Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa.

BACKGROUND: The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. METHODS: Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. RESULTS: The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95-100% similarity to the isolates from the gene bank. CONCLUSION: The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

Evaluation of the safety and efficacy of the novel Mycoplasma gallisepticum vaccine, Vaxsafe MG304, after spray-vaccination of 1-day-old specific pathogen-free chicks.

Mycoplasma gallisepticum causes chronic respiratory disease in poultry. A novel vaccine, Vaxsafe MG304 (the ts-304 strain), has greater protective efficacy in chickens than the Vaxsafe MG (strain ts-11) vaccine when delivered by eye drop at 3 weeks of age. Applying this vaccine in the hatchery to 1-day-old birds, using mass administration methods, would improve animal welfare and reduce labour costs associated with handling individual birds. This study assessed the protection provided by vaccination with Vaxsafe MG304 after administration to 1-day-old chicks. Chicks were administered a single dose of the vaccine to assess the efficacy of either a high dose (107.0 colour changing units, CCU) or a low dose (105.7 CCU) after eye drop or spray (in water or gel) administration against experimental challenge with virulent M. gallisepticum strain Ap3AS at 7 weeks of age. The vaccine was able to colonise the palatine cleft of chicks after vaccination by eye drop (at both doses) or by spray (in water or gel) (at the high dose). The high dose of vaccine, when delivered by eye drop or spray, was shown to be safe and induced a serological response and protective immunity (as measured by tracheal mucosal thickness and air sac lesion scores) against challenge. Vaccination of 1-day-old chicks with Vaxsafe MG304 by eye drop induced protective immunity equivalent to vaccination at 3 weeks of age. Vaxsafe MG304 was also protective when applied by both coarse- and gel spray methods at the higher dose and is therefore a suitable live attenuated vaccine for use in 1-day-old chicks.

Immune Evasion of Mycoplasma gallisepticum: An Overview.

Mycoplasma gallisepticum is one of the smallest self-replicating organisms. It causes chronic respiratory disease, leading to significant economic losses in poultry industry. Following M. gallisepticum invasion, the pathogen can persist in the host owing to its immune evasion, resulting in long-term chronic infection. The strategies of immune evasion by mycoplasmas are very complex and recent research has unraveled these sophisticated mechanisms. The antigens of M. gallisepticum exhibit high-frequency changes in size and expression cycle, allowing them to evade the activation of the host humoral immune response. M. gallisepticum can invade non-phagocytic chicken cells and also regulate microRNAs to modulate cell proliferation, inflammation, and apoptosis in tracheal epithelial cells during the disease process. M. gallisepticum has been shown to transiently activate the inflammatory response and then inhibit it by suppressing key inflammatory mediators, avoiding being cleared. The regulation and activation of immune cells are important for host response against mycoplasma infection. However, M. gallisepticum has been shown to interfere with the functions of macrophages and lymphocytes, compromising their defense capabilities. In addition, the pathogen can cause immunological damage to organs by inducing an inflammatory response, cell apoptosis, and oxidative stress, leading to immunosuppression in the host. This review comprehensively summarizes these evasion tactics employed by M. gallisepticum, providing valuable insights into better prevention and control of mycoplasma infection.

Unmasking the dynamics of Mycoplasma gallisepticum: deciphering HD11 macrophage polarization for innovative infection control strategies.

Mycoplasma gallisepticum (MG) is a highly contagious avian respiratory pathogen characterized by rapid spread, widespread distribution, and long-term persistence of infection. Previous studies have shown that chicken macrophage HD11 cells play a critical role in the replication and immunomodulation of MG. Macrophages are multifunctional immunomodulatory cells that polarize into different functions and morphologies in response to exogenous stimuli. However, the effect of MG infection on HD11 polarization is not well understood. In this study, we observed a time-dependent increase in both the expression of the MG-related virulence protein pMGA1.2 and the copy number of MG upon MG infection. Polarization studies revealed an upregulation of M1-type marker genes in MG-infected HD11 cells, suggesting that MG mainly induces HD11 macrophages towards M1-type polarization. Furthermore, MG activated the inflammatory vesicle NLRP3 signaling pathway, and NLRP3 inhibitors affected the expression of M1 and M2 marker genes, indicating the crucial regulatory role of the NLRP3 signaling pathway in MG-induced polarization of HD11 macrophages. Our findings reveal a novel mechanism of MG infection, namely the polarization of MG-infected HD11 macrophages. This discovery suggests that altering the macrophage phenotype to inhibit MG infection may be an effective control strategy. These findings provide new perspectives on the pathogenic mechanism and control measures of MG.

