Baicalin ameliorates Mycoplasma gallisepticum-induced inflammatory injury in the chicken lung through regulating the intestinal microbiota and phenylalanine metabolism.

Baicalin shows excellent protective effects against Mycoplasma gallisepticum (MG) induced inflammatory injury as discussed in our previous studies. However, the physiological effects of baicalin are notable in contrast to its low bioavailability, and the critical mechanism for the protective effects of baicalin against MG infection is still unclear. The main objective of this study was to investigate whether baicalin alleviates MG-induced lung inflammatory injury through regulating gut microbiota. Using an MG infection model, results showed that baicalin treatment significantly reduced MG colonization and ameliorated the abnormal pathological changes in the lung. Baicalin treatment also reduced the level of proinflammatory cytokines and suppressed proinflammatory protein expression. Notably, MG infection changed the gut microbiota composition, however, the abnormal gut microbiota composition was partially alleviated by baicalin treatment. Baicalin significantly enriched the commensal bacterium Bacteroides fragilis, and gavaged with Bacteroides fragilis alleviating MG infection-induced inflammatory injury in the lung. In addition, baicalin reversed peripheral accumulation of phenylalanine induced by MG infection. Importantly, increased phenylalanine induced excessive necroptosis through the modulation of gga-miR-190a-3p-Fas-associated protein with death domain (FADD) axis in HD11 macrophages. Together, our findings highlighted the role of gut microbiota and phenylalanine metabolism in MG infection and confirmed that baicalin could effectively inhibit MG-induced inflammatory injury in the lung by remodeling the gut microbiota and phenylalanine metabolism.

Transcriptional and Pathological Host Responses to Coinfection with Virulent or Attenuated Mycoplasma gallisepticum and Low-Pathogenic Avian Influenza A Virus in Chickens.

The avian pathogen Mycoplasma gallisepticum, the etiological agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a copathogen such as low-pathogenic avian influenza virus (LPAIV). To further investigate the intricacies of this copathogenesis, chickens were monoinfected or coinfected with either virulent M. gallisepticum strain Rlow or LPAIV H3N8 (A/duck/Ukraine/1963), with assessment of tracheal histopathology, pathogen load, and transcriptomic host responses to infection by RNA sequencing. Chickens coinfected with M. gallisepticum Rlow followed by LPAIV H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening than chickens infected with LPAIV H3N8 alone and greater viral loads than chickens infected first with H3N8 and subsequently with M. gallisepticum Rlow Recovery of live M. gallisepticum was significantly higher in chickens infected first with LPAIV H3N8 and then with M. gallisepticum Rlow, compared to chickens given a mock infection followed by M. gallisepticum Rlow The transcriptional responses to monoinfection and coinfection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as Toll-like receptor 15, Toll-like receptor 21, and matrix metallopeptidase 1. Pathway and gene ontology analyses of these differentially expressed genes suggest that coinfection with virulent M. gallisepticum and LPAIV induces decreases in the expression of genes related to ciliary activity in vivo and alters multiple immune-related signaling cascades. These data aid in the understanding of the relationship between M. gallisepticum and LPAIV during copathogenesis in the natural host and may contribute to further understanding of copathogen infections of humans and other animals.

Metabolite profiling of Mycoplasma gallisepticum mutants, combined with bioinformatic analysis, can reveal the likely functions of virulence-associated genes.

Mycoplasma gallisepticum is an economically important pathogen of commercial poultry. An improved understanding of M. gallisepticum pathogenesis is required to develop better control methods. We recently identified a number of M. gallisepticum mutants with defects in colonization and persistence in chickens using signature-tagged transposon mutagenesis. Loss of virulence was associated with mutations in a putative oligopeptide/dipeptide (opp/dpp) ATP-binding cassette (ABC) transporter (where the transposon was inserted into the MGA_0220 (oppD1) gene and two hypothetical proteins (encoded by MGA_1102 and MGA_0588), one of which (MGA_1102) contains a putative peptidase motif. To further characterise the function of these proteins, we compared the metabolome of each transposon mutant with that of wild type bacteria. Two independent LC/MS analyses revealed consistent significant decreases in the abundances of several amino acids and the dipeptide alanyl-glycine (Ala-Gly) in the MGA_0220 mutant, consistent with this protein being a peptide transporter. Similarly, lysine and Ala-Gly were significantly decreased in the MGA_1102 mutant, consistent with our bioinformatic analysis suggesting that MGA_1102 encodes a membrane-located peptidase. Few differences were observed in metabolite levels in the MGA_0588 mutant, suggesting that the disrupted protein has a non-metabolic role. Overall, this study indicates that metabolomics is a useful tool in the functional analysis of mutants.

House finch responses to Mycoplasma gallisepticum infection do not vary with experimentally increased aggression.

