Comparative Genome Analysis of Two Streptococcus suis Serotype 8 Strains Identifies Two New Virulence-Associated Genes.

Streptococcus suis is an important zoonotic pathogen that can cause meningitis and septicemia in swine and humans. Among numerous pathogenic serotypes, S. suis serotype 8 has distinctive characteristics such as a high detection rate and causing multi-host infection. There is no complete genome of serotype 8 strains so far. In this study, the complete genome of two S. suis serotype 8 strains, virulent strain 2018WUSS151 and non-virulent strain WUSS030, were sequenced. Comparative genomic analysis showed that the homology of the two genomes reaches 99.68%, and the main difference is the distinctive prophages. There are 83 genes unique to virulent strain 2018WUSS151, including three putative virulence-associated genes (PVGs). Two PVGs, padR and marR, are passenger genes in ISSsu2 family transposons that are able to form circular DNA intermediates during transposition, indicating the possibility of horizontal transmission among S. suis strains. The deletion mutant of PVGs marR or atpase attenuated the virulence of serotype 2 virulent SC070731 in a mouse infection model, confirming their role in S. suis virulence. These findings contribute to clarifying the genomic characterization of S. suis serotype 8 and S. suis pathogenesis.

Characteristics, Whole-Genome Sequencing and Pathogenicity Analysis of Escherichia coli from a White Feather Broiler Farm.

Avian colibacillosis, caused by avian Escherichia coli (E. coli), has historically been one of the most prevalent infectious diseases in large-scale poultry production, causing growth delays and mortality in chickens, resulting in huge economic losses. In recent years, the widespread use of antibiotics has led to the emergence of multidrug resistance in E. coli as a significant global problem and long-term challenge. Resistant E. coli can be transmitted to humans through animal products or the environment, which presents significant public health concerns and food safety issues. In this study, we analyzed the features of 135 E. coli strains obtained from a white feather broiler farm in Shandong, China, including antimicrobial susceptibility tests, detection of class 1 integrons, drug resistance genes, virulence genes, and phylogenetic subgroups. It is particularly worrying that all 135 E. coli strains were resistant to at least five antibiotic agents, and 100% of them were multidrug-resistant (MDR). Notably, the resistance genes of blaTEM, blaCTX-M, qnrS, aaC4, tetA, and tetB exhibited a high prevalence of carriage among the tested resistance genes. However, mcr-2~mcr-9 were not detected, while the prevalence of mcr-1 was found to be 2.96%. The most common virulence genes detected were EAST1 (14.07%, encoding enterotoxins) and fyuA (14.81%, encoding biofilm formation). Phylogenetic subgroup analysis revealed that E. coli belonging to groups B2 and D, which are commonly associated with high virulence, constituted 2.22% and 11.11%, respectively. The positive rate of class 1 integrons was 31.1%. Whole-genome sequencing (WGS) and animal experiments were performed on a unique isolated strain called 21EC78 with an extremely strong membrane-forming capacity. The WGS results showed that 21EC78 carried 11 drug resistance genes and 16 virulence genes. Animal experiments showed that intraperitoneal injection with 2 × 105 CFU could cause the death of one-day-old SPF chickens in 3 days. However, the mortality of Luhua chickens was comparatively lower than that of SPF chickens. This study reports the isolation of multidrug-resistant E. coli strains in poultry, which may pose a potential threat to human health via the food chain. Furthermore, the findings of this study enhance our comprehension of the frequency and characteristics of multidrug-resistant E. coli in poultry farms, emphasizing the urgent need for improved and effective continuous surveillance to control its dissemination.

Prioritized antibiotics screening based on comprehensive risk assessments and related management strategy in various animal farms.

Antibiotic pollution in the environment caused by animal breeding has become a serious issue. The persistent release of antibiotics with animal waste may lead to antibiotic resistances in the environment, which poses a threat to human health. This study tries to provides a practical method for screening prioritized antibiotics via a comprehensive risk assessment and determination of their major sources, and put forward corresponding regulatory measures for animal industries. We investigated the occurrence and distribution of 20 antibiotics belonging to eight classes, spanning the areas of animal feed, drinking water, and animal feces on 59 animal farms in Shandong Province, China. The results showed that antibiotic contamination was prevalent in different environmental mediums (feed, feces, and drinking water) on these farms. Tetracyclines typically exhibited higher concentrations than the other classes in all samples, and the majority of antibiotics had greater concentrations in cattle feces than in pig- or chicken feces. For the antibiotic ecological risks in feces, doxycycline, tetracycline, and enrofloxacin exhibited much more toxic effects on terrestrial organisms (e.g., wheat, cucumber, and rice). Ciprofloxacin, enrofloxacin, ofloxacin, and tetracycline levels in drinking water samples can lead to high risk of antibiotic resistance, while no antibiotic posed obvious risks to human health. Based on compressive risk assessments, 11 antibiotics were prioritized to control in the animal breeding environment. Based on the survey of feeds, drinking water and animal waste from the farm, roxithromycin in the feces mainly originated from the feeds, while most prioritized antibiotics, were from extra addition in the animal breeding process (including injection and other oral routes). The key point of local antibiotic management in animal farms should be adjusted from the feed factory to the extra addition of antibiotics in animal breeding processes.

