In order to limit the spread of the coronavirus, several protective measures have been put in place in the community, in private and public residences and in health care centers. Some measures have a negative impact on communication. They include physical distancing, the use of face masks and shields as well as the increased use of telephone and videoconferencing for distance communication. The effects of COVID-19 are particularly harsh on older adults. Consequently, older adults, especially those with hearing loss, are particularly at risk of experiencing communication breakdowns and increased social isolation. Health care professionals should learn about and be encouraged to use communication strategies to maintain good interactions with their patients. This article proposes practical suggestions to health professionals who interact with older adults, especially those who have difficulty understanding speech. The goal of this article is to inform on the prevalence of hearing loss, the hearing difficulties experienced by older adults, the manifestations of hearing problems, the effects of pandemic protection measures on communication and the strategies that can be used to optimize professional-patient communication during a pandemic. Coronaviruses are known to cause respiratory damage. A new strain of coronavirus, SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), was identified in December 2019. The term COVID-19 refers to the infection caused by the virus, Coronavirus Disease 2019. Current scientific data shows that the virus is transmitted mainly by droplets of respiratory secretions . In order to limit the spread of the virus, several protective measures have been put in place in the community, private and public residences, as well as in health care centers. In addition to hand washing, social isolation and telecommuting, physical distancing and the use of a face mask, face shield and transparent partitions are recommended by public health agencies around the world [Older individuals with hearing loss, whether they use hearing aids or not, learn to employ a variety of strategies to optimize speech understanding and communication –4. For eIn times of pandemic, special consideration should be given to communication among the population of older individuals with hearing loss. Communication with COVID-19 protection measures is problematic. Health care professionals should be aware of this and use strategies designed to optimize professional-patient communication. Effective communication is a shared responsibility; it is not problematic only for persons with hearing loss.This article is intended for all health care professionals who interact and provide services to older adults that experience difficulties understanding speech. The goal of this article is to provide practical strategies that can be used to optimize communication during the pandemic. The prevalence of hearing loss, the communication difficulties, the other manifestations of hearing problems and the effects of pandemic protection measures on communication are addressed. Although the information provided in the article is relevant for all adults with hearing loss, we will concentrate specifically on older adults.According to the World Health Organization , there aPresbycusis, or age-related hearing loss (ARHL), is defined as an age-related elevation of hearing thresholds . This heMany older adults report negative psychosocial and emotional consequences attributable to their hearing loss. For example, they may experience frustration and a reduced self-esteem due to the difficulties associated with understanding speech ,19. BecaThe LIME model is a simplified illustration of the speech communication chain . In lineA communication breakdown occurs when the listener (L), a person with hearing loss, does not understand the intended spoken information transmitted by the interlocutor (I). To understand speech, the listener must detect and recognize the relevant speech elements, use working memory to store the information and apply other cognitive functions to decode the information and generate an appropriate response . The lisThe message itself (M) can constitute the source of a communication breakdown. For example, a communication breakdown would occur if the listener is not knowledgeable of the vocabulary , language structure or cognitive concept being expressed by the interlocutor. Simplifying the language is a helpful strategy to use when communicating with an older person who has difficulty understanding the intended message.Finally, and very importantly with regards to communication, the environment (E) in which a conversation takes place may constitute a facilitator or an obstacle to a fluent verbal exchange. For example, an appropriate source of lighting directed toward the interlocutor’s face will make it easier for the listener to extract the visual speech cues from the communication partner . On the other hand, a source of light emanating from behind the interlocutor may create a lighting contrast that makes it difficult to see the details of the talker’s face.Related to speech communication, the presence of background acoustic noise typically constitutes the single most important obstacle to fluent conversations. Noise may be defined as any unwanted signal. For example, when two or more persons are having a conversation in a cafeteria, the sound generated by the other persons conversing and the clanging of utensils and dishes are considered to be sources of noise. The primary effect of background noise is a decrement in the speech signal-to-noise ratio (SNR), that is the difference in the level of the intended acoustic signal relative to the level of the background noise. For young normal hearing adults, a SNR of at least +1 dB is required to ensure good speech understanding . Older aAll communication breakdowns can be accounted for by an obstacle that occurs at one or more of the fours elements of the LIME model of speech communication. Similarly, strategies that are effective in avoiding or repairing a communication breakdown can be associated with one of the four elements of the LIME model.With the COVID-19 outbreak in countries around the world, the WHO and public health agencies recommend the use of many measures to reduce the transmission of the virus [Physical distancing  is not optimal for communication, especially in the presence of background noise and when one of the communication partners has hearing loss or visual impairment. The level of a sound decreases as a function of the distance from the source of the sound. In a free field , the level of an acoustic signal decreases by 6 dB each time the distance from the sound source is doubled . Under nThe results of recent investigations have revealed that the surgical mask acts as an acoustical low-pass filter, reducing the level of high-frequency sounds (between 2 and 7 kHz) ,39. The Also, the use of face masks may have detrimental psychological effects, which could impact communication as well as the quality of health services provided. For instance, Metha et al. (2020) reported that face masks decrease the feeling of trust between the patient and the health care provider . FurtherThree of the strategies most recommended for countering the deleterious effects of background noise are: 1) eliminate or reduce the background noise , 2) provide visual cues so that the listener can use speechreading to complement the less than ideal acoustic speech signal and, 3) reduce the distance between the two interlocutors. However, pandemic protective measures, such as the use of a face mask and physical distancing, preclude the use of some of those strategies.With the COVID-19 pandemic, there has been a sudden increase in the use of telehealth. Many health care professionals now rely on the telephone or videoconferencing to provide health care services to their patients. Although these communication technologies serve to reduce the risk of transmitting an infectious disease, they can be obstacles to effective communication for older adults, especially for those with hearing difficulties. The primary obstacles to distance communication include (but may not be limited to): the quality of the communication device  and Internet connection, the quality of the acoustic signal , a poor temporal synchrony between the acoustic and the visual signals when videoconferencing and the lack of access to visual cues while using the telephone. These factors may interfere with the fluency of speech communication and lead to communication breakdowns. It is also important to keep in mind that although older adults report mainly positive attitudes toward the use of technologies , they maAs mentioned previously, communication is a shared responsibility between the listener and the interlocutor. Given this premise, using appropriate communication strategies is the responsibility of all communication partners. Communication strategies may be defined as the set of attitudes, knowledge, actions and requests that can be used to promote more effective communication . A commuAnticipation strategies are employed to avoid or minimize communication breakdowns. They aim to anticipate difficult situations. For example, closing the door leading to a noisy hallway before conversing with an older adult with hearing loss would be an appropriate anticipation strategy. Likewise, verifying that a patient’s hearing aids are functioning properly before conducting a medical case history would be classified as an anticipatory strategy. Typical anticipation strategies include informing others about difficulty hearing, modifying the physical environment and informing about the subject and the terminology before the conversation. VIGNETTE 1Mrs. A was recently hired by a nursing home. She previously worked as a nurse in a private clinic, but since the COVID-19 outbreak, she decided to re-orient her career. Today, she must attend to Mr. B, a male patient of 95 years-old, who requires blood work. Before entering Mr. B’s room, Mrs. A puts on her complete protective gear, including a surgical face mask and a plastic face shield. Mr. B is watching television, with is back to the door. The lights are lightly dimmed, and Mr. B is not wearing is hearing aids, which are on the night table. While she prepares the equipment needed, Mrs. A explains to Mr. B that she will draw blood. Mrs. A then takes a seat beside Mr. B in order to begin the blood test. It is only then that Mr. B, notices her presence in the room. He is surprised and startled. He does not understand what this woman, wearing a complete protective attire, is doing in his room. Because of the face mask and shield, Mrs. A's speech is muffled and incomprehensible.1. Flashed the lights to warn Mr. B that someone was entering the room or walked in his line of sight and greet him good morning.2. Turned-on the lights to ensure good lighting, closed the bedroom door and turned off the television to reduce ambient noise as much as possible.3. Asked Mr. B to verify if the hearing aid batteries were operational and then put on his hearing aids (assisting him if help was needed).4. Asked if Mr. B understood correctly or if he needed clarifications.To optimize communication in this setting, Mrs. A could have used anticipation strategies:Repair strategies are actions taken to restore the conversational flow after a communication breakdown occurs. For example, repeating the message while using louder and clearer speech is a form of repair strategy used to overcome a communication breakdown. Clear speech is characterized