Development of molecular assays for the analysis of genetic relationships of Mycoplasma iowae.

Mycoplasma iowae is a worldwide spread and economically important avian pathogen that mostly infects turkeys. Currently, multi-locus sequence typing (MLST) serves as the gold standard method for strain identification in M. iowae. However, additional robust genotyping methods are required to effectively monitor M. iowae infections and conduct epidemiological investigations. The first aim of this study was to develop genotyping assays with high resolution, that specifically target M. iowae, namely a multiple-locus variable number of tandem-repeats analysis (MLVA) and a core genome multi-locus sequence typing (cgMLST) schema. The second aim was the determination of relationships among a diverse selection of M. iowae strains and clinical isolates with a previous and the newly developed assays. The MLVA was designed based on the analyses of tandem-repeat (TR) regions in the six serotype reference strains (I, J, K, N, Q and R). The cgMLST schema was developed based on the coding sequences (CDSs) common in 95% of the examined 99 isolates. The samples were submitted for a previously published MLST assay for comparison with the developed methods. Out of 94 TR regions identified, 17 alleles were selected for further evaluation by PCR. Finally, seven alleles were chosen to establish the MLVA assay. Additionally, whole genome sequence analyses identified a total of 676 CDSs shared by 95% of the isolates, all of which were included into the developed cgMLST schema. The MLVA discriminated 19 distinct genotypes (GT), while with the cgMLST assay 79 sequence types (ST) could be determined with Simpson's diversity indices of 0.810 (MLVA) and 0.989 (cgMLST). The applied assays consistently identified the same main clusters among the diverse selection of isolates, thereby demonstrating their suitability for various genetic analyses and their ability to yield congruent results.

Isolation of Mycoplasma iowae in turkey flocks with skeletal abnormalities: a retrospective study.

Mycoplasma iowae, a pathogen affecting the turkey industry, is commonly associated with decreased hatchability and leg abnormalities in young progeny. This Mycoplasma was in the spotlight more in the past than today since its prevalence has been decreasing over time. Reports of M. iowae in turkeys showing reduced growth performances, leg problems and skeletal abnormalities are scarce although there is no report whether this pathogen has been completely eradicated in commercial turkeys. Additionally, there are no comprehensive epidemiological data available on M. iowae prevalence in any country. Therefore, we carried out a retrospective study to evaluate the prevalence of the infection and any correlation between necropsy findings and M. iowae presence in Italian turkeys between 2011 and 2012. Necropsy was performed on 101 dead turkey submissions presented for diagnostic purposes. Fifty-six submissions (55.4%) tested positive for M. iowae, most of which (69.6%) were between 4 and 7 weeks of age. Skeletal abnormalities were observed in 36 cases (35.6%). The logistic regression analysis revealed that the probability of finding a M. iowae-positive submission was four times higher if the animals showed skeletal abnormalities (OR = 4.48, IC 95%: 1.66-12.15). This is the first retrospective, cross-sectional study on M. iowae field outbreaks in commercial turkeys. These results suggest that M. iowae should be considered as a differential diagnosis when skeletal abnormalities are observed. RESEARCH HIGHLIGHTSM. iowae was found in more than half of the turkey groups analysed.M. iowae was likely to be detected if skeletal abnormalities were present in the studied turkeys.

Molecular Differentiation of Mycoplasma gallisepticum Outbreaks: A Last Decade Study on Italian Farms Using GTS and MLST.

Mycoplasma gallisepticum (MG) infects many avian species and leads to significant economic losses in the poultry industry. Transmission of this pathogen occurs both horizontally and vertically, and strategies to avoid the spread of MG rely on vaccination and the application of biosecurity measures to maintain breeder groups as pathogen-free. Two live attenuated MG vaccine strains are licensed in Italy: 6/85 and ts-11. After their introduction, the implementation of adequate genotyping tools became necessary to distinguish between field and vaccine strains and to guarantee proper infection monitoring activity. In this study, 40 Italian MG isolates collected between 2010-2019 from both vaccinated and unvaccinated farms were genotyped using gene-targeted sequencing (GTS) of the cythadesin gene mgc2 and multilocus sequence typing (MLST) based on six housekeeping genes. The discriminatory power of GTS typing ensures 6/85-like strain identification, but the technique does not allow the identification ts-11 strains; conversely, MLST differentiates both vaccine strains, describing more detailed interrelation structures. Our study describes MG genetic scenario within a mixed farming context. In conclusion, the use of adequate typing methods is essential to understand the evolutionary dynamics of MG strains in a particular area and to conduct epidemiological investigations in the avian population.

