Investigation of the safety and protective efficacy of an attenuated and marker M. bovis-BoHV-1 combined vaccine in bovines.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry worldwide; it is caused by multiple bacterial or viral coinfections, of which Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens. Although live vaccines have demonstrated better efficacy against BRD induced by both pathogens, there are no combined live and marker vaccines. Therefore, we developed an attenuated and marker M. bovis-BoHV-1 combined vaccine based on the M. bovis HB150 and BoHV-1 gG-/tk- strain previously constructed in our lab and evaluated in rabbits. This study aimed to further evaluate its safety and protective efficacy in cattle using different antigen ratios. After immunization, all vaccinated cattle had a normal rectal temperature and mental status without respiratory symptoms. CD4+, CD8+, and CD19+ cells significantly increased in immunized cattle and induced higher humoral and cellular immune responses, and the expression of key cytokines such as IL-4, IL-12, TNF-α, and IFN-γ can be promoted after vaccination. The 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- combined strain elicited the most antibodies while significantly increasing IgG and cellular immunity after challenge. In conclusion, the M. bovis HB150 and BoHV-1 gG-/tk- combined strain was clinically safe and protective in calves; the mix of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its low amount of shedding and highest humoral and cellular immune responses compared with others. This study introduces an M. bovis-BoHV-1 combined vaccine for application in the cattle industry.

Diversity and antigenic potentials of Mycoplasmopsis bovis secreted and outer membrane proteins within a core genome of strains isolated from North American bison and cattle.

Mycoplasmopsis bovis is a worldwide economically important pathogen of cattle that can cause or indirectly contribute to bovine respiratory disease. M. bovis is also a primary etiological agent of respiratory disease in bison with high mortality rates. A major challenge in the development of an efficacious M. bovis vaccine is the design of antigens that contain both MHC-1 and MHC-2 T-cell epitopes, and that account for population level diversity within the species. Publicly available genomes and sequence read archive libraries of 381 M. bovis strains isolated from cattle (n = 202) and bison (n = 179) in North America were used to identify a core genome of 575 genes, including 38 that encode either known or predicted secreted or outer membrane proteins. The antigenic potentials of the proteins were characterized by the presence and strength of their T-cell epitopes, and their protein variant diversity at the population-level. The proteins had surprisingly low diversity and varying predictive levels of T-cell antigenicity. These results provide a reference for the selection or design of antigens for vaccine testing against strains infecting North American cattle and bison.

The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Enhanced Pathogenesis Caused by Influenza D Virus and Mycoplasma bovis Coinfection in Calves: a Disease Severity Linked with Overexpression of IFN-γ as a Key Player of the Enhanced Innate Immune Response in Lungs.

Bovine respiratory disease (BRD) is a major disease of young cattle whose etiology lies in complex interactions between pathogens and environmental and host factors. Despite a high frequency of codetection of respiratory pathogens in BRD, data on the molecular mechanisms and pathogenesis associated with viral and bacterial interactions are still limited. In this study, we investigated the effects of a coinfection with influenza D virus (IDV) and Mycoplasma bovis in cattle. Naive calves were infected by aerosol with a French IDV strain and an M. bovis strain. The combined infection shortened the incubation period, worsened the disease, and led to more severe macroscopic and microscopic lesions compared to these parameters in calves infected with only one pathogen. In addition, IDV promoted colonization of the lower respiratory tract (LRT) by M. bovis and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The gamma interferon (IFN-γ) gene was shown to be the gene most statistically overexpressed after coinfection at 2 days postinfection (dpi) and at least until 7 dpi, which correlated with the high level of lymphocytes in the LRT. Downregulation of the PACE4 and TMPRSS2 endoprotease genes was also highlighted, being a possible reason for the faster clearance of IDV in the lungs of coinfected animals. Taken together, our coinfection model with two respiratory pathogens that when present alone induce moderate clinical signs of disease was shown to increase the severity of the disease in young cattle and a strong transcriptomic innate immune response in the LRT, especially for IFN-γ. IMPORTANCE Bovine respiratory disease (BRD) is among the most prevalent diseases in young cattle. BRD is due to complex interactions between viruses and/or bacteria, most of which have a moderate individual pathogenicity. In this study, we showed that coinfection with influenza D virus (IDV) and Mycoplasma bovis increased the severity of the respiratory disease in calves in comparison with IDV or M. bovis infection. IDV promoted M. bovis colonization of the lower respiratory tract and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The IFN-γ gene in particular was highly overexpressed after coinfection, correlated with the disease severity, immune response, and white cell recruitment in the lungs. In conclusion, we showed that IDV facilitates coinfections within the BRD complex by modulating the local innate immune response, providing new insights into the mechanisms involved in severe respiratory diseases.