by a spoken signal that is slightly louder, articulated precisely, uttered at a slower than normal rate, and it includes the insertion of natural pauses between words, phrases and sentences. Although all of those modifications generate a signal that optimizes speech intelligibility, it is important not to exaggerate any of the suggested speech manipulations. The most appropriate repair strategies to use will depend on the source or the cause of the communication breakdown. Applying the LIME model  outlined earlier may be a good way to identify possible sources of a communication breakdown. For example, if it is determined that the breakdown is attributable to an inattentive listener, an efficient repair strategy would be to capture the listener’s attention before repeating the intended message. Typical repair strategies include asking to specify, clear speech, reformulating and using a synonym, elaborating, using non-verbal and body language, giving the context of the conversation and spelling or writing. VIGNETTE 2Mr. M, a 70-year-old male with a severe hearing loss, comes in the pharmacy to pick up his prescribed medicine. The pharmacist (Ms. F) is wearing a face mask and is standing behind a plexiglass partition. The pharmacist reads Mr. M’s prescription and asks if he is familiar with the medication and if he has taken this medication before. Because of the background noise, the mask and the partition, the pharmacist’s voice is distorted and too soft for Mr. M to understand what she asked. He replies, somewhat inappropriately, that it was his family physician that prescribed this medication for him. Given his response, the pharmacist repeats (verbatim) what she asked him initially. After several unsuccessful repetitions (and slightly annoyed), the pharmacist finally gives up, and heads for the laboratory counter to prepare the medication. Upon her return, she explains what the medication is for and instructs Mr. M on the daily dosage he should take. Mr. M has difficulty understanding the technical terms used by the pharmacist. Furthermore, he misunderstands the instructions regarding the daily dosage. He understands that he should take two pills in the morning and at night. In fact, the pharmacist had instructed him to “take one pill in the morning, but not in the evening.” At the cash register, Mr. M does not hear when the pharmacist informs him of the cost of the medication. He simply gives her his credit card rather than ask her to repeat.1. Used clear speech  when providing information to Mr. M.2. Stood facing Mr. M and used body language  to communicate more effectively with the face mask.3. Used a pen and paper to write down the questions and instructions. Also, she could have pointed the window of the cash register to indicate the cost of the medication.4. Avoided jargon or technical language when giving information to Mr. M about his medication, and used short and simple sentences.5. Reformulated the instructions to facilitate Mr. M’s understanding, rather than simply repeating the same thing.6. Ensure that Mr. M understood the instructions by asking him what dosage of medication he should take each day.To optimize communication in this setting, the pharmacist could have used repair strategies:Finally, maintenance strategies are used to demonstrate a person’s interest and active participation in the ongoing conversation. Nodding to signal to the communication partner that the message was understood is a common and effective maintenance strategy to use during a conversation. Common maintenance strategies include physical proximity, visual contact, facial expressions and providing confirmation and feedback. VIGNETTE 3Mrs. Y is a general nurse-practitioner. Because of the pandemic, in order to reduce social contacts and limit the spread of the COVID-19, many of her appointments are conducted by telephone. She conducts her interviews from her hospital office, which is adjacent to a general waiting area shared by several practitioners. Today is a particularly busy day in the clinic and the level of noise in the waiting room is quite high. Mrs. Y's 10-o’clock appointment is with Mr. D, a 65-year-old male with a known age-related hearing loss. Wanting to show her open attitude toward her patients and co-workers, Mrs. Y typically leaves her office door open even during appointments. Mrs. Y uses the hand-free option on her cellphone to call her patient. This allows her to take notes on the computer at the same time as she conducts the interview. Because she is running late in her appointments, Mrs. Y goes straight to the point and questions Mr. D on his symptoms. Mr. D struggles to understand Mrs. Y’s questions, because there seems to be a lot of echo and background noise coming from the telephone. Mrs. Y talks too quickly for Mr. D. to understand her. Consequently, for almost every question he is asked, Mr. D. needs several repetitions before he understands and answers the question.1. Shut the door of her office to reduce background noise and reverberation, that is being transmitted over the telephone line (anticipation strategy).2. Used earphones with an integrated microphone with her telephone, or at least, not use the hand-free function in order to reduce the reverberation and the background noise (anticipation strategy).3. Introduced herself before starting the interview, verified that Mr. D. could hear her well and then ask what the general reason for the consultation was (anticipation strategy).4. Used alternate ways to seek the information sought rather than always repeating exactly the same thing every time Mr. D. asked for a repetition (repair strategy).5. Used clear speech (repair strategy).6. Considered using videoconferencing  (maintenance strategy).7. Verified with Mr. D that he was hearing well the questions and asked him to provide confirmation during the interview that he understood well (maintenance strategy).To optimize communication in this setting, Mrs. Y could have used multiple strategies:Based on the LIME model, In the context of a pandemic, effective communication may be more at risk than usual because protective and preventive measures are applied while providing health care services. Protective measures that may negatively impact speech communication include physical distancing, the use of face masks and shields as well as the increased use of telephone and videoconferencing for distance communication. Older adults, especially those with hearing loss, are particularly at risk of experiencing communication breakdowns and increased social isolation. Using hearing aids may help overcome the loss of signal intensity. However, they do not restore normal hearing and do not improve the loss of clarity. Communication strategies  should be used by all partners involved in the communication situation, including the health care professional, in order to maintain good interactions and prevent communication breakdowns. In almost every country, there are professionals trained to provide hearing health care services . Some of these professionals are responsible for the prevention, assessment and treatment of hearing disorders and provide hearing rehabilitation services for individuals with hearing difficulties. For more information concerning any issues related to hearing and communication, it is suggested that the health care professional consult the accredited hearing health care professional in their jurisdiction.


Some patients still felt mild hearing difficulty although their PTA results were normal, but there was no clear relationship between the VAS score, wave I amplitude and speech recognition scores. The present findings point to the possible existence of cochlear synaptopathy in ears that have completely recovered from unilateral sudden sensorineural hearing loss. We suggest that the causes of cochlear synaptopathy and of idiopathic sudden hearing loss may have something in common.Cochlear synaptopathy refers to a subclinical hearing pathology which could potentially explain hearing difficulties within the normal hearing threshold; it is also called “hidden hearing loss”. We hypothesized that a temporary threshold shift in sudden sensorineural hearing loss (ISSNHL) also affects the function in the synapse. The aim of the study was to evaluate the presence of cochlear synaptopathy in patients who had completely recovered from unilateral SSNHL Nineteen patients who had completely recovered from ISSNHL from January 2018 to June 2021 were assessed. Complete recovery was established by pure tone audiometry (PTA) 3 months after treatment, according to the American Academy of Otolaryngology–Head and Neck Surgery criteria. Subjects completed the pure tone audiometry, speech audiometry and auditory brain stem response (ABR) test, and completed a questionnaire regarding hearing loss after hearing recovery. The ABR amplitudes of wave I and wave V, and the ratio of wave I/V of both ears  were assessed. A visual analog scale (VAS) and a hidden hearing loss questionnaire were used to evaluate subjective hearing difficulty. The ABR waves I of the recovered ears had a significantly lower amplitude ( Cochlear synaptopathy refers to a subclinical hearing pathology which could potentially explain hearing difficulties within the normal hearing threshold, and is also called “hidden hearing loss” . PatientAnimal studies have revealed a decrease in the number of ribbon synapses between inner hair cells and auditory nerve fibers as a result of repetitive exposure to noise, or aging, and such cochlear synaptopathy is considered a main cause of hidden hearing loss ,6. It ocIdiopathic sudden sensorineural hearing loss (ISSNHL) is defined as a sudden increase of 30dB or more in the hearing threshold across three or more consecutive frequencies, and it considered an emergent condition needing prompt management . It is kThe aim of the present study was to investigate the possibility of cochlear synaptopathy by measuring ABR amplitude in patients who had recovered completely from unilateral SSNHL.Among consecutive ISSNHL patients from January 2018 to June 2021, ISSNHL patients with complete recovery were selected and assessed for hidden hearing loss. Based on the 2019 American Academy of Otolaryngology–Head and Neck Surgery (AAO–HNS) guidelines, SSNHL was defined as hearing loss of ≥30 dB at 3 consecutive frequencies within 3 days without an identifiable cause. All patients were treated with uniform management protocol of high-dose oral steroid and salvage intratympanic steroid injection. Complete recovery was defined as hearing returning to within 10 dB of the contralateral  ear at 3 months after treatment . In addiFinally, a total of 19 patients were included. After complete hearing recovery, identified by pure tone audiometry (PTA), patients underwent word recognition test and ABR test. Additionally, subjective hearing status after hearing recovery was evaluated via a hearing questionnaire. In addition, medical records including basic characteristics of the patients, underlying diseases, associated symptoms and treatment methods, were also reviewed.Written informed consent was obtained from all patients, and the investigation was approved by the Institutional Review Board (IRB) of Hanyang University Guri Hospital and performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines (IRB FILE No: 2021-10-002).All eligible patients underwent PTA, a word recognition test and ABR tests after hearing recovery. PTA was performed in the frequency range of 250–8000 Hz, and thresholds were recorded and calculated as the average thresholds at frequencies of 0.5, 1, 2 and 4 kHz. The word recognition score (WRS) was evaluated using the words in Korean Standard Monosyllabic Word Lists for adults (KS-MWL-A). A total of 50 words were randomly presented to listeners at most comfortable level of loudness and the percentage of correct responses were calculated. According to WRS, we divided the subjects into WRS of 100% group and WRS of less than 100% group. The ABR test was performed in a soundproofed room using Biologic Navigator Pro . The test side ear was stimulated by clicks of 90 dB nHL intensity and 100 μs duration repeated at 13.30/s, with alternating polarity. Potentials were amplified with a gain of 100,000 using a low-pass filter of 300 Hz and high-pass filter of 3 kHz. The peaks of each wave were annotated manually by a single senior audiologist by averaging data from two trials. The noise level was kept at zero during the measurement process, and the sweep number was standardized at 1024. In addition, contralateral masking used at a 50 dB HL for counterbalance. The ABR measurements were concentrated on the peaks of wave I and wave V, and amplitude was defined as the vertical distance from the positive peak of each wave to the baseline  15]. Wi. Wi15]. The hearing questionnaire was divided into two domains: the visual analogue scale (VAS) score and the hearing index. The VAS score used a 6-point scale, allowing patients to judge the level of hearing difficulty that they felt on a scale from 0 to 5.