Are Purple Finches (Haemorhous purpureus) the Next Host for a Mycoplasmal Conjunctivitis Epidemic?

Ever since 1994, when the bacterial pathogen Mycoplasma gallisepticum jumped from poultry to wild birds, it has been assumed that the primary host species of this pathogen in wild North American birds was the house finch (Haemorhous mexicanus), in which disease prevalence was higher than in any other bird species. Here we tested two hypotheses to explain a recent increase in disease prevalence in purple finches (Haemorhous purpureus) around Ithaca, New York. Hypothesis 1 is that, as M. gallisepticum evolved and became more virulent, it has also become better adapted to other finches. If this is correct, early isolates of M. gallisepticum should cause less-severe eye lesions in purple finches than in house finches, while more-recent isolates should cause eye lesions of similar severity in the two species. Hypothesis 2 is that, as house finch abundance declined following the M. gallisepticum epidemic, purple finches around Ithaca increased in abundance relative to house finches and purple finches are thus more frequently exposed to M. gallisepticum-infected house finches. This would then lead to an increase in M. gallisepticum prevalence in purple finches. Following an experimental infection with an early and a more-recent M. gallisepticum isolate, eye lesions in purple finches were more severe than in house finches. This did not a support Hypothesis 1; similarly, an analysis of Project Feeder Watch data collected around Ithaca did not show differences in changes in purple and house finches' abundance since 2006, a result which does not support Hypothesis 2. We conclude that purple finch populations will, unlike those of house finches, not suffer a severe decline because of a M. gallisepticum epidemic.

¿Son los pinzones purpúreos (Haemorhous purpureus) los próximos huéspedes de una epidemia de conjuntivitis por micoplasma? Desde el año 1994, cuando el patógeno bacteriano Mycoplasma gallisepticum saltó de las aves comerciales a las aves silvestres, se ha supuesto que la principal especie huésped de este patógeno en las aves silvestres de América del Norte era el pinzón mexicano (Haemorhous mexicanus), en el que la prevalencia de la enfermedad era mayor que en cualquier otra especie aviar. En este estudio se analizaron dos hipótesis para explicar un aumento reciente en la prevalencia de la enfermedad en los pinzones purpúreos (Haemorhous purpureus) alrededor de Ithaca, en Nueva York. La hipótesis 1 es que, a medida que M. gallisepticum evolucionó y se volvió más virulento, también se adaptó mejor a otros pinzones. Si esto es correcto, los aislamientos tempranos de M. gallisepticum deberían causar lesiones oculares menos graves en los pinzones purpúreos que en los pinzones mexicanos, mientras que los aislamientos más recientes deberían causar lesiones oculares de gravedad similar en las dos especies. La hipótesis 2 es que, a medida que la abundancia de pinzones mexicanos disminuyó después de la epidemia de M. gallisepticum, los pinzones purpúreos alrededor de Ithaca aumentaron en abundancia en relación con los pinzones mexicanos y, por lo tanto, los pinzones morados están expuestos con mayor frecuencia a los pinzones caseros infectados con M. gallisepticum. Esto conduciría a un aumento de la prevalencia de M. gallisepticum en los pinzones purpúreos. Después de una infección experimental con un aislamiento temprano y uno más reciente de M. gallisepticum, las lesiones oculares en los pinzones purpúreos fueron más graves que en los pinzones mexicanos. Esto no apoyó la Hipótesis 1; de manera similar, un análisis de los datos del Proyecto Feeder Watch recopilados alrededor de Ithaca no mostró diferencias en los cambios de la abundancia de pinzones purpúreos y mexicanos desde 2006, un resultado que no respalda la Hipótesis 2. Se concluye que las poblaciones de pinzones purpúreos, a diferencia de las de los pinzones mexicanos, no sufrieron un declive severo a causa de una epidemia de M. gallisepticum.

Baicalin ameliorates Mycoplasma gallisepticum-induced inflammatory injury via inhibiting STIM1-regulated ceramide accumulation in DF-1 cells.