Effects of inflammatory stimuli on responses of macrophages to Mycoplasma bovis infection.

Inflammation in the respiratory tract is thought to worsen the disease response to Mycoplasma bovis infection. This study investigated the cells involved in this response with a focus on proteases and cytokines as harmful effector mechanisms. By immunohistochemistry, Mac387-positive macrophages were the main cell type comprising the foci of caseous necrosis in cattle with M. bovis pneumonia. Thus, the study evaluated how priming of different types of macrophages with bacterial lysate (or pro-inflammatory cytokines induced by the bacterial lysate) affected their responses to M. bovis infection. Inducible responses were detected in monocyte-derived macrophages (M1-MDMs and M2-MDMs), whereas pulmonary alveolar macrophages (PAMs) were minimally affected by priming or infection. M. bovis-infected MDMs secreted MMP-12 and SPLA2, and priming with pro-inflammatory cytokines increased the secretion of cathepsin B in response to M. bovis infection. Of these, there were higher concentrations of cathepsin B and SPLA2 in lungs with M. bovis pneumonia compared to healthy lungs, and these are potential mechanisms for macrophage-induced lung damage in M. bovis infection. Priming of MDMs with either bacterial lysate or with pro-inflammatory cytokines caused an enhanced response to M. bovis infection with respect to IL-8 and IL-1ß secretion. The findings of this study suggest proteases, lipases and cytokines derived from monocyte-derived macrophages as possible mediators by which prior inflammation in the respiratory tract worsen disease outcomes from M. bovis infection.

Transcriptome analysis of Mycoplasma bovis stimulated bovine peripheral blood mononuclear cells.

Mycoplasma bovis is a pathogenic bacterium in bovines that causes huge global economic losses. Numerous factors play important roles in M. bovis pathogenesis; however, the host immune response involved in M. bovis infection has not been fully elucidated. We aimed to determine the characteristics of the host immune response to Mycoplasma infection. We evaluated the responsiveness of bovine peripheral blood mononuclear cells (PBMCs) stimulated with M. bovis via microarray analysis. The transcriptional abundance of innate immune-related genes IL-36A, IL-27, IFN-γ, and IL-17 in PBMCs increased after M. bovis exposure. Upon M. bovis infection, there was increased expression of the lymphocyte activated genes basic leucine zipper transcription factor (BATF) and signaling lymphocytic activation molecule family members 1 and 7 (SLAMF 1 and SLAMF 7) in PBMCs compared with that in unstimulated cells. The study revealed that the transcriptional abundance of innate immunity genes in PBMCs increased during M. bovis infection. This induced the activation of PBMCs, giving rise to an immune response, which is followed by the development of the inflammatory response. The results from this study could be used as the basis for the development of novel vaccine candidates against M. bovis.

Mycoplasma bovis and other Mollicutes in replacement dairy heifers from Mycoplasma bovis-infected and uninfected herds: A 2-year longitudinal study.

