MS Strains Research Articles

Pathogenic bacteria associated with outbreaks of respiratory disease in Iranian broiler farms.

Multi-causal respiratory infections are more commonly observed than uncomplicated cases with single agents in the commercial poultry industry. Recently, increased mortality rates associated with respiratory clinical signs have been reported in Iranian broiler farms. The present study aimed to determine the spectra of avian mycoplasmas (Mycoplasma gallisepticum, MG and Mycoplasma synoviae, MS) and Ornithobacterium rhinotracheale (ORT) in the broiler farms with the multi-causal respiratory disease (MCRD) from 2017 to 2020. Trachea and lung tissue samples were collected from 70 broiler flocks presenting increased mortality and acute respiratory disease. MG, MS, and ORT were detected by performing polymerase chain reaction with primers complementary to the 16S rRNA, vlhA, and 16S rRNA genes, respectively. Genetic materials of MG, MS, and ORT were detected in five, three, and five of the 70 flocks. Based on the phylogenetic analysis of the complete mgc2 coding sequences, all MG strains formed a distinct cluster along with other Iranian MG isolates. According to the phylogenetic analysis of the partial vlhA gene of MS strains, two isolates were located along with Australian and European strains. In addition, one of them displayed an out-group association with MS isolates from Jordan. Phylogenetic analysis of Iranian ORT strains using a partial sequence of the 16S rRNA gene showed a distinct group among the other ORT strains. The results indicate that MG, MS, and ORT are not predominantly responsible for the MCRD. However, continuous monitoring of poultry flocks could be significant for obtaining valuable information related to different MG, MS, and ORT strains and designing effective control strategies.

Lateral metabolome study reveals the molecular mechanism of cytoplasmic male sterility (CMS) in Chinese cabbage

BackgroundChinese cabbage is one of the most widely grown leafy vegetables in China. Cytoplasmic male sterility (CMS) is a maternally inherited trait that produces abnormal pollen during anther development, which is commonly seen in cruciferous vegetables. However, the molecular mechanism of Chinese cabbage CMS is not clear. In this study, the metabolome and hormone profiles of Chinese cabbage male sterile line (CCR20000) and sterile maintainer line (CCR20001) were analyzed in flower buds during normal stamen development and abnormal stamen development, respectively.ResultsA total of 556 metabolites were detected based on UPLC-MS/MS detection platform and database search, and the changes of hormones such as auxin, cytokinins, abscisic acid, jasmonates, salicylic acid, gibberellin acid and ethylene were analyzed. The results showed that compared with the male fertile line (MF), the male sterile line (MS) significantly decreased the content of flavonoids and phenolamides metabolites in the stamen dysplasia stage, accompanied by a large accumulation of glucosinolate metabolites. Meanwhile, the contents of GA9, GA20, IBA, tZ and other hormones in MS were significantly lower than those in MF strains. Further, by comparing the metabolome changes of MF and MS during stamen dysplasia, it was found that flavonoid metabolites and amino acid metabolites were distinctly different.ConclusionsThese results suggest that flavonoids, phenolamides and glucosinolate metabolites may be closely related to the sterility of MS strains. This study provides an effective basis for further research on the molecular mechanism of CMS in Chinese cabbage.

Investigation on eggshell apex abnormality (EAA) syndrome in France: isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum

BackgroundMycoplasma synoviae (MS) is known to cause Eggshell Apex Abnormality (EAA) syndrome characterized by an altered shell surface with increased translucency on the apex. However, no large-scale studies have been conducted to obtain prevalence data of EAA and MS isolates associated to this syndrome. This manuscript reports the results of two field studies performed in the French poultry industry (2015–2017): focusing mainly on investigation of presence and prevalence of EAA in different types of laying hen flocks (phase 1), and isolation of MS strains from EAA-infected flocks (phase 2).ResultsThe first survey included 77 farms of commercial layers in three French egg-production regions, hosting 40 flocks in alternative systems (ALT) and 56 in furnished cages (FC). Seven flocks (4 FC and 3 ALT) presented EAA clinical signs, giving a prevalence of 7.3% in this studied sample. A second independent field study was conducted to identify MS by in vitro cultivation and PCR in samples from 28 flocks with clinical signs of EAA. Different types of biological specimens were collected in EAA-affected flocks and submitted to the laboratory. M. synoviae was detected in 25/28 flocks, from both production systems (5/5 ALT and 20/23 FC). Detection of MS was significantly higher in tracheal swabs (59%) than in cloacal (10.5%), albumen (3.6%) and egg yolk (1.1%) swabs. It is worth to mention that attempts to clone MS from positive samples were often hampered by the presence of another Mycoplasma species, which showed fast growing behaviour in the selective media used in this study (Frey Medium 4 and Frey Medium 4 supplemented with erythromycin). The use of MALDI-TOF mass spectrometry in combination with next-generation sequencing (NGS) results allowed the identification of this fast growing mycoplasma as Mycoplasma pullorum, which was detected in 14 of the 25 (56%) MS-positive flocks.ConclusionsThese results confirmed the presence of the EAA syndrome in MS-positive flocks of layers in France, reared in different regions and in different production systems (ALT and FC). Studies need to be conducted to test whether M. pullorum may influence the expression of clinical signs of EAA in MS-infected layer farms.

Analysis of Transcriptome of Drosophila melanogaster Strains with Disrupted Control of gypsy Retrotransposon Transposition

To study the causes of impaired control of the activity of mobile genetic elements in the strains with the flamenco phenotype SS (w, flamenco mutant) and MS (w, flamenco mutant, active copy of gypsy), sequencing of these transcriptomes was performed. The D32 strain was used as control (laboratory wild type strain). An algorithm was developed for the search for amino acid substitutions in high-throughput RNA sequencing data using a triplet code for analysis. With the help of this algorithm, seven nonsense mutations were detected. The allele-specific PCR method confirmed the presence of five of the seven nonsense mutations found in silico. However, the detected nonsense mutations are not associated with the flamenco phenotype. A search for mutations in 89 genes of the RNA interference system in the SS and MS strains relative to the reference BDGP6 genome and the wild type D32 strain was carried out. No deletions, insertions, nonsense codons, and other disorders that can unambiguously lead to a change in the function of the gene are detected. To identify genes with specific expression for the strains with the flamenco phenotype, the transcriptomes of the SS and MS strains were compared with the control strains D-32, OregonR, and w1118. A set of 25 genes with differential expression was identified, among which two genes, sosie and CR45822, significantly changed the expression in the SS and MS strains. Both genes, directly or indirectly, are involved in oogenesis. Thus, the expression of the sosie and CR45822 genes can be used as a marker of the flamenco phenotype in the SS and MS strains.

Identification and characterization of linear B cell epitopes on the nucleocapsid protein of porcine epidemic diarrhea virus using monoclonal antibodies

The nucleocapsid (N) protein of porcine epidemic diarrhea virus (PEDV), the most important pathogen causing severe diarrhea in piglets, is a highly conserved structural protein. In this study, 5 monoclonal antibodies (McAbs) against the PEDV N-protein were prepared and identified. Three new epitopes, 56QIRWRMRRGERI67, 318GYAQIASLAPNVAALLFGGNVA VRE342 and 398HEEAIYDDV406, were firstly identified in the viral N-protein, by using McAbs 3F10, 6A11, and 1C9. The epitope 398HEEAIYDDV406 was deleted in SH strain (isolated by our lab) and different between CV777 and YZ strain (isolated by our lab). To study the characters of this epitope, four peptides were synthesized according to the sequence of SH and CV777 and used in the study. The result showed that the 398th amino acid maybe an important amino acid of the epitope. Biological information analysis showed that the three B cell linear epitopes are highly conserved among different PEDV isolates. In addition, McAb 1C9, which attached to the epitope 398HEEAIYDDV406, showed variant reactivity with PEDV CV777, SH, YZ and MS strains. McAb 1C9 reacted with PEDV strains CV777 and YZ, but not with SH which had a deletion from 399 to 410 amino acids in N-protein (No. MK841494). Among the three McAbs, 6A11, 3F10 and 1C9, only 6A11 reacted with porcine transmissible gastroenteritis virus (TGEV) in immunofluorescence assay, therefore the other two could be used to distinguish TGEV and PEDV. These mAbs and their defined epitopes may provide useful tool for the study of the PEDV N-protein structure and function, and facilitate the development of diagnostic methods for PEDV.