Mycoplasma gallisepticum (MG) is dependent on its host for many nutrients due to the loss of many important metabolic pathways. Ceramide is a sphingolipid that regulates multiple cellular processes in eukaryotic cell. Several studies highlighted the crucial role of ceramide on the pathogenesis of various pathogens. This study aimed to determine whether ceramide plays a crucial role in the pathogenesis of MG. Based on an MG infection model in DF-1 cells, the results revealed that MG infection induced ceramide accumulation in DF-1 cells. Inhibiting the de novo synthesis of ceramide significantly inhibited MG proliferation and inflammatory injury caused by MG in DF-1 cells. Meanwhile, MG infection led to endoplasmic reticulum stress, and pharmacologic inhibition of endoplasmic reticulum stress prevented ceramide accumulation and MG proliferation in DF-1 cells, alleviating the inflammatory injury caused by MG. In addition, MG infection significantly promoted expression level of stromal interaction molecule 1 (STIM1), thus induced calcium overload and oxidative stress. Furthermore, inhibition of STIM1 expression partially restored calcium homeostasis and mitigated oxidative stress, thus alleviated endoplasmic reticulum stress. Importantly, the inflammatory injury caused by MG were partially ameliorated by baicalin treatment (20 µg/mL) through downregulating STIM1 expression. In summary, these results suggests that ceramide accumulation through the de novo pathway plays an important role to promote MG proliferation and baicalin can alleviate MG infection induced inflammatory injury via regulating STIM1-related oxidative stress, endoplasmic reticulum stress and ceramide accumulation in DF-1 cells.

Hydroxytyrosol mitigates Mycoplasma gallisepticum-induced pulmonary injury through downregulation of the NF-κB/NLRP3/IL-1ß signaling pathway in chicken.

In this study, the anti-inflammatory and antiapoptotic effects of hydroxytyrosol (HT) in Mycoplasma gallisepticum (MG)-infected chicken were investigated, and the underlying molecular mechanisms were explored. The results revealed severe ultrastructural pathological changes after MG infection in the lung tissue of chicken, including inflammatory cell infiltration, thickening of the lung chamber wall, visible cell swelling, mitochondrial cristae rupture, and ribosome shedding. MG possibly activated the nuclear factor κB (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin (IL)-1ß signaling pathway in the lung. However, HT treatment significantly ameliorated MG-induced pathological damage of the lung. HT reduced the magnitude of pulmonary injury after MG infection by reducing apoptosis and releasing the proinflammatory factors. Compared with the MG-infected group, the HT-treated group exhibited significant inhibition of the expression of NF-κB/NLRP3/IL-1ß signaling-pathway-related genes; for example, the expressions of NF-κB, NLRP3, caspase-1, IL-1ß, IL-2, IL-6, IL-18, and TNF-α significantly decreased (P < 0.01 or <0.05). In conclusion, HT effectively inhibited MG-induced inflammatory response and apoptosis and protected the lung by blocking the activation of NF-κB/NLRP3/IL-1ß signaling pathway and reducing the damage caused by MG infection in chicken. This study revealed that HT may be a suitable and effective anti-inflammatory drug against MG infection in chicken.

Quercetin alleviates Mycoplasma gallisepticum-induced inflammatory damage and oxidative stress through inhibition of TLR2/MyD88/NF-κB pathway in vivo and in vitro.

Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) in chickens leads to enormous economic damage to the poultry industry yearly. The active components and mechanism of action of the traditional herbal remedy Ephedra houttuynia powder (EHP), which had been approved for clinical treatment against MG infection in China, remain unknown. In this study, the active components of EHP against MG were screened using a network pharmacological method, additionally, we studied the mechanism of action of the screened results (quercetin (QUE)). The findings demonstrated that QUE was an essential element of EHP against MG infection, effectively attenuating MG-induced oxidative stress and activation of the TLR2/MyD88/NF-κB pathway. Following QUE therapy, IL-1, IL-6, and TNF-α content and expression were downregulated, whereas IL-4 and IL-10 expression were upregulated, eventually suppressing the inflammatory response both in vitro and in vivo. Together, this study presents a strong rationale for using QUE as a therapeutic strategy to inhibit MG infection-induced inflammatory damage and oxidative stress.

