Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.     

The effect of an experimentally induced acute mastitis on the test results of an Ostertagia ostertagi milk ELISA

Antibody levels against Ostertagia ostertagi in the milk are a promising parameter to identify dairy cows or herds with production losses due to gastrointestinal nematodes. However, milk antibody levels can be influenced by udder-related factors. In this study, the effect of an experimentally induced mastitis on the test results (ODR) of a milk O. ostertagi ELISA was investigated. Twenty-five cows that were naturally infected with gastrointestinal nematodes, were inoculated in their left udder quarters with Escherichia coli P4:O32 and quarter milk samples were collected at several intervals from 24 h before until 144 h after experimental infection. The effect of the contribution of milk from one or more infected quarters on the bulk tank milk ODR was estimated, based on a titration experiment. The mean O. ostertagi antibody levels of the infected udder quarters were significantly (P < 0.001) higher than those of the uninfected udder quarters at each sampling time post-infection. The largest difference was observed at 24 h post-infection with a mean difference of 0.251 ODR (95%CI: 0.172; 0.330). There was also a significant increase (P < 0.001) in total IgG levels with the largest difference being observed at 24 h post-infection. Highly significant (P < 0.005) correlation coefficients were observed between O. ostertagi ODR, total IgG ODR, Na+ and Cl- ion concentration and log transformed somatic cell counts at 24 h post-infection. The results demonstrate that an acute mastitis causes a flow of specific and non-specific antibodies from serum to milk with a subsequent increase in the O. ostertagi ODR values. The effect of the contribution of milk from infected quarters on the bulk tank milk O. ostertagi ODR was estimated to be minor if the relative number of infected quarters is small (< 3%).     

Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vaccine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n = 14) or used as unvaccinated control (group B, n = 8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration.     

Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd

This report describes an outbreak of Mycoplasma bovis mastitis affecting 45 cows in a herd of 122 dairy cattle in Northern Italy. Clinically, the outbreak was characterized by agalactia, multiple swollen and painless quarters, high milk somatic cell count and unresponsiveness to conventional antibiotic therapy. M. bovis was isolated from the milk samples of all the 32 affected cows tested and from the mammary tissue of three affected cows that underwent necropsy. No other pathogens were isolated from these samples. Lesions in two of the necropsied cows were characterized by mild chronic suppurative mastitis and galactophoritis. The other necropsied cow showed a chronic necrosuppurative and pyogranulamaous galactophoritis, a condition not previously associated with M. bovis. M. bovis was detected immunohistochemically in the lumen of the affected mammary ducts suggesting that ascending infection via the teat canal was the likely route of transmission. No other intralesional pathogens were demonstrated microscopically.     

The kinetics of inflammation and phagocytosis during bovine mastitis induced by Streptococcus agalactiae bearing the protein X

The protein X of Streptococcus agalactiae is a surface antigen borne by a high proportion of strains isolated from bovine mastitis. We have tested the capacity of two strains of X-bearing Streptococcus agalactiae to induce mastitis in dairy cows. The reference X-strain (411.07) produced an intramammary infection with local clinical signs in the three inoculated quarters. Another X-bearing strain (443.31) of bovine origin produced infection in all 11 quarters inoculated with only 25 or 85 colony-forming units. In naive cows, strain 433.31 induced less exudation of plasma into the milk, shedding of bacteria, macroscopic alteration, and a lower somatic cell count (SCC) than did the reference strain. Only one quarter spontaneously eliminated the infection before antibiotic treatment 9 days after inoculation.The serum of all the cows contained naturally acquired or induced antibodies to the challenge strain (443.31) and possessed opsonic activity. Before inflammation occurred, the milk was almost devoid of antibody or opsonic activities. The early phase of infection was characterized by rapid multiplication of streptococci in the milk, followed by a sharp drop in bacterial counts concomitant with the onset of inflammation.Three cows immunized with protein X displayed higher SCC and bactericidal activity in milk from the inoculated quarter at the onset of inflammation than non-immunized cows. Two of the three immunized cows underwent an early and transient febrile episode and eliminated the infection.     

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T_h1 and T_h2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.     

