RESEARCH HIGHLIGHTS:The co-infection rate of infectious tumour disease in chickens is 37% in China.The incidence rate is the highest in Dezhou City.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a serious threat to the swine industry in China. As a major pig-producing province, Shandong requires continuous epidemiological monitoring of PRRSV. To elucidate the molecular epidemiology of the virus, 1621 clinical samples were collected from suspected cases across different regions of Shandong Province between 2023 and 2025, primarily from Tai’an, Linyi, Jining, and Liaocheng. RT-qPCR detection showed that the positive rate for PRRSV-2 was 20.05% (325/1621). Genetic analysis based on ORF5 and NSP2 genes indicated that Sublineage L1C (NADC30-like) was the dominant strain for 38.38% of ORF5 gene and 72.73% of NSP2 sequencing results. This was followed by Sublineage L8E and L1A and L5A strains. Key virulence-related mutations were identified at residues R13 and R151 in the GP5 protein, which are associated with enhanced pathogenicity. Additionally, variations in neutralizing epitope and the number of N-glycosylation sites (ranging from 2 to 5 per strain) suggested potential immune evasion. Notably, 26.79% (15/56) of sequenced samples showed discordant ORF5 and NSP2 genotyping results, indicating widespread recombination among PRRSV strains in Shandong Province. These finding demonstrated that the genetic diversity, high recombination frequency, and key amino acid variations in circulating PRRSV strains collectively undermine vaccine effectiveness. This study highlights the need to optimize vaccination strategies, enhance biosecurity measures, and implement effective disease control and elimination programs to reduce the impact of PRRSV in Shandong Province.

Multiple infections, either single or mixed, involving pathogens such as serotype 4 fowl adenovirus (FAdV-4) and serotype 8b fowl adenovirus (FAdV-8b), have been observed in numerous laying hens in China, leading to severe liver damage. Thus, fowl adenovirus (FAdVs) is speculated to cause and inflammatory response. In this study, single infections with FAdV-4 and FAdV-8b were confirmed through RT-PCR and Enzyme-linked Immunosorbent Assay (ELISA) in specific pathogen-free (SPF) chickens exhibiting severe liver damage. Following this, the two reference strains, FAdV-4 and FAdV-8b, were inoculated into cardiomyocytes (CM) and cardiac fibroblasts (CF) to assess their immune responses. Additionally, the replication dynamics of FAdV-4 and FAdV-8b, as well as the expression levels of immune-related cytokines, were evaluated. The results demonstrated that FAdV-4 significantly enhanced viral replication in the heart, CM, and CF cells. The transcriptional levels of IL-1β, TNFα, IL-6, and IL-8 were markedly increased in cells infected with either FAdV-4 or FAdV-8b. These findings confirmed the in vitro and in vivo infection of FAdV-4 and FAdV-8b, elucidating their pathogenic mechanisms and providing new insights into the viral interactions and immune responses.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortion and respiratory disease in swine, hindering the development of the pig farming industry worldwide. However, at present, there are no effective vaccines or drugs for PRRSV control. In this study, we evaluated the inhibitory effect of hyperoside on PRRSV replication in vitro and in vivo and explored the underlying mechanisms. Our results revealed that hyperoside significantly inhibited PRRSV infection in MARC-145 and porcine alveolar macrophages (PAMs). This inhibition was linked to the hyperoside-induced attenuation of pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) upregulation induced by PRRSV infection, which was mediated by the suppression of the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-kB) signaling pathway. Moreover, hyperoside alleviated the autophagy induced by PRRSV via p62/Nrf2/Keap1 signaling pathway activation. In vivo, hyperoside treatment led to an obvious decrease in PRRSV replication in piglets. Therefore, hyperoside may be a useful antiviral agent against PRRSV.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes abortion and respiratory disease in swine, which induces huge economic losses every year. However, there have been no effective vaccines or drugs for PRRSV control until now. Our present study found that the inhibitory effect of hyperoside on PRRSV replication in vitro and in vivo. Furthermore, we demonstrate that hyperoside inhibits PRRSV proliferation via inhibiting inflammation and autophagy through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) and p62-Nrf2-Keap1 signaling pathways. Hence, we believe that hyperoside may be a useful antiviral agent to control PRRSV.