Research Note: Oxidative stress and immune response following the administration of live attenuated Mycoplasma gallisepticum vaccination in backyard chicken.

In the present study, we investigated a possible relationship between the immune response and the oxidative stress (OS) state trend in a group of 12 chickens after intraocular administration of an attenuated Mycoplasma gallisepticum (MG) vaccine. Blood samples were collected at the vaccination time (T0), after 14 (T1) and 21 d (T2). White blood cell count (WBC), differential leucocyte count, and anti-MG antibodies titer (S/P) were studied as immune response indexes. As plasmatic OS biomarkers levels, we considered malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), reactive oxygen metabolites derived compounds (d-ROMs), the ferric reducing ability of plasma (FRAP), and superoxide anion (O2-). After antigenic stimulation, it was observed a significant decrease in monocythemia and a significant increase in thrombocythemia, S/P, MDA, and SOD. Furthermore, subjects with high d-ROMs levels at T0 tended to develop higher cellular mobilization with increases in WBC and lymphocytes accompanied by lower antibody release. It was also observed that the antioxidant components FRAP and SOD were moderately positively correlated to the entity of antibody response.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey's medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Mycoplasma gallisepticum escapes the host immune response via gga-miR-365-3p/SOCS5/STATs axis.

A disruption in the expression of gga-miR-365-3p was confirmed in the Mycoplasma gallisepticum (MG)-infected Chicken primary alveolar type II epithelial (CP-II) cells based on previous sequencing results, but the role it plays in the infection was unclear. In the present study, we demonstrate that MG evaded cellular host immunity via a gga-miR-365-3p/SOCS5-JAK/STATs negative feedback loop. Specifically, we found that at the initial stage of MG infection in cells, gga-miR-365-3p was rapidly increased and activated the JAK/STAT signaling pathway by inhibiting SOCS5, which induced the secretion of inflammatory factors and triggered immune response against MG infection. Over time, though, the infection progressed, MG gradually destroyed the immune defences of CP-II cells. In late stages of infection, MG escaped host immunity by reducing intracellular gga-miR-365-3p and inhibiting the JAK/STAT pathway to suppress the secretion of inflammatory factors and promote MG adhesion or invasion. These results revealed the game between MG and host cell interactions, providing a new perspective to gain insight into the pathogenic mechanisms of MG or other pathogens. Meanwhile, they also contributed to novel thoughts on the prevention and control of MG and other pathogenic infections, shedding light on the immune modulating response triggered by pathogen invasion and their molecular targeting.

Host resistance to Mycoplasma gallisepticum infection is enhanced by inhibiting PI3K/Akt pathway in Andrographolide-treating chickens.

Mycoplasma gallisepticum (MG) is a pathogenic microorganism that causes chronic respiratory disease (CRD). MG infection has a serious negative impact on the poultry industry. Andrographolide (AG) is known to regulate immune responses, antimicrobial infections, and anti-inflammatory responses. However, the underlying molecular mechanisms of AG action in MG-infected chickens remain unclear. Hence, we constructed models of MG infection by using chickens and chicken macrophage-like (HD11) cells in vivo and in vitro, respectively. The results showed that AG significantly inhibited the mRNA and protein expression of the toxic adhesion protein pMGA1.2 in vivo and in vitro. Meanwhile, AG treatment significantly decreased the mRNA expression of pro-inflammatory such as interleukin-6 (IL-6) and interleukin- 1ß (IL-1ß), and increased the mRNA expression of an anti-inflammatory such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß) in vivo and in vitro. Furthermore, AG treatment down-regulated inflammasome NLRP3 and apoptosis genes caspase3 and caspase9, and up-regulated autophagy protein light chain 3 (LC3) by regulating the PI3K/Akt signaling pathway in vitro. Our results suggest that AG can reduce the expression of NLRP3 and alleviate the inflammatory response from MG infection by inducing autophagy, probably by modulating PI3K/Akt signaling pathway. This study demonstrates that AG can be used as a specific target to prevent and treat MG infection effectively.

Role of DNA modifications in Mycoplasma gallisepticum.

The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene.