Genetic comparison of the Mycoplasma gallisepticum 6/85 vaccine strain and 6/85-like field isolates.

Mycoplasma gallisepticum infection in poultry leads to disease and pathology that can reduce producer profits. Live attenuated vaccines are available that can limit or completely prevent the effects of infection. Field isolates that are genetically related to the attenuated vaccine strains have been isolated, raising the question of whether the attenuation of the vaccine strains is limited and can lead the strains to revert to more virulent forms. The 6/85 live attenuated vaccine is derived from a field isolate collected in the United States. Analysis of the genome of sequenced M. gallisepticum strains revealed a cluster of 10 6/85-like strains that group with the 6/85 vaccine strain. Four genomic regions were identified that allowed for strain differentiation. The genetic differences between strains points toward nine of the ten strains most likely being sister strains to the 6/85 vaccine strain. Insufficient differences are present in the tenth strain to make a definitive conclusion. These results suggest that most if not all strains similar to the live attenuated vaccine strain are field isolates of the parent strain used to derive the live attenuated vaccine.

Transcriptomic analysis of the effects of tylosin on the protective immunity provided by the Mycoplasma gallisepticum vaccine Vaxsafe MG ts-304.

The antimicrobial tylosin is commonly used to control mycoplasma infections, sometimes in combination with vaccination. However, the efficacy of a live mycoplasma vaccine, when combined with subsequent antimicrobial treatment, against the effects of subsequent infection with a virulent strain is unknown. This study employed differential gene expression analysis to evaluate the effects of tylosin on the protection provided by the live attenuated Vaxsafe MG ts-304 vaccine, which has been shown to be safe and to provide long-term protective immunity against infection with Mycoplasma gallisepticum. The transcriptional profiles of the tracheal mucosa revealed significantly enhanced inflammation, immune cell proliferation and adaptive immune responses in unvaccinated, untreated birds and in unvaccinated birds treated with tylosin 2 weeks after infection with virulent M. gallisepticum. These responses, indicative of the typical immune dysregulation caused by infection with M. gallisepticum, were less severe in the unvaccinated, tylosin-treated birds than in the unvaccinated, untreated birds. This was attributable to the effect of residual levels of tylosin in the tracheal mucosa on replication of virulent M. gallisepticum. These responses were not detected in vaccinated, tylosin-treated birds or in vaccinated, untreated birds after infection. The tracheal mucosal transcriptional profiles of these birds resembled those of unvaccinated, untreated, uninfected birds, suggesting a rapid and protective secondary immune response and effective vaccination. Overall, these results show that, although tylosin treatment reduced the duration of immunity, the initial protective immunity induced by Vaxsafe MG ts-304 lasted for at least 22 weeks after vaccination, even after the administration of tylosin for 16 weeks following vaccination.

Inflammatory responses and barrier disruption in the trachea of chicks following Mycoplasma gallisepticum infection: a focus on the TNF-α-NF-κB/MLCK pathway.

Mycoplasma gallisepticum (MG) can induce persistent inflammatory damage to the tracheal mucosa of poultry and cause chronic respiratory diseases in chickens. To further investigate the mechanism of MG-induced injury to the tracheal mucosa, we used chick embryo tracheal organ culture (TOC) as a model to study the invasion and reproduction of MG, the effect of MG on tracheal morphology, and the potential factors that promote MG tissue invasion. The results showed that MG infection significantly damaged the tracheal epithelial structure and weakened tracheal epithelial barrier function; MG also increased the occurrence of bacterial displacement, with a significant (p < 0.05) increase in the bacterial load of the infected TOCs at 5 and 7 days post-infection. In addition, MG significantly (p < 0.05) increased the expression levels of inflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß), and IL-6, and activated the NF-κB signalling pathway, leading to increased nuclear translocation of NF-κB p65. Simultaneously, the map kinase pathway (MAPK) was activated. This activation might be associated with increased myosin light chain (MLC) phosphorylation, which could lead to actin-myosin contraction and disruption of tight junction (TJ) protein function, potentially compromising epithelial barrier integrity and further catalysing MG migration into tissues. Overall, our results contribute to a better understanding of the interaction between MG and the host, provide insight into the mechanisms of damage to the tracheal mucosa induced by MG infection, and provide new insights into the possible pathways involved in Mycoplasma gallisepticum infection in vivo.

