss pig production. In fact, during the last few years the number of EP cases has slightly increased. Therefore, genotyping of the involved M. hyopneumoniae strains was conducted in order to elucidate possible sources and routes of infection. All available and typeable samples from totally 22 cases during the period 2014–2016 were investigated by extended multilocus sequence typing (MLST). A total of 16 cases, including eight from 2014, five from 2015 and three from 2016 could thereby be included in the study. MLST revealed that the majority of cases in 2014/2015 were due to two major spread scenarios, i.e. two M. hyopneumoniae sequence types, each scenario involving six individual production farms in five to six different Cantons (states), respectively. Moreover, by comparison of archived sequences some sequence types were observed over ten years demonstrating their persistence over a long time and the possible partial failure of elimination measures in Switzerland. Insufficient sanitation on affected farms and subsequent animal transport of symptomless infected pigs could lead to recurrent cases. Wild boar harbor identical strains found with EP but solid data are missing to assign a role as reservoir to this wild animal. Implementing a monitoring scheme for M. hyopneumoniae in wild boar in combination with genotyping of all available samples from domestic pigs could direct responsible authorities to possible gaps and deficiencies of control measures taken for combating enzootic pneumonia. With the newly installed PubMLST database sequence types for M. hyopneumoniae are now available and allow tracing back strains on the international level.

Impact of growth matrix on pharmacodynamics of antimicrobial drugs for pig pneumonia pathogens

BackgroundThe most widely used measure of potency of antimicrobial drugs is Minimum Inhibitory Concentration (MIC). MIC is usually determined under standardised conditions in broths formulated to optimise bacterial growth on a species-by-species basis. This ensures comparability of data between laboratories. However, differences in values of MIC may arise between broths of differing chemical composition and for some drug classes major differences occur between broths and biological fluids such as serum and inflammatory exudate. Such differences must be taken into account, when breakpoint PK/PD indices are derived and used to predict dosages for clinical use. There is therefore interest in comparing MIC values in several broths and, in particular, in comparing broth values with those generated in serum. For the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, MICs were determined for three drugs, florfenicol, oxytetracycline and marbofloxacin, in five broths [Mueller Hinton Broth (MHB), cation-adjusted Mueller Hinton Broth (CAMHB), Columbia Broth supplemented with NAD (CB), Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB)] and in pig serum.ResultsFor each drug, similar MIC values were obtained in all broths, with one exception, marbofloxacin having similar MICs for three broths and 4–5-fold higher MICs for two broths. In contrast, for both organisms, quantitative differences between broth and pig serum MICs were obtained after correction of MICs for drug binding to serum protein (fu serum MIC). Potency was greater (fu serum MIC lower) in serum than in broths for marbofloxacin and florfenicol for both organisms. For oxytetracycline fu serum:broth MIC ratios were 6.30:1 (P. multocida) and 0.35:1 (A. pleuropneumoniae), so that potency of this drug was reduced for the former species and increased for the latter species. The chemical composition of pig serum and broths was compared; major matrix differences in 14 constituents did not account for MIC differences. Bacterial growth rates were compared in broths and pig serum in the absence of drugs; it was concluded that broth/serum MIC differences might be due to differing growth rates in some but not all instances.ConclusionsFor all organisms and all drugs investigated in this study, it is suggested that broth MICs should be adjusted by an appropriate scaling factor when used to determine pharmacokinetic/pharmacodynamic breakpoints for dosage prediction.