Insights into growth-promoting, anti-inflammatory, immunostimulant, and antibacterial activities of Toldin CRD as a novel phytobiotic in broiler chickens experimentally infected with Mycoplasma gallisepticum.

Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) leads to impaired broiler growth performance and significant economic losses worldwide. The utilization of essential oils (EOs) as natural alternatives to antibiotics to control CRD outbreaks is not completely clarified yet. Thus, we investigated the effect of a commercial EOs mixture (toldin CRD), in comparison to tilmicosin antibiotic, on the clinical observations, growth performance, immunity, digestive enzymes, gut barrier functions, and bacterial loads in broilers experimentally infected with MG. A total of 400 one-day-old broiler chicks were assigned into four groups; negative control (NC), positive control (PC), tilmicosin, and toldin CRD treated groups. All groups except NC were experimentally infected with MG at 14 d of age. Our data showed that birds treated with toldin CRD showed significant enhancement in the body weight gain (BWG) and feed conversion ratio (FCR) (P = 0.001 each) over the whole experimental period. Likely, improved digestibility and intestinal barrier functions in the toldin CRD treated group was evidenced by the significant upregulation (P < 0.05) of cholecystokinin (CCK), alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP), junctional adhesion molecule-2 (JAM-2), occludin, and mucin-2 (MUC-2) genes. Moreover, toldin CRD exhibited immunostimulant and ant-inflammatory activities via significant downregulation (P < 0.05) of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 genes, significant reduction of lysozyme (LYZ), myeloperoxidase (MPO), and nitric oxide (NO) levels (P = 0.03, 0.02, and 0.001, respectively) and significant increase in the immunoglobulin G (IgG) level (P = 0.03). Notably, immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR) results showed prominent reductions (P < 0.05) in the levels of MG antigens and MG loads in the toldin CRD treated group, which were evidenced by relieving the clinical picture of MG experimental infection. In conclusion, we recommend the utilization of toldin CRD as a potential candidate for controlling MG infection in broiler chickens.

The expression of GapA and CrmA correlates with the Mycoplasma gallisepticum in vitro infection process in chicken TOCs.

Mycoplasma (M.) gallisepticum is the most pathogenic mycoplasma species in poultry. Infections cause mild to severe clinical symptoms associated with respiratory epithelial lesion development. Adherence, biofilm formation, and cell invasion of M. gallisepticum contribute to successful infection, immune evasion, and survival within the host. The important M. gallisepticum membrane-bound proteins, GapA and CrmA, are key factors for host cell interaction and the bacterial life-cycle, including its gliding motility, although their precise role in the individual infection step is not yet fully understood. In this study, we investigated the correlation between the host-pathogen interaction and the GapA/CrmA expression in an environment that represents the natural host's multicellular compartment. We used an in vitro tracheal organ culture (TOC) model, allowing the investigation of the M. gallisepticum variants, Rlow, RCL1, RCL2, and Rhigh, under standardised conditions. In this regard, we examined the bacterial adherence, motility and colonisation pattern, host lesion development and alterations of mucociliary clearance. Compared to low virulent RCL2 and Rhigh, the high virulent Rlow and RCL1 were more efficient in adhering to TOCs and epithelium colonisation, including faster movement from the cilia tips to the apical membrane and subsequent cell invasion. RCL2 and Rhigh showed a more localised invasion pattern, accompanied by significantly fewer lesions than Rlow and RCL1. Unrelated to virulence, comparable mucus production was observed in all M. gallisepticum infected TOCs. Overall, the present study demonstrates the role of GapA/CrmA in virulence factors from adherence to colonisation, as well as the onset and severity of lesion development in the tracheal epithelium.

Extracellular HMGB1 as Inflammatory Mediator in the Progression of Mycoplasma Gallisepticum Infection.