Quadrivalent hemagglutinin and adhesion expressed on Saccharomyces cerevisiae induce protective immunity against Mycoplasma gallisepticum infection and improve gut microbiota.

Mycoplasma gallisepticum (MG) infection causes infectious respiratory diseases in poultry, causing economic losses to the poultry industry. Therefore, this study aims to develop a safe, convenient, and effective multivalent recombinant Saccharomyces cerevisiae vaccine candidate and to explore its potential for oral immunization as a subunit vaccine. Mycoplasma gallisepticum Cytadhesin (MGC) and variable lipoprotein and hemagglutinin (vlhA) are associated with the pathogenesis of MG. In this study, a quadrivalent recombinant Saccharomyces cerevisiae (ST1814G-MG) displaying on MGC2, MGC3, VLH5, and VLH3, proteins was innovatively constructed, and its protective efficiency was evaluated in birds. The results showed that oral immunization with ST1814G-MG stimulates specific antibodies in chickens, reshapes the composition of the gut microbiota, reduces the Mycoplasma loading and pulmonary disease injury in the lungs. In addition, we found that oral ST1814G-MG had better protection against MG infection than an inactivated vaccine, and co-administration with the inactivated vaccine was even more effective. The results suggest that ST1814G-MG is a potentially safer and effective agent for controlling MG infection.

STAT5-mediated transcription of miR-33-5p in Mycoplasma gallisepticum-infected DF-1 cells.

RESEARCH HIGHLIGHTS: MG-HS regulates the expression of transcription factor STAT5.Transcription factor STAT5 can target miR-33-5p promoter element.MG-influenced STAT5 regulates miR-33-5p and its target gene expression.

Male pathology regardless of behaviour drives transmission in an avian host-pathogen system.

Host sex is an important source of heterogeneity in the severity of epidemics. Pinpointing the mechanisms causing this heterogeneity can be difficult because differences in behaviour among sexes (e.g. greater territorial aggression in males) can bias exposure risk, obfuscating the role of immune function, which can lead to differences in pathology, in driving differential susceptibility between sexes. Thus, sex-biased transmission driven by differences in immune function independent of behaviour is poorly understood, especially in non-mammalian systems. Here we examine the previously unexplored potential for male-biased pathology to affect transmission using an avian host-pathogen system. We employ a sex-dependent multistate transmission model parameterized with isolated, individual-based experimental exposures of domestic canaries and experimental transmission data of house finches. The experiment revealed that male birds have shorter incubation periods, longer recovery periods, higher pathogen burdens and greater disease pathology than females. Our model revealed that male-biased pathology led to epidemic size rapidly increasing with the proportion of male birds, with a nearly 10-fold increase in total epidemic size from an all-female to an all-male simulation. Our results demonstrate that female-biased resistance, independent of male behaviour, can drive sex-dependent transmission in wildlife, indicating that sex-based differences in immune function, not just differences in exposure risk, can shape epidemic dynamics.

Pharmacokinetic/pharmacodynamic integration of amphenmulin: a novel pleuromutilin derivative against Mycoplasma gallisepticum.