Application of different laboratory techniques to monitor the behaviour of a Mycoplasma synoviae vaccine (MS-H) in broiler breeders

BackgroundMycoplasma synoviae (MS) is a major poultry pathogen which causes severe economic losses in all the productive sectors. The prevalence of MS in European countries has increased in the last few years, leading to greater attention to the available methods to prevent its spread. The main strategy currently applied for its containment is the development and maintenance of MS-free breeder flocks. A live MS vaccine (MS-H) obtained by mutagenizing an Australian field strain has recently been introduced in Italy. The aim of the present study was to evaluate the vaccine behaviour in broiler breeder groups at different production stages and the effectiveness of the available laboratory tests in discriminating the MS-H from a field strain.ResultsThe vaccine diffused extensively through the population, shown by the wide serological response (over 80% of positive samples in RSA and 85% in ELISA), the high serological titres, the positivity of all the tracheal samples collected during the production phase by MS PCR and the positivity by cultivation from tracheal swabs at the end-point (55 weeks after vaccination). In contrast, only one swab from a sternal bursa was positive in MS PCR, while all the joint and oviduct samples were negative. There was no evidence of vertical transmission. Different genotyping techniques were used to achieve a clear classification of the MS positive samples. The vlhA and the obg gene analysis showed that most of the strains were homologous with the vaccine, but some ambiguous samples were further investigated with the multi locus sequence typing (MLST) scheme which confirmed the homology.ConclusionsThe development of a multi-technique approach to monitor vaccinated avian flocks, based both on serological and biomolecular methods, is advised as well as the use of effective genotyping techniques to analyse the MS strains circulating in high densely populated poultry areas.

Methionine sulfoxide reductase A of Salmonella Typhimurium interacts with several proteins and abets in its colonization in the chicken

Defending phagocyte generated oxidants is the key for survival of Salmonella Typhimurium (S. Typhimurium) inside the host. Met residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO). Methionine sulfoxide reductase (Msr) can repair Met-SO to Met thus restoring the function(s) of Met-SO containing proteins. Using pull down method we have identified several MsrA interacting proteins in the S. Typhimurium, one of them was malate synthase (MS). MS is an enzyme of glyoxylate cycle. This cycle is essential for survival of S. Typhimurium inside the host under nutrient limiting conditions. By employing in vitro cross-linking and blot overlay techniques we showed that purified MsrA interacted with pure MS. Treatment of pure malate synthase with H2O2 resulted in reduction of MS activity. However, MsrA along with thioredoxin-thioredoxin reductase system partially restored the activity of oxidized MS. Our mass spectrometry data demonstrated H2O2 mediated oxidation and MsrA mediated repair of Met residues in MS. Further in comparison to S. Typhimurium, the msrA gene deletion (∆msrA) strain showed reduced (60%) malate synthase specific activity. Oral inoculation with wild type, ∆msrA and ∆ms strains of S. Typhimurium resulted in colonization of 100, 0 and 40% of the poultry respectively.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5′ end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

Children with severe early childhood caries: streptococci genetic strains within carious and white spot lesions