High-mobility group box 1 (HMGB1), a member of damage-associated molecular patterns (DAMPs), is involved in the immune regulation of several infectious diseases. Mycoplasma gallisepticum (MG) infection is proved to cause an abnormal immune response, but the role of HMGB1 in MG-induced chronic respiratory disease (CRD) is unclear. In this study, we found that HMGB1 was released from the nucleus to the extracellular in macrophages upon infection with MG. Extracellular HMGB1 bound to TLR2 activating the NF-κB pathway triggering a severe inflammatory storm and promoting the progression of MG infection. More importantly, TLR4 could be activated by HMGB1 to trigger immune disorders after TLR2 was silenced. This disease process could be interrupted by ethyl pyruvate (EP) inhibition of HMGB1 release or glycyrrhizic acid (GA). Furthermore, treatment of MG-infected chickens with GA significantly alleviated immune organ damage. In conclusion, we demonstrate that HMGB1 is secreted extracellularly to form an inflammatory environment upon MG infection, triggering a further cellular inflammatory storm in a positive feedback approach. Blocking MG-induced HMGB1 release or suppression downstream of the HMGB1-TLR2/TLR4 axis may be a promising novel strategy for the treatment of CRD. Furthermore, this study may provide a theoretical reference for understanding non-LPS-activated TLR4 events.

Survey of respiratory disease associated with Mycoplasma gallisepticum in British gamebirds (2016-2019): In vitro antibiotic sensitivity, pathology and detection of other pathogens.

BACKGROUND: The causes of respiratory disease in British gamebirds were investigated during 2016-2019 following concerns about poorer responses to antibiotic treatment. Emphasis was placed on Mycoplasma gallisepticum, but other possible bacterial and viral causes were included, along with gross and histopathological examination. METHODS: Clinical respiratory disease outbreaks were investigated. RESULTS: Mycoplasma gallisepticum was detected by PCR in 65 of 69 outbreaks in pheasants and partridges and isolated from 56 of these. Partial mgc2 gene sequences from 28 M. gallisepticum isolates were compared, and 26 proved identical, suggesting the prevalence of a dominant sequence type. Minimum inhibitory concentration values for tiamulin, tylosin, tylvalosin, doxycycline and tetracycline were significantly higher than the reference strain but could not be correlated with treatment failures. Other bacterial species were isolated from sinuses but were not consistently correlated with disease. RT-PCRs detected coronaviruses in 18% of 49 outbreaks and avian metapneumovirus in 8%. Histopathological lesions were typical of M. gallisepticum sinusitis and significantly associated with M. gallisepticum PCR outbreak positivity. CONCLUSION: Mycoplasma gallisepticum remains an important cause of respiratory disease in gamebirds. Synergism with other pathogens may have played a role in some outbreaks. Specific reasons for variable responses to antibacterial treatment were not identified.

Seroprevalence of major respiratory diseases of chickens in central Ethiopia in different chicken production systems.

In Ethiopia, most chicken disease outbreaks and mortalities are attributed to a respiratory syndrome known as "fengil" with variable clinical signs and undefined etiology. The main goal of this study was to determine whether key respiratory pathogens that could contribute to the fengil syndrome circulate in Ethiopia. Specifically, we aimed to determine the seroprevalence of infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (Mg), and avian metapneumovirus (aMPV). A cross-sectional survey was conducted in 158 scavenging and 42 small and medium-scale intensive chicken holdings in the East, West and North Shewa Zones of central Ethiopia. Blood from 495 chickens was collected and serological tests were used to determine exposure to these pathogens. Vaccination against NDV was the only immunization practiced with a significantly higher vaccination rate in the intensive than the scavenging system. Serological evidence of a high level of exposure to all pathogens was detected, including the first report on the seroprevalence of aMPV, ILTV, and IBV in the East Shewa Zone. The chicken and holding seroprevalence rates were respectively 91% and 94% for IBV, 34% and 57% for aMPV, 47% and 66% for Mg, 27% and 51% for ILTV and in unvaccinated flocks, 39% and 53% for NDV. These pathogens could contribute to the fengil syndrome, commonly ascribed to NDV. The seroprevalence of aMPV and ILTV was higher in chickens under the scavenging system. Exposure to multiple pathogens was common, with more than 50% of chickens positive for three or more pathogens in the scavenging system. This was reflected in significant positive associations between seropositivity to ILTV, Mg, ILTV, and IBV. The role of these pathogens in the causation of respiratory disease in the field requires further investigation.