Amphenmulin is a novel pleuromutilin derivative with great anti-mycoplasma potential. The present study evaluated the action characteristics of amphenmulin against Mycoplasma gallisepticum using pharmacokinetic/pharmacodynamic (PK/PD) modeling approaches. Following intravenous administration, amphenmulin exhibited an elimination half-life of 2.13 h and an apparent volume of distribution of 3.64 L/kg in healthy broiler chickens, demonstrating PK profiles of extensive distribution and rapid elimination. The minimum inhibitory concentration (MIC) of amphenmulin against M. gallisepticum was determined to be 0.0039 µg/mL using the broth microdilution method, and the analysis of the static time-kill curves through the sigmoid Emax model showed a highly correlated relationship (R ≥ 0.9649) between the kill rate and drug concentrations (1-64 MIC). A one-compartment open model with first-order elimination was implemented to simulate the in vivo anti-mycoplasma effect of amphenmulin, and it was found that bactericidal levels were reached with continuous administration for 3 days at doses exceeding 0.8 µg/mL. Furthermore, the area under the concentration-time curve divided by MIC (AUC/MIC) correlated well with the anti-mycoplasma effect of amphenmulin within 24 h after each administration, with a target value of 904.05 h for predicting a reduction of M. gallisepticum by 1 Log10CFU/mL. These investigations broadened the antibacterial spectrum of amphenmulin and revealed its characteristics of action against M. gallisepticum, providing a theoretical basis for further clinical development.IMPORTANCEMycoplasma has long been recognized as a significant pathogen causing global livestock production losses and public health concerns, and the use of antimicrobial agents is currently one of the mainstream strategies for its prevention and control. Amphenmulin is a promising candidate pleuromutilin derivative that was designed, synthesized, and screened by our laboratory in previous studies. Moreover, this study further confirms the excellent antibacterial activity of amphenmulin against Mycoplasma gallisepticum and reveals its action characteristics and model targets on M. gallisepticum by establishing an in vitro pharmacokinetic/pharmacodynamic synchronization model. These findings can further broaden the pharmacological theoretical basis of amphenmulin and serve as data support for its clinical development, which is of great significance for the discovery of new antimicrobial drugs and the control of bacterial diseases in humans and animals.

Quercetin and AMPK: A Dynamic Duo in Alleviating MG-Induced Inflammation via the AMPK/SIRT1/NF-κB Pathway.

Mycoplasma gallisepticum (MG) is recognized as a principal causative agent of avian chronic respiratory disease, inflicting substantial economic losses upon the poultry industry. However, the extensive use of conventional antibiotics has resulted in the emergence of drug resistance and various challenges in their clinical application. Consequently, there is an urgent need to identify effective therapeutic agents for the prevention and treatment of mycoplasma-induced respiratory disease in avian species. AMP-activated protein kinase (AMPK) holds significant importance as a regulator of cellular energy metabolism and possesses the capacity to exert an anti-inflammatory effect by virtue of its downstream protein, SIRT1. This pathway has shown promise in counteracting the inflammatory responses triggered by pathogenic infections, thus providing a novel target for studying infectious inflammation. Quercetin possesses anti-inflammatory activity and has garnered attention as a potential alternative to antibiotics. However, there exists a gap in knowledge concerning the impact of this activation on MG-induced inflammatory damage. To address this knowledge gap, we employed AlphaFold2 prediction, molecular docking, and kinetic simulation methods to perform a systematic analysis. As expected, we found that both quercetin and the AMPK activator AICAR activate the chicken AMPKγ1 subunit in a similar manner, which was further validated at the cellular level. Our project aims to unravel the underlying mechanisms of quercetin's action as an agonist of AMPK against the inflammatory damage induced by MG infection. Accordingly, we evaluated the effects of quercetin on the prevention and treatment of air sac injury, lung morphology, immunohistochemistry, AMPK/SIRT1/NF-κB pathway activity, and inflammatory factors in MG-infected chickens. The results confirmed that quercetin effectively inhibits the secretion of pro-inflammatory cytokines such as IL-1ß, TNF-α, and IL-6, leading to improved respiratory inflammation injury. Furthermore, quercetin was shown to enhance the levels of phosphorylated AMPK and SIRT1 while reducing the levels of phosphorylated P65 and pro-inflammatory factors. In conclusion, our study identifies the AMPK cascade signaling pathway as a novel cellular mediator responsible for quercetin's ability to counter MG-induced inflammatory damage. This finding highlights the potential significance of this pathway as an important target for anti-inflammatory drug research in the context of avian respiratory diseases.