Pharmacokinetic/pharmacodynamic integration and modelling of florfenicol for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida

Pharmacokinetic-pharmacodynamic (PK/PD) integration and modelling were used to predict dosage schedules for florfenicol for two pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Pharmacokinetic data were pooled for two bioequivalent products, pioneer and generic formulations, administered intramuscularly to pigs at a dose rate of 15 mg/kg. Antibacterial potency was determined in vitro as minimum inhibitory concentration (MIC) and Mutant Prevention Concentration in broth and pig serum, for six isolates of each organism. For both organisms and for both serum and broth MICs, average concentration:MIC ratios over 48 h were similar and exceeded 2.5:1 and times greater than MIC exceeded 35 h. From in vitro time-kill curves, PK/PD modelling established serum breakpoint values for the index AUC24h/MIC for three levels of inhibition of growth, bacteriostasis and 3 and 4log10 reductions in bacterial count; means were 25.7, 40.2 and 47.0 h, respectively, for P. multocida and 24.6, 43.8 and 58.6 h for A. pleuropneumoniae. Using these PK and PD data, together with literature MIC distributions, doses for each pathogen were predicted for: (1) bacteriostatic and bactericidal levels of kill; (2) for 50 and 90% target attainment rates (TAR); and (3) for single dosing and daily dosing at steady state. Monte Carlo simulations for 90% TAR predicted single doses to achieve bacteriostatic and bactericidal actions over 48 h of 14.4 and 22.2 mg/kg (P. multocida) and 44.7 and 86.6 mg/kg (A. pleuropneumoniae). For daily doses at steady state, and 90% TAR bacteriostatic and bactericidal actions, dosages of 6.2 and 9.6 mg/kg (P. multocida) and 18.2 and 35.2 mg/kg (A. pleuropneumoniae) were required. PK/PD integration and modelling approaches to dose determination indicate the possibility of tailoring dose to a range of end-points.

Pharmacokinetic/pharmacodynamic integration and modelling of oxytetracycline for the porcine pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida.

Pharmacokinetic–pharmacodynamic (PK/PD) integration and modelling were used to predict dosage schedules of oxytetracycline for two pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in broth and porcine serum. PK/PD integration established ratios of average concentration over 48 h (C av0–48 h)/MIC of 5.87 and 0.27 μg/mL (P. multocida) and 0.70 and 0.85 μg/mL (A. pleuropneumoniae) for broth and serum MICs, respectively. PK/PD modelling of in vitro time–kill curves established broth and serum breakpoint values for area under curve (AUC 0–24 h)/MIC for three levels of inhibition of growth, bacteriostasis and 3 and 4 log10 reductions in bacterial count. Doses were then predicted for each pathogen, based on Monte Carlo simulations, for: (i) bacteriostatic and bactericidal levels of kill; (ii) 50% and 90% target attainment rates (TAR); and (iii) single dosing and daily dosing at steady‐state. For 90% TAR, predicted daily doses at steady‐state for bactericidal actions were 1123 mg/kg (P. multocida) and 43 mg/kg (A. pleuropneumoniae) based on serum MICs. Lower TARs were predicted from broth MIC data; corresponding dose estimates were 95 mg/kg (P. multocida) and 34 mg/kg (A. pleuropneumoniae).

Comprehensive Analysis of Tiamulin Metabolites in Various Species of Farm Animals Using Ultra-High-Performance Liquid Chromatography Coupled to Quadrupole/Time-of-Flight.