Evaluation of culturing and molecular assay for detection of Mycoplasma gallisepticum in chicken suffering from chronic respiratory disease.

Mycoplasma gallisepticum (M. gallisepticum) is a bacterium that causes chronic respiratory disease (CRD) and infectious sinusitis (IS) in chicken and turkeys .Therefore, rapid and immediate diagnosis or regular detection of Mycoplasma may be of great help to early detection. 120 chicken layers, Within Karbala city was carried out during their laying period on breeding flocks. The study proposed a promising method for isolation of M. gallisepticum, 120 tracheal swabs and blood samples from chicken in different dairy farms were used to analyze M. gallisepticum utility of PCR and culture. Compared with ELISA anti-IgG M. gallisepticum, the clinical specificity of PCR detection is 89.66%, the sensitivity is 86.36%, and the kappa coefficient is 0.817. Compared with the culture method, the specificity is 100%, the specificity is 45%, and the kappa coefficient is 0.543. Demonstrate the method's effectiveness and applicability as a standard method for mycoplasmas field diagnosis.

Mycoplasma gallisepticum induced exosomal gga-miR-193a to disturb cell proliferation, apoptosis, and cytokine production by targeting the KRAS/ERK signaling pathway.

Mycoplasma gallisepticum (MG) is the main pathogen of chronic respiratory disease (CRD), an infectious disease in chickens with high morbidity. Exosomal miRNAs are emerging as important regulators in host immune response to microbial invasion. Previously, we found that gga-miR-193a was significantly up-regulated in exosomes from MG-infected primary chicken type II pneumocytes (CP-IIs). Therefore, the purpose of this study was to investigate the role of exosomal gga-miR-193a in MG infection. Exosomes were isolated and identified via ultracentrifugation, transmission electron microscopy, and nanoparticle-tracking analysis. Real-time quantitative PCR and Western blot were used to detect the gene expression. Enzyme-linked immunosorbent assay was used to examine the levels of the inflammatory cytokines. CCK-8 and flow cytometry assays were applied to analyze the cell functions. The results showed that MG infection induced high expression of gga-miR-193a in exosomes from CP-IIs. Moreover, exosomes secreted by MG-infected CP-IIs could selectively transport gga-miR-193a into DF-1 cells. Exosomal gga-miR-193a internalized by DF-1 cells inhibited cell proliferation, promoted apoptosis, and increased interleukin-1ß and tumor necrosis factor-α secretions by targeting the RAS/ERK signaling pathway. These results suggest that MG induced the secretion of gga-miR-193a by exosomes to damage the life activities of normal cells, which partially interpreted the mechanism of MG establishing systemic chronic infection in the body.

Detection of Mycoplasma gallisepticum in broiler chickens by PCR.

Background: Mycoplasma is a significant microorganism of poultry, which can cause respiratory infections and synovial inflammation, bringing about huge financial misfortunes to poultry workmanship worldwide. Aim: The goal of existing research was to determine the infection rate of Mycoplasma gallisepticum (MG) from chronic respiratory disease cases among broilers fields in Mosul/ Iraq using the polymerase chain reaction (PCR) technique. Methods: All 92 lungs samples were collected from broilers with classical respiratory signs in different regions of the Nineveh governorate for 3 months from February to April 2021. Results: PCR tests were performed using two couple primers, one for the qualitative amplification of 16S rRNA genes (285 base pairs) in Mycoplasma spp. and the second couple for the detection of M. gallisepticum (580 base pairs). Among the samples obtained from broilers, 87 (94.7%) were positive for Mycoplasma and 79 (85.9%) were positive for M. gallisepticum. Conclusion: Our results showed that MG infection in broiler chickens leads to serious clinical symptoms and severe lesions. The rate of Mycoplasma isolation in this study is high despite the short lifespan of broiler chickens.

Lnc90386 Sponges miR-33-5p to Mediate Mycoplasma gallisepticum-Induced Inflammation and Apoptosis in Chickens via the JNK Pathway.