(0)No hearing difficulty;(1)Mild hearing difficulty;(2)Moderate hearing difficulty;(3)Moderate to severe hearing difficulty;(4)Severe hearing difficulty;(5)Cannot hear at all.In addition, since there are no established guidelines for diagnosing hidden hearing loss, the authors developed four questions based on the signs that may appear in hidden hearing loss, and used them as a hearing index. The hearing index was calculated by adding up the scores (0 to 4) for which the patient answered ‘yes’ to the four following questions.(1)Do you have trouble hearing when there is noise in the background?(2)Do you have a problem hearing on the telephone?(3)Is it difficult for you to follow a conversation when people talk at once?(4)Do you feel people you are talking to seem to mumble?t-test were used to compare them, and categorical variables were expressed as frequencies and percentages. A two-tailed p-value < 0.05 was considered statistically significant.Statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) for Windows 26.0 . Continuous variables were described by means ± standard deviations and data normality was confirmed with a p–p plot. Paired Student’s Of the 19 patients who completely recovered from SSNHL, 10 were male (52.6%) and the mean age was 45.7 ± 16.3 years. A total of 2 (10.5%) patients had hypertension, 5 (26.3%) had diabetes mellitus and 5 (26.3%) had cardiovascular disease. SSNHL was present on the right and left side in 31.6% (6 patients) and 68.4% (13 patients) of the patients, respectively. Associated symptoms included vertigo (15.8%), tinnitus (89.5%) and ear fullness (78.9%). The mean time from onset of symptoms to treatment was 3.5 ± 2.4 days. A total of 17 (89.5%) patients were treated with a high dose of steroids, of which 13 (68.4%) also received an intratympanic steroid injection. A total of 2 (10.5%) patients only received the intratympanic steroid injection because systemic steroids were contraindicated. The mean initial hearing threshold of the affected ear was 65.7 ± 17.8 dB, which was restored to 12.6 ± 7.8 dB after treatment. The hearing thresholds of the affected ears at each frequency before and after treatment are shown in p = 0.002). On the other hand, the mean ABR amplitude of wave V was 0.21 ± 0.08 μV on the recovered side and 0.24 ± 0.10 μV on the healthy side (p = 0.985). In addition, the wave amplitude ratio I/V was 0.94 ± 0.73 on the recovered side and 0.97 ± 0.66 on the healthy side (p = 0.107)  .p = 0.873). According to the hearing index, the mean amplitude of ABR wave I in the 0-point group without any discomfort was 0.15 ± 0.05 μV, which again was not statistically different from the amplitude in the group complaining of at least one discomfort . In addition, the amplitude of the ABR I wave in the word recognition score of the 100% group and less than the 100% group did not show any statistical significance , whereas the amplitude of ABR wave V and the ratio of amplitudes I/V were not significantly different. (2) According to the VAS score and hearing index, 26.3% (5/19) and 36.8% (7/19) of the patients, respectively, complained of mild hearing discomfort even after complete recovery, but this was not associated with a decrease in the amplitude of ABR wave I.In previous studies, cochlear synaptopathy has been considered a major mechanism of hidden hearing loss. Our intention in this study was to evaluate the possibility of cochlear synaptopathy in patients who have completely recovered from SSNHL. The results of the study can be summarized as follows. (1) The amplitude of ABR wave I on the recovered side was significantly lower than that on the healthy side (The cause of hearing impairment can involve a defect of the cochlea, auditory nerve, or various neurotransmitters in synapses. Defects of the outer hair cells, which are considered the main cause of sensorineural hearing loss, disrupt cochlear amplification . On the The ABR is a recording of the electric potential generated by activation of the auditory nerve and brainstem through the scalp electrode. Wave I represents the compound action potential of the distal cochlear nerve in contact with the inner hair cell, and wave V represents the potentials generated by the auditory brainstem . TherefoThe presumed causes of SSNHL include autoimmune inner ear diseases, viral infections, vascular disorders, ototoxic medication, genetic disorders, trauma, and acoustic tumors, but in most cases, the etiology is uncertain ,22. As oIn this study, we found no correlation between amplitude of ABR wave I and subjective symptoms obtained via hearing questionnaire. This means that some patients may experience perceptual difficulty but have a normal amplitude of ABR wave I, or that other patients may feel they have normal hearing but show a decrease in the amplitude of ABR wave I, implying the possibility of cochlear synaptopathy. This may be because patients perceive sounds from both sides, and the contralateral hearing is normal. In addition, it is thought that subjective symptoms are expressed differently depending on the individual, because there is a complex reciprocal relationship between the peripheral and central auditory nervous systems in the auditory cognitive process.The present study has several limitations. First, only a small number of patients were included in this study because we included only patients who had completely recovered from SSNHL. In addition, since there was no established tool for diagnosing hidden hearing loss, the authors investigated subjective symptoms through a self-designed questionnaire. Studies with a larger number of patients and a more detailed questionnaire are needed. Another limitation is the possibility that comparing the ABR amplitudes on the affected and contralateral sides may not be accurate because the amplitudes of the ABR wave on the affected side before the onset of hearing loss are unknown. Finally, further studies are required using electrocochleography or hearing in noise test, the alternative diagnostic tools for cochlear synaptopathy. Nevertheless, the present study has strength in that it is the first to demonstrate the possibility of cochlear synaptopathy in SSNHL.The decrease in amplitude of the ABR wave I in ears that have completely recovered from SSNHL suggests that cochlear synaptopathy may be involved in the pathogenesis of SSNHL.


Beauveria bassiana is an entomopathogenic fungus that is used for the biological control of different agricultural pest insects. B. bassiana is traditionally cultivated in submerged fermentation and solid-state fermentation systems to obtain secondary metabolites with antifungal activity and infective spores. This work presents the design and characterization of a new laboratory-scale biofilm bioreactor for the simultaneous production of oosporein and aerial conidia by B. bassiana PQ2. The reactor was built with materials available in a conventional laboratory. LaK was determined at different air flows (1.5–2.5 L/min) by two different methods in the liquid phase and in the exhaust gases. The obtained values showed that an air flow of 2.5 L/min is sufficient to ensure adequate aeration to produce aerial conidia and secondary metabolites by B. bassiana. Under the conditions studied, a concentration of 183 mg oosporein per liter and 1.24 × 109 spores per gram of support was obtained at 168 h of culture. These results indicate that the biofilm bioreactor represents a viable alternative for the production of products for biological control from B. bassiana. The production of spores and metabolites, such as antibiotics, enzymes, and pigments from filamentous fungi, has taken on global importance for the biological control of pests and biotechnological purposes ,2,3,4,5.Beauveria bassiana is an entomopathogen and endophyte fungi used as a biocontrol agent against pest insects as a spore formulation −1 using nitrogen as the nebulizing gas and helium as the damping gas. The parameters of the ion source were as follows: spray voltage of 5.0 kV, capillary voltage of 90.0 V, and temperature of 350 °C. Full scan spectra were acquired in the m/z range 100–2000, and, subsequently, the MS/MS analyses were performed on a series of selected ions. The data were collected and processed using MS Workstation software (V 6.9).The filtered total aqueous extract at the final point of fermentation was analyzed by reversed-phase high-performance liquid chromatography (HPLC) equipped with an autosampler , ternary pump , and PDA detector . A sample (5 μL) was injected into a Denali C-18 column . The oven temperature was 30 °C. The elution gradient was formic acid (0.2 % p = 0.05) for comparing the significant differences between airflow rates.The biofilm reactor setup experiments were performed with three repetitions, and the results are presented as mean ± SD. A post hoc analysis was carried out by Tukey test . The maximal production rate was 80.59 mg CO2 mL−1  began biomass formation on the inert support at 50 h. Then, an exponential phase was observed followed by constant sugar consumption until the end of fermentation (168 h) .In regard to the respiration activity of CO2 mL−1  at 168 hCO2 mL−1 . It coul9 conidia/gram of support at 168 h. Although part of the biomass was attached to the walls or internal parts of the biofilm bioreactor, its concentration was not considered in this study. The sterile conditions were confirmed, as there was no contamination of the culture medium. The production of aerial conidia did not present any problems using the metal structured packing as an inert support , allowin−1 at the end of the fermentation. The whole process reached an oosporein productivity of 1.09 mg/L/h  is a parameter that allows characterizing the capacity of a bioreactor to supply oxygen and is very important for the design, operation, and scaling of bioreactors [LaK of the biofilm bioreactor was determined by two methods, one physical method and one chemical method. Under the operating conditions studied, a LaK value of 0.99 to 2.10 min−1 was obtained by the chemical method, while the physical method obtained values of 