Tiamulin is an antimicrobial widely used in veterinary practice to treat dysentery and pneumonia in pigs and poultry. However, knowledge about the metabolism of tiamulin is very limited in farm animals. To better understand the biotransformation of tiamulin, in the present study, in vitro and in vivo metabolites of tiamulin in rats, chickens, swine, goats, and cows were identified and elucidated using ultra-high performance liquid chromatography coupled to quadrupole/time-of-flight. As a result, a total of 26 metabolites of tiamulin, identified in vitro and in vivo, and majority of metabolites were revealed for the first time. In all farm animals, tiamulin undergoes phase I metabolic routes of hydroxylation in the mutilin part (the ring system), S-oxidation and N-deethylation on side chain, and no phase II metabolite was detected. Among these, 2β- and 8α-hydroxylation and N-deethylation were the main metabolic pathways of tiamulin in farm animals. In addition, we have put forward that 8a-hydroxy-tiamulin and 8a-hydroxy-N-deethyl-tiamulin could be hydroxylated into 8a-hydroxy-mutilin, the marker residue of tiamulin in swine. Furthermore, a significant interspecies difference was observed on the metabolism of tiamulin among various farm animals. The possible marker residues for tiamulin in swine were 8α-hydroxy-tiamulin, N-deethyl-tiamulin, and 8α-hydroxy-N-deethyl-tiamulin, which were consistent with the hypothesis proposed by the European Agency for the Evaluation of Medicinal Products. However, results in present study indicated that three metabolites (2β-hydroxy-tiamulin, N-deethyl-tiamulin, and 2β-hydroxy-N-deethyl-tiamulin) of tiamulin in chickens had larger yields, which implied that 2β-hydroxy-mutilin or N-deethyl-tiamulin was more likely to be regarded as the potential marker residue of tiamulin in chickens.

Roles of the Putative Type IV-like Secretion System Key Component VirD4 and PrsA in Pathogenesis of Streptococcus suis Type 2.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen causing septic infection, meningitis and pneumonia in pigs and humans. SS2 may cause streptococcal toxic shock syndrome (STSS) probably due to excessive release of inflammatory cytokines. A previous study indicated that the virD4 gene in the putative type IV-like secretion system (T4SS) within the 89K pathogenicity island specific for recent epidemic strains contributed to the development of STSS. However, the functional basis of VirD4 in STSS remains unclear. Here we show that deletion of virD4 led to reduced virulence as shown by about 65% higher LD50, lower bacterial load in liver and brain, and lower level of expression of inflammatory cytokines in mice and cell lines than its parent strain. The ΔVirD4 mutant was more easily phagocytosed, suggesting its role as an anti-phagocytic factor. Oxidative stress that mimic bacterial exposure to respiratory burst of phagocytes upregulated expression of virD4. Proteomic analysis identified 10 secreted proteins of significant differences between the parent and mutant strains under oxidative stress, including PrsA, a peptidyl-prolyl isomerase. The SS2 PrsA expressed in E. coli caused a dose-dependent cell death and increased expression of proinflammatory IL-1β, IL-6 and TNF-α in murine macrophage cells. Our data provide novel insights into the contribution of the VirD4 factor to STSS pathogenesis, possibly via its anti-phagocytic activity, upregulation of its expression upon oxidative stress and its involvement in increased secretion of PrsA as a cell death inducer and proinflammatory effector.

Activity of florfenicol for Actinobacillus pleuropneumoniae and Pasteurella multocida using standardised versus non-standardised methodology

Four indices of antimicrobial potency were determined for florfenicol and the pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), mutant prevention concentration (MPC) and time–kill curves were determined in two matrices, broth and pig serum. Five overlapping sets of two-fold dilutions were used to increase accuracy of the measurements. MIC and MBC serum:broth ratios for A. pleuropneumoniae were 0.96:1 and 1.07:1, respectively, and corresponding values for P. multocida were 0.72:1 and 0.50:1. The percentage binding of florfenicol to serum protein was 65.4%, and fraction unbound (fu) serum MICs were significantly lower, by 2.71-fold and 3.82-fold, respectively, than predicted for free serum concentrations for A. pleuropneumoniae and P. multocida. Similar culture medium differences were obtained for MBC and MPC. MICs in serum and broth were increased significantly and progressively for high, medium and low initial inoculum counts. Serum MPC:MIC ratios for A. pleuropneumoniae and P. multocida were 12.5:1 and 13.6:1, respectively; ratios for broth were similar. The killing action of florfenicol had the characteristics of concentration dependency for both species in both growth media. These data indicate the value of using a biological medium, when determining microbiological potency indices, to predict dosage for clinical use.

Pathogenesis of Korean SapelovirusA in piglets and chicks.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.