Comparative Antigenicity and Pathogenicity of Two Distinct Genotypes of Highly Pathogenic Avian Influenza Viruses (H5N8) From Wild Birds in China, 2020-2021.

To date, there have been three epidemic waves of H5N8 avian influenza worldwide. The current third epidemic wave began in October 2020 and has expanded to at least 46 countries. Active and passive surveillance were conducted to monitor H5N8 viruses from wild birds in China. Genetic analysis of 10 H5N8 viruses isolated from wild birds identified two different genotypes. Animal challenge experiments indicated that the H5N8 isolates are highly pathogenic in chickens, mildly pathogenic in ducks, while pathogenicity varied in BALB/c mice. Moreover, there were significant differences in antigenicity as compared to Re-11 vaccine strain and vaccinated chickens were not completely protected against challenge with the high dose of H5N8 virus. With the use of the new matched vaccine and increased poultry immune density, surveillance should be intensified to monitor the emergence of mutant strains and potential worldwide spread via wild birds.

Cross-sectional study on risk factors associated with porcine epidemic diarrhea virus infection in pig farms in Junan county, China.

Porcine epidemic diarrhea (PED) is a highly contagious, intestinal infectious disease in pigs, characterized by severe diarrhea, vomiting and dehydration. PED is widely epidemic in China as well as in many eastern Asian and America countries, causing tremendous losses in pig industry. However, little was known about the disease frequency and the associated risk factors of PED in pig farms. A cross-sectional study was conducted to estimate the herd prevalence and to identify the potential risk factors of porcine epidemic diarrhea virus (PEDV) infection of pig farms (≥100 pigs) in Junan county, China. A two-stage random sampling strategy was adopted, and a total of 751 fecal samples from suckling piglets of 82 farms were collected and tested by RT-PCR and sequencing for PEDV. Meanwhile, information on the putative risk factors of PEDV infection of those farms were collected in forms of questionnaires, followed by a descriptive analysis, univariable and multivariable logistic regression analysis. The results showed that the herd-level true prevalence of PEDV infection of pig farms in Junan was 35.16 % (95 %CI: 22.91-53.89); there were two variables significantly associated with PEDV infection, which were 'having more than 1000 slaughter pigs per annum' (OR = 5.42, 95 %CI: 1.19-24.72), and 'Weaning at 21-25 days' old' (OR = 4.50, 95 %CI:1.25-16.20). The research suggested that PED was highly endemic in pig farms in Junan county; larger herd size and weaning at an earlier age were potential risk factors associated with PEDV infection in pig farms in Junan. This study set an example in the research on herd-level prevalence of PEDV infection and risk factors associated with PEDV infection, and the results were of practical significance for the future planning of prevention and control of PED in Junan or other areas of China.

Diversity of porcine reproductive and respiratory syndrome virus in Shandong, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant pathogens in swine industry of China. To study infection and genetic variation of PRRSV, 637 tissue samples were collected from diseased pigs in Shandong, and then subjected to detection of PRRSV. The nsp2 and ORF5 genes were sequenced for investigation of variations and phylogenetic analysis. The results showed that positive rate of PRRSV was 9.58% in the collected samples. Phylogenetic analysis of GP5 showed that these strains were clustered into two lineages (1 and 8) indicating different genotypes of PRRSV were circulating in Shandong province. Meanwhile, sequence analysis Of nsp2 showed that the PRRSV strains with 30 amino acids deletions were dominant. Moreover, novel pattern of recombination/deletion and insertion in nsp2 was observed in these strains, indicating that novel PRRSV strains with different patterns of deletions or insertions in nsp2 are emerging in China. All the results suggested that continuous surveillance of PRRSV in China is warranted. Keywords: PRRSV; GP5; nsp2; genetic analysis; Shandong.

Pathological analysis and genetic characterization of the first outbreak H5N8 subtype avian influenza virus isolated from wild swan in Shandong, China.