0.51 to 3.66 min−1. These values are close to those obtained for a circulating-bed biofilm reactor (0.017 s−1) [−1) [LaK values obtained for the biofilm reactor built for this work are within the values (9.5–208 h−1) measured for different commercial stirred-tank bioreactors used for the production of B bassiana blastospores [Oxygen availability is a critical factor for oduction ,31,32,33reactors . Therefo017 s−1)  and high−1) [−1) . Furthertospores .B. bassiana PQ2 was carried out using the aeration flow rate of 2.5 L/min, which also contributed to the high availability of oxygen at the liquid–gas interface [Hence, the bioprocess using nterface .B. bassiana PQ2 when adapting to the fermentation conditions , the aerial conidia were harvested from the metal solid support . The yiez et al.  for sporg et al.  using a bstrates  and 5.0 ice husk . These rBeauveria bassiana under submerged fermentation, which shows antimicrobial and insecticidal activities. The yield obtained (183 mg/L−1) is close to the 270 mg/L−1 reported by Strasser et al. [B. brongniartii in submerged culture. Amin et al. [B. bassiana in submerged fermentation, reaching a yield of up to 480 mg/L. In addition, a combination of spores and pigment increases the insecticidal activity, which suggests that the use of the biofilm bioreactor developed in the present research would help to obtain infective units and metabolites in a single step for their possible use in the biological control of pests in further in vitro experiments. The production dynamics of oosporein  by Trichoderma reesei [Aspergillus clavatus in a biofilm reactor have been shown to be effective in mosquito (Culex quinquefasciatus) control [The results of this research represent a viable and novel way to produce aerial conidia and oosporein from a reesei . In addi control . LaK of the biofilm bioreactor showed high oxygen transfer in both liquid and gaseous phases. The designed bioreactor ensured the development of SmF and SSF at the same time and the production of both aerial conidia and oosporein by Beauveria bassiana PQ2. The CO2 production dynamic allowed determining the fungal growth in the whole process. To our knowledge, this is the first report on the biomass estimation of B. bassiana through respirometry and the production of oosporein in a biofilm bioreactor. It represents a viable alternative for obtaining value-added products from filamentous fungi.The


We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies.Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for This article has an associated First Person interview with the first author of the paper. Summary: Characterisation of a novel loss-of-function zebrafish model for CDKL5 deficiency disorder, containing a nonsense mutation, demonstrates its utility for investigating disease aetiology and allowing high-throughput screening for potentially effective therapies. CDKL5). Patients with CDD often display a heterogeneous array of clinical phenotypes, including early infantile epilepsy, delayed motor function and intellectual disability  is a severe neurodegenerative disease caused by mutations in cyclin-dependent kinase-like-5  mice recapitulate many features of CDD, exhibiting severe neuronal impairment leading to deficits in learning, memory and social behaviour . These din vivo analysis of neurological development and CDD progression. Notably, the optical clarity and power of live imaging, combined with the accessibility of developing zebrafish larvae, provide novel ways for monitoring disease states. Development of both the brain and spinal cord is highly conserved between humans and zebrafish, as well as many signalling pathways involved in their function. In zebrafish, the skeletal muscle makes up ∼80% of the body mass, is functional as early as 20 h post fertilization (hpf) and, after 2 days post fertilization (dpf), the fish swim spontaneously. Muscle contractions controlling early spontaneous motor activity are likely to be directly modulated by embryonic central nervous system activity. At this time, the upper motor neurons in the primary cortex send their projections to the developing spinal cord, and it is only after the fast muscle is formed at ∼52 hpf that the lower motor neurons project to the myotome  are produced during zebrafish development, which differ in the inclusion of exon 16  and the notochord (or developing spine) at 3 dpf . We also used a sense cdkl5 mRNA probe and observed low and non-specific expression, unlike that observed for the antisense cdkl5 probe . The brain, spinal cord, eyes, muscle and kidney represent tissues that are affected in patients carrying CDKL5 mutations  creating a premature termination codon leading to a truncated and non-functional gene product  compared to both +/−cdkl5 (1.055±0.0647 mean±s.e.m.) and +/+cdkl5 fish (1.00±0.0469 mean±s.e.m.) (−/−cdkl5 fish (1.108±0.0892 mean±s.e.m.) compared to their siblings  . However, we did detect a significant decrease in the normalised duration spent swimming by −/−cdkl5 (0.774±0.0663 mean±s.e.m.) compared to +/+cdkl5 fish (1.00±0.0622 mean±s.e.m.), suggesting the reduced distance swum by −/−cdkl5 fish is due to reduced activity (−/−cdkl5 compared to +/+cdkl5 fish (Patients carrying ±s.e.m.) A. Intere±s.e.m.) B. We alsactivity C. We ana+/+ fish D,E at 2 +/−cdkl5 fish to a Tg(islet1:EGFP) zebrafish transgenic strain, labelling all of the motor neurons and ventral interneurons with enhanced green fluorescent protein (EGFP) (islet1:EGFP);−/−cdkl5 (244.6±2.65 mean±s.e.m.) compared to Tg(islet1:EGFP);+/+cdkl5 fish (246.2±5.96 mean±s.e.m.) (islet1:EGFP);−/−cdkl5 (7.004±0.626 mean±s.e.m.) compared to Tg(islet1:EGFP);+/+cdkl5 fish (9.600±0.499 mean±s.e.m.) .We then crossed n (EGFP) . Using t±s.e.m.) A,B, we o±s.e.m.) A,C. Thiscdkl5 mutants display seizures during early embryonic development, we used a Tg(HuC:H2B-GCaMP6s) transgenic strain, in which the genetically encoded calcium indicator GCaMP6s is expressed in mature neurons (HuC:H2B-GCaMP6s);−/−cdkl5 (0.875±0.214 mean±s.e.m.) compared to Tg(HuC:H2B-GCaMP6s);+/+cdkl5 fish (0.296±0.115 mean±s.e.m.) (−/−cdkl5 and +/+cdkl5 fish with a 20 mM pentylenetetrazole (PTZ) solution, a convulsant that affects GABA and glutamate receptors, which has previously been used as a seizure model in zebrafish (HuC:H2B-GCaMP6s);−/−cdkl5 (4.083±0.360 mean±s.e.m.) compared to Tg(HuC:H2B-GCaMP6s);+/+cdkl5 fish (2.974±0.342 mean±s.e.m.) (HuC:H2B-GCaMP6s);−/−cdkl5 fish and 1.031±0.004 for Tg(HuC:H2B-GCaMP6s);+/+cdkl5 fish; mean±s.e.m.] and after PTZ treatment   D, associ±s.e.m.) A-C. To eebrafish . Followi±s.e.m.) F over a ±s.e.m.] E,G.Fig.Fig. S4A). Based on these observations, we selected 2.0 ng MO to use in further experiments. To confirm that cdkl5 mRNA levels are reduced in Cdkl5 morphants, we performed RT-PCR analyses at 2 and 6 dpf and found that cdkl5 mRNA is decreased compared to standard control MO-injected fish . We performed locomotion assays at 6 dpf and found a significant decrease in swimming performance in Cdkl5 morphants (0.678±0.0336 mean±s.e.m.) compared to standard control MO-injected fish (1.00±0.0664 mean±s.e.m.) . We did not observe any defects in muscle patterning in standard control-MO injected fish or Cdkl5 morphants at 2 and 6 dpf , consistent with −/−cdkl5 fish.To confirm that a reduction in Cdkl5 causes defects in motor function and muscle innervation, we used a morpholino (MO) targeting the splice donor site of exon 5 (Cdkl5 ex5 MO) to knockdown Cdkl5 levels. To determine an effective dose for knockdown, we injected the MO at 0.5 ng, 1.0 ng or 2.0 ng into wild-type embryos and performed RT-PCR. We were able to detect a number of smaller sized amplicons by RT-PCR only in the 2.0 ng injected samples . However, in Tg(islet1:EGFP);Cdkl5 knockdown fish, there appeared to be a reduction in the density of axonal projections , recapitulating the pathologies observed in −/−cdkl5 fish  compared to +/+cdkl5 fish (3.793 mm±0.022 mean±s.e.m.)  compared to +/+cdkl5 fish  at 6 dpf ] (P<0.01) in total brain volume in Tg(HuC:EGFP)−/−;cdkl5  compared to Tg(HuC:EGFP)+/+;cdkl5 fish  (P<0.01) in the cerebellar volume of Tg(HuC:EGFP)−/−;cdkl5 (4.880±0.0153 mean±s.e.m.) compared to Tg(HuC:EGFP)+/+;cdkl5 fish (4.948±0.0162 mean±s.e.m.) .CDD patients have been reported to exhibit low muscle tone and a small head size, with some demonstrating cortical atrophy identified through brain imaging . To deteat 6 dpf A, and no±s.e.m.) A,B, therat 6 dpf A,C,D, reC:EGFP)] . We reco±s.e.m.) A,B, in l±s.e.m.) C,D. Addi±s.e.m.) C. We sta−/−cdkl5 and +/+cdkl5 fish. The cranial cartilage of the zebrafish is the first skeletal structure present and forms at ∼3 dpf. As zebrafish grow, the bones continue to ossify, with the first osteoblasts surrounding the cartilage and forming bone matrix by 7 dpf  compared to +/+cdkl5 fish  at 7 dpf  (HuC:EGFP) (HuC:H2B-GCaMP6s) (cdkl5 mutant line (sa21938) obtained from the Zebrafish International Resource Centre. For cdkl5 genotyping, Kompetitive allele-specific PCR technology (Geneworks) was used. For MO injections, the Cdkl5 ex5 (5′-AGATATAAACACTGTCATACCTCTG-3′) and standard control (5′-CCTCTTACCTCAGTTACAATTTATA-3′) MOs (GeneTools) were diluted in distilled water and co-injected with Cascade Blue-labelled dextran (Molecular Probes) into one- to two-cell wild-type  embryos. MO concentrations were calibrated according to Zebrafish were maintained according to standard protocols . Zebrafit1:EGFP) , Tg(HuC:uC:EGFP) , Tg(HuC:GCaMP6s)  and an Nin situ hybridization was carried out as described previously .Whole-mount eviously . Probes +/−cdkl5 incross, for three independent biological replicates. The tails were used for genotyping and the heads were used for RNA extraction, with 20 embryos per genotype per replicate. For MO experiments, whole embryos were analysed at either 2 or 6 dpf. Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies). cDNA was synthesised from 1 μg of each RNA sample in a 20 μl reaction using a Protoscript first strand cDNA synthesis kit (New England Biosciences), oligo(dT)20 and Random Primer Mix primers following the supplier's instructions. Primers used for RT-PCR were cdkl5RTF, 5′-CTCCGTACGCTCAAACAAGAC-3′, and cdkl5RTR, 5′-TGAGCTCTCCCAAAATACACC-3′; cdkl5shortF, 5′-CGCTTTTATGCTCAATGCAC-3′, and cdkl5shortR, 5′-AGCATCTTCAGCTCCCGTAA-3′; cdkl5longF, 5′-GCCAGAAGGTGGCAATAGTT-3′, and cdkl5longR, 5′-CCCGTCCCTAAGTTTCCATA-3′; cdkl5MOF, 5′-CTCCGTACGCTCAAACAAGAC-3′, and cdkl5MOR, 5′-TGAGCTCTCCCAAAATACACC-3′; βActF, 5′-GCATTGCTGACCGTATGCAG-3′, and βActR, 5′-GATCCACATCTGCTGGAAGGTGG-3′; and ef1αF, 5′-CTGGAGGCCAGCTCAAACAT-3′, and ef1αR, 5′-ATCAAGAAGAGTAGTACCGCTAGCATTAC-3′.For the qRT-PCR experiment, head and tails were separated from 3 dpf zebrafish, the progeny of a Locomotion assays were performed on 6 dpf zebrafish as per Immunofluorescence was performed on 2, 3 and 6 dpf zebrafish as per +/−cdkl5 fish were crossed to the Tg(HuC:H2B-GCaMP6s) line and raised to adulthood. Tg(HuC:H2B-GCaMP6s)+/−;cdkl5 fish were crossed to +/−cdkl5 fish, and their progeny were raised in embryo medium  containing 200 µM N-phenylthiourea  from 12 hpf to suppress melanocyte formation, changing medium every 24 h. Embryos were sorted for EGFP fluorescence at 2 dpf. At 4 dpf, fish were anaesthetised using 0.0016% tricaine methanesulfonate (Sigma-Aldrich) in embryo medium, and DNA was extracted from a small tail clipping and used to genotype embryos (as described above).0) was calculated from the first image taken for each fish in standard embryo medium. The final fluorescence values (F/F0) were normalised by dividing the mean fluorescence intensity (F) by the baseline value (F0).At 6 dpf, embryos were again anaesthetised and immobilised in 0.5% low-melting-point agarose in embryo medium. Embryos were first imaged for 5 min in standard embryo medium, after which point PTZ  dissolved in standard embryo medium  from 12 h to suppress melanocyte formation, changing the medium every 24 h. At 7 dpf, embryos were fixed in 4% paraformaldehyde and left overnight in methanol at 4°C. Embryos were rinsed in acid alcohol (70% EtOH:1% HCl) and stained overnight at room temperature in 0.1% Alcian Blue (80% EtOH:20% Acetic Acid). Embryos were washed for 6 h in acid alcohol and then rinsed in PBS plus Tween 20 before imaging.