Flow cytometry follow-up analysis of peripheral blood leukocyte subpopulations in calves experimentally infected with field isolates of Mycoplasma bovis

Changes in peripheral blood leukocyte subpopulations were investigated in calves challenged intratracheally with three different Mycoplasma bovis isolates in Groups E1, E2, and E3. The controls received a placebo. Blood samples were collected before challenge and then at days 1 to 7, 14, 21 and 28. White blood cells (WBC), polymorphonuclear leukocytes (PMNLs), lymphocytes (LYMs), monocytes, eosinophils and basophils (mid-size cells, MID), as well as CD2+, CD4+, CD8+, WC4+ lymphocyte subsets with CD4:CD8 ratio were also analysed. A transient increase of WBC and PMNLs in all challenged calves was observed on day 1. Increased LYM counts were observed in E1 throughout the study, whereas in E2 the LYM counts were higher only between days 14 and 28, and consistently lower in E3. The MID count had broadly comparable values for all groups. Stimulation of the CD2+ response was observed in E2 and E3 in contrast with E1 which had a lower CD2+ throughout. The CD4+ response was dominant in E1 and E2, whereas in E3 a parallel CD4+ and CD8+ stimulation was observed. The B-cell response (WC4+) and an increased CD4:CD8 ratio was most apparent in E1. The main host responses to M. bovis infections are a stimulation of CD4+ cells and an enhancement of the WC4+ response

VNTR analysis reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of contagious agalactia

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma agalactiae </it>is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. Previous studies of <it>M. agalactiae </it>have shown it to be unusually homogeneous and there are currently no available epidemiological techniques which enable a high degree of strain differentiation.</p> <p>Results</p> <p>We have developed variable number tandem repeat (VNTR) analysis using the sequenced genome of the <it>M. agalactiae </it>type strain PG2. The PG2 genome was found to be replete with tandem repeat sequences and 4 were chosen for further analysis. VNTR 5 was located within the hypothetical protein MAG6170 a predicted lipoprotein. VNTR 14 was intergenic between the hypothetical protein MAG3350 and the hypothetical protein MAG3340. VNTR 17 was intergenic between the hypothetical protein MAG4060 and the hypothetical protein MAG4070 and VNTR 19 spanned the 5' end of the pseudogene for a lipoprotein MAG4310 and the 3' end of the hypothetical lipoprotein MAG4320.</p> <p>We have investigated the genetic diversity of 88 <it>M. agalactiae </it>isolates of wide geographic origin using VNTR analysis and compared it with pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. Simpson's index of diversity was calculated to be 0.324 for PFGE and 0.574 for VNTR analysis. VNTR analysis revealed unexpected diversity within <it>M. agalactiae </it>with 9 different VNTR types discovered. Some correlation was found between geographical origin and the VNTR type of the isolates.</p> <p>Conclusion</p> <p>VNTR analysis represents a useful, rapid first-line test for use in molecular epidemiological analysis of <it>M. agalactiae </it>for outbreak tracing and control.</p

<i>Mycoplasma ovipneumoniae </i>- A Primary Cause of Severe Pneumonia Epizootics in the Norwegian Muskox (<i>Ovibos moschatus</i>) Population

The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004-2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection

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

A Novel Rapid DNA Microarray Assay Enables Identification of 37 Mycoplasma Species and Highlights Multiple Mycoplasma Infections

Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment

Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae isolated from seven European countries during 2015-2016

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease, causing significant economic losses. Results from the 2015-2016 MycoPath pan-European antimicrobial susceptibility monitoring survey of M. hyopneumoniae are presented. In total, 147 M. hyopneumoniae porcine isolates from Belgium, France, Germany, Great Britain, Hungary, Italy, and Spain were tested. One isolate per farm was retained from pigs that had not been recently treated with antimicrobial agents. The minimal inhibitory concentration (MIC) of 13 antimicrobial agents was determined in a central laboratory using a broth microdilution method, with Friis Medium, incubated at 35 +/- 1 degrees C for 5-12 days. M. hyopneumoniae NCTC 10110 was used as Quality Control. MIC50/MIC90 (mg/L) values were: enrofloxacin 0.06/1; marbofloxacin 0.06/2; spiramycin 0.06/0.25; tulathromycin <= 0.001/0.004; gamithromycin 0.06/0.5; tylosin 0.016/0.06; tilmicosin 0.06/0.5; florfenicol 0.5/1; doxycycline 0.25/1; oxytetracycline 0.25/2; lincomycin 0.06/0.25; tiamulin 0.016/0.06 and valnemulin <= 0.001/0.004. Compared with the data from 2010 to 2012 MycoPath study (50 isolates), MIC50/90 results were similar and the majority were within +/- two dilution steps, except for the MIC50 of oxytetracycline which is more than two dilution steps higher in the present study. Between-country comparisons show some differences in the MIC values for the fluoroquinolones, tulathromycin and tylosin, but the limited sample size per country precludes performing meaningful country comparisons for several countries. Standardized laboratory methods and interpretive criteria for MIC testing of veterinary mycoplasmas are clearly needed; there are currently no clinical breakpoints available to facilitate data interpretation and correlation of MICs with in vivo efficacy

Mycoplasma bovis: mechanisms of resistance and trends in antimicrobial susceptibility

Mycoplasma bovis is a cell-wall-less bacterium and belongs to the class Mollicutes. It is the most important etiological agent of bovine mycoplasmoses in North America and Europe, causing respiratory disease, mastitis, otitis media, arthritis, and reproductive disease. Clinical disease associated with M. bovis is often chronic, debilitating, and poorly responsive to antimicrobial therapy, resulting in significant economic loss, the full extent of which is difficult to estimate. Until M. bovis vaccines are universally available, sanitary control measures and antimicrobial treatment are the only approaches that can be used in attempts to control M. bovis infections. However, in vitro studies show that many of the current M. bovis isolates circulating in Europe have high minimum inhibitory concentrations (MIC) for many of the commercially available antimicrobials. In this review we summarize the current MIC trends indicating the development of antimicrobial resistance in M. bovis as well as the known molecular mechanisms by which resistance is acquired