Genomic Relatedness, Antibiotic Resistance and Virulence Traits of Campylobacter jejuni HS19 Isolates From Cattle in China Indicate Pathogenic Potential

Although campylobacteriosis is a zoonotic foodborne illness, high-risk isolates from animal sources are rarely characterized, and the pathogenic potential of zoonotic strains remains an obstacle to effective intervention against human infection. HS19 has been acknowledged as a maker serotype represented by Campylobacter jejuni (C. jejuni) isolates from patients with post-infection Guillain-Barré syndrome (GBS), which is circulation in developed countries. However, a previous serotype epidemiological study of C. jejuni isolates in an animal population revealed that HS19 was also prevalent in isolates from cattle in China. In this study, to investigate the hazardous potential of zoonotic strains, 14 HS19 isolates from cattle were systematically characterized both by genotype and phenotype. The results showed that all of these cattle isolates belonged to the ST-22 complex, a high-risk lineage represented by 77.2% HS19 clinical isolates from patients worldwide in the PubMLST database, indicating that the ST-22 complex is the prominent clonal complex of HS19 isolates, as well as the possibility of clonal spread of HS19 isolates across different regions and hosts. Nevertheless, these cattle strains clustered closely with the HS19 isolates from patients, suggesting a remarkable phylogenetic relatedness and genomic similarity. Importantly, both tetracycline genes tet(O) and gyrA (T86I) reached a higher proportional representation among the cattle isolates than among the human clinical isolates. A worrying level of multidrug resistance (MDR) was observed in all the cattle isolates, and two MDR profiles of the cattle isolates also existed in human clinical isolates. Notably, although shared with the same serotype HS19 and sequence type ST-22, 35.7% of cattle isolates induced severe gastrointestinal pathology in the IL-10–/– C57BL/6 mice model, indicating that some bacteria could change due to host adaptation to induce a disease epidemic, thus the associated genetic elements deserve further investigation. In this study, HS19 isolates from cattle were first characterized by a systematic evaluation of bacterial genomics and in vitro virulence, which improved our understanding of the potential zoonotic hazard from food animal isolates with high-risk serotypes, and provided critical information for the development of targeted C. jejuni mitigation strategies.

Implication of bats in the transmission of zoonotic strains of Salmonella in Benin

Bats play a very important role in the transmission of zoonosis, including Salmonella. Salmonella are responsible for salmonellosis, which is a major public health concern. They are the cause of many hospitalizations and deaths worldwide. The objective of this study was to contribute to the im-provement of the control of zoonotic strains of Salmonella in Benin. To do so, a collection of 400 bats was made and after slaughter and dissection, bacteriological analyses were made on the gut to isolate and identify the different strains of Salmonella carried by these bats. The resistance profile and the presence of specific virulence gene such invA, spvR, spvC and stn were studied. The strain Salmonella Typhimurium ATCC 14028 was used as a positive control. Of the 400 bats slaughtered, 14 isolate of Salmonella spp were identified by API gallery. Therefore, the prevalence of Salmonella strains in bats was 3.5%. Salmonella spp strains isolated showed total re-sistance to amoxicillin, clavulanic acid and also to first and second genera-tions of cephalosporins. The stn and invA genes have been found in the DNA of all strains of Salmonella isolated. The consumption of bats being a com-mon practice in Benin, the risk of virulent Salmonella strains transmission must be taken very seriously and people must be sensitized to this in order to slow down the risk of infection.

Presumptive Identification of Smooth Brucella Strain Antibodies in Canines

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus. There are four Brucella strains of zoonotic importance in our domestic species, subdivided by their culture phenotypes: Brucella abortus (B. abortus), B. melitensis, B. suis (smooth strains) and B. canis (rough strain). Dogs can serve as hosts for all four of the zoonotic strains; however, routine serologic testing in dogs has been limited to the identification of B. canis antibodies. The aim of our study was to identify smooth Brucella strain antibodies in canines. We hypothesize that the Brucella abortus Fluorescence Polarization Assay would be successful in identifying smooth Brucella strain antibodies in canines. Ninety-five dogs, including forty-five hog hunting dogs were screened for circulating antibodies to any of the four zoonotic strains of the bacteria utilizing a combination of Canine Brucella Slide Agglutination Test (CBSA), Brucella canis Agar Gel Immunodiffusion II test (AGIDII), Brucella abortus Card Agglutination Test (BCA), and the Brucella abortus Fluorescence Polarization Assay (FPA). Test interpretation results yielded a 0% (0/95) smooth Brucella strain seropositivity rate, with 2% (2/95) of dogs yielding inconclusive rough Brucella strain serology results (0–2% rough strain seropositivity rate). Additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of Brucella by testing previously banked canine serum samples stored at Cornell's Veterinary Diagnostic Laboratory from 2018 to 2019 via Brucella abortus FPA. Of the 769 serum samples tested, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, 30/769 (4%) yielded a positive FPA test result, and 1/769 (0.1%) had to be excluded due to insufficient sample remaining to perform the diagnostic test. Of the 30 FPA positive canine serum samples, 97% (29/30) also tested positive on the CBSA test. Additionally, there was a statistically significant (p < 0.0001) likelihood of altered (spayed/neutered) and mixed breed dogs to be FPA positive when compared to intact, purebred dogs, respectively.