+/−cdkl5 fish were crossed to the Tg(HuC:EGFP) line and raised to adulthood. Tg(+/−HuC:EGFP);cdkl5 fish were crossed to +/−cdkl5 fish, and their progeny were raised in embryo medium containing 200 µM PTU (Sigma-Aldrich) from 12 hpf to suppress melanocyte formation, changing the medium every 24 h. Embryos were sorted for EGFP fluorescence at 2 dpf. At 4 dpf, fish were anaesthetised using 0.0016% tricaine methanesulfonate in embryo medium, and DNA was extracted from a small tail clipping and used to genotype embryos (as described above). At 6 dpf, embryos were again anaesthetised and set in 1% low-melting-point agarose in embryo medium containing tricaine in 0.8 mm fluorinated ethylene propylene tubing (Bola). Images were taken using a Thorlabs confocal microscope with an Olympus 20× water dipping 1.0 NA objective . Brain image registration and total brain and cerebellar volume analysis was performed as per +/−cdkl5 fish were crossed to the Tg(islet1:EGFP) line and raised to adulthood. Tg(islet1:EGFP)+/−;cdkl5 fish were crossed to +/−cdkl5 fish, and their progeny were sorted for EGFP fluorescence at 2 dpf. At 3 dpf, fish were anaesthetised using 0.0016% tricaine methanesulfonate in embryo medium, and DNA was extracted from a small tail clipping and used to genotype embryos (as described above). At 6 dpf, embryos were again anaesthetised and set in 1% low-melting-point agarose in embryo medium containing tricaine, and imaged using an LSM 710 confocal microscope (Zeiss) equipped with a 20× water dipping 1.0 NA objective and a 488 nm laser. Images were taken from comparable regions along the spinal cord for each fish. The total number of EGFP+ motor neuron cell bodies in Tg(islet1:EGFP) zebrafish were counted from a maximum projection image using the cell counter function in Fiji. The axonal density was determined by quantifying the surface of fluorescence above background. Each maximum projection Tg(islet1:EGFP) zebrafish image was cropped to remove any cell bodies. The background was normalised across images, and an upper threshold level of 64,000 and lower threshold level of 9000 was used for each image. The images were converted to a mask image and the percentage area of EGFP+ neuronal projections was determined using Fiji.+/+cdkl5 siblings. All statistical analyses were performed in GraphPad Prism 7 using a one-way ANOVA for qRT-PCR . Data in +/+cdkl5 embryo 4 sample from replicate 4), which was excluded from the analysis.For all experiments, the investigators were blinded to genotype during data capture and once the analyses were completed the genotypes of the fish were resolved. For swimming analyses, all values were normalised to the average value of  qRT-PCR D and cdkg assays A,B, a twanalyses C, seizuranalyses D-F, growurements B,C, braiurements B,C, Alciurements C and Cdk


Mycoplasma hyopneumoniae was evaluated on three pig farms.The field efficacy of a bivalent vaccine containing porcine circovirus type 2b (PCV2b) and M. hyopneumoniae infections between 84 and 126 days of age while concurrent porcine circovirus‐associated disease and clinical M. hyopneumoniae infection between 70 and 105 days of age. Each farm vaccinated pigs with a single dose of a bivalent vaccine at 10 days of age while unvaccinated pigs were administered a single dose of phosphate buffered‐saline at the same age.Three pig farms were used, two of which had a history of subclinical PCV2 and clinical p < .05) when compared with unvaccinated animals. The amount of PCV2d loads in blood and M. hyopneumoniae loads in nasal swabs of vaccinated animals were also significantly lower (p < .05) when compared with unvaccinated animals. Immunologically, vaccinated groups elicited a significantly higher (p < .05) level of protective immunity against PCV2d such as neutralizing antibodies and interferon‐γ secreting cells (IFN‐γ‐SC), as well as protective immunity against M. hyopneumoniae such as IFN‐γ‐SC when compared with unvaccinated animals. Pathologically, vaccination significantly lowered (p < .05) the scores of M. hyopneumoniae‐induced pneumonia and PCV2‐associated lymphoid lesions when compared with unvaccinated animals.Vaccination improved growth performance and reduced clinical scores significantly (M. hyopneumoniae infection under field conditions.The evaluated bivalent vaccine provided good protection against PCV2d and  M. hyopneumoniae infection under field conditions.Bivalent vaccine was effective in improving growth performance and protecting pigs against PCV2d and  PCV2 is a very small DNA virus which can be divided into at least eight different genotypes (‘a’ to ‘h’) based on its sequence identity in open reading frame 2 (ORF2)  and Mycoplasma hyopneumoniae are the smallest self‐replicating bacteria and are distinguished by the lack of a cell wall that is present in most bacteria  was first introduced into the Asian market. In particular, this bivalent vaccine is clinically interesting because it contains the PCV2b genotype. Genetically, PCV2b and PCV2d  are more closely related than PCV2a and PCV2d .Coinfection with PCV2 and 22.1M. hyopneumoniae, while all piglets from this production system were immunized against both pathogens. Farms A and C were selected based on history of concurrent subclinical PCV2 infection and enzootic pneumonia. Herds approximately 12–18 weeks of age displayed subclinical PCV2 infection and enzootic pneumonia characterized by respiratory signs and growth retardation which were mainly associated with a marked increase in the mortality rate  from 21 to 140 days of age. Lungs were examined at the slaughterhouse where it was confirmed that 12 of 20 Farm A pigs and 15 of 20 Farm C pigs had mycoplasmal pneumonia lesions based on scoring from a previous method  at approximately 13–18 weeks of age. A lung examination was performed at the slaughterhouse, which confirmed that 8/10 pigs had mycoplasmal pneumonia lesions. Submitted cases from Farm B met the definition of PCVAD  between May and October of 2019. Farms A, B and C were each a 380‐sow, 160‐sow and 430‐sow (respectively), farrow‐to‐finish swine farm with an all‐in‐all‐out production system. All three farms are located in Chungcheongnam‐do. Sows from these selected farms had not received vaccines against PCV2 or AD Chae,  includin2.2M. hyopneumoniae  at 10 days of age. An equal volume of phosphate‐buffered saline  was injected in the same anatomical location in pigs of the unvaccinated  groups. Both vaccinated and unvaccinated pigs were comingled before they were randomly distributed into four different pens kept within one room. Each pen contained 10 pigs with a similar proportion of each treatment per pen. Pens were identical in design and equipment which included free access to a feed and water trough. Standard farm procedures were followed regarding the feeding and watering of study animals. Blood and nasal swabs were collected at 0 (10 days of age), 18 (28 days of age), 39 (49 days of age), 81 (91 days of age) and 102 (112 days of age) days post‐vaccination (dpv). All methods used in this study were approved by the Seoul National University Institutional Animal Care and Use Committee.The experimental design for the field study strictly adhered to the guidelines set by QIA. QIA guidelines required that 20 piglets  were selected and assigned to each group of vaccinated and unvaccinated animals. In an effort to minimize sow variation, four to six 7‐day‐old piglets were randomly selected from each sow and assigned evenly to either the vaccinated or unvaccinated group using the random number generation function in Excel . The pigs in the vaccinated  groups were injected intramuscularly in the right side of the neck with 1.0 ml of the bivalent vaccine containing PCV2b and 2.3Pigs were monitored and scored weekly for clinical signs as previously described , 70 (60 dpv), 112 (102 dpv) and 175 (165 dpv) days of age. The average daily weight gain  was analysed over three time periods: (1) between 10 and 70 days of age, (2) between 70 and 112 days of age and (3) between 112 and 175 days of age. ADWG during the different production stages was calculated as the difference between the starting and final weight divided by the duration of the stage. Data for dead or removed pigs were included in the calculation.2.5DNA was extracted from serum samples by use of a commercial kit . Real‐time PCR was used to quantify the number of genomic DNA copies for PCV2d . Real‐time PCR was used to quantify the number of genomic DNA copies for 2.7M. hyopneumoniae antibodies with commercial ELISA kits . Serum samples were considered as positive for anti‐PCV2 antibodies if the reciprocal ELISA titre was > 350 and as positive for M. hyopneumoniae antibody if the sample‐to‐positive (S/P) ratio was ≥ 0.4 in accordance with the manufacturer's instructions for each kit. Serum samples were also tested for neutralizing antibodies (NA) against PCV2d  was used to test for both PCV2 and 2.8M. hyopneumoniae and PCV2d‐specific interferon‐γ secreting cells (IFN‐γ‐SC). Peripheral blood mononuclear cells (PBMC) were stimulated using the aforementioned challenge M. hyopneumoniae and PCV2d strains  at the Seoul National University  to estimate the percentage of the lung affected by pneumonia. Scoring was performed out of 100 total possible points over the entire lung as follows: 10 points each to the right cranial lobe, right middle lobe, left cranial lobe and left middle lobe; 27.5 points each to the right caudal lobe and left caudal lobe and 5 points to the accessory lobe . The severity of peribronchiolar and perivascular lymphoid tissue hyperplasia was assessed by scoring mycoplasmal pneumonia lesions (0 to 6)  to obtain the quantitative data. For the analysis of PCV2, 10 fields were randomly selected, and the number of positive cells per unit area (0.95 mm2) was determined as previously described  clinical scores between 25 and 74 dpv when compared with the unvaccinated group. Clinical scores were significantly lower (p < .05) in the Farm B vaccinated group between 32 and 74 dpv, and at 95 and 102 dpv when compared with the unvaccinated group. Farm C clinical scores were significantly lower (p < .05) between 32 and 95 dpv in the vaccinated group when compared with the unvaccinated group  in the ADWG was observed on Farm A between vaccinated and unvaccinated group between 10 and 70, between 70 and 112 and between 112 and 175 days of age. The ADWG in the Farm B vaccinated group during the 10–70 and 112–175 days period was significantly higher (p < .05) than that of the unvaccinated group. The overall growth performance of all three farms (10 to 175 days of age) of vaccinated groups was significantly higher (p < .05) than that of unvaccinated group  number of genomic copies of PCV2d in their blood at 39 dpv compared with that of unvaccinated animals. Vaccinated pigs from all three farms had a significantly lower (p < .05) number