Exosomal microRNA/miRNA Dysregulation in Respiratory Diseases: From Mycoplasma-Induced Respiratory Disease to COVID-19 and Beyond.

Respiratory diseases represent a significant economic and health burden worldwide, affecting millions of individuals each year in both human and animal populations. MicroRNAs (miRNAs) play crucial roles in gene expression regulation and are involved in various physiological and pathological processes. Exosomal miRNAs and cellular miRNAs have been identified as key regulators of several immune respiratory diseases, such as chronic respiratory diseases (CRD) caused by Mycoplasma gallisepticum (MG), Mycoplasma pneumoniae pneumonia (MMP) caused by the bacterium Mycoplasma pneumoniae, coronavirus disease 2019 (COVID-19), chronic obstructive pulmonary disease (COPD), asthma, and acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Consequently, miRNAs seem to have the potential to serve as diagnostic biomarkers and therapeutic targets in respiratory diseases. In this review, we summarize the current understanding of the functional roles of miRNAs in the above several respiratory diseases and discuss the potential use of miRNAs as stable diagnostic biomarkers and therapeutic targets for several immune respiratory diseases, focusing on the identification of differentially expressed miRNAs and their targeting of various signaling pathways implicated in disease pathogenesis. Despite the progress made, unanswered questions and future research directions are discussed to facilitate personalized and targeted therapies for patients with these debilitating conditions.

Avian safety guardian: Luteolin restores Mycoplasma gallisepticum-induced immunocompromise to improve production performance via inhibiting the IL-17/NF-kB pathway.

Mycoplasma gallisepticum (MG) is a major pathogen causing chronic respiratory disease (CRD) in chickens. Exposure to MG poses a constant threat to chicken health and causes substantial economic losses. Antibiotics are the main treatment for MG infections, but have to struggle with antibiotic residues and MG resistance. To date, no safe and more effective prevention or treatment for MG infections has been identified. Luteolin (Lut) is a natural flavonoid compound known for its excellent anti-viral, anti-bacterial, immunoregulatory, and anti-inflammatory pharmacological activities. Herein, we established an MG-infected model using partridge shank chickens and chicken-like macrophages (HD11 cells) to investigate the effect and potential mechanism of Lut against MG-induced immune damage. According to our findings, Lut significantly inhibited the expression of MG adhesion protein (pMGA1.2) in vivo and in vitro. Lut effectively mitigated the MG-induced decrease in body weight gain, feed conversion ratio, survival rate, and serum IgG and IgA levels. Lut directly repaired MG-induced spleen and thymus damage by histopathological analysis. Furthermore, network pharmacology analysis revealed that Lut most probably resisted MG infection through the IL-17/NF-kB pathway. In vivo and in vitro experiments, Lut significantly suppressed the increase in key protein IL-17A, TRAF6, p-p65, and p-IkBα in the IL-17/NF-kB pathway. Meanwhile, Lut markedly alleviated MG-induced the increase of pro-inflammatory cytokines TNF-α, IL-6, IL-1ß, pro-apoptotic genes caspase3 and caspase9, while promoting the expression of anti-apoptotic genes Bcl-2 and Bcl-XL. In summary, Lut effectively suppressed MG colonization, alleviated MG-induced the production performance degradation, inflammatory responses, and immune damage by inhibiting the IL-17/ NF-kB pathway. This study indicates Lut can serve as a safe and effective antibiotic alternative drug for preventing and treating MG-induced CRD. It also provides new evidence to explore the molecular mechanisms of MG infection.

Immune Response and Histological Changes in Broilers Chickens Vaccinated with Mycoplasma gallisepticum Vaccines.