African swine fever (ASF) is a devastating swine infectious disease that poses a serious threat to the global swine industry. Since ASF was first reported in China in 2018, this disease has caused significant economic losses in China. To characterize the histopathological changes induced by ASF virus (ASFV) in pigs and elucidate its pathogenic mechanism, this study used the ASFV HLJ/18 strain to infect specific pathogen-free piglets. The thymus, bone marrow, spleen, tonsil, lymph node, heart, liver, lung, and kidney were collected within the tenth day post-infection for histopathology, immunohistochemistry (IHC), TdT-mediated dUTP nick-end labeling, and Martius Scarlet Blue staining tests. The results showed that ASFV was widely distributed in various organs. Hemorrhage, cellular degeneration, and necrosis were observed in all examined organs, accompanied by a significant loss of lymphocytes in the immune organs, along with a large number of necrotic and apoptotic cells. Indirect immunofluorescence assay and IHC further showed that ASFV can infect monocytes and macrophages, epithelial cells, and hepatocytes. Apoptosis was prominently observed, particularly in lymphocytes within the immune organs, but it is worth noting that ASFV induced apoptosis in many different cell types in addition to lymphocytes. Furthermore, fibrin was observed in the blood vessels of multiple tissues, which may be associated with disseminated intravascular coagulation. These results demonstrate that the ASFV HLJ/18 strain was highly pathogenic to piglets, leading to severe pathological damage. This study provides valuable insights for deeply analyzing the pathogenicity mechanisms of ASFV. IMPORTANCE:Since African swine fever (ASF) first appeared in China in 2018, it has seriously jeopardized the healthy development of China's swine industry and caused huge economic losses. The characteristic of ASF is extensive hemorrhage in the organs, accompanied by immune suppression in the infected pigs. This study examined the damage caused by the virus and related mechanisms in histopathology and immunopathology. ASFV targets the macrophages, induces massive cell necrosis, and the formation of vascular microthrombi in multiple systems, leading to severe tissue damage and high mortality. Additionally, apoptosis and necrosis occurring in immune organs lead to an imbalance in the number and proportion of lymphocytes, further developing immune suppression. In conclusion, this study provides comprehensive insight into the immunopathology of ASFV infection.