The pathogen Mycoplasma dispar Shows High Minimum Inhibitory Concentrations for Antimicrobials Commonly Used for Bovine Respiratory Disease.

Mycoplasma dispar is an overlooked pathogen often involved in bovine respiratory disease (BRD), which affects cattle around the world. BRD results in lost production and high treatment and prevention costs. Additionally, chronic therapies with multiple antimicrobials may lead to antimicrobial resistance. Data on antimicrobial susceptibility to M. dispar is limited so minimum inhibitory concentrations (MIC) of a range of antimicrobials routinely used in BRD were evaluated using a broth microdilution technique for 41 M. dispar isolates collected in Italy between 2011-2019. While all isolates had low MIC values for florfenicol (<1 µg/mL), many showed high MIC values for erythromycin (MIC90 ≥8 µg/mL). Tilmicosin MIC values were higher (MIC50 = 32 µg/mL) than those for tylosin (MIC50 = 0.25 µg/mL). Seven isolates had high MIC values for lincomycin, tilmicosin and tylosin (≥32 µg/mL). More, alarmingly, results showed more than half the strains had high MICs for enrofloxacin, a member of the fluoroquinolone class considered critically important in human health. A time-dependent progressive drift of enrofloxacin MICs towards high-concentration values was observed, indicative of an on-going selection process among the isolates.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma.

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL)-mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 L(d). Increase of H-2 L(d) expression by cDNA transfection (Sp6/B7/L(d)) raised tumour immune protection and shifted most CTL responses towards H-2 L(d)-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 L(d)-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/L(d) cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost.

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10(4)-10(-1)) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10(2) and 10(3)MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6weeks by real-time PCR.

Multiplexed typing of Mycobacterium avium subsp. paratuberculosis types I, II, and III by Luminex xMAP suspension array.

Differentiation among types I, II, and III is the primary step in typing Mycobacterium avium subsp. paratuberculosis. We propose an innovative approach based on detection of gyrase B (gyrB) gene polymorphisms by suspension array technology, with high discriminatory power and high-throughput potential.

Induction of an antitumour adaptive immune response elicited by tumour cells expressing de novo B7-1 mainly depends on the anatomical site of their delivery: the dose applied regulates the expansion of the response.

De novo expression of costimulatory molecules in tumours generally increases their immunogenicity, but does not always induce a protective response against the parental tumour. This issue was addressed in the mouse Sp6 hybridoma model, comparing different immunization routes (subcutaneous, intraperitoneal and intravenous) and doses (0.5 x 10(6) and 5 x 10(6) cells) of Sp6 cells expressing de novo B7-1 (Sp6/B7). The results can be summarized as follows. First, de novo expression of B7-1 rendered Sp6 immunogenic, as it significantly reduced the tumour incidence to < or =15% with all delivery routes and doses tested, whereas wild-type Sp6 was invariably tumorigenic (100% tumour incidence). Second, long-lasting protection against wild-type Sp6 was mainly achieved when immunization with Sp6/B7 was subcutaneous: a dose of 0.5 x 10(6) Sp6/B7 cells elicited protection that was confined to sites in the same anatomical quarter as the immunizing injection. Repeated injections of the same dose extended protection against wild-type Sp6 to other anatomical districts, as well as a single injection of a 10-fold higher dose (5 x 10(6) cells). Finally, Sp6-specific cytotoxic T-lymphocyte activity was detected in draining lymph nodes, and the splenic expansion of Sp6-specific cytotoxic T-lymphocyte precursors quantitatively correlated with the dose of antigen. We conclude that activation of a protective immune response against Sp6 depends on the local environment where the immunogenic form of the 'whole tumour cell antigen' is delivered. The antigen dose regulates the anatomical extent of the protective response.

Myelin basic protein epitopes secreted by human T cells encounter natural autoantibodies in the serum.

A previously isolated and characterized IgM monoclonal antibody (mAb 1H6.2) specific to myelin basic protein (MBP) and to MBP epitopes expressed by nonneural cells was used to immunoprecipitate and investigate the expression of MBP epitopes by human T cells. Peripheral T lymphocytes secreted MBP epitopes, and secretion increased in time after mitogen stimulation. Conversely, thymocytes secreted these proteins independently on mitogen stimulation. Specific antibody reactivity (primarily due to IgG3) towards immunoprecipitated MBP epitopes was found in all tested sera from healthy donors and from multiple sclerosis patients as well as in sera from normal human cord blood. Collectively, these data provide insights into the immunological mechanisms leading to central and peripheral tolerance to MBP products.