Mycoplasma is unique among prokaryotes because of its small size, small genomes, and complete lack of cell walls, which makes them cell wall-less prokaryotes. This study aimed to evaluate the effect of vaccinating one-day-old chicks with inactivated and live vaccines (CRDF) of Mycoplasma gallisepticum (MG) on their humoral immune response and immune organs. The Enzyme-Linked Immunosorbent Assay was used to measure Ab titers and investigate histopathological changes. A total of 130 one-day-old broiler chicks were randomly divided into four groups of 30. The groups were treated as follows: G1 included the chicks vaccinated with live F-strain MG vaccine (on eye drop of 0.03ml/dose), G2 included the chicks vaccinated with inactivated MG (0.3 ml s.c) vaccine, G3 included the chicks vaccinated with inactivated and live MG vaccines, and G4 was considered the control group, in which the chicks were not vaccinated. Blood samples were collected on days 21 and 35 of the chick's life to measure the titers of specific antibodies. On day 35, the chicks were dissected, and the bursa of Fabricius, as well as the spleen, were removed for histological evaluations. On day 21, the results showed a significant difference (P≤0.05) between all vaccinated groups in Ab titers, compared to G4, with the highest mean in G3, followed by G2 and G1, in descending order. On day 35, there was a significant difference (P≤0.05) between G3 and other vaccinated groups (G2 and G1), as well as G4. In addition, there was a significant increase in all vaccinated groups on day 35, compared to day 21. In G1, histopathological examination results showed a moderate lymphocytic hyperplasia bursal follicle. In G2, varying degrees of lymphoproliferative were observed in the major bursal follicle, and in G3, a marked lymphocytic hyperplasia bursal follicle was observed. In G4, on the other hand, no obvious histopathological findings were recorded. The results of the spleen histopathological evaluation showed various degrees of lymphoproliferative and moderate neutrophilic infiltrate in the red pulp in G1, and mild sinus congestion with scattered lymphocytes was recorded in the lumen in G2. In the spleen of the chicks in G3, reactive lymphoid hyperplasia was observed. In contrast to the groups mentioned above, in G4, the spleen structure showed a typical structure. It was concluded that the chicks vaccinated with inactivated and live MG vaccines experienced increased production of Ab titers and the immune stimulation of immune organs.

Bacillus subtilis KC1 prevents Mycoplasma gallisepticum-induced lung injury by enhancing intestinal Bifidobacterium animalis and regulating indole metabolism in chickens.

It has been reported that dietary administration of Bacillus subtilis KC1 is effective in alleviating lung injury induced by Mycoplasma gallisepticum (MG) infection in chickens. However, the underlying molecular mechanism of B. subtilis KC1 against MG infection is still unclear. The purpose of this study was to determine whether B. subtilis KC1 could alleviate MG infection-induced lung injury in chickens by regulating their gut microbiota. The results of this study indicate that B. subtilis KC1 supplementation has the potential to alleviate MG infection-induced lung injury as reflected by reduced MG colonization, reduced pathologic changes, and decreased production of pro-inflammatory cytokines. In addition, B. subtilis KC1 supplementation was partially effective in alleviating the gut microbiota disorder caused by MG infection. Importantly, B. subtilis KC1 enriched the beneficial Bifidobacterium animalis in gut and thus reversed indole metabolic dysfunction caused by MG infection. B. subtilis KC1 supplementation increased levels of indole, which enhanced aryl hydrocarbon receptor activation, improving barrier function and alleviating lung inflammation caused by MG. Overall, this study indicates that B. subtilis KC1 has a "gut-lung axis" mechanism that can reduce the severity of MG infection by enriching intestinal B. animalis and regulating indole metabolism.

Rapid adaptation to a novel pathogen through disease tolerance in a wild songbird.

Animal hosts can adapt to emerging infectious disease through both disease resistance, which decreases pathogen numbers, and disease tolerance, which limits damage during infection without limiting pathogen replication. Both resistance and tolerance mechanisms can drive pathogen transmission dynamics. However, it is not well understood how quickly host tolerance evolves in response to novel pathogens or what physiological mechanisms underlie this defense. Using natural populations of house finches (Haemorhous mexicanus) across the temporal invasion gradient of a recently emerged bacterial pathogen (Mycoplasma gallisepticum), we find rapid evolution of tolerance (<25 years). In particular, populations with a longer history of MG endemism have less pathology but similar pathogen loads compared with populations with a shorter history of MG endemism. Further, gene expression data reveal that more-targeted immune responses early in infection are associated with tolerance. These results suggest an important role for tolerance in host adaptation to emerging infectious diseases, a phenomenon with broad implications for pathogen spread and evolution.