Background and objectivesMutans streptococci (MS) are one of the major microbiological determinants of dental caries. The objectives of this study are to identify distinct MS and non-MS streptococci strains that are located at carious sites and non-carious enamel surfaces in children with severe early childhood caries (S-ECC), and assess if cariogenic MS and non-cariogenic streptococci might independently exist as primary bacterial strains on distinct sites within the dentition of individual children.DesignDental plaque from children (N=20; aged 3–6) with S-ECC was collected from carious lesions (CLs), white spot lesions (WSLs) and non-carious enamel surfaces. Streptococcal isolates (N=10–20) from each site were subjected to polymerase chain reaction (PCR) to identify MS, and arbitrarily primed-PCR for assignment of genetic strains. Primary strains were identified as ≥50% of the total isolates surveyed at any site. In several cases, strains were characterized for acidurity using ATP-driven bioluminescence and subjected to PCR-determination of potential MS virulence products. Identification of non-MS was determined by 16S rRNA gene sequencing.ResultsSixty-four independent MS or non-MS streptococcal strains were identified. All children contained 1–6 strains. In many patients (N=11), single primary MS strains were identified throughout the dentition. In other patients (N=4), primary MS strains were identified within CLs that were distinct from primary strains found on enamel. Streptococcus gordonii strains were identified as primary strains on enamel or WSLs in four children, and in general were less aciduric than MS strains.ConclusionsMany children with S-ECC contained only a single primary MS strain that was present in both carious and non-carious sites. In some cases, MS and non-cariogenic S. gordonii strains were found to independently exist as dominant strains at different locations within the dentition of individual children, and the aciduric potential of these strains may influence susceptibility in the development of CLs.

Analysis of genetic complementation between the flamenco and piwi loci in Drosophila melanogaster

Previous studies indicate that the heterochromatic flamenco locus, which regulates transposition of mobile genetic element gypsy, is the source of antisense RNA interacting with the piwi locus. The Piwi protein in Drosophila melanogaster is an essential component in the RNA interference mechanism. Interestingly, mutations in piwi and flamenco produce the same phenotype: enhanced gypsy transcription and frequency of transposition. To clarify the relationship between piwi and flamenco, we performed a genetic complementation screen between MS and SS drosophila strains, mutant for flamenco, and piwi3 strain, heterozygous for piwi gene. Measurements of gypsy transcript levels in gonads of hybrid offspring uncovered complementation between the flamenco and piwi alleles in the testes. In addition, we found that gypsy transcription is regulated differently in the testis and ovaries in the SS and MS parental strains. We propose that this effect is due to a difference in activity of Piwi-interacting RNA clusters in the male and female gonads.

The effect of galactan content on the mechano-sorptive strain in loblolly pine

Abstract Mechano-sorptive (MS) strains were measured in torsion on eight loblolly pine specimens as a function of galactan content. The strains were generated by an increase of environmental humidity from 38% to 84% and were measured during an anticlockwise and a clockwise loading scheme that caused the specimen to recover its original dimensions at the end of the test. The magnitude of the MS strains was found to be independent of galactan concentration. The specimens with high and low galactan contents had different MS strains but the average of the measured data was the same. The number of observations (eight) was too small for a reliable statistical analysis to identify the regressors that are the main sources of the strain variations. The cell structure of the specimens was visualised by X-ray tomography.

Retrotransposons IDEFIX and ZAM are responsible for transposition in Drosophila melanogaster MS strain mutant for the flamenco gene

Transposition activity of Drosophila melanogaster gypsy retrotransposon is controlled by the flamenco locus. Transposition activity of the gypsy, ZAM, Idefix, springer, nomad, rover, Quasimodo, 17.6, 297, and Tirant retrotransposons was investigated in isogenic SS and MS strains of D. melanogaster mutant for the flamenco gene. It has been shown that gypsy, ZAM, and Idefix have different genomic surrounding in the studied strains that evidences to their transposition in these strains.