Protective effect of CpG-DNA against mastitis induced by Escherichia coli infection in a rat model

A mastitis model in rats, induced by Escherichia coli infection, was established and the protective effect of Cytosine-phosphate-Guanosine (CpG)-DNA was determined. An E. coli suspension containing either 2 x 10(3) colony forming units (CFU)mL(-1)(EL group), 2 x 10(5)CFU mL(-1) (EH group), or (as controls) 100 microL phosphate buffer saline (CON group), was inoculated into the mammary glands 72 h after parturition. The rats were euthanased 24 h post-infection. The histopathological changes in mammary tissue in the EL group were mild, whereas the structural changes in the EH group were severe and polymorphonuclear leukocytes (PMNs) had accumulated in the mammary alveoli. Interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and N-acetyl-beta-d-glucosaminidase (NAGase) were significantly increased in the mammary tissue from the EH group but not significantly changed in the EL group. On the basis of these findings, the potential protective effect of CpG-DNA on mammary glands was tested using a 2 x 10(5)CFU mL(-1) suspension. An intramuscular injection of either CpG-DNA (200 microg) or PBS (100 microL) was given immediately after parturition. At 72 h post-partum, 2 x 10(5)CFU mL(-1)E. coli (100 microL) were inoculated into the mammary glands of all rats. At pre-infection (0 h), and 8, 16, 24, 48 and 72 h after inoculation six rats were euthanased. CpG-DNA induced more rapid migration of PMNs from the blood to mammary tissue at the initial stage of infection, stimulated the secretion of IL-6 and TNF-alpha at different time points, reduced viable E. coli in mammary tissues and decreased the activity of NAGase. CpG-DNA also promoted the expression of its specific receptor TLR-9 mRNA in mammary tissue. The study showed that CpG-DNA protected against E. coli mastitis in this rat model.     

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen

The nature of the local immune response was assessed studying the distribution of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes, IgM⁺ B cells, IL-4⁺ and IFN-γ⁺ cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-γ⁺ lymphoid cells was absent or very occasional in HLN where the number of IL-4⁺ cells was higher than that of IFN-γ, suggesting a polarized Th2 response in immunised and in infected control group.     

The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells

An investigation was undertaken to assess whether polyclonal convalescent and hyperimmune sera obtained from pigs inhibit Mycoplasma hyopneumoniae induced increases in intracellular calcium [Ca2+](i) in ciliated porcine tracheal cells. Basal [Ca2+](i) in the tracheal cells was 97+/-13 nM (n=22 cells in four experiments) and after exposure to M. hyopneumoniae (300 micro g/mL or 10(11) CCU/mL), [Ca2+](i) increased by 246+/-56 nM within 100 s. After pre-treatment with hyperimmune or convalescent serum, M. hyopneumoniae increased [Ca2+](i) by 196+/-43 and 223+/-65 nM, respectively. It was found that neither hyperimmune nor convalescent serum significantly prevented the increase in [Ca2+](i) compared with M. hyopneumoniae alone. It was concluded that polyclonal antibodies produced by mycoplasma vaccination or exposure to the pathogen do not prevent M. hyopneumoniae-induced increase in [Ca2+](i).     

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology,persistence of variable surface protein antigens and local immune response

Background

Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.

Methods

Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4⁺ and CD8⁺ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.

Results

Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4⁺ and CD8⁺ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.

Conclusions

The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.     

Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)₃. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine.     

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4⁺ and CD8⁺ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.     

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.     

Stimulation and analysis of the immune response in calves from vaccinated pregnant cows

The effect of vaccinating pregnant cows with an inactivated vaccine against Mannheimia haemolytica, BRSV and PI3V infections on selected immune responses in their offspring was examined. Blood samples were collected weekly for 12 weeks from six newborn calves from each of vaccinated (experimental) and unvaccinated (control) dams. Specific antibodies to M. haemolytica, BRSV and PI3V and mean values of IgA, IgG concentrations were significantly higher in the experimental calves compared with the controls. However, specific antibody titres to adenovirus type 3, BHV1 and BVDV in the experimental calves had constant levels while the control group levels changed. The IgM, Hp and SAA concentrations generally increased until week 8 in the experimental group, but the control group titres became higher after week 9. This study demonstrates that specific immunisation of cows pre-partum significantly stimulated parameters associated with immunity and it also controlled the acute phase response intensity in their offspring. Therefore the vaccination of dams may provide additional antibody protection against infection to their offspring.     

The efficacy of valnemulin (Econor) in the control of disease caused by experimental infection of calves with Mycoplasma bovis

Mycoplasma bovis infection was experimentally induced in groups of six young calves. A further group was uninfected and served as a control. Ten days after infection, medication with either enrofloxacin (Baytril, Bayer) or valnemulin (Econor, Novartis) was instituted via the milk replacer for a further 10 days, after which all calves were killed. Infection resulted in depression, pyrexia, inappetance and prominent respiratory signs. Arthritis occurred in two animals and two (unmedicated) animals died. At post-mortem examination extensive lesions were present in the lungs and M. bovis was re-isolated from infected unmedicated calves' lungs. Medication with either enrofloxacin or valnemulin resulted in a rapid diminution of clinical signs, restoration of appetite and reversal of weight loss. Isolation of Pasteurella multocida from the calves' lungs was suppressed by both medicaments. Valnemulin resulted in a more rapid reduction of clinical scores and eliminated M. bovis from the lungs more effectively than enrofloxacin.     

