Antigenic comparison of H3N8 equine influenza viruses belonging to Florida sublineage clade 1 between vaccine strains and North American strains isolated in 2021-2022.

Equine influenza virus strains of Florida sublineage clade 1 (Fc1) have been circulating in North America. In this study, virus neutralization assays were performed to evaluate antigenic differences between Fc1 vaccine strains and North American Fc1 strains isolated in 2021-2022, using equine antisera against A/equine/South Africa/4/2003 (a vaccine strain recommended by the World Organisation for Animal Health) and A/equine/Ibaraki/1/2007 (a Japanese vaccine strain). Antibody titers against four North American Fc1 strains isolated in 2021-2022 were comparable to those against the homologous vaccine strains. These results suggest that current Fc1 vaccine strains are effective against North American strains from 2021-2022.

Campylobacter coli Clade 3 Isolates Induce Rapid Cell Death In Vitro.

Campylobacter bacteria are major human enteropathogens. Campylobacter coli shows less genetic diversity than C. jejuni and clusters into three clades, of which clade 1 includes most human and farm animal isolates, while environmental C. coli isolates mainly belong to clades 2 and 3. Recently, we sequenced the whole genomes of eight C. coli clade 2 and 3 isolates cultivated from water, and here we studied their interaction with human HT-29 colon cancer cells compared to that of clinical clade 1 isolates. All C. coli clade 3 isolates already caused cell necrosis 1 to 2 h after inoculation, whereas none of the clade 1 and 2 isolates analyzed induced cell death. Isolates from clades 2 and 3 adhered to epithelial cells better than clade 1 isolates, but all isolates induced similar levels of interleukin-8 (IL-8). Alignment and phylogenetic analysis of the translated putative virulence genes cadF, flpA, iamA, ciaB, and ceuE revealed clade-specific protein sequence variations, with clade 1 and 2 sequences being more closely related and clade 3 sequences being further apart, in general. Moreover, when RNA levels were measured, clade 3 isolates showed significantly lower levels of expression of cadF, iamA, and ceuE than clade 2 isolates, while flpA expression levels were higher in clade 3 isolates. The cytolethal distending toxin genes were also expressed in clades 2 and 3, although there was no difference between clades. Our findings demonstrate differences between the effects of C. coli clade 1, 2, and 3 isolates on human cells and suggest that C. coli clade 3 might be more virulent than clade 2 due to the observed cytotoxicity.IMPORTANCECampylobacter coli is a common zoonotic cause of gastroenteritis in humans worldwide. The majority of infections are caused by C. coli clade 1 isolates, whereas infections due to clade 2 and 3 isolates are rare. Whether this depends on a low prevalence of clade 2 and 3 isolates in reservoirs important for human infections or their lower ability to cause human disease is unknown. Here, we studied the effects of C. coli clade 2 and 3 isolates on a human cell line. These isolates adhered to human cells to a higher degree than clinical clade 1 isolates. Furthermore, we could show that C. coli clade 3 isolates rapidly induced cell death, suggesting differences in the virulence of C. coli The exact mechanism of cell death remains to be revealed, but selected genes showed interesting clade-specific expression patterns.

Epidemiological and virological findings during an outbreak of equine influenza in Uruguay in 2018.

Equine influenza is one of the major respiratory infectious diseases in horses. In 2018, equine influenza virus (EIV) was confirmed as the cause of outbreaks of respiratory disease in horses in Chile and Argentina. In the same year, for the first time in Uruguay, EIV infection was confirmed by isolation and molecular analysis to be the cause of respiratory disease among hundreds of clinically affected thoroughbred horses in training and racing facilities. The virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A realtime reverse transcription polymerase chain reaction (rRT-PCR). The partial nucleotide sequence of the haemagglutinin 1 (HA1 ) gene (994 base pairs) was determined and analysed phylogenetically using MEGA X software. Amino acid sequence alignments were constructed, and serum samples were tested by haemagglutination inhibition and single radial haemolysis. The diagnosis of EIV was confirmed by rRT-PCR, virus isolation and serological testing. The phylogenetic analysis of the partial HA1 gene sequence of the isolated virus indicated that it belongs to clade 1 of the Florida sub-lineage of the American lineage and is closely related to viruses isolated in the recent past. Study of the HA1 region (331 amino acids) of the virus identified in horses in racing facilities in Uruguay displayed the highest amino acid sequence identity with viruses detected in Argentina, Chile and the United Kingdom in 2018. The surveillance data reported illustrate the international spread of EIVs and support the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel to include viruses of the Florida sub-lineage in vaccines.

