Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

Occurrence and severity of lung lesions in slaughter pigs vaccinated against Mycoplasma hyopneumoniae with different strategies

Different vaccination strategies against Mycoplasma hyopneumoniae have been adopted worldwide. Reports from the field indicate varying levels of protection among currently available vaccines. The goal of the present study was to compare the efficacies of three widespread commercial vaccination strategies against M. hyopneumoniae under field conditions. 20 farms were included. 14 farms used different single dose vaccines (vaccine 1 [V1], 8 herds; vaccine 2 [V2], 6 herds); another 6 farms (V3) used a two dose vaccination strategy. Gross lesions of 854 lungs and histopathology from 140 lungs were quantified, and a quantitative PCR was applied to detect M. hyopneumoniae and porcine circovirus 2 (PCV2) DNA in lung tissue (n = 140). In addition, porcine reproductive and respiratory disease virus (PRRSV), swine influenza virus (SIV), Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida were tested by qualitative PCR. 53% of lungs were positive for M. hyopneumoniae. 55.9% of lungs showed macroscopic enzootic pneumonia (EP)-like lesions. Lung lesion scores (P < 0.001) and M. hyopneumoniae-loads (P < 0.008) differed significantly among the vaccination groups, with the most severe cases and highest amounts occurring in V1. Histological alterations differed (P < 0.001) between V1 and V3. Lung lesion scores and histopathological changes were significantly correlated, with prevalence and load of M. hyopneumoniae indicating that the applied diagnostic tools are valuable in confirming the prevalence and severity of M. hyopneumoniae infections. Comparing different vaccination strategies against M. hyopneumoniae indicates varying levels of protection. M. hyopneumoniae is still a major problem despite the widely applied vaccination.     

Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45

The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease.     

Seroprevalence and risk factors of Mycoplasma suis infection in pig farms in central China

Mycoplasma suis, the causative agent of porcine infectious anemia, causes large economic losses to the swine industry worldwide. A questionnaire-based survey was conducted in 69 pig farms in Hubei Province, China, from November 2011 to August 2013 to ascertain the prevalence and associated risk factors of M. suis. Four thousand and four blood samples from pigs of all the age groups were tested for M. suis antibodies using the established rMSG1–ELISA assay. Among these 4004 samples, 1615 blood samples from multiparous sows were examined to identify the association between seroprevalence and different seasons. Information on risk factors collected from farmers or attending veterinarians was recorded on a pre-designed questionnaire. The overall test seroprevalence of M. suis infection at the animal level was 31.9% (1277/4004; 95% CI: 30.5%, 33.4%), whereas at the farm level, this value was 95.65% (66/69; 95% CI: 87.8%, 99.1%). The seroprevalence of M. suis was higher in replacement gilts (40.6%; 95% CI: 35.1%, 46.3%), multiparous sows (48.2%; 95% CI: 45.8%, 50.7%) and boars (44.4%; 95% CI: 34.5%, 54.8%), as compared to piglets (13.0%; 95% CI: 9.4%, 17.3%), weaned-piglets (10.8%; 95% CI: 8.9%, 13.0%), and growing-finishing pigs (25.0%; 95% CI: 22.0%, 28.3%). In terms of seasons, the prevalence of M. suis in pigs was significantly higher in summer (65.3%; 95% CI: 61.0%, 69.5%) and autumn (65.0%; 95% CI: 59.0%, 70.6%) compared to spring (30.1%; 95% CI: 26.0%, 34.4%) and winter (36.4%; 95% CI: 31.4%, 41.5%). Farm-level risk factors were identified by multivariable logistic regression analysis. The associated factors retained in the final multivariable logistic regression model were drug treatment, presence of mosquitoes and flies, and frequency of disinfection. Drug treatment (OR = 0.24; 95% CI: 0.07, 0.88; P = 0.031) and frequency of disinfection (OR = 0.23; 95% CI: 0.06, 0.90; P = 0.035) were protective factors, and the presence of mosquitoes and flies (OR = 5.994; 95% CI: 1.56, 23.00; P = 0.009) was a risk factor for M. suis infection on farms. The results of the present study provide the first insight into the impact of associated determinants on M. suis infection in central China.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production,oxidative stress,and caspase-3 activation

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.     