Replacement dairy heifers exposed to Mycoplasma bovis as calves may be at risk of future clinical disease and pathogen transmission, both within and between herds; however, little information is available about these risks. We conducted a 2-yr longitudinal (panel) study starting with 450 heifer calves reared to weaning in 8 herds (7 M. bovis infected with clinical disease, 1 uninfected) under the same ownership. After weaning, heifers were commingled and managed with non-study heifers at a single heifer rearing facility. Nose, conjunctival, and vaginal swabs were collected along with a blood sample at weaning, prebreeding, precalving, and approximately 1 mo postcalving. Additionally, a colostrum sample was collected upon calving and a composite milk sample was collected 1 mo postcalving. The swabs, colostrum, and milk samples were cultured for Mycoplasma spp., and serum from the blood was evaluated for serological evidence of exposure to M. bovis using an ELISA. Despite a high M. bovis ELISA seroprevalence at weaning in the heifers from the 7 M. bovis-infected herds with clinical disease [72% (289/400); range by herd: 28-98%], M. bovis was isolated from only 4% (16/400) of the same heifers at the same time. In heifers from the uninfected herd at weaning, M. bovis seroprevalence was 2% (1/50) and M. bovis was not detected by culture. Mycoplasma bovis was isolated from 0.5% (2/414) of heifers at prebreeding, 0% (0/374) of heifers at precalving, and 0.3% (1/356) of heifers 1 mo postcalving. The nose was the predominant anatomical site of M. bovis colonization (74%; 14/19 culture positives). A single heifer (from an M. bovis-infected herd with clinical disease) was repeatedly detected with M. bovis in its nose at weaning, prebreeding, and postcalving samplings. This demonstrates the possibility, albeit rare, of a long-term M. bovis carrier state in replacement heifers exposed to M. bovis as calves, up to at least 1 mo after entry into the milking herd. No M. bovis clinical disease was detected in any heifer from weaning through to the end of the study (approximately 1 mo after calving). Acholeplasma spp. were commonly isolated throughout the study. Mycoplasma bovigenitalium, Mycoplasma bovoculi, and Mycoplasma bovirhinis were isolated infrequently. Mycoplasma bovis seroprevalences at prebreeding, precalving, and postcalving samplings were 27% (112/414), 12% (46/374), and 18% (65/356), respectively. Overall, the results show that replacement heifers from groups exposed to M. bovis preweaning can become colonized with M. bovis and that colonization can, uncommonly, be present after their first calving. For groups of 50 or more heifers exposed to M. bovis preweaning, there is at least a nontrivial probability that the group will contain at least 1 shedding heifer postcalving.

Development and application of a colloidal carbon test strip for the detection of antibodies against Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is an important bovine mycoplasma implicated in economically important clinical diseases, such as respiratory diseases, otitis media, and mastitis. The prevalence of M. bovis-associated mastitis in both cattle and buffaloes has been increasingly recognized as a global problem. High morbidity rates and consequential economic losses have been devastating to the affected cattle and buffalo farms, especially those in developing countries. Therefore, a rapid and accurate method is urgently needed to detect M. bovis. In this study, a rapid and simple lateral flow strip for detecting antibodies against M. bovis was established that used carbon nanoparticles (CNPs) as the labelled materials. The results from the test strip were highly consistent with those from ELISA. The test showed high specificity (100%) and no cross-reaction with other bovine pathogens. The detection sensitivity of the test was also relatively high (97.67%). All the results indicated that the colloidal carbon test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for detecting antibodies against M. bovis at cattle farms.

Cytokine levels of peripheral blood mononuclear cells in the clinical cases of Holstein calves infected with Mycoplasma bovis.

The immune related factors of peripheral blood mononuclear cells (PBMC) were analyzed in the clinical cases with Mycoplasma (M.) bovis infection. Seventy-eight Holstein calves in one farm were used. These calves were divided into three groups; the calves with M. bovis infection of poor outcome after treatment (Non-Recovery Group), the calves with M. bovis infection recovered (Recovery Group) and clinically healthy calves (Control Group). Blood samples were collected at days of the first medical treatment and the final treatment or euthanasia. IL-17A levels in the Non-Recovery Group were higher than those in the Recovery Group on both days. Our result suggested that the IL-17A of PBMC is an important factor to affect outcome of the calves with M. bovis infection.

Mycoplasma bovis: Interactions with the Immune System and Failure to Generate an Effective Immune Response.

Host responses are often ineffective at clearing Mycoplasma bovis infection and may contribute to the pathogenesis of disease. M bovis possesses a surprisingly large repertoire of strategies to evade and modulate host responses. Unopsonized M bovis impairs phagocytosis and killing by neutrophils and macrophages. Apoptosis of neutrophils and lymphocytes is enhanced, whereas it is delayed in macrophages. Both proinflammatory and antiinflammatory cytokines are stimulated during M bovis infection depending on the cell type and location, and overall systemic responses tend to have a T-helper 2 bias. M bovis reduces proliferation of T cells and, in chronic infection, causes T-cell exhaustion.

A European inter-laboratory trial to evaluate the performance of three serological methods for diagnosis of Mycoplasma bovis infection in cattle using latent class analysis.

BACKGROUND: Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer's instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. RESULTS: The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. CONCLUSIONS: The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.

Antibodies Specific to Membrane Proteins Are Effective in Complement-Mediated Killing of Mycoplasma bovis.