La grippe équine est l'une des principales maladies respiratoires infectieuses affectant les équidés. En 2018, il a été confirmé que des foyers de maladie respiratoire enregistrés chez des chevaux au Chili et en Argentine étaient dus au virus de la grippe équine. Cette même année en Uruguay, pour la première fois dans ce pays, il a été établi par isolement viral et par des méthodes moléculaires que le virus de la grippe équine était l'agent causal d'une maladie respiratoire affectant cliniquement des centaines de chevaux pur-sang dans des centres d'entraînement et des hippodromes. La détection du virus s'est faite à partir d'écouvillons prélevés par voie naso-pharyngée en appliquant une technique d'amplification en chaîne par polymérase couplée à une transcription inverse en temps réel (rRT­PCR) à large spectre pour les virus influenza de type A. Une séquence nucléotidique partielle correspondant au gène de l'hémagglutinine 1 (HA1) (994 paires de bases) a fait l'objet d'une analyse phylogénétique au moyen du programme informatique MEGA X. Il a été procédé à la construction d'une matrice d'alignements de ces séquences d'acides aminés. D'autre part, des prélèvements de sérum issus de chevaux atteints ont été soumis à l'épreuve d'inhibition de l'hémagglutination et à une hémolyse radiale unique. Aussi bien la rRT­PCR que l'isolement viral et l'analyse sérologique ont confirmé le diagnostic de l'infection par le virus de la grippe équine. Il ressort de l'analyse phylogénétique du fragment de séquence du gène HA1 du virus isolé que ce dernier appartient au clade 1 de la sous-lignée Florida de la lignée américaine et qu'il est étroitement apparenté à des virus isolés au cours des dernières années. L'étude de la région HA1 (331 acides aminés) du virus détecté chez des chevaux de course en Uruguay a montré que les virus qui présentaient la plus grande similitude avec cette séquence d'acides aminés étaient ceux détectés en Argentine, au Chili et au Royaume-Uni en 2018. Les données de surveillance rapportées illustrent la propagation à l'échelle internationale des virus de la grippe équine et renforcent la recommandation émise par le Groupe d'experts de l'Organisation mondiale de la santé animale (OIE) chargé de la surveillance de la composition des vaccins contre la grippe équine d'inclure les virus de la sous-lignée Florida dans la composition de ces vaccins.

La gripe equina es una de las principales infecciones respiratorias que afectan al caballo. En 2018 se confirmó que el virus de la gripe equina era la causa de diversos brotes de afección respiratoria que habían afectado a caballos de Chile y Argentina. Ese mismo año, por primera vez en el Uruguay, se confirmó por aislamiento y análisis molecular que el virus de la gripe equina era la causa de una infección respiratoria que, acompañada de manifestaciones clínicas, afectó a cientos de caballos purasangre de hipódromos y centros de adiestramiento. El virus fue detectado en muestras de frotis nasales mediante una técnica de reacción en cadena de la polimerasa acoplada a transcripción inversa en tiempo real (rRT­PCR, por sus siglas en inglés) que reacciona ante todos los virus gripales de tipo A. Tras secuenciar parcialmente el gen de la hemaglutinina 1 (HA1 ) (994 pares de bases), se procedió a su análisis filogenético empleando el programa informático MEGA X. Además de crear una matriz de alineamiento de secuencias de aminoácidos, se sometieron muestras de suero a pruebas de inhibición de la hemaglutinación y hemólisis radial simple. Así, el diagnóstico que apuntaba al virus de la gripe equina fue confirmado por rRT­PCR, aislamiento vírico y análisis serológico. El análisis filogenético de la secuencia parcial del gen HA1 del virus aislado puso de manifiesto que pertenece al clado 1 del sublinaje Florida del linaje americano y guarda estrecho parentesco con otros virus aislados en fechas recientes. El estudio de la región HA1 (331 aminoácidos) del virus detectado en caballos de hipódromos uruguayos reveló que el mayor nivel de concordancia de su secuencia de aminoácidos se daba con virus detectados en Argentina, Chile y el Reino Unido en 2018. Los datos de vigilancia comunicados dan fe de la propagación internacional de los virus de la gripe equina y avalan la recomendación formulada por el Panel de expertos en vigilancia de la composición de las vacunas contra la gripe equina de la Organización Mundial de Sanidad Animal (OIE), que aboga por incluir virus del sublinaje Florida en las vacunas.