Mycoplasma synoviae cell invasion: Elucidation of the Mycoplasma pathogenesis in chicken

Fluorochrome-labelled cells of two field isolates and Mycoplasma synoviae (Ms) were inoculated onto monolayer cultures of fluorochrome-labelled HEp-2 cells and monitored by confocal laser scanning microscopy (CLSM). Ms was detected initially adhered to and subsequently inside the host cells. Between 24 and 48 h of infection, Ms was detected in the perinuclear region, and after 72 h of infection was confirmed by gentamicin invasion assay. High and low passage Ms strains showed no differences in adherence or invasion. The morphology and the actin filaments of the infected HEp-2 cells were preserved throughout the study period. The observed invasion by Ms is consistent with the biology of Mollicutes, and could explain the difficulties in recovering field isolates of the mycoplasma and in controlling the infection in birds even after long-term antibiotic treatment.

Comparative Evaluation of Tigecycline and other Antimicrobials in the United States Against Multi-drug Resistant (MDR) Staphylococcus aureus

Objectives: Worldwide methicillin-resistant S.aureus (MRSA) are increasing dramatically with corresponding resistance to multiple drug classes (MDR). Tigecycline, a new glycylcycline offers the potential of enhanced activity against MDR S. aureus. The tigecycline evaluation surveillance trial (T.E.S.T.) evaluated the activity of tigecycline and comparators to MDR S. aureus(including MR + MS strains) isolated in the United States. Methods: 193 hospital sites in the United States, between 2004 and 2007, collected 5280 clinically significant S. aureus. MICs were performed as specified by CLSI at each site. Results: MIC90 of tigecycline and comparators to S. aureus resistant groups 0–3+ are shown in the following table: Conclusions: Tigecycline demonstrated in vitro activity comparable to that of linezolid and vancomycin against all multidrug resistant groups of S. aureus. Tigecycline in vitro activity is unaffected by multidrug resistant phenotypes of S. aureus and offers an alternative in the treatment of infections due to S. aureus where approved indications apply.

A comparative study of host response to three Mycobacterium tuberculosis PE_PGRS proteins

Three Mycobacterium tuberculosis proteins, PE_PGRS 16 (Rv0977), PE_PGRS 26 (Rv1441c) and PE_PGRS 33 (Rv1818c), were expressed in Mycobacterium smegmatis and used to investigate the host response to members of this unique protein family. Following infection of macrophages with the recombinant M. smegmatis (Ms) strains, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 were significantly more persistent (4.4 and 4.2 log c.f.u.) compared with Ms-PE_PGRS 16 (3.4 log c.f.u.) at day 6. Similarly, after infection of mice, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 persisted at significantly higher levels in the spleen (3.5 and 3.2 log c.f.u.) and liver (3 and 2.6 log c.f.u.) compared with Ms-PE_PGRS 16 in the spleen (2 log c.f.u.) and in the liver (1 log c.f.u.) at day 10. Increased persistence of Ms-PE_PGRS 33 and Ms-PE_PGRS 26 was associated with cell death and increased release of lactate dehydrogenase in macrophage cultures as well as increased levels of IL-10 and, in contrast, lower levels of IL-12 and NO both in vitro and in mouse splenocytes. Conversely, poor survival of Ms-PE_PGRS 16 was associated both in macrophage cultures and in vivo with higher levels of NO and IL-12. All three PE_PGRS proteins were found to be cell-surface antigens, but immunization of mice with these PE_PGRS antigens as DNA vaccines showed no protection in a TB aerosol challenge model. In general, the results suggest that variable expression of different PE_PGRS proteins within host cells can affect either the fate of the mycobacterial pathogen or that of the host during infection and point to the importance of studying the expression and function of individual members of the PE_PGRS gene family of M. tuberculosis.