The metabolic inhibition (MI) test is a classic test for the identification of mycoplasmas, used for measuring the growth-inhibiting antibodies directed against acid-producing mycoplasmas, although their mechanism still remains obscure. To determine the major antigens involved in the immune killing of Mycoplasma bovis, we used a pulldown assay with anti-M. bovis antibodies as bait and identified nine major antigens. Among these antigens, we performed the MI test and determined that the growth of M. bovis could be inhibited effectively in the presence of complement by antibodies against specifically membrane protein P81 or UgpB in the presence of complement. Using a complement killing assay, we demonstrated that M. bovis can be killed directly by complement and that antibody-dependent complement-mediated killing is more effective than that by complement alone. Complement lysis and scanning electron microscopy results revealed M. bovis rupture in the presence of complement. Together, these results suggest that the metabolic inhibition of M. bovis is antibody-dependent complement-mediated killing. This study provides new insights into mycoplasma killing by the complement system and may guide future vaccine development studies for the treatment of mycoplasma infection. Furthermore, our findings also indicate that mycoplasmas may be an appropriate new model for studying the lytic activity of membrane attack complex (MAC).

Mycoplasma bovis delay in apoptosis of macrophages is accompanied by increased expression of anti-apoptotic genes, reduced cytochrome C translocation and inhibition of DNA fragmentation.

Bacterial pathogens have evolved to manipulate host cell death and survival pathways for their intracellular persistence. Understanding the ability of a bacterium to induce or inhibit cell death is essential for elucidating the disease pathogenesis and suggesting potential therapeutic options to manage the infection. In recent years, apoptosis inhibition by different bacteria has been suggested as a mechanism of survival by allowing the pathogen to replicate and disseminate in the host. Mycoplasma bovis has evolved mechanisms to invade and modulate apoptosis of bovine peripheral blood mononuclear cells (PBMC), red blood cells (RBCs), primary macrophages and monocytes. To date, these mechanisms are poorly understood. Using apoptosis assays such as Annexin V binding, caspases activity, reactive oxygen species production, DNA fragmentation and differential gene expression we set out to determine how M. bovis modulates macrophage survival. Using the BoMac cell line, we report a significant reduction in STS-induced apoptosis through caspase dependent manner. Besides activating the NF-kß pathway and inhibiting caspases 3, 6 and 9, M. bovis strain Mb1 also inhibits production of reactive oxygen species and DNA fragmentation of the host cell. We also report a significant up-regulation of the anti-apoptotic genes Bcl-2 and Bcl-XL upon infection. Our results indicate that M. bovis strain Mb1 inhibits the intrinsic pathway of apoptosis and up-regulate survival genes in BoMac cells.

Mycoplasma bovis antibody dynamics in naturally exposed dairy calves according to two diagnostic tests.

BACKGROUND: Inexpensive and convenient diagnostic tests for use in clinical work and for the surveillance of infection with Mycoplasma bovis are in demand. The objective of this longitudinal field study was to gain knowledge about the dynamics of antibodies against M. bovis in sera from naturally exposed calves with and without different clinical signs, measured by two different ELISA tests. RESULTS: A total of 83 calves were subject to between one and five blood samples and clinical examinations using a standard protocol during five herd visits to each of four outbreak dairy herds. The blood samples were analysed for the presence of antibodies against M. bovis using the commercial IgG ELISA test BioX K302 (BioX) and an in-house indirect IgG ELISA test (MilA ELISA). Linear mixed models were used to describe and compare the antibody dynamics as measured by the two tests in relation to the disease status and age of the animals. The BioX ELISA response was below the recommended cut-off (37 ODC%) for the entire study period in many of the calves. The estimated mean ODC% increased slowly but did not reach the recommended individual animal cut-off in three of the four herds. The highest estimated ODC% was not reached until the calf was 110-130 days old. The MilA ELISA response rose above the recommended cut-off (135 antibody units (AU)) in almost all calves, and in two herds, the estimated mean was above the individual animal cut-off shortly after the birth of the calf. The highest estimated antibody concentration was reached when the calf was approximately 60 days old. Disease status of the calf was not significantly associated with the results of either test. CONCLUSIONS: We conclude that the BioX ELISA cannot be recommended for use in calves below 3 months of age. The MilA ELISA was able to detect antibodies shortly after birth (i.e. from approximately 3 weeks of age and onwards) and is therefore a more sensitive test for M. bovis exposure in young calves. Neither ELISA seemed able to differentiate between calves with arthritis and/or otitis media, and respiratory disease.