In January 2021, an outbreak of H5N8 subtype avian influenza was identified from wild mute swans (Cygnus olor) in an overwintering site located within Yellow River Delta Nature Reserve in Shandong, China, for the first time. We sequenced the hemagglutinin (HA) and neuraminidase (NA) genome segments of the virus, named A/mute swan/Shandong/1/2021 (H5N8), and explored the genetic characterization and comparative phylogenetic analysis to track its origin. The wild swans prior to death showed nervous signs accompanied by a twisted neck. The major pathological changes of the heart, lung, trachea, kidney, spleen, and glandular stomach were hemorrhage, and extensive necrotic lesions in the liver and pancreas were also observed. Further analysis of the HA and NA phylogenetic trees generated by H5N8 strains isolated worldwide revealed that the isolate has a close genetic relationship with the H5N8 subtype avian influenza virus (AIV) recently isolated in Korea and Japan from 2020 to 2021, suggesting the potential role of mute swans in the global dissemination of the H5N8 subtype AIVs along the migration routes. Therefore, enhanced active surveillance in wild and domestic birds is required to monitor the introduction and spread of the H5N8 subtype AIV by migratory birds in order to decrease the risk of H5N8 outbreaks.

Genetic characterization, mechanisms and dissemination risk of antibiotic resistance of multidrug-resistant Rothia nasimurium.

Rothia nasimurium is part of the commensal flora of humans and other animals and has recently received increasing attention for its multidrug-resistance (MDR) and pathogenicity. Currently, no systematic reports characterize the genetics, mechanisms, and dissemination risks of antibiotic resistance in MDR R. nasimurium. Here, we present the first report outlining a MDR strain of R. nasimurium, E1706032a, isolated from ducks exhibiting clinical sickness. Phylogenetic analysis indicates that E1706032a mostly likely originated in the commensal bacteria of Amazona aestiva in Florida. E1706032a is resistant to beta-lactams, aminoglycosides, macrolides, sulfonamides, fluoroquinolones, rifamycins, tetracyclines, lincosamides and chloramphenicol. Genetic sequences related to drug resistance were detected, including resistance genes (aac(6')-Ib, ant(3″)-Ia, sul1, dfrA7, erm(X)), efflux pumps (tetZ, qacEΔ1, cmx, phosphate ABC transporter ATP-binding protein), and resistance-related proteins (hydrolase of the metallo-beta-lactamase (MBLs), mycinamicin resistance protein (myrA), DNA-directed RNA polymerase subunit beta (rpoB) variants, etc). E1706032a carries an IS481-like element, IS5564 and IS6-like elements, and IS6100 along with several novel transposases of the IS3 family. E1706032a also carries the class 1 integron gene IntI1, which is downstream adjacent to the gene cassettes aac(6')-Ib, tetZ, dfrA27, ant(3″)-Ia, qacEΔ1, sul1, cmx and upstream adjacent to gene tnpA of IS6100. Genetic analysis suggests that E1706032a carries wide antibiotic resistance and dissemination potential through movable elements and thus has the potential to cause difficult-to-treat infections in animals and humans. The dissemination of E1706032a from parrots in Florida to ducks in eastern China indicates a cross-regional public health infection risk that should be evaluated for risk of global spreading.

Recombinant Lactobacillus acidophilus expressing S1 and S2 domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), which is characterized by a high mortality rate in piglets. Since 2012, a remarkable growth in PED outbreaks occurred in many pig farms in China, landing a heavy blow on the pig industry. In order to develop a new effective vaccine for the current PEDV, oral vaccines were generated by transferring eukaryotic expression recombinant plasmids carrying the S1 and S2 (antigenic sites of the S protein) epitopes of PEDV into a swine-origin Lactobacillus acidophilus (L. acidophilus). After oral immunization of the BALB/c mice, higher levels of anti-PEDV specific IgG and SIgA antibodies and cellular immune responses were detected in mice orally administered with the recombinant L. acidophilus-S1 compared to the L. acidophilus-S2. Furthermore, L. acidophilus-S1 was used to inoculate the pregnant sows orally and the results showed that the recombinant L. acidophilus-S1 could elicit a specific systemic and mucosal immune response. In summary, our study demonstrated that oral immunization with L. acidophilus-S1 could improve the humoral and mucosal immune levels in sows and would be a promising candidate vaccine against PEDV infection in piglets.

Allelic variants of PRNP in 16 Chinese local sheep breeds.

Here, polymorphisms of the ovine prion protein gene were analyzed in 486 Chinese sheep from 16 main local breeds. Polymorphisms R or H at codons 154 and four polymorphisms at codon 171 encoding Q, R, H, or K were identified. The A/V polymorphism at codon 136 was not observed, and all sheep were homozygous for A at this position. In addition, ten polymorphisms at codons 21, 101, 112, 127, 138, 141, 143, 146, 153 and 189 were detected. The predominant Q allele occurred at codon 171, with a high frequency of 88.68 %, implying a risk of scrapie in China.