of genomic copies of PCV2d in their blood between 81 and 102 dpv compared with that of unvaccinated animals  at all three farms. Vaccinated pigs from Farms A and C had a significantly lower  number of M. hyopneumoniae genomic copies in their nasal swabs when compared with unvaccinated animals at 39 and 81 dpv (Farm A), and 81 dpv (Farms B and C)  Figure .3.6p < .05) in the vaccinated group when compared with the unvaccinated group at 18–102 dpv in three farms. The mean frequencies of PCV2‐specific IFN‐γ‐SC remained at basal levels (< 20 cells/106 PBMC) in both groups until 0 dpv. Thereafter, the mean number of PCV2‐specific IFN‐γ‐SC was significantly higher (p < .05) in the vaccinated group when compared with the unvaccinated group from 18 to 81 dpv (Farms A and C) and from 18 to 102 dpv at Farm B , significant differences between vaccinates and non‐vaccinates were not detected at any of the three farms in regard to anti‐PCV2 antibodies. The PCV2 ELISA titres Figure  and PCV2B Figure .3.7M. hyopneumoniae antibodies were not detected in any of the groups or farms. Vaccinated animals from Farms A and C had significantly higher (p < .05) M. hyopneumoniae ELISA S/P ratios at 39 and 81 dpv (respectively) when compared with unvaccinated animals. Farm B vaccinated animals had a significantly higher (p < .05) M. hyopneumoniae ELISA S/P ratios at 18 and 39 dpv when compared with unvaccinated animals. The mean number of M. hyopneumoniae‐specific IFN‐γ‐SC remained at basal levels (< 20 cells/106 PBMC) in both groups until 18 dpv  in the vaccinated groups when compared with the unvaccinated groups from 18 to 81 dpv (Farm A) and from 39 to 81 dpv (Farms B and C) , anti‐v Figure . The mea) Figure .3.8p < .05) of M. hyopneumoniae‐induced pneumonia and PCV2‐associated lymphoid lesions when compared with unvaccinated pigs. Vaccinated pigs on three farms also had significantly lower numbers of PCV2 antigen‐positive cells within lymphoid lesions when compared with unvaccinated pigs.The results of lung and lymphoid lesion score are summarized in Table 4M. hyopneumoniae resulted in a statistically significant improvement in growth performance when compared with unvaccinated pigs in all three farms, regardless of whether the farm was experiencing a subclinical PCV2 and clinical M. hyopneumoniae concurrent infection (Farms A and C), or concurrent PCVAD and clinical M. hyopneumoniae infection (Farm B). Although Farm B received a PCV2a vaccine, PCVAD was not eradicated. Commercial PCV2a‐based vaccines have proven to provide cross‐protection against PCV2d  could interfere with piglets’ active seroconversion after vaccination  or NA (> 7 log2) titres seemed to show interference with the development of the humoral immune response after vaccination. Most of the piglets (> 80% in three Farms) had lower ELISA (< 9 log2) or NA (< 7 log2) titres at the time of vaccination (data not shown). The data presented in this study support that the bivalent vaccine can elicit PCV2‐specific NA and IFN‐γ‐SC even in the presence of MDA. It is therefore concluded that a negative MDA effect on active immunization after vaccination could not have impacted the efficacy of the bivalent vaccine.The most common age of piglet PCV2 vaccination occurs either at weaning or immediately after weaning (around 3–4 weeks of age). In contrast, the evaluated bivalent vaccine containing PCV2b and M. hyopneumoniae also continues to be debated because of the interference of MDA. M. hyopneumoniae may infect pigs within the first 3 weeks of life . Therefore, an earlier vaccination against M. hyopneumoniae at less than 21 days old may be necessary in order to vaccinate piglets to prevent the onset of a natural infection. A number of studies have been conducted to evaluate early piglet vaccination. One experimental challenge study proved that vaccination of pigs at 7 days old with M. hyopneumoniae was effective in reducing lung lesions even in the presence of MDA at a titre considerably higher than what is typically seen in the field  of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine and by the BK 21 FOUR Future Veterinary Medicine Leading Education and Research Center.All of the methods were previously approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee. Sample collection was carried out according to the animal welfare code of Korea.https://publons.com/publon/10.1002/vms3.420.The peer review history for this article is available at


Unveiling the correlations among molecular structures, morphological characteristics, macroscopic properties and device performances is crucial for developing better photovoltaic materials and achieving higher efficiencies. To achieve this goal, a comprehensive study is performed based on four state-of-the-art non-fullerene acceptors (NFAs), which allows to systematically examine the above-mentioned correlations from different scales. It’s found that extending conjugation of NFA shows positive effects on charge separation promotion and non-radiative loss reduction, while asymmetric terminals can maximize benefits from both terminals. Another molecular optimization is from alkyl chain tuning. The shortened alkyl side chain results in strengthened terminal packing and decreased π-π distance, which contribute high carrier mobility and finally the high charge collection efficiency. With the most-acquired benefits from molecular structure and macroscopic factors, PM6:BTP-S9-based organic photovoltaics (OPVs) exhibit the optimal efficiency of 17.56% (certified: 17.4%) with a high fill factor of 78.44%, representing the best among asymmetric acceptor based OPVs. This work provides insight into the structure-performance relationships, and paves the way toward high-performance OPVs via molecular design. Understanding correlations between molecular structures and macroscopic properties is critical in realising highly efficient organic photovoltaics. Here, the authors conduct a comprehensive study based on four non-fullerene acceptors revealing how the extended conjugation, asymmetric terminals and alkyl chain length can affect device performance. Especially for nonfullerene acceptors (NFAs), it has been evolved in several generations from early twisted molecules based on diketopyrrolopyrrole (DPP) or perylene diimide (PDI) units7, to efficient acceptor-donor-acceptor (A-D-A) type planar acceptors, typically ITIC8, then to state-of-the-art A-DA’D-A type Y-series molecules, like Y69. Each evolution of NFAs has been accompanied with the better understanding on the relationships between material designs and morphological or macroscopic factors. For example, ITIC resolved the paradox between suitable phase separation and efficient charge transport by combining bulky D unit with small but strong A terminal, and Y6 enabled multiple charge transport channels and stronger luminescence efficiencies by introducing an electron-deficient core13. To push OPVs toward a higher efficiency, it’s necessary to build the structure-performance correlations among molecular structures, morphological characteristics , macroscopic factors , and the device performances. For this purpose, NFAs with delicately tailored molecular structures should be designed to perform the systematic studies. Compared with symmetric NFAs, asymmetric electron acceptors with two different terminals, e.g., A1-D-A2 type molecules, have more possibilities in achieving more functionalities by combining or compromising diverse merits from two terminals16. For example, our group previously reported asymmetric electron acceptors, BTP-S1 and BTP-S2, by introducing halogenated indandione (A1) and halogenated 3-dicyanomethylene-1-indanone (A2) as two different terminals, leading to high electroluminescence quantum efficiency and efficient charge separation simultaneously16. Thus, together with symmetric electron acceptors, asymmetric electron acceptors provide an extra dimension to tailor the molecular structure for the correlation studies.Organic photovoltaics (OPVs) have now entered a period of prosperity with power conversion efficiencies (PCEs) growing up to over 18%, due to the innovations of photoactive materialsVoc), short-circuit current density (Jsc), and fill factor (FF), which ultimately determine the overall PCE. Generally, the molecular structures and the subsequent morphological characteristics of active layer are closely related to the macroscopic properties of the thin films, and can further determine the photovoltaic parameters. Currently, single correlation is gradually established. For example, it’s recognized that the radiative loss below bandgap and nonradiative loss, which are correlated with energetic disorder and electroluminescence quantum efficiency (EQEEL), respectively, are two main channels limiting the open-circuit voltage22. In principle, a nanoscale phase-separated blend morphology is required to match the exciton diffusion length for efficient charge separation, and dominant π–π stacking orientation of NFAs is also needed for vertical charge transport26. In NFA systems, the OPVs have been observed to work efficiently at a small exciton separation driving force, and thus carrier dynamics of NFA systems with small driving forces, including hole transfer and exciton lifetime, is also worthy of exploring30. Nevertheless, comprehensive understanding from molecular structure to device performance is still required to unveil the fundamentals of OPVs and guide the community to higher device performance.Device performances of OPVs are composed of three photovoltaic parameters of open-circuit voltage , density functional theory (DFT) calculations at B3LYP/6-31 G were performed and the results were presented in Supplementary Fig. Normally, terminal tuning will affect the energy levels. To compare the energy levels of these four electron acceptors, cyclic voltammetry (CV) method was firstly applied Fig. . As expe40. The absorption spectra of the four electron acceptors in chloroform (CF) solutions and thin films were measured and compared  and 16.76% (BTP-S7), while those based on two asymmetric electron acceptors could achieve higher PCEs of 17.33% (BTP-S8) and 17.56% (BTP-S9). A certified efficiency of 17.4% was achieved for PM6:BTP-S9-based OPVs from National Institute of Metrology (NIM), China  measurements were performed to examine the properties of photocurrent generation, and the relevant results were illustrated in Fig. Light stability and thermal stability of OPVs based on the four NFAs were examined and compared Fig. . After l−4 cm2 V−1 s−1  method. The results were illustrated in Fig. J–V curves under various light intensities (Plight)41. The relationship between Voc and Plight can be described as Voc ∝ nkT/qln(Plight), in which k is the Boltzmann constant, T is the Kelvin temperature, and q is the elementary charge. From the Voc − Plight relationship, we could judge whether bimolecular recombination (if n close to 1) or trap-assisted recombination (if n close to 2) was the dominant charge recombination form. As shown in Supplementary Fig. Jsc − Plight relationship, which could be described as Jsc ∝ Plightα. The more closer to 1 of α value is, the less bimolecular recombination it is. The α values were calculated as 0.996 for PM6:BO-4Cl-based OPVs, 0.995 for both PM6:BTP-S7 and PM6:BTP-S8-based OPVs, and 0.999 for PM6:BTP-S9-based OPVs  of a device, the nonradiative loss can be easily calculated through the following formula:k is the Boltzmann constant, T is the temperature in Kelvin. Relevant characterizations to calculate the energy loss can be found in Fig. According to Shockley-Queisser (SQ) limit, energy loss of a device contains three parts, as shown in the following formulaE1, it’s unavoidable and all four types of OPVs showed the similar values of ΔE1 due to their similar band gaps. For ΔE2, BTP-S7 and BTP-S8-based OPVs had smaller ΔE2 than BO-4Cl and BTP-S9-based OPVs, which is related with their energetic disorder at the tail-state absorption. The degree of energetic disorder can be quantified with the Urbach energy (EU). The relationship between tail-state absorption (α(E)) and EU follows the Urbach rule described by the following formula43:0 and E0 are two constants and E is the photon energy. Thus, the smaller EU means the lower energetic disorder. By measuring the high-resolution Fourier transform photocurrent spectroscopy EQE spectra (FTPS-EQE) or absorption coefficients, we were able to derive EU through exponential fitting. The Urbach energy values of OPV devices based on the four NFAs exhibit negligible differences, but get decreased compared to those of pristine NFA films as shown in Supplementary Fig. EU were calculated as 21.37, 21.27, 20.79, and 21.39 meV for devices based on PM6:BO-4Cl, PM6:BTP-S7, PM6:BTP-S8, and PM6:BTP-S9, respectively. BTP-S7 and BTP-S8-based devices indeed possessed smaller EU values and less energetic disorder than BO-4Cl and BTP-S9-based devices, even though polymer donor PM6 was introduced. To the best of our knowledge, the EU value of 20.79 meV for PM6:BTP-S8-based device is the lowest in highly efficient OPVs. Therefore, lowering energetic disorder is beneficial for reducing radiative loss below bandgap. For ΔE3, BTP-S7-based OPVs possessed the lowest ΔE3, BO-4Cl-based OPVs owned the highest ΔE3, while BTP-S8 and BTP-S9-based OPVs had the in-between ΔE3. Finally, the relationships in total energy losses were similar to the relationships in ΔE3, representing that nonradiative loss was still the main factor limiting the energy loss.For ΔBlend morphology is one of the key factors in affecting device performances. Atomic force microscopy (AFM) technology was firstly utilized to study the upper surface topographies. For a preferred blend morphology, bicontinuous interpenetrating networks and nanoscale phase separations are required. From the AFM images Fig. , the abo−1 (d = 3.59 Å) was presented for BTP-S7, BTP-S8, and BTP-S9 in the out-of-plane (OOP) direction, but at q = 1.77 Å−1 (d = 3.55 Å) for BO-4Cl. In the in-plane (IP) direction, a lamellar peak at q = 0.39 Å−1 (d = 16.10 Å) was observed for BO-4Cl, BTP-S8 and BTP-S9, but at q = 0.37 Å−1 (d = 16.97 Å) for BTP-S7. All four electron acceptors showed face-on orientations. For PM6:NFA blends  from polymer donor PM6 in the OOP direction. However, the lamellar peaks of acceptors in the IP direction also moved to q = 0.30 Å−1, under the influences of polymer donor PM6, indicating strong influence of PM6 on the molecular packing of NFAs. In addition, a backbone ordering peak at q = 0.40 Å−1 (d = 15.70 Å) in the IP direction appeared for the PM6:BTP-S9 blend. As for the (010) π–π stacking peak, it was located at q = 1.73 Å−1 (d = 3.63 Å) for both PM6:BTP-S7 and PM6:BTP-S8 blends, but at q = 1.74 Å−1 (d = 3.61 Å) for PM6:BO-4Cl blend and q = 1.75 Å−1 (d = 3.59 Å) for PM6:BTP-S9 blend. Above distinct features of molecular packing from the acceptors in the blends might also reveal the molecular packing information in the single crystals of relevant NFAs, as demonstrated in previous works44. Comparing with neat acceptor films, only PM6:BTP-S9 blend could maintain the position of π–π stacking peak as that of neat acceptor, after introducing polymer donor PM6, while the other three blends would all be affected. Thus, the tightest π–π stacking was achieved in PM6:BTP-S9 blend, and the retention of the pristine NFA crystalline structure indicates more pure phase in blend. Besides, higher crystallinity was also observed in PM6:BTP-S9 blend, than those in PM6:BTP-S7 and PM6:BTP-S8 blends. Thereby, the tighter molecular packing and higher crystallinity enabled PM6:BTP-S9 blend possessing the highest electron mobility among the four blends as shown above.To further study the crystallinity, molecular packing and orientation in films, grazing-incidence wide-angle X-ray scattering (GIWAXS) characterization was performed. For neat acceptor films Fig. , a dominnds Fig. , all fouDifferent molecular aggregation behaviors presented in AFM images possibly led to different domain sizes. To quantify the domain sizes, grazing-incidence small-angle X-ray scattering (GISAXS) characterization was also performed. The results were displayed in Fig. Photoinduced charge carrier dynamics is also crucial in determining the OPV efficiency. First, we used time-resolved photoluminescence spectroscopy (TRPL) to measure the lifetimes of neat NFA and PM6:NFA blend films  for electron transfer, while HOMO offsets were all less than 0.3 eV, thus we here mainly focused on the hole transfer dynamics. Since the absorption of PM6 and NFAs are well separated, 750 nm excitation was chosen to selectively excite the NFAs. The TA spectra of neat BTP-S9 and PM6:BTP-S9 blend are shown in Fig. 1 and τ2, in which τ1 represents the ultrafast exciton dissociation time at the interfaces and τ2 represents the slower exciton diffusion assisted interfacial hole transfer process45. As displayed in Fig. 1 = 0.152 ± 0.015 ps for PM6:BO-4Cl, 0.144 ± 0.014 ps for PM6:BTP-S7, 0.133 ± 0.013 ps for PM6:BTP-S8, 0.160 ± 0.016 ps for PM6:BTP-S9). Although PM6:BTP-S7 blend had smaller HOMO offset than PM6:BO-4Cl, faster hole transfer speed was observed in PM6:BTP-S7 blend, indicating NC-2F with more delocalized electron cloud distribution , which were related with charge transport and phase separation properties. Thus, by combining the values of τ1 and τ2 and their relevant proportions, we could acquire the total average times for hole transfer process (τh) as 1.69 ± 0.17 ps, 0.89 ± 0.09 ps, 1.31 ± 0.13 ps, and 1.16 ± 0.12 ps for PM6:BO-4Cl, PM6:BTP-S7, PM6:BTP-S8, and PM6:BTP-S9 blends, respectively.The hole transfer process can be best described by two time constants of τIn this section, we will first analyze the relationship between molecular structures and morphological characteristics, then try to correlate molecular structures with macroscopic factors, and finally discuss the echoing between macroscopic factors and device performances. The information of molecular structures, morphological characteristics, macroscopic factors, and device performances is summarized in Fig. (010)) and domain size of pure acceptor phase (in the D:A blends) are selected as the morphological characteristics for comparison , v (Voc/VSQ), and f (FF/FFSQ), and these further can be divided into two categories of photo-carrier generation management representing with j and charge recombination management representing with v × f47. The first term j quantifies property of utilizing photon for generating current, while the latter one v × f numerically illustrates the carrier recombination behaviors in the whole working regions, i.e., from 0 V to Voc. With similar Jsc values and band gaps, these OPVs demonstrate semblable situations of photo-carrier generation management except PM6:BTP-S7. Whereas PM6:BTP-S9-based OPV shows better carrier recombination property than other three OPVs, due to the concerted benefits from Voc and FF as analyzed above. These results determine PM6:BTP-S9 as the best-performance system among the four NFAs, stressing that the molecular asymmetry might be one of the options for well balancing parameters trade-off. A further step in efficiency is possible if carrier generation and recombination can be better manipulated. As results, the above analysis illustrates the correlations among molecular structures, morphological characteristics, macroscopic factors, and device performances, and has great implication for developing better-performing OPVs.Finally, correlation between the macroscopic properties and device performances is depicted Fig. . PhotovoIn conclusion, we performed a multidimensional study based on state-of-the-art NFA systems to build correlations among molecular structures, morphological characteristics, macroscopic properties, and device performances. Four NFAs involved in three molecular structure features of extending conjugation, asymmetric terminals, and alkyl chain length tuning were selected or synthesized. After correlation analysis, it was found that extending conjugation at the terminals of NFAs could have a positive effect on boosting charge separation and lowering nonradiative loss, thus beneficial for a high open-circuit voltage. It was also indicated that asymmetric terminals could better balance the three device parameters for a high efficiency. It should be noticed that the length of alkyl side chain affects the molecular packing and electron mobility. Shortening the alkyl side chain could enhance the molecular packing strength and decrease the π–π distance, thus enhancing the electron mobility, finally leading to a higher FF. As a result, with the most-acquired benefits from molecular structure and macroscopic factors, the OPV based on the designed asymmetric acceptor BTP-S9 with shorter alkyl side chain achieved the optimal PCE of 17.56% with a high FF of 78.44%. The correlations revealed in this work provide a guideline for optimizing specific factors via molecular design, thus pave the way toward developing better photoactive materials for higher device performances.PM6 and BO-4Cl were purchased from Solarmer Materials Inc. The detailed synthesis procedures of BTP-S7, BTP-S8, and BTP-S9 are described in 2, as defined by the overlapping area of ITO and Ag. A mask with an area of 5.979 mm2  was used to measure the efficiencies. The J–V measurement was performed via the solar simulator  along with AM 1.5 G spectra at 100 mV/cm2, that was calibrated by the certified standard silicon solar cell  with KG-2 filter. Devices were tested in N2-filled glovebox. The scan direction is −0.2 to 1.2 V, with a scan step of 0.01 V and dwell time is 1 ms. The EQE data were obtained by using the solar-cell spectral-response measurement system .Organic photovoltaics were fabricated on glass substrates commercially pre-coated with a layer of ITO with the conventional structure of ITO/PEDOT:PSS/Active Layer/PFN-Br/Ag. Prior to fabrication, the substrates were cleaned using detergent, deionized water, acetone and isopropanol consecutively for 15 min in each step, and then treated in an ultraviolet ozone generator for 15 min before being spin coated at 4500 rpm with a layer of 20 nm thick PEDOT:PSS (Baytron P AI4083). After baking the PEDOT:PSS layer in air at 170 °C for 20 min, the substrates were transferred to a glovebox. Then the active layer was spin coated from chloroform solution  at relevant speed  for 30 s to form the active layers. Notably, the PM6:BTP-S9 solution should be heated at 60 °C for 1 h before spin coating. Then an extra preannealing at 80 °C (PM6:BTP-S7 and PM6:BTP-S9) or 100 °C (PM6:BO-4Cl and PM6:BTP-S8) for 10 min was performed. Then a 5 nm thick PFN-Br film was deposited as the cathode buffer layer by the spin coating of a solution of 0.5 mg/mL PFN-Br in methanol. Finally, the Ag (100 nm) electrode was deposited by thermal evaporation to complete the device with an active area of 9.25 mmFurther information on research design is available in the Supplementary InformationPeer Review FileSolar Cells Reporting Summary