Aggression can alter infectious disease dynamics through two, non-exclusive mechanisms: 1) increasing direct contact among hosts and 2) altering hosts' physiological response to pathogens. Here we examined the latter mechanism in a social songbird by manipulating intraspecific aggression in the absence of direct physical contact. We asked whether the extent of aggression an individual experiences alters glucocorticoid levels, androgen levels, and individual responses to infection in an ecologically relevant disease model: house finches (Haemorhous mexicanus) infected with Mycoplasma gallisepticum (MG). Wild-caught male finches were housed in one of three settings, designed to produce increasing levels of aggression: 1) alone, with no neighbor ("no neighbor"), 2) next to a sham-implanted stimulus male ("sham neighbor"), or 3) next to a testosterone-implanted stimulus male ("testosterone neighbor"). Following one week of social treatment, focal males were experimentally infected with MG, which causes severe conjunctivitis and induces sickness behaviors such as lethargy and anorexia. While social treatment increased aggression as predicted, there were no differences among groups in baseline corticosterone levels, total circulating androgens, or responses to infection. Across all focal individuals regardless of social treatment, pre-infection baseline corticosterone levels were negatively associated with the severity of conjunctivitis and sickness behaviors, suggesting that corticosterone may dampen inflammatory responses in this host-pathogen system. However, because corticosterone levels differed based upon population of origin, caution must be taken in interpreting this result. Taken together, these results suggest that in captivity, although aggression does not alter individual responses to MG, corticosterone may play a role in this disease.

MalF is essential for persistence of Mycoplasma gallisepticum in vivo.

There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonization of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonized birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonization. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and could not be reisolated even when inoculated by itself into a group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.

Phenolic constituents, antioxidant and preliminary antimycoplasmic activities of leaf skin and flowers of Aloe vera (L.) Burm. f. (syn. A. barbadensis Mill.) from the Canary Islands (Spain).

The methanol extracts of leaf skins and flowers of Aloe vera from the Canary Islands were analyzed for their phenolic profiles and screened for their antioxidant and antimycoplasmic activities. The use of reversed phase high performance liquid chromatography (RP-HPLC) allowed the identification of 18 phenolic constituents. Leaf skin extracts were characterized by the abundance of catechin, sinapic acid and quercitrin. Gentisic acid, epicatechin and quercitrin were the most prominent phenolic compounds of the flowers. The in vitro antioxidant activities determined by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric antioxidant reducing power (FRAP) assays revealed that both extracts exhibited antioxidant activity, being the leaf skin extract the most active fraction. The leaf skin extract was also found to be active against the microbial strains tested. Therefore, A. vera extracts from leaf skin and flowers can be considered as good natural antioxidant sources.

[Adaptation of mycoplasmas to stressful factors: nucleotide sequences nonregistered in vegetative forms of M. galisepticum S6 cells are revealed in nonculturable forms of the mycoplasma].

Adaptation of M. gallisepticum S6 to unfavorable environmental conditions is connected with transformation of the vegetative forms of the mycoplasma cells to the viable but non-culturable (VBNC) forms. The vegetative forms and the VBNC forms differ in the spectrum of the PCR-products that was forming due to amplification of the nucleotide sequence of the pvpA-gene coding the able cytoadhesion protein. As to vegetative forms of the mycoplasma the only amplicon, containing one open reading frame (1086 b.p.) with a high homology (97%) to the pvpA-gene of M. gallisepticum R and Pendik is detected. In the case of VBNC forms of M. gallisepticum S6, the additional amplicons besides those indicating the pvpA-gene of the mycoplasma are observed. In the nucleotide sequences of the additional amplicons, the open reading frames are detected that are not registered in the database of the complete sequence of the mycoplasma genome. A high homology (54-55%) of the nucleotide sequences of the pvpA-gene and the additional pvpA-amplicons allows to suggest that thepvpA-gene sequence seems to be a basis for forming new regions within the mycoplasma genome during adaptation of the bacterium to unfavorable environmental conditions.

Atomic force microscopy investigation of DNA extracted from the vegetative forms and the viable but nonculturable forms of Mycoplasma gallisepticum S6.

Recent studies show that mycoplasmas have various programs of life. This means that changes in morphology and genome expression may occur once the environment of these microorganisms becomes extremely altered. In this article, we report on changes in the DNA molecule obtained from the vegetative forms and the viable but nonculturable (VBNC) forms of Mycoplasma gallisepticum S6. Atomic force microscopy studies show that the above-mentioned forms of the mycoplasma have different values of DNA parameters (height: 0.461 +/- 0.141 and 0.236 +/- 0.069 nm; width: 2.221 +/- 0.286 and 1.291 +/- 0.705 nm for the vegetative and the VBNC forms, respectively). We suppose that the observed phenomenon may be connected with the process of adaptation of these bacteria to severe environments.

[Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions: changes in morphological and physiological characteristics].