Analysis of the immune response of calves to various saponin-based adjuvants for an experimental Mycoplasma bovis vaccine.

Mycoplasma bovis is a primary infectious agent of many disorders in cattle including bovine respiratory disease. No commercial vaccines against M. bovis are available in Europe. The immune response of calves to three saponin-based adjuvants combined with a field Polish M. bovis strain was evaluated. Four groups of six calves each were injected subcutaneously with the M. bovis strain combined with either saponin, saponin + Emulsigen®, saponin + Emulsigen® + alphatocopherol acetate, or with phosphate-buffered saline as control group. Blood and nasal swab samples were collected up to day 84 post injection. All formulations effectively stimulated the humoral and the cellular immune response of the calves, but the course of the response depended on the adjuvant formulation. These immunological data provide additional information supporting the findings of previous M. bovis saponin and Emulsigen® vaccine challenge studies to facilitate the development of successful M. bovis vaccines.

Isolation of Mycoplasma spp. and serological responses in bulls prior to and following their introduction into Mycoplasma bovis-infected dairy herds.

With the common use of bulls for breeding following a period of artificial insemination in seasonally bred dairy herds, it is important to consider the potential role of the bull in transmission of Mycoplasma spp. within and between herds. This study aimed to assess the prevalence of Mycoplasma spp. in a population of bulls before and after use in Mycoplasma bovis-infected herds. The frequency of subclinical infection was also measured serologically postbreeding, and the association of Mycoplasma spp. on semen quality was evaluated. Mycoplasma bovis was isolated from 4 of 118 bulls after use in 4 herds infected with M. bovis. In the bulls, M. bovis seroprevalence increased from 9% prebreeding to 46% postbreeding with a total seroconversion rate of 44% across the 4 herds, with no evidence of clinical disease. There was no association of Mycoplasma spp. in the bulls' semen and abnormal palpation characteristics (enlarged or nodular) of seminal vesicular glands or poor semen quality attributes such as semen mass activity, sperm motility, and morphology. These results demonstrate a high degree of subclinical exposure of the bulls to M. bovis in infected herds and highlight the potential for bulls to be mycoplasma carriers within and between herds. Herd biosecurity protocols and control programs should take into account the potential role of bulls in the introduction and spread of Mycoplasma spp.

A longitudinal observational study of the dynamics of Mycoplasma bovis antibodies in naturally exposed and diseased dairy cows.

Mycoplasma bovis is an important pathogen causing disease and substantial economic losses in cattle. However, knowledge of the dynamics of antibody responses in individual cows in the face of an outbreak is currently extremely limited. The use of commercial antibody tests to support clinical decision-making and for surveillance purposes is therefore challenging. Our objective was to describe the dynamics of M. bovis antibody responses in 4 Danish dairy herds experiencing an acute outbreak of M. bovis-associated disease, and to compare the antibody dynamics between dairy cows with different disease manifestations. A total of 120 cows were examined using a standardized clinical protocol and categorized into 4 disease groups: "mastitis," "systemic," "nonspecific," and "none." Paired blood and milk samples were collected and tested using a commercial M. bovis antibody-detecting ELISA. Plots of raw data and generalized additive mixed models with cow and herd as random effects were used to describe serum and milk antibody dynamics relative to the estimated time of onset of clinical disease. Cows with mastitis had high optical density measurement (ODC%) of antibodies in both milk and serum at disease onset. The estimated mean ODC% in milk was below the manufacturer's cut-off for the other groups for the entire study period. The estimated mean serum ODC% in the "systemic" group was high at onset of disease and stayed above the cut-off until 65 d after disease onset. However, the lower 95% confidence interval (CI) for the mean ODC% was only above the manufacturer's cut-off between 7 and 17 d after onset of disease. The CI of the "systemic" and "none" groups did not overlap at any time between the day of disease onset and 65 d after disease onset, and the estimated mean ODC% for both the "nonspecific" and "none" groups were generally below the cut-off for the majority of the study period. In conclusion, the serum antibody responses were highly dynamic and showed a high level of variation between individual cows. This strongly suggests that serology is unlikely to be useful for individual diagnosis of M. bovis-associated disease in dairy cows. However, it might still be useful for herd- or group-level diagnosis. Antibodies in milk were only increased in cows with M. bovis mastitis, indicating that milk antibody measurements only have diagnostic utility for cows with mastitis.