Florfenicol (FLO) is a chemically synthesized broad-spectrum antimicrobial agent of amide alcohols for animals, which is one of the most widely used antimicrobials in livestock, poultry, and aquaculture. With the use of FLO, more and more attention has been paid to its hematopoietic toxicity, immunotoxicity, genotoxicity, and embryotoxicity. In this study, SPF chicks at the age of 3 d began to drink water with the FLO at a dose of 100 mg L-1 for six consecutive days, and the growth performance of chicks was monitored, the effect of FLO on immune organs was detected by pathological examination and TUNEL apoptosis staining. In order to evaluate the level of organism immunity, the level of Newcastle disease virus antibody in serum was detected by hemagglutination inhibition test, the content of cytokines (IL-1, IL-2, IL-6, TNF-α, IFN-γ) in serum was detected by ELISA, and the transcription of interferon-related genes (IRF-7, 2’-5’OAS, Mx1) and cytokine genes (IL-6, TNF-α, IFN-γ) in immune organs were detected by real time fluorescence quantitative PCR. The results showed that the early application of FLO could inhibit the growth and development of chicks, and the body weight and immune organ index of the treatment group were lower than those of the control group. Histopathological examination showed that there was a decrease in the number of lymphocytes in the bursa of Fabricius in the treatment group in the early stage of drug withdrawal, and the results of TUNEL apoptosis staining in the bursa of Fabricius showed that obvious lymphocyte apoptosis occurred in the FLO treatment group. Compared with the control group, the transcription levels of interferon-related genes IRF-7, 2’-5’OAS, and cytokine genes IL-6, TNF-α and IFN-γ in FLO treatment group decreased to a certain extent, while the transcription level of Mx1 gene had no significant difference at all time points. The level of serum NDV antibody and the contents of cytokines IL-1, IL-2 and IFN-γ in the FLO treatment group were significantly lower than those in the control group in the early stage of drug withdrawal, but recovered gradually in the later stage. This study showed that FLO has a certain degree of effect on the immune function of chicks, and the results of the study laid the foundation for further research on the mechanism of FLO-induced immunotoxicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of porcine reproductive and respiratory syndrome (PRRS); a disease of pigs, which results in great economic losses in the pork industry. The non-structural protein 4 (Nsp4), a 3C-like serine protease responsible for most non-structural protein processing, plays an essential role in PRRSV infection. We used label-free quantitative proteomics to elucidate the Nsp4 interactome and SRCAP was identified as one of the interactors. SRCAP facilitated PRRSV infection by activating non-canonical Notch signaling. The ATPase I-IV domain in SRCAP and the (122)VITEA(126) in Nsp4 were identified as the interacting sites. The infection of recovered mutant rTA-12/5A ((122)AAAAA(126)) could not activate Notch signaling. The results indicated that (122)VITEA(126) in Nsp4 were key sites to determine the function of SRCAP and their interaction. A function of Nsp4 in activating the Notch signaling pathway was discovered. Block Notch signaling pathway could inhibit PRRSV infection both in vitro and in vivo which may lead to the development of novel therapeutic antiviral strategies. IMPORTANCE In the present study, the interactome of the NSP4 originating from PRRSV was studied and SRCAP was confirmed as one of the interactors. Mechanism study showed the interaction of Nsp4 and SRCAP was found to facilitate PRRSV infection by activating non-canonical Notch signaling. ATPase I -IV domain in SRCAP and the (122)VITEA(126) in Nsp4 were identified as the interacting sites that demined the activating of Notch signaling. Block Notch signaling pathway could inhibit PRRSV infection in vitro and in vivo which may be a new target for antiviral drug development.

Chickens infected with FAdV-4 and FAdV-8b both exhibit hepatic lesions characterized by hemorrhagic necrosis and intranuclear inclusion body formation. However, only FAdV-4 induces pericardial effusion and acute mortality in chickens. To investigate the similarities and differences in the pathogenicity of HPS and IBH, this study intends to compare the infectivity and pathogenicity of FAdV-4 and FAdV-8b, 2 serotypes of fowl adenovirus isolated in our laboratory. The 2 viruses were respectively inoculated subcutaneously into SPF chicks at the neck. The clinical manifestations and pathological changes in these infected groups of chickens differed to some extent. Chickens infected with FAdV-4 exhibit evident depression and acute mortality, with a mortality rate of 60%; while those infected with FAdV-8b only display mild depression. Postmortem examination reveals serosanguinous effusion in the pericardial sac, spot-like hemorrhage, and focal necrosis in the liver of chickens infected with FAdV-4. Additionally, various degrees of edema are observed in organs such as the lungs, spleen, kidneys, and pancreas. In contrast, chickens infected with FAdV-8b exhibit spot-like hemorrhage and focal necrosis in the liver but do not display pericardial effusion or widespread organ edema. Histopathological examination demonstrates that both FAdV-4 and FAdV-8b can induce inflammatory reactions of varying degrees in the kidneys, pancreas, and duodenum of chickens, while reducing the necrosis of bursa of Fabricius, thymus, and spleen lymphocytes. Our data preliminarily reveal that both FAdV-4 and FAdV-8b can induce strong pathogenicity in chickens.