ОСОБЛИВОСТІ ПАТОМОРФОЛОГІЧНИХ ЗМІН ЗА АСОЦІАТИВНОГО ПЕРЕБІГУ МІКОПЛАЗМОЗУ

Coplasma pneumonia of pigs is a chronic infectious disease of pigs of all age groups, characterized by exudative–proliferative inflammation of the lungs, intermittent fever, cough and delayed growth and development of piglets, and complications – progressive weight loss. Mycoplasmosis increases the susceptibility of pigs to secondary infections, making it more difficult and often leads to death of animals.Conducted a comprehensive laboratory study of the use of bacteriological, serological and coprologic research methods pigs aged 1.5 and 3 months. The studies were conducted in the conditions of farms for growing and fattening pigs in the Poltava region.Characteristic of all cases of death of animals was uneven redness and slight thickening of the skin in the abdomen. The increase and uneven coloration pdsalvy, tracheal, inguinal lymph nodes.Lungs with signs of catarrhal pneumonia with lesions predominantly cranial lobes. Some areas of dark red color with a bluish tint, more dense consistency, fall on a common surface, in the lumen of the bronchial – mucous mass. The other part of the body gets mild tuberosity at the age of 1.5 months and more intelligible – at the age of 3 months. In all cases of death of pigs, the pericardium and the pleura with hemorrhage. The spleen is diffusely dark red. The buds are light brown with areas of bluish color. At the age of 1.5 months on the mucosa of a thick intestine was rounded multiple small formations, protruding into the lumen of the intestine.

Enhancement of innate immunity with granulocyte colony-stimulating factor did not mitigate disease in pigs infected with a highly pathogenic Chinese PRRSV strain

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for one of the most economically important diseases in swine worldwide. It causes reproductive failure in sows and pneumonia in pigs that predisposes them to secondary bacterial infections. Methods to control PRRSV and/or limit secondary bacterial infections are desired to reduce the impact of this virus on animal health. Neutrophils play a major role in combatting infection; they can act as phagocytes as well as produce and release lytic enzymes that have potent antimicrobial effects leading to the destruction and clearance of bacterial pathogens. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that controls the production, differentiation and function of granulocytes (including neutrophils) from the bone marrow. Recent work from our laboratory has shown that encoding porcine G-CSF in a replication-defective adenovirus (Ad5-G-CSF) and delivering a single dose to pigs induced a neutrophilia lasting more than two weeks. As secondary bacterial infection is a common occurrence following PRRSV infection, particularly following challenge with highly pathogenic (HP)-PRRSV, the aim of the current study was to evaluate the effectiveness of a single prophylactic dose of adenovirus-encoded G-CSF to mitigate secondary bacterial disease associated with HP-PRRSV infection. Administration of Ad5-G-CSF induced a significant neutrophilia as expected. However, between 1 and 2days following HP-PRRSV challenge the number of circulating neutrophils decreased dramatically in the HP-PRRSV infected group, but not the non-infected Ad5-G-CSF group. Ad5-G-CSF administration induced monocytosis as well, which was also reduced by HP-PRRSV challenge. There was no difference in the progression of disease between the Ad5-G-CSF and Ad5-empty groups following HP-PRRSV challenge, with pneumonia and systemic bacterial infection occurring in both treatment groups. Given the impact of HP-PRRSV infection on the neutrophilia induced by the Ad5-G-CSF administration, additional studies are warranted to evaluate the timing of Ad5-G-CSF induced neutrophilia and multiple G-CSF inoculations on protection against secondary bacterial infection following PRRSV infection. Nevertheless, this study may provide insight into the pathogenesis of HP-PRRSV.

Complete Genome Sequence of Highly Virulent Haemophilus parasuis Serotype 11 Strain SC1401.