Interactive effects of dexamethasone and opsonized Mycoplasma bovis on bovine neutrophil function in vitro.

Previously we had reported that exposure to high levels of glucocorticoids, and to unopsonized Mycoplasma bovis, has a negative interactive effect on bovine neutrophil function in vitro, and this interactive effect was a function of M. bovis strain differences. Here we hypothesized that in vitro treatment of bovine neutrophils by glucocorticoid would impair phagocytosis of opsonized M. bovis compared to non-treated neutrophils and such impairment would be a function of M. bovis strain differences. Neutrophils isolated from 20 mid-lactation cows were treated with immunosuppressive dose of 5 × 10-4 M dexamethasone or placebo and incubated with one of four opsonized M. bovis strains that had been isolated from bovine origin. After incubation neutrophil function measured included: percentage reduction in log10 of M. bovis CFU/ml, percentage of phagocytizing neutrophils, phagocytized M. bovis per neutrophil, and killed M. bovis per neutrophil. Least square means of all neutrophil groups were contrasted using linear mixed-effects models. Effects due to strain, treatment, and their interaction on neutrophil function measured by the number of phagocytized M. bovis per neutrophil and number of killed M. bovis per neutrophil were different (P < 0.05). However, no significant strain by treatment interaction effect on percentage reduction in log10 of M. bovis CFU/ml was found. Neither a strain nor a strain by treatment interaction was found to affect the percentage phagocytizing neutrophils. These findings might explain in part the association of stressful events with subsequent outbreaks of Mycoplasma bovis associated bovine diseases.

Association of Circulating Transfer RNA fragments with antibody response to Mycoplasma bovis in beef cattle.

BACKGROUND: High throughput sequencing allows identification of small non-coding RNAs. Transfer RNA Fragments are a class of small non-coding RNAs, and have been identified as being involved in inhibition of gene expression. Given their role, it is possible they may be involved in mediating the infection-induced defense response in the host. Therefore, the objective of this study was to identify 5' transfer RNA fragments (tRF5s) associated with a serum antibody response to M. bovis in beef cattle. RESULTS: The tRF5s encoding alanine, glutamic acid, glycine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with antibody response against M. bovis. tRF5s encoding alanine, glutamine, glutamic acid, glycine, histidine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with season, which could be attributed to calf growth. There were interactions (P < 0.05) between antibody response to M. bovis and season for tRF5 encoding selenocysteine (anticodon UGA), proline (anticodon CGG), and glutamine (anticodon TTG). Selenocysteine is a rarely used amino acid that is incorporated into proteins by the opal stop codon (UGA), and its function is not well understood. CONCLUSIONS: Differential expression of tRF5s was identified between ELISA-positive and negative animals. Production of tRF5s may be associated with a host defense mechanism triggered by bacterial infection, or it may provide some advantage to a pathogen during infection of a host. Further studies are needed to establish if tRF5s could be used as a diagnostic marker of chronic exposure.

Short communication: Shedding of Mycoplasma bovis and antibody responses in cows recently diagnosed with clinical infection.

Mycoplasma bovis can have significant consequences when introduced into immunologically naïve dairy herds. Subclinically infected carrier animals are the most common way that M. bovis is introduced into herds. Although M. bovis udder infections can be detected by milk sampling lactating animals before their introduction, currently, no definitive way of identifying M. bovis carrier animals that are nonlactating (i.e., calves, heifers, dry cows, or bulls) is available. Understanding the prevalence of M. bovis shedding from various body sites in clinically infected animals could inform strategies for the detection of subclinical infection in nonlactating stock. The mucosal surfaces of the nose, eye, and vagina of 16 cows with recent clinical mastitis caused by M. bovis were examined for the presence of M. bovis shedding. Blood was collected for serological evaluation by a commercially available ELISA. Mycoplasma bovis was isolated from the vagina of only 3 (18.8%) of the cows and was not detected from the noses or eyes of any of the cows. Fifteen of the 16 (93.8%) cows were seropositive to the ELISA. With such low prevalence of detection of M. bovis from the vagina and no detections from the noses or eyes of recently clinically infected animals, it is very likely that sampling these sites would be ineffective for detecting subclinical infection in cattle. Serology using the ELISA may have some use when screening animals for biosecurity risk assessment. However, more information regarding time to seroconversion, antibody longevity, and test diagnostic sensitivity and specificity are required to define the appropriate use of this ELISA for biosecurity purposes.