Swine influenza viruses (SIVs) have been circulating in swine globally and are potential threats to human health. During the surveillance of SIVs in Shandong Province, China, from 2019 to 2022, 21 reassortant G4 genotype Eurasian avian-like (EA) H1N1 subtypes containing genes from the EA H1N1 (HA and NA), 2009 pandemic (pdm/09) H1N1 virus (PB2, PB1, PA, NP, and M), and classical swine (CS) H1N1 (NS) lineages were isolated. The analysis of the key functional amino acid sites in the isolated viruses showed that two mutation sites (190D and 225E) that preferentially bind to the human α2-6 sialic acid receptor were found in HA. In PB2, three mutation sites (271A, 590S, and 591R) that may increase mammalian fitness and a mutation site (431M) that increases pathogenicity in mice were found. A typical human signature marker that may promote infection in humans, 357K, was found in NP. The viruses could replicate efficiently in mouse lungs and turbinates, and one of the H1N1 isolates could replicate in mouse kidneys and brains without prior adaption, which indicates that the viruses potentially pose a threat to human health. Histopathological results showed that the isolated viruses caused typical bronchopneumonia and encephalitis in mice. The results indicate that G4 genotype H1N1 has potential transmissibility to humans, and surveillance should be enhanced, which could provide important information for assessing the pandemic potential of the viruses.

Chicken infectious anemia (CIA) is a vertical transmission infectious chicken disease caused by the chicken infectious anemia virus (CAV). The disease can induce stunting and immunosuppression in chicks by infecting bone marrow-derived stem cells, causing huge economic losses for the poultry industry. To determine the prevalence of CIA in Shandong Province, China, 854 suspected CIA samples were collected and analyzed in 13 cities in Shandong from 2020 to 2022. The PCR results showed that a total of 115 CAV were isolated. The CAV-positive rates were 17.21% (26/151) in 2020, 12.23% (35/286) in 2021, and 12.94% (54/417) in 2022, with severe mixed infections. Among them, CAV and fowl adenovirus (FAdV) were the most common, accounting for 40.86%. VP1 gene homology analysis showed that isolated strains shared 96.1-100% homology with the previously reported CAV strains. Genetic variation analysis showed that most of the isolated CAV strains were located in genotype A. These results indicate that CIA infection in Shandong chickens in recent years has been prevalent and mixed infections are common, but there were no significant genetic variations. Our results extend the understanding of the prevalence and genetic evolution of CIA in Shandong Province. They will offer new references for further study of the epidemiology and virus variation and the prevention and control of this disease.

The novel avian influenza viruses (AIVs) can cause serious economic losses to the poultry industry. Systematic surveillance of novel AIVs in poultry is essential for control of avian influenza. In the present research, we isolated H3N3 subtypes AIVs from chicken farms reporting illness in China during 20222023. All of these chickens showed respiratory disease signs and a 10-40% reduction in egg production. We conducted epidemiological surveys, virus isolation and identification, sequence analysis, and chicken infection experiments. The results showed that: 1) In December 2022, H3N3 AIVs was first found in a layer chicken flock in Eastern China, and the virus was transmitted to multiple Provinces with high density chicken populations in a short time. 2) Sequence analysis showed that the novel H3N3 AIVs were evolving as a triple reassortment event, which bears H3N8-derived HA gene, H10N3-derived NA gene and H9N2-derived internal genes. 3) The novel H3N3 AIVs were highly susceptible to SPF chickens. The virus replicated efficiently in the concha nasalis, trachea, lungs and Harders glands and infected chickens showed pathological damage. 4) H3N3 viruses were airborne transmission among chickens, whereas H3N8 viruses were not. In conclusion, the novel reassortment H3N3 virus showed pathogenicity and airborne transmissibility in chickens. Therefore, comprehensive surveillance of H3N3 AIVs in domestic poultry is imperative and control of the virus endemic is needed.