Adaptation of Mycoplasma gallisepticum to unfavorable growth conditions results in altered morphological and physiological characteristics of the cells. M. gallisepticum populations in a complete nutrient medium contain pear-shaped vegetative cells (d approximately 0.3 microm; l approximately 0.8 microm) with pronounced polar and cytoskeleton-like structures. Such mycoplasma cells are able to induce damage in a bacterial genome, causing an SOS response of the test strain (Escherichia coli PQ37). In a starvation medium, M. gallisepticum produces nanoforms, small coccoid cells (d approximately 0.15-0.2 microm) without either polar or cytoskeleton-like structures. Unlike vegetative cells, nanoforms do not induce genome damage. Alleviation of unfavorable growth conditions results in a reversion of nanoforms to typical vegetative cells.

Effects of supplemental dietary phytase and 25-hydroxycholecalciferol on the blood characteristics of commercial layers inoculated before or at the onset of lay with the F-strain of Mycoplasma gallisepticum.

In 3 trials, the effects of dietary supplementation with phytase (PHY) and 25-hydroxycholecalciferol on BW and the blood characteristics of commercial layers that were inoculated prelay (12 wk of age) or at the onset of lay (22 wk of age) with F-strain Mycoplasma gallisepticum were assessed at 34, 50, and 58 wk of age. Experimental layer diets, which included either a basal control diet or the same diet supplemented with 0.025% PHY and 25-hydroxycholecalciferol, were fed from 20 through 58 wk of age. The supplemented diet decreased blood hematocrit values across bird age, inoculation type (sham vs. F-strain M. gallisepticum), and age of inoculation (prelay vs. onset of lay). Phytase- and 25-hydroxycholecalciferol-supplemented diets reduced bird BW in sham-inoculated control birds across bird age and age of inoculation. This effect was not observed in F-strain M. gallisepticum-inoculated birds. Furthermore, across diet (control vs. supplemented) and inoculation type, total plasma protein concentration at 34 wk of age was higher in birds that were inoculated at the onset of lay compared with those inoculated prelay. Diet, inoculation type, and inoculation age had no effect on mortality, reproductive organ histopathological lesion scores, or serum cholesterol and Ca concentrations. In conclusion, throughout lay, the supplementation of commercial layer diets with PHY may lower hematocrit, and inoculation with F-strain M. gallisepticum prelay or at the onset of lay may ameliorate the depressing effects of dietary PHY and 25-hydroxycholecalciferol supplementation on hen BW.

Dynamics of a novel pathogen in an avian host: Mycoplasmal conjunctivitis in house finches.

In early 1994, a novel strain of Mycoplasma gallisepticum (MG)--a poultry pathogen with a world-wide distribution--emerged in wild house finches and within 3 years had reached epidemic proportions across their eastern North American range. The ensuing epizootic resulted in a rapid decline of the host population coupled with considerable seasonal fluctuations in prevalence. To understand the dynamics of this disease system, a multi-disciplinary team composed of biologists, veterinarians, microbiologists and mathematical modelers set forth to determine factors driving and influenced by this host-pathogen system. On a broad geographic scale, volunteer observers ("citizen scientists") collected and reported data used for calculating both host abundance and disease prevalence. The scale at which this monitoring initiative was conducted is unprecedented and it has been an invaluable source of data for researchers at the Cornell Laboratory of Ornithology to track the spread and magnitude of disease both spatially and temporally. At a finer scale, localized and intensive field studies provided data used to quantify the effects of disease on host demographic parameters via capture-mark-recapture modeling, effects of host behavior on disease and vice-versa, and the biological and genetic profiles of birds with known phenotypic characteristics. To balance the field-based component of the study, experiments were conducted with finches held in captivity to describe and quantify the effects of experimental infections on hosts in both individual and social settings. The confluence of these various elements of the investigation provided the foundation for construction of a general compartmentalized epidemiological model of the dynamics of the house finch-MG system. This paper serves several purposes including (i) a basic review of the pathogen, host, and epidemic cycle; (ii) an explanation of our research strategy; (iii) a basic review of results from the diverse multi-disciplinary approaches employed; and (iv) pertinent questions relevant to this and other wildlife disease studies that require further investigation.

Persistence of Mycoplasma gallisepticum in chickens after treatment with enrofloxacin without development of resistance.

The ability of the avian pathogen Mycoplasma gallisepticum to persist despite fluoroquinolone treatment was investigated in chickens. Groups of specific pathogen free chickens were experimentally infected with M. gallisepticum and treated with enrofloxacin at increasing concentrations up to the therapeutic dose. When M. gallisepticum could no longer be re-isolated from chickens, birds were stressed by inoculation of infectious bronchitis virus or avian pneumovirus. Although M. gallisepticum could not be cultured from tracheal swabs collected on several consecutive sampling days after the end of the enrofloxacin treatments, the infection was not eradicated. Viral infections reactivated the mycoplasma infection. Mycoplasmas were isolated from tracheal rings cultured for several days, suggesting that M. gallisepticum persisted in the trachea despite the enrofloxacin treatment. The minimal inhibitory concentration (MIC) of enrofloxacin for most of the re-isolated mycoplasmas was the same as that of the strain with which the birds were inoculated. Furthermore, no mutation could be detected in the fluoroquinolone target genes. These results suggest that M. gallisepticum can persist in chickens without development of resistance despite several treatments with enrofloxacin.