Haemophilus parasuis, a normal Gram-negative bacterium, may cause Glässer’s disease and pneumonia in pigs. This study aims to identify the genes related to natural competence of the serotype 11 strain SC1401, which frequently shows competence and high pathogenicity. SC1401 shows many differences from strains without natural competence within the molecular basis. We performed complete genome sequencing together with restriction modification system analysis to lay the foundation for later study.

Induction of mycoplasmal pneumonia in experimentally infected pigs by means of different inoculation routes.

The purpose of this study was to assess the effect of three different inoculation routes into mycoplasmal pneumonia (MP) in pigs challenged with Mycoplasma hyopneumoniae (M. hyopneumoniae). Thirty six-week-old M. hyopneumoniae seronegative piglets were randomly assigned to four groups: three challenged groups with experimentally inoculated pigs by either the endotracheal (ET; n = 8), intranasal (IN; n = 8) or aerosol (AE; n = 8) routes and one uninfected group (Control; n = 6). Blood samples were collected 1 day before challenge and at necropsy, 28 days post-inoculation (dpi), to assess seroconversion. Laryngeal swabs were collected at −1, 7, 14, 21 and 28 dpi in order to evaluate colonization. At necropsy, lung lesions were scored and lung tissue was collected for histopathological studies and M. hyopneumoniae DNA detection. Broncho-alveolar lavage fluid (BALF) was also obtained to detect M. hyopneumoniae DNA, specific IgA antibodies and cytokines. MP was observed in all inoculated groups, but the ET group displayed a significantly higher number of animals affected by MP as well as a higher mean lung lesion score. These results were paralleled with an earlier seroconversion and upper respiratory tract colonization of M. hyopneumoniae. Additionally, in the ET group, higher levels of pro-inflammatory cytokines and specific IgA antibodies in BALF were found. Under the conditions of the present study, MP was reproduced by the three evaluated inoculation routes. Obtained results suggest that the ET route is the most effective in order to induce MP in pigs experimentally challenged with M. hyopneumoniae.

Preparation, characterization and pharmacokinetics of doxycycline hydrochloride and florfenicol polyvinylpyrroliddone microparticle entrapped with hydroxypropyl-β-cyclodextrin inclusion complexes suspension

In order to effectively control the bacterial pneumonia in pigs, doxycycline hydrochloride (DoxHcl) and florfenicol (FF) microparticle suspension together with inclusion complexes was prepared by using hydroxypropyl-β-cyclodextrin (HP-β-CD) as host molecules, polyvinylpyrroliddone (PVP) as polymer carriers and hydroxypropyl methyl cellulose (HPMC) as suspending agents. In vitro antibacterial activity, properties, stability and pharmacokinetics of the suspension were studied. The results demonstrated that DoxHcl and FF had a synergistic or additive antibacterial activity against Streptococcus suis, Actinobacillus pleuropneumoniae and Haemophilus parasuis. The size, polydispersity index and zeta potential of microparticles were 1.46±0.06μm, 0.30±0.02 and 1.53±0.04mV, respectively. The encapsulation efficiency (EE) of DoxHcl and FF was 45.28%±3.30% and 89.69%±2.71%, respectively. The re-dispersed time and sedimentation rate of the suspension were 1min and 1. The suspension went through the 9-gage needle smoothly with withdrawal volume of 9.12±0.87mL/min. The suspension showed good stability when stored away from light, no irritation at the injection site and sustained release in PBS buffer. After intramuscular administration to pig, DoxHcl and FF could maintain over 0.15μg/mL for 72h. Compared to the control injection, the suspension increased the elimination half-life (T½ke) as well as mean residence time (MRT) of DoxHcl from 5.73 to 9.77h and from 12.02 to 18.81h, and those of FF from 12.02 to 26.19h and from12.02 to 28.16h, respectively. The suspension increased the bioavailability of DoxHcl and FF by 1.74 and 1.13-fold, respectively. These results suggest that the compound suspension is a promising formulation for pig pneumonia therapy.