Infectious bronchitis virus (IBV) and H9N2 avian influenza virus (AIV) are frequently identified in chickens with respiratory disease. However, the role and mechanism of IBV and H9N2 AIV co-infection remain largely unknown. Specific-pathogen-free (SPF) chickens were inoculated with IBV 2 days before H9N2 virus inoculation (IBV/H9N2); with IBV and H9N2 virus simultaneously (IBV+H9N2); with H9N2 virus 2 days before IBV inoculation (H9N2/IBV); or with either IBV or H9N2 virus alone. Severe respiratory signs, pathological damage, and higher morbidity and mortality were observed in the co-infection groups compared with the IBV and H9N2 groups. In general, a higher virus load and a more intense inflammatory response were observed in the three co-infection groups, especially in the IBV/H9N2 group. The same results were observed in the transcriptome analysis of the trachea of the SPF chickens. Therefore, IBV might play a major role in the development of respiratory disease in chickens, and secondary infection with H9N2 virus further enhances the pathogenicity by inducing a severe inflammatory response. These findings may provide a reference for the prevention and control of IBV and H9N2 AIV in the poultry industry and provide insight into the molecular mechanisms of IBV and H9N2 AIV co-infection in chickens. Coronaviruses are important infectious pathogens affecting humans and animals and are classified into four genera: α, β, γ, and δ . In receMycoplasma gallisepticum, avian metapneumovirus, and infectious laryngotracheitis virus, is common in chickens  chicken model and found that IBV could increase the pathogenicity of H9N2 AIV. We also demonstrated that co-infection with IBV and H9N2 virus could exacerbate inflammation in the trachea, lung, and kidneys. Furthermore, the transcriptome analysis of the trachea of the SPF chickens showed that co-infection with IBV and H9N2 virus could induce a stronger inflammatory response. These findings expand our understanding of the synergistic infection of IBV and H9N2 viruses in chickens.All animal experiments involved in this study were approved by the South China Agricultural University Experimental Animal Welfare Ethics Committee and were performed in strict accordance with the approved guidelines.50) of these viruses were determined as described previously. One-day-old SPF chickens were purchased from Guangdong Wens Dahuanong Biotechnology Co., Ltd (production license: SCXK [Yue] 2018–0019) and housed in isolator cages.The current epidemic H9N2 AIV (A/chicken/ Hunan/HN/2015) belonged to the h9.4.2.5 lineage, as described previously . The IBV6 EID50 H9N2 virus and IBV, respectively  as the control group. Oropharyngeal and cloacal swabs were collected from each chicken and suspended in 1000 μL PBS at 3, 5, 7, 9, 11, and 13 dpi. At 3, 5, and 7 dpi, three chickens in each group were euthanized. Gross lesions were recorded as described previously  and RNAfast200 , respectively, according to the manufacturer's instructions. Viral loads of H9N2 AIV and IBV in the tissues and swabs samples were detected using the HiScript II One Step qRT-PCR SYBR Green Kit . To investigate the mRNA expression of inflammatory cytokines, RNA from tested tissues samples was reverse-transcribed to cDNA using the HiScript II Q RT SuperMix for qPCR + gDNA wiper (Vazyme) and determined with qRT-PCR using SYBR® Premix Ex Taq™ (Takara). The primers are shown in To calculate the viral load, we generated a standard curve for each virus, as described previously. Briefly, the same region of the N gene of IBV and M gene of H9N2 virus was amplified and cloned into the pMD-19T vector (Takara) as the standard plasmid. We plotted the copy number of the standard plasmid for each dilution against the cycle threshold (CT) to generate a standard curve.The expression of NLPR3 protein in the trachea, lungs, and kidneys was detected using western blot, as described previously. The rabbit anti-NLRP3 antibody was provided by Professor Zhangyong Ning . The expression of IL-1β in the trachea, lungs, and kidneys was also detected using a chicken IL-1β ELISA kit .Twenty 5-day-old SPF chickens were randomly divided into four groups, namely, the H9N2, IBV, IBV/H9N2, and control groups, and the operation of the viral-infection method for each group was conducted as described in 4.3. At 5 dpi, three chickens in each group were euthanized, and tracheal samples were taken and sent to Health Time Gene Co., Ltd. , for transcriptome analysis as described previously .P < 0.05 was considered statistically significant. The “a” was used to indicated a significant difference (P < 0.05) between the three co-infection groups and the IBV group, while “b” was used to indicated a significant difference (P < 0.05) between the three co-infection groups and the H9N2 group.All data were analyzed using two-way analysis of variance with GraphPad Prism8.0 software. The data were expressed as means ± standard deviations, and a To evaluate the clinical signs caused by co-infection with IBV and H9N2 AIV in chickens, four groups of 19 one-day-old chickens were inoculated intranasally with the H9N2 virus, IBV, or both. The morbidity was 40 and 30% in the single-infection H9N2 and IBV groups, respectively. However, the morbidity was 60, 60, and 70% in the IBV+H9N2, H9N2/IBV, and IBV/H9N2 groups, respectively . Only miSignificant swelling of the kidneys and signs of a “spotted kidney” were noted at necropsy in the IBV+H9N2, IBV/H9N2, and H9N2/IBV groups but not in the IBV and H9N2 group, while kidney swelling was observed in the IBV group . NotablyCompared with the control group, obvious pathological changes in the trachea and kidneys in the co-infection groups were noted, especially in the IBV/H9N2 group. Varying degrees of epithelial degeneration of ciliated cells, hemorrhage, mucosal injury, and mononuclear cell infiltration could be observed in the tracheas of chickens in the co-infection groups . SimilarP < 0.05) in the IBV/H9N2 group at 5 and 7 dpi, as well as in the IBV+H9N2 group at 3 dpi and 7 dpi, respectively (P < 0.05) in the following: the trachea at 5 and 7 dpi, and the lungs and kidneys at 5 dpi in the IBV/H9N2 group; the lungs and kidneys at 5 dpi, and the trachea at 7 dpi in the IBV+H9N2 group; and the lungs and kidneys at 5 dpi in the H9N2/IBV group  for virus titration. Compared with the single infection IBV group, the IBV loads in the trachea and kidneys were significantly higher (ectively . CompareBV group .P < 0.05) than those in the IBV group at 3, 7, and 9 dpi (P < 0.05) IBV shedding titers were also observed in the oropharyngeal region in the IBV+H9N2 group at 3 and 1 dpi, and in the IBV/H9N2 group at 7 and 9 dpi. Meanwhile, the shedding titers of IBV in the cloacal tissue in the IBV/H9N2 group were also significantly higher (P < 0.05) than those in the IBV group at 3, 5, 7, and 11 dpi (P < 0.05) than those in the H9N2 group  in the IBV/H9N2 group than in the IBV and H9N2 groups (P < 0.05) in the IBV+H9N2 group than in the single-infection IBV and H9N2 groups. The expression levels of IFN-β at 7 dpi and IL-18 at 5 dpi were significantly higher (P < 0.05) in the IBV+H9N2 group than in the H9N2 group; those of IL-6 and NLRP3 at 7 dpi were significantly higher (P < 0.05) in the IBV+H9N2 group than in the IBV group. The expression levels of IFN-α and IL-8 at 7 dpi were significantly higher (P < 0.05) in the H9N2/IBV group than in the IBV and H9N2 groups. The IFN-β, IL-1β, and IL-18 mRNA expression at 5 dpi and the IL-6 mRNA expression at 5 and 7 dpi were significantly upregulated (P < 0.05) in the H9N2/IBV group, compared with the H9N2 group.Previous studies have demonstrated that the inflammatory response plays an important role in viral infection. In this study, we investigated the expression of inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, IL-18, NLR family pyrin domain containing 3 (NLRP3), tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), and IFN-β, in the trachea, lungs, and kidneys. In the trachea, the transcriptional expression levels of inflammatory cytokines at 5 dpi  and at 7 dpi (including IFN-α and IL-6) were significantly higher (2 groups . The expP < 0.05) in the IBV/H9N2 group than in the IBV and H9N2 groups (P < 0.05) in the IBV/H9N2 group, compared with the H9N2 group. The expression of IL-6 and IL-18 at 5 dpi, IL-1β and TNF-α at 7 dpi, as well as IFN-α at 5 and 7 dpi were significantly higher (P < 0.05) in the IBV+H9N2 group than in the H9N2 group; the expression levels of IFN-α at 5 and 7 dpi, IL-18 at 5 dpi, as well as IL-1β, NLRP3, and TNF-α at 7 dpi were also significantly higher (P < 0.05) in the H9N2 /IBV group. The expression levels of IFN-β, IL-6, and IL-8 at 7 dpi were also significantly higher (P < 0.05) than in the IBV and H9N2 groups.In the lungs, the expression levels of IFN-α, IFN-β, and IL-18 at 5 dpi and IL-8 at 7 dpi were significantly higher (2 groups . The IL-P < 0.05) in the IBV/H9N2 group than in the IBV and H9N2 groups (P < 0.05). Overall, these results indicated that co-infection with IBV and H9N2 virus could induce a severe inflammatory response in the tissues of chickens.In the kidneys, the expression levels of IL-1β and TNF-α at 5 dpi, IFN-β at 7 dpi, and IL-6, IL-18, and NLRP3 at 5 and 7 dpi were significantly higher  were significantly higher in the tested tissues, including the trachea, lungs, and kidneys, in the co-infection groups than in the IBV or H9N2 group, indicating that co-infection promoted the propagation of IBV and H9N2 viruses. In a previous study, Haghighat-Jahromi et al. showed that the live IBV vaccine strain H120 could enhance the replication of H9N2 AIV (A/chicken/Iran/SH-110/99) . Some reThe pathogenicity of infectious microorganisms mainly depends on the replication capacity and host immune response, such as the inflammatory response. The inflammatory response is a double-edged sword: while fighting infection, it is also accompanied by tissue damage. If the immune system is activated increasingly without control, it will cause the expression of a large number of cytokines and aggregation of immune cells, thereby damaging the host tissues and increasing morbidity and mortality , 55. ForThe proinflammatory cytokine IL-1β is a key mediator of the inflammatory response . The matWe found that co-infection with IBV and H9N2 AIV could induce worse clinical outcomes and high morbidity and mortality in chickens. Furthermore, primary infection with IBV might play a major role in the development of respiratory disease in chickens, and secondary infection with H9N2 virus might further enhance pathogenicity. Co-infection with IBV and H9N2 virus might increase the pathogenicity by inducing a strong inflammatory response. Therefore, these findings suggest that the co-infection with IBV and H9N2 deserves more attention and the threat should not be ignored. Moreover, we lay a foundation for the further study of the synergistic infection mechanism of IBV and H9N2 AIV and provide a scientific basis for the prevention of mixed infections by these viruses in clinical practice.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by South China Agricultural University Experimental Animal Welfare Ethics Committee.LK, RY, LC, and TR designed the experiments. LK, DZ, RY, and BX performed the experiments. LC, QY, JL, and QL assisted with the animal experiments. LK and RY drafted the manuscript. LC, TR, ML, and CD reviewed and revised the manuscript. All authors have read and approved the final manuscript.This research was funded by the National Key Research and Development Program of China (2018YFD0500100) and the Scientific and Technological Research Project of Foshan (2020001000151).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.