Seroepidemiological survey on pigs and cattle for novel K88 (F4)-like colonization factor detected in human enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that express various fimbrial or nonfimbrial colonization factors and enterotoxins are critical causes of diarrheal diseases. Human ETEC serotype O169:H41 (O169) has been a representative of epidemic ETEC worldwide; the organism shows massive adherence to HEp-2 cells similar to enteroaggregative E. coli. Previously, we determined the complete sequence of the unstable virulence plasmid, pEntYN10. The plasmid included a unique set of genes encoding a novel colonization factor (CF) resembling K88 (F4) of porcine ETEC, in addition to CS6, a well-known representative CF of human ETEC, and another novel CF similar to CS8 (CFA/III) of human ETEC. To determine whether the K88-like CF (after this, K88 O169) allows the organisms to infect domestic animals like the original K88-harboring strains that can cause diarrhea in piglets, samples were tested for antibodies against recombinant proteins of possible paralogous adhesins, FaeG1 and FaeG2, from K88O169 and the FaeG of typical K88 (F4). The seroepidemiological study using recombinant antigens (two paralogs FaeG1 and FaeG2 from K88O169) showed reactivity of porcine (18.0%) and bovine (17.1%) sera to K88O169 FaeG1 and/or FaeG2 antigens on indirect ELISA tests. These results suggest that E. coli with K88O169 adhesin can infect various hosts, including pigs and cattle. This is the first report of domestic animals having antibodies to K88O169 of human ETEC. Although human ETEC had been thought to be distinguished from those of domestic animals based on colonization factors, zoonotic strains may conceal themselves among human ETEC organisms. The concept of One Health should be adopted to intervene in ETEC infections among animals and humans.

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Flu Pandemic Preparedness: Detection of the Potentially Pandemic G4 EA Avian-Like H1N1 Strains Using QIAstat-Dx Respiratory SARS-CoV-2 Panel

A recent publication by Sun H. and colleagues in the Proceedings of the National Academy of Science (PNAS) has drown global attention to the predominant genotype “G4” Eurasian (EA) avian-like H1N1 Influenza A virus that has been spreading among pigs in China since 2016, which is predicted to have a significant pandemic potential. Since pigs are hosts for the generation of pandemic Influenza A viruses (IAVs), surveillance and preparedness are critical to prevent pandemics. In this regards, one distinguishing feature of the QIAstat-Dx® Respiratory SARS-CoV-2 panel is the double target approach Influenza A detection of seasonal strains affecting humans, by amplification of a generic Influenza A assay plus one of the specific assays discriminating H3, H1 and H1N1pdm09 subtypes. The generic Influenza A assay is designed to amplify any IAV, a key feature for preparedness. Here we report an initial in-silico analysis that predicts that the G4 EA avian-like H1N1 strains tested in the QIAstat-Dx® Respiratory SARS-CoV-2 Panel would be detected yielding a positive result for the generic Influenza A assay and negative results for the seasonal H3, H1 and H1N1pdm09 assays. This prediction was confirmed in-vitro using dsDNA fragments mixed to mimic the genomes of the different reported G4 EA IAV strains. In conclusion, the QIAstat-Dx® Respiratory SARS-CoV-2 Panel could be a useful tool to differentially diagnose zoonotic strains from the seasonal Influenza A strains commonly affecting humans.

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Antibacterial Activity of Bacteriocinogenic Commensal Escherichia coli against Zoonotic Strains Resistant and Sensitive to Antibiotics

Antibiotic resistance concerns various areas with high consumption of antibiotics, including husbandry. Resistant strains are transmitted to humans from livestock and agricultural products via the food chain and may pose a health risk. The commensal microbiota protects against the invasion of environmental strains by secretion of bacteriocins, among other mechanisms. The present study aims to characterize the bactericidal potential of bacteriocinogenic Escherichia coli from healthy humans against multidrug-resistant and antibiotic-sensitive strains from pigs and cattle. Bacteriocin production was tested by the double-layer plate method, and bacteriocin genes were identified by the PCR method. At least one bacteriocinogenic E. coli was detected in the fecal samples of 55% of tested individuals, adults and children. Among all isolates (n = 210), 37.1% were bacteriocinogenic and contained genes of colicin (Col) Ib, ColE1, microcin (Mcc) H47, ColIa, ColM, MccV, ColK, ColB, and single ColE2 and ColE7. Twenty-five E. coli carrying various sets of bacteriocin genes were further characterized and tested for their activity against zoonotic strains (n = 60). Strains with ColE7 (88%), ColE1-ColIa-ColK-MccH47 (85%), MccH47-MccV (85%), ColE1-ColIa-ColM (82%), ColE1 (75%), ColM (67%), and ColK (65%) were most active against zoonotic strains. Statistically significant differences in activity toward antibiotic-resistant strains were shown by commensal E. coli carrying MccV, ColK-MccV, and ColIb-ColK. The study demonstrates that bacteriocinogenic commensal E. coli exerts antagonistic activity against zoonotic strains and may constitute a defense line against multidrug-resistant strains.

Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines

ABSTRACT With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2′O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2′O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2′O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2′O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2′O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2′O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.

Combination attenuation offers strategy for live-attenuated coronavirus vaccines

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live-attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2’O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the SARS-CoV mouse model, a NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2’O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2’O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live-attenuated coronavirus vaccines.SignificanceEmergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations like the aged and immune-compromised. This study illustrates that despite success in young models, the NSP16 mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the NSP16 mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for NSP16 attenuation as a major component of future live-attenuated coronavirus vaccines.