Immunoregulatory effects of Taishan Pinus massoniana pollen polysaccharide on chicks co-infected with avian leukosis virus and Bordetella avium early in ovo

In recent years, co-infection of chicken embryos with immunosuppressive viruses and bacteria occurs with an annually increasing frequency. Consequently, studies on new and safe immunoregulators, especially plant polysaccharides, have become a popular topic in the poultry industry. In the present study, we selected 300 specific pathogen free embryonated eggs, which were injected with subgroup B avian leukosis virus (ALV-B) and Bordetella avium (B. avium) to establish an artificial co-infection model. The chicks that hatched from these co-infected embryonated eggs were treated with Taishan Pinus massoniana pollen polysaccharide (TPPPS). Results indicated that relevant indices in the co-infection group were significantly lower than that in B. avium-only group. Furthermore, pathogenicity of B. avium was exacerbated, with the chicks exhibiting decreased body weights. The TPPPS groups exhibited gradual improvements in immune function and developmental status. Therefore, in terms of improving immunologic function and production performance, TPPPS could be used as immunoregulator for immune responses.     

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ of the fungal isolates and observed after 48 h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10⁸, 10⁷ or 10⁶ conidia mg feces⁻¹. In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27 ± 0.5 °C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10⁸ conidia ml⁻¹. For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p < 0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p < 0.05) of the insect at every concentration of conidia.     

Pathogens of zoonotic and biological importance in roe deer (Capreolus capreolus): Seroprevalence in an agro-system population in France

Antibody prevalence for several infectious and parasitic diseases in a population of roe deer (Capreolus capreolus) inhabiting a mixed agricultural landscape (south of France) has been analyzed. Serological analyses with ELISA in 245 animals captured from 2008 to 2012 has been performed. We found a high prevalence of Toxoplasma gondii (46.4%), Chlamydophila abortus (17.27%) and Coxiella burnetii (11.26%) compared to other studies in Europe. Seroprevalence varied strongly among years for T. gondii (27–91%), C. abortus (0–42%) and C. burnetii (0–27%). T. gondii prevalence was lower in juvenile females, compared to juvenile males and adults of both sexes. Other pathogens had low prevalences: Neospora caninum (1.56%), Bovine herpesvirus 1 (1.17%, 2008/09; 1.1%, 2010/11), Mycoplasma agalactiae (1.45%, 2009/10), Mycobacterium avium subsp. paratuberculosis (0.9%) and Slow viruses (CAEV–MVV) (0.15%, 2008/10; 0%, 2011/12). Antibodies to bluetongue virus and pestiviruses were not found in any individual.     

Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.     

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: evaluation of cell respiration

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.     

Trypanosoma evansi Assessment in Equines: A Study in One Decade in an Endemic Area of the Canary Islands,Spain

Trypanosoma evansi was diagnosed in a camel in the Canary Islands for the first time in 1997, and the parasite is still present in a little area of the Archipelago. Since then, the disease has exclusively affected camel herds, and clinical evidence of the infection has not been demonstrated in any other animal hosts. In the frame of a control plan of the disease in the Archipelago, 864 equines located in the infected area were examined during one decade (2001-2010), of which a total of 26 (3%) were serologically positive but showed negative results at parasitological and molecular examinations. FreeCalc, an epidemiological tool to detect presence/absence of disease, showed that the population would be diseased at a confidence level of 99.916%. These results must suppose to implement the control plan against the disease including the equine population present in the surrounding area of the infected camel farms.     

Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress

In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC₅₀ being 13.8 ± 1.11 μg/mL. The effect of S-EtOH (1 μg/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55 ± 0.03 to 47.32 ± 2.25 (p < 0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90 ± 0.72 μM to 18.79 ± 0.32 μM (p < 0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95 ± 0.48 to 33.34 ± 1.66 (p < 0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5 ± 49.44 to 407.4 ± 12.61 (p < 0.01). Additionally, S-EtOH (14 μg/mL, 24 h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.