Porcine Sapelovirus Uses α2,3-Linked Sialic Acid on GD1a Ganglioside as a Receptor.

ABSTRACTThe receptor(s) for porcine sapelovirus (PSV), which causes diarrhea, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs, remains largely unknown. Given the precedent for other picornaviruses which use terminal sialic acids (SAs) as receptors, we examined the role of SAs in PSV binding and infection. Using a variety of approaches, including treating cells with a carbohydrate-destroying chemical (NaIO4), mono- or oligosaccharides (N-acetylneuraminic acid, galactose, and 6′-sialyllactose), linkage-specific sialidases (neuraminidase and sialidase S), lectins (Maakia amurensis lectin and Sambucus nigra lectin), proteases (trypsin and chymotrypsin), and glucosylceramide synthase inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and phospholipase C), we demonstrated that PSV could recognize α2,3-linked SA on glycolipids as a receptor. On the other hand, PSVs had no binding affinity for synthetic histo-blood group antigens (HBGAs), suggesting that PSVs could not use HBGAs as receptors. Depletion of cell surface glycolipids followed by reconstitution studies indicated that GD1a ganglioside, but not other gangliosides, could restore PSV binding and infection, further confirming α2,3-linked SA on GD1a as a PSV receptor. Our results could provide significant information on the understanding of the life cycle of sapelovirus and other picornaviruses. For the broader community in the area of pathogens and pathogenesis, these findings and insights could contribute to the development of affordable, useful, and efficient drugs for anti-sapelovirus therapy.IMPORTANCE The porcine sapelovirus (PSV) is known to cause enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. However, the receptor(s) that the PSV utilizes to enter host cells remains largely unknown. Using a variety of approaches, we showed that α2,3-linked terminal sialic acid (SA) on the cell surface GD1a ganglioside could be used for PSV binding and infection as a receptor. On the other hand, histo-blood group antigens also present in the cell surface carbohydrates could not be utilized as PSV receptors for binding and infection. These findings should contribute to the understanding of the sapelovirus life cycle and to the development of affordable, useful and efficient drugs for anti-sapelovirus therapy.

Increased fucosyl glycoconjugate by Mycoplasma hyopneumoniae enhances adherences of Pasteurella multocida type A in the ciliated epithelial cells of the respiratory tract

BackgroundThe objective of this study was to elucidate the pathogenic mechanisms of how Mycoplasma hyopneumoniae enhances secondary Pasteurella multocida type A infection which leads to porcine enzootic pneumonia in infected pigs. Sixteen pigs were experimentally infected with M. hyopneumoniae and then euthanized at 7, 14, 21 and 28 days post inoculation. In situ hybridization for M. hyopneumoniae DNA and Ulex europaeus agglutinin-I (UEA-I) lectin histochemistry for fucosyl glycoconjugate, was performed in serial lung sections to determine alteration of fucosyl glycoconjugate in M. hyopneumoniae-infected bronchial and bronchiolar epithelium. Bacterial overlay assay was performed to determine the affinity of P. multocida type A with L-fucose.ResultsThe luminal surface of bronchial and bronchiolar epithelial cells that were stained with UEA-I always showed hybridization signals for M. hyopneumoniae but it was negative in the unaffected parts of the lung from M. hyopneumoniae-infected pigs and in lung from negative control pigs. Colocalization of M. hyopneumoniae and UEA-I was especially prominent in the luminal surface of bronchial and bronchiolar epithelial cells in serial section of lung. The mean number of M. hyopneumoniae-positive cells correlated with the mean number of UEA-I-positive cells in lungs from infected pigs throughout the experiment. All eight P. multocida type A isolates from naturally occurring enzootic pneumonia, bound strongly at levels of 2 μg and 5 μg of L-fucose.ConclusionsThe results of the present study demonstrate that M. hyopneumoniae increases the L-fucose composition to enhance adherence of P. multocida type A to the bronchial and bronchiolar epithelial cells.