Low let-7d microRNA levels in chick embryos enhance innate immunity against Mycoplasma gallisepticum by suppressing the mitogen-activated protein kinase pathway.

Chick embryos are a valuable model for studying immunity and vaccines. Therefore, it is crucial to investigate the molecular mechanism of the Mycoplasma gallisepticum (MG)-induced immune response in chick embryos for the prevention and control of MG. In this study, we screened for downregulated let-7d microRNA in MG-infected chicken embryonic lungs to explore its involvement in the innate immune mechanism against MG. Here, we demonstrated that low levels of let-7d are a protective mechanism for chicken embryo primary type II pneumocytes (CP-II) in the presence of MG. Specifically, we found that depressed levels of let-7 in CP-II cells reduced the adhesion capacity of MG. This suppressive effect was achieved through the activated mitogen-activated protein kinase phosphatase 1 (MKP1) target gene and the inactivated mitogen-activated protein kinase (MAPK) pathway. Furthermore, MG-induced hyperinflammation and cell death were both alleviated by downregulation of let-7d. In conclusion, chick embryos protect themselves against MG infection through the innate immune molecule let-7d, which may result from its function as an inhibitor of the MAPK pathway to effectively mitigate MG adhesion, the inflammatory response and cell apoptosis. This study may provide new insight into the development of vaccines against MG.

Isolation and characterization of an atypical Mycoplasma gallisepticum strain showing a new mgc2 variant.

Mycoplasma gallisepticum (MG) is an important pathogen of the poultry industry able to cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite the application of biosecurity measures and the availability of vaccines for chickens, monitoring systems routinely applied for MG detection are still essential for infection control. Pathogen isolation is time-consuming and not suitable for rapid detection, albeit it is a compulsory step for genetic typing and antimicrobial susceptibility evaluation of single strains. The mgc2 gene is a species-specific molecular target adopted by most of the PCR protocols available for MG diagnosis, which are also included in the WOAH Terrestrial Manual. We describe the case of an atypical MG strain, isolated in 2019 from Italian turkeys, characterized by an mgc2 sequence not detectable by common endpoint PCR primers. Considering the potential risk of false negative results during diagnostic screenings with the endpoint protocol, the authors propose an alternative mgc2 PCR endpoint protocol, named MG600, which should be considered as a further diagnostic tool.

EFFECTS OF MYCOPLASMA GALLISEPTICUM INFECTION ON PREENING BEHAVIORS AND FEATHER QUALITY IN HOUSE FINCHES (HAEMORHOUS MEXICANUS).

Infections can have far-reaching sublethal effects on wildlife, including reduced maintenance of external structures. For many wildlife taxa, daily maintenance of external structures (termed preening in birds) is critical to fitness, but few studies have examined how infections alter such maintenance. Mycoplasma gallisepticum is a common pathogen in free-living House Finches (Haemorhous mexicanus), where it causes mycoplasmal conjunctivitis. Despite documented behavioral changes associated with M. gallisepticum infections in finches, no studies have examined how preening behavior may change with infection and how potential differences in preening may affect feather quality. To test this, we experimentally inoculated captive House Finches with M. gallisepticum or a control treatment, and we collected behavioral and feather quality data to detect potential changes in feather maintenance due to infection. We found that finches infected with M. gallisepticum preened significantly less often, and within the infected treatment, birds with the highest conjunctivitis severity preened the least often. However, there was no difference in the quality scores for secondary flight feathers collected from control versus infected birds. We also assayed feather water retention and found that the degree of water retention correlated with our feather quality scores, such that feathers with poor scores retained more water. However, as with quality scores, feather water retention did not differ with infection; this may be due to the controlled environment that the birds experienced while in captivity. Our data suggest that, in addition to sickness behaviors previously observed in finches, M. gallisepticum infection decreases other behaviors critical to survival, such as preening. While the consequences of reduced preening on feather maintenance were not apparent in captive conditions, further work is needed to determine whether House Finches in the wild that are infected with M. gallisepticum experience a fitness cost, such as increases in ectoparasite loads, due to this reduced feather maintenance.