The recovery of mechano-sorptive creep strains

Mechano-sorptive strain was generated in strips of rectangular section stressed in torsion with the humidity cycled between 38% and 84%. After a number of humidity cycles the specimen was unloaded, the applied stress was reversed and further humidity cycles were imposed. All the initial strain was recovered and reversed strain was developed. Tests were carried out at two maximum shear stress levels, 1.12 MPa and 5.05 MPa. After seven humidity cycles at the higher stress a large mechano-sorptive strain of about 0.06 was developed, which was nearly nine times the initial elastic strain. During the development of this strain no significant change of the shear modulus occurred, from which it is concluded that the large MS strain produces no permanent change of the features of the cell wall structure that determine the shear modulus. There are close parallels between the mechanism generating the MS strains and that responsible for the large plastic strains reported by Keckes et al. in wet compression wood loaded in tension. In both cases it is suggested that the shear strains arise from the breaking and reforming of hydrogen bonds between parallel polymer chains.

Peroxidase Reaction as a Parameter for Discrimination of Streptococcus mutans and Streptococcus sobrinus

425 strains of mutans streptococci and 12 reference strains were investigated by membrane fatty acid spectra (MFAS) and peroxidase reaction (PR) after aerobic and anaerobic incubation. 423 strains were identified as Streptococcus mutans. The remaining 2 strains were identified as Streptococcus sobrinus. The PR of 29 strains was doubtful; immediately after anaerobic incubation a negative PR changed into a slightly positive PR. To test the diagnostic value of PR the strains were additionally investigated by means of species–specific polymerase chain reactions (PCR). The species–specific PCRs were developed on the basis of the respective genes of 16S rRNA of the pathogens S. mutans and S. sobrinus. Specificity and sensitivity were tested on reference strains (n = 17) and negative control strains (n = 39). The results of this investigation showed that an anaerobic incubation regime could lead to false–positive (S. mutans) or false–negative (S. sobrinus) PR. The 425 MS strains were classified as either S. mutans (n = 420) or S. sobrinus (n = 5). The findings on the reference strains required a reclassification of S. mutans V 100 into S. sobrinus V 100. Summarising, it is possible now to differentiate strains of mutans streptococci by MFAS and PR after aerobic incubation.

In vitro evaluation of various antimicrobials against Mycoplasma gallisepticum and Mycoplasma synoviae by the micro‐broth method, and comparison with a commercially‐prepared test system

The efficacy of danofloxacin, a new quinolone antimicrobial agent, was tested in vitro by the micro-broth method with nine field strains of Mycoplasma gallisepticum (Mg) and eight of M. synoviae (Ms) and comparison was made with oxytetracycline and tylosin tartrate. The virulent S6 strain of Mg was also included for reference. All Mycoplasma strains, including a strain of Mg that was resistant to tylosin tartrate, were susceptible to danofloxacin with minimal inhibitory concentrations ranging from pound 0.008 to 0.5 microg/ml. A commercially produced test system (Sensititre), using micro-plates whose wells were predosed with antimicrobial agents and then dried, was also investigated. Results with the same three antimicrobials were in agreement with those obtained by the micro-broth method. With the exception of the tylosin resistant strain, the Mg strains were more susceptible to erythromycin than the Ms strains. Conversely, Ms strains were susceptible to apramycin, while the Mg strains appeared to be resistant.

Soil temperature affects infection of potato and tomato by mixtures of DM and MS strains of Verticillium albo-atrum

When potato and tomato plants were inoculated with a mixture of the DM and MS strains ofVerticillium albo-atrum at 16, 22, and 27 C soil temperatures, the two components were recovered as individual strains, or as a mixture depending on the soil temperature used. The DM strain was the component most often recovered throughout the range of 16 – 27 C while the frequency of recovery of the MS strain alone tended to increase above 22 C. Simultaneous infection by both strains ofV. albo-atrum occurred at the three soil temperatures in tomato but only at 22 and 27 C in potato. The fact that both strains could be recovered from the same infected plant indicated that they were not mutually exclusive in the host plant.