Duck Tembusu virus (DTMUV) has caused significant economic losses to the global duck industry since its outbreak in 2010. The macrophages act as the key immune cell, and its polarization in different functional states is very important for host's immune responses and microbial infections. Avian macrophages are the main target cells of DTMUV, its polarization induced by DTMUV and the underlying mechanisms were explored in this study. Through quantitative real-time PCR, nitrite assay, and flow cytometry analysis, we found that DTMUV caused severe inflammatory responses in chicken macrophage line HD11 by reprogramming the expression of M1- and M2-associated genes, leading to the polarization of HD11 macrophage to M1-type. In term of mechanism, transcriptomics was performed to analyze the M1-type polarization triggered by DTMUV, it was found that most differential genes were implicated in biological processes, and DTMUV infection significantly activated innate immune signaling pathways, including cytokine-cytokine receptor interaction, MAPK signaling pathway. Moreover, transcription factors NF-κB and AP1 also be activated after viral infection. However, further validation analysis by inhibitors and siRNAs of NF-κB and AP1 showed that NF-κB molecule was essential for DTMUV-induced M1 polarization in HD11 cell, but not AP1. Additionally, the inhibiting assays targeting MyD88 and TRIF molecules were conducted to determine their effect on NF-κB and M1-associated genes upregulated by DTMUV. The results showed that although the inhibition of both MyD88 and TRIF significantly downregulated the mRNA level of NF-κB, but the expression of M1-associated genes such as CD86 was lower in MyD88 inhibition group than in the other group, indicating that the role of MyD88 in mediating M1 polarization induced by DTMUV was more important. Overall, these results demonstrated that DTMUV infection induces M1-type polarization in chicken macrophage HD11 through MyD88-NF-κB signaling pathways. This finding will lay the foundation for further study the pathogenesis of DTMUV, and provide new insights into the prevention and control of this disease.

鸭疫里默氏杆菌病是由鸭疫里默氏杆菌(RA)引起的一种鸭急性传染病.2021年4月,山东省菏泽市某养鸭场21日龄肉鸭突然出现食欲减退、共济失调等症状,剖检可见明显纤维素性心包炎和肝周炎,为确定发病原因,本试验采集了病鸭的心脏、肝脏和大脑等组织,对病鸭的心脏和肝脏组织进行病理学观察,从病鸭大脑和肝脏组织中分离细菌,通过革兰染色、血清型分析和PCR方法对分离细菌进行鉴定,并对分离菌株进行耐药性和耐药基因分析.结果显示,病鸭心脏血管高度扩张,充满红细胞,心包膜外有明显的纤维素渗出物,肝细胞脂肪变性;通过细菌分离鉴定共获得了 3株鸭疫里默氏杆菌(分别命名为RA-HZ01、RA-HZ02和RA-HZ03),其中血清型6型1株、7型2株.药敏试验结果显示,RA-HZ01和RA-HZ03对头孢噻肟、万古霉素、美罗培南和恩诺沙星敏感,对克林霉素中介,对卡那霉素、多西环素、红霉素、复方新诺明和多黏菌素耐药,而RA-HZ02对美罗培南、恩诺沙星和多黏菌素敏感,对头孢噻肟和卡那霉素中介,对其他抗菌药耐药.耐药基因检测结果显示,RA-HZ01和RA-HZ03携带氨基糖苷类耐药基因aac(6')-Ⅰ、aph(3')-Ⅵ和aac(6')-Ⅰb,RA-HZ02携带氨基糖苷类耐药基因aac(6')-Ⅰ和aph(3')-Ⅵ、喹诺酮类耐药基因qnrS以及消毒剂耐药基因qacE△1.结果表明,引起该鸭场此次发病的病原主要是鸭疫里默氏杆菌,且其具有多重耐药表型.本试验结果对本地区鸭场鸭疫里默氏杆菌病的诊断和防治具有一定的指导意义.

H9N2 avian influenza virus（AIV） has widely circulated in poultry worldwide and sporadic infections in humans and mammals. During our surveillance of chicken from 2019 to 2021 in Shandong Province, China, we isolated 11 H9N2AIVs. Phylogenetic analyses showed that the eight gene segments of the 11 isolates were closely related to several sublineages of Eurasian lineage: BJ/94-like clades（HA and NA genes）, G1-like clades（PB2 and M genes）, and SH/F/98-like clades（PB1, PA, NP and NS genes）. The isolates showed mutation sites that preferentially bind to humanlike receptors（HA） and mammalian fitness sites（PB2, PB1 and PA）, as well as mutations in antigen and drug resistance sites. Moreover, studies with mice revealed four isolates with varying levels of pathogenicity. The average antibody titer of the H9N2 AIVs was 8.60 log 2 . Based on our results, the epidemiological surveillance of H9N2 AIVs should be strengthened.