Mycoplasma gallisepticum (MG) is one of the most important pathogens, that causes chronic respiratory disease (CRD) in chickens. Long non-coding RNAs (lncRNAs) are emerging as new regulators for many diseases and some lncRNAs can function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we found that miR-33-5p was significantly up-regulated both in MG-infected chicken embryonic lungs and chicken embryo fibroblast cells (DF-1), and Lnc90386 negatively correlated with miR-33-5p. miR-33-5p, as a new regulator for MG infection, repressed apoptosis, inflammatory factors in DF-1 cells by targeting JNK1. Further analyses showed that Lnc90386 sponged miR-33-5p to weaken its inhibitory effect on JNK1, forming the ceRNA regulatory network. Furthermore, knockdown of Lnc90386 significantly inhibited apoptosis and inflammatory factors, and promoted DF-1 cells proliferation. However, co-treatment with miR-33-5p inhibitor and Lnc90386 siRNA showed that knockdown of Lnc90386 could partially eliminate the inhibiting effect of miR-33-5p inhibitor on inflammation, cell apoptosis and proliferation. In conclusion, Lnc90386 sponges miR-33-5p to defend against MG infection by inhibiting the JNK signaling pathway.

Measures of tracheal lesions are more discriminatory and reproducible indications of chronic respiratory disease caused by Mycoplasma gallisepticum in poultry.

Mycoplasma gallisepticum is the primary causative agent of chronic respiratory disease in poultry, and vaccination is the measure most commonly used for its control. Pathological changes caused by M. gallisepticum are mainly observed in the trachea and air sacs, but assessment of air sac lesions is subjective. Standardized parameters for evaluation of pathological changes, and their reproducibility and discrimination in uninfected and infected groups, are critical when assessing the efficacy of M. gallisepticum vaccination. This study reviewed and critically appraised the published literature on evaluation of vaccine efficacy against pathological changes caused by M. gallisepticum in poultry in the trachea and air sacs. A search of four electronic databases, with subsequent manual filtering, identified 23 eligible papers published since 1962 describing the assessment of histopathological changes in the trachea using tracheal lesion scores and/or measurement of tracheal mucosal thicknesses and assessment of gross air sac lesions using lesion scores. Measurement of tracheal lesions proved a more reliable and robust method of assessing disease induced by M. gallisepticum when compared to assessment of air sac lesions, highlighting the importance of including assessment of tracheal lesions as the primary outcome variable in vaccine efficacy studies. In addition, this study also identified the necessity for use of a standardized model for evaluation and reporting on M. gallisepticum vaccines to minimize variations between vaccine efficacy studies and to allow direct comparisons between them.RESEARCH HIGHLIGHTS Tracheal and air sac lesions have been used to assess M. gallisepticum vaccine efficacy.The specific parameters and statistical tests used to compare tracheal and air sac lesions vary greatly.Measures of tracheal lesions are more discriminatory than measures of air sac lesions.A standardized model is needed to evaluate vaccines against infection with M. gallisepticum.

Targeted sequencing analysis of Mycoplasma gallisepticum isolates in chicken layer and breeder flocks in Thailand.

Mycoplasma gallisepticum (MG) is one of the most economically important pathogens worldwide. MG affects the respiratory system and impairs growth performance in poultry. In developing countries, the most widely used technique to identify MG is the conventional PCR assay. In this study, 24 MG isolates collected from Thailand farms with unvaccinated chickens during 2002-2020 were characterized by gene-targeted sequencing (GTS), followed by phylogenetic analysis using unweighted pair group method with arithmetic mean. These 24 Thai MG isolates differed from vaccine strains, including the F, ts-11 and 6/85 strains. One isolate showed 99.5-100% genetic similarity to the F strain with 4 partial gene analyses. This result may have been due to contamination from vaccinated flocks because the F strain is the most commonly used vaccine strain in Thailand. However, the GTS analysis using the partial MG genes in this study showed that the isolates could be grouped into different patterns based on individual gene sequences. The phylogenetic analysis of partial mgc2, gapA, pvpA and lp gene sequences classified the Thai MG isolates into 7, 11, 7 and 2 groups, respectively. In conclusion, at least 2 partial MG genes, especially partial gapA and mgc2 genes, are needed to differentiate MG isolates.