Protective immune responses induced by in ovo immunization with recombinant adenoviruses expressing spike (S1) glycoprotein of infectious bronchitis virus fused/co-administered with granulocyte-macrophage colony stimulating factor

Infectious bronchitis virus (IBV) causes tremendous economic losses associated with production inefficiencies and mortality in poultry industry worldwide. In the present report, the recombinant adenoviruses expressing chicken granulocyte-macrophage colony stimulating factor (GM-CSF) and S1 gene of nephropathogenic IBV were constructed and characterized. Then, the immunological efficacy and protection against homologous IBV challenge were assessed in specific pathogen free (SPF) chickens. The results showed that the chickens vaccinated in ovo with rAd-S1, rAd-GM-S1 (GM-CSF fused with S1 using glycine linkers) and rAd-GM-CSF plus rAd-S1 (co-administered) developed specific anti-IBV HI antibodies. Moreover, the fusion of the GM-CSF markedly increased spleen cell proliferation and IFN-γ production while mild increased in IL-4 production, which demonstrated the enhancement of cell-mediated immune responses. Following challenge with IBV, the chickens in the group vaccinated with rAd-S1 fused or co-administered with GM-CSF had fewer nephropathic lesions and showed 100% protection as compared to that of rAd-S1 alone which showed 70% protection. It indicated that the single dose in ovo vaccination of the GM-CSF fused or co-administered with S1 of IBV could enhance significantly the humoral, cellular immune responses and provide complete protection against nephropathogenic IBV challenge. This finding may provide basic information for effective in ovo vaccines design against IBV.     

Comparison of the immune responses induced by oral immunization of mice with Lactobacillus casei-expressing porcine parvovirus VP2 and VP2 fused to Escherichia coli heat-labile enterotoxin B subunit protein

The major structural protein VP2 of porcine parvovirus (PPV) was used as the model parvovirus antigen, which has been expressed in Lactobacillus casei fusing with Escherichia coli heat-labile enterotoxin B subunit (LTB) as mucosal adjuvant. The VP2-LTB DNA fragment was cloned into vector pPG611 or pPG612 to generated inducible surface-displayed and secretion expression systems based on xylose promoter, designated as rLc:pPG611-VP2-LTB (recombinant L. casei) and rLc:pPG612-VP2-LTB, respectively. Expression of the fusion protein was verified by SDS-PAGE, Western blot immunofluorescence and electron microscopy. It was observed that the level of IgG or sIgA from mice orally immunized with VP2-LTB was higher than that from mice received VP2 and negative control, which demonstrated significantly statistically different. Especially, the titer of IgG or sIgA in mice immunized with rLc:pPG612-VP2-LTB is the highest in this study. In summary, LTB as mucosal adjuvant was able to effectively facilitate induction of mucosal and systemic immunity by L. casei-expressing VP2 fusion protein.     

Canine babesiosis caused by Babesia canis vogeli in rural areas of the State of Minas Gerais, Brazil and factors associated with its seroprevalence

This epidemiological survey on canine babesiosis was carried out in three distinct rural regions (Lavras, Belo Horizonte and Nanuque) of the State of Minas Gerais, Brazil. Ticks and blood samples were collected during a dry season (Lavras, n = 92; Belo Horizonte, n = 50; Nanuque, n = 102) and the subsequent rainy season (Lavras, n = 71; Belo Horizonte, n = 28; Nanuque, n = 66) from dogs living on farms. Plasma samples were analyzed by the indirect fluorescent antibody test for detection of anti-Babesia canis vogeli antibodies. DNA was extracted from blood of serologically positive dogs and molecular characterization of Babesia species was performed. Rhipicephalus sanguineus, Amblyomma cajennense and Boophilus microplus were the tick species identified in all regions. In Lavras, in addition to those tick species, A. tigrinum and A. ovale were also identified. The most prevalent tick species was A. cajennense (35.3%), followed by R. sanguineus (19%) and B. microplus (4.0%). Dogs living in Nanuque region were more heavily infested with ticks than dogs living in Belo Horizonte and Lavras regions. The overall frequency of anti-B. c. vogeli antibodies in the canine population in rural areas of Minas Gerais was 28.7%, with prevalence rates of 49.0% in Nanuque, 34.0% in Belo Horizonte and 3.3% in Lavras. The age of the animals and tick infestation were associated with seroprevalence of B. c. vogeli. The sequence analysis showed that B. c. vogeli was the only Babesia species present in all three regions. This study showed different rates of prevalence and incidence of canine babesiosis among the three rural regions sampled in Minas Gerais State. The results point to the importance of canine babesiosis in rural areas and to the need for further studies related to its transmission and maintenance in nature.