当前猪价持续下滑,深不见底;饲料原料持续暴涨,饲料成本居高不下;非洲猪瘟、蓝耳病等重大疫病广泛流行,疫病防控压力大.猪价低迷时期,由于资金短缺,很多养猪企业在实行降本增效,其中不乏从防疫方面进行降本的乱象.本文对当前我国中小规模家庭猪场的防疫现状进行浅析,供大家参考.

猪呼吸与繁殖综合征(PRRS)俗称猪蓝耳病,在我国出现至今已有20多年,一直没有得到很好的控制,该病对我国生猪养殖造成了严重的危害.本文对猪蓝耳病的流行情况和综合防治措施进行了详细论述.

猪繁殖与呼吸综合征俗称猪蓝耳病,是由猪繁殖与呼吸综合征病毒引起的一种高度接触性传染病,其主要特征为母猪发热、流产和产死胎、木乃伊胎,仔猪和肥育猪发生呼吸道疾病.2022年3月山东省肥城市某小规模自繁自养猪场,母猪零星发生流产,保育猪大量发生咳喘及死亡现象,对送检的病死猪进行了剖检诊断并取材进行实验室检测,结合猪只临床病症、病理组织学检查和分子生物学检测结果,最终确定为猪繁殖与呼吸综合征病毒感染所导致的疾病,在此基础上制定了综合防控措施,及时遏制了疾病的传播,减少了养殖户的经济损失.

African swine fever (ASF) caused by ASF virus (ASFV) is a fatal disease in pigs and results in great economic losses. Due to the lack of available vaccines and treatments, serological diagnosis of ASF plays a key role in the surveillance program, but due to the lack of knowledge and the complexity of the ASFV genome, the candidate target viral proteins are still being researched. False negativity is still a big obstacle during the diagnostic process. In this study, the high antigenic viral proteins p30, p54 and p72 were screened to find the antigenic dominant domains and the tandem His–p30–54–72 was derived. An indirect enzyme–linked immunosorbent assay (iELISA) coated with His–p30–54–72 was developed with a cut–off value of 0.371. A total of 192 clinical samples were detected by His–p30–54–72–coated indirect ELISA (iELISA) and commercial ASFV antibody kits. The results showed that the positive rate of His–p30–54–72–coated iELISA was increased by 4.7% and 14.6% compared with a single viral protein–based commercial ASFV antibody kits. These results provide a platform for future ASFV clinical diagnosis and vaccine immune effect evaluation.

Background Swine influenza A virus (IAV-S) is a common cause of respiratory disease in pigs and poses a major public health threat. However, little attention and funding have been given to such studies. The aim of this study was to assess the prevalence of the Eurasian avian-like H1N1 (EA H1N1), 2009 pandemic H1N1 (pdm/09 H1N1), and H3N2 subtype antibodies in unvaccinated swine populations through serological investigations. Such data are helpful in understanding the prevalence of the IAV-S. Methods A total of 40,343 serum samples from 17 regions in China were examined using hemagglutination inhibition (HI) tests against EA H1N1, pdm/09 H1N1, and H3N2 IAV-S from 2016 to 2021. The results were analyzed based on a reginal distribution, seasonal distribution, and in different breeding stages. Results A total of 19,682 serum samples out of the 40,343 were positive for IAV-S (48.79%). The positivity rates to the EA H1N1 subtype, pdm/09 H1N1 subtype, and H3N2 subtype were 24.75% (9,986/40,343), 7.94% (3,205/40,343), and 0.06% (24/40,343), respectively. The occurrences of coinfections from two or more subtypes were also detected. In general, the positivity rates of serum samples were related to the regional distribution and feeding stages. Conclusions The results of this study showed that the anti-EA H1N1 subtype and pdm/09 H1N1 subtype antibodies were readily detected in swine serum samples. The EA H1N1 subtype has become dominant in the pig population. The occurrences of coinfections from two or more subtypes afforded opportunities for their reassortment to produce new viruses. Our findings emphasized the need for continuous surveillance of influenza viruses.