Clostridium difficile isolates derived from Czech horses are resistant to enrofloxacin; cluster to clades 1 and 5 and ribotype 033 predominates.

Clostridium difficile has been recovered from the faeces of several animal species as well as horses. Between April 2015 and October 2016, 213 samples of faeces from non-hospitalized (n = 138) and hospitalized horses (n = 75) were investigated and eighteen C. difficile isolates were cultured using an enrichment method. Sixteen C. difficile positive samples were identified from hospitalised horses (p < 0.01). Molecular typing revealed seven ribotypes and sequence types (RT033/ST11 n = 8, 44.4%; RT081/ST9 n = 4, 22.2%; RT009/ST3 n = 2, 11.1%; RT003/ST12 n = 1, 5.6%; RT010/ST15 n = 1, 5.6%; RT012/ST54 n = 1, 5.6%; RT039/ST26 n = 1, 5.6%). Seven identified STs clustered to two clades (1 and 5). All C. difficile isolates were susceptible to amoxicillin, metronidazole, moxifloxacin, and vancomycin. One isolate (RT039) exhibited a high level of resistance to erythromycin and clindamycin (256 mg/L) and carried the ermB, adenine methylase gene. Five isolates were resistant to clindamycin at lower minimal inhibitory concentrations (MICs = 8-16 mg/L) and were susceptible to erythromycin and also ermB negative. All isolates were resistant to enrofloxacin (MICs ranged between 4 and 32 mg/L). Eight isolates were resistant to tetracycline (MICs 12-32 mg/L). Of them, four isolates carried the tetM gene and four isolates the tetW gene. In addition, the tetracycline resistance determinants identified were: tetA (P) (n = 4); tetB (P); and tetL (n = 1 each). The presence of tetW or tetM, together with other tet-class mechanisms, lead to an increase in the MICs to tetracycline. C. difficile isolates derived from Czech horses are identical to the ribotypes identified in humans and carry acquired antimicrobial resistance genes whose dissemination from veterinary healthcare sector to humans should be monitored by the "One health" approach.

Characterisation of the epidemic strain of H3N8 equine influenza virus responsible for outbreaks in South America in 2012.

BACKGROUND: An extensive outbreak of equine influenza occurred across multiple countries in South America during 2012. The epidemic was first reported in Chile then spread to Brazil, Uruguay and Argentina, where both vaccinated and unvaccinated animals were affected. In Brazil, infections were widespread within 3months of the first reported cases. Affected horses included animals vaccinated with outdated vaccine antigens, but also with the OIE-recommended Florida clade 1 strain South Africa/4/03. METHODS: Equine influenza virus strains from infected horses were isolated in eggs, then a representative strain was subjected to full genome sequencing using segment-specific primers with M13 tags. Phylogenetic analyses of nucleotide sequences were completed using PhyML. Amino acid sequences of haemagglutinin and neuraminidase were compared against those of vaccine strains and recent isolates from America and Uruguay, substitutions were mapped onto 3D protein structures using PyMol. Antigenic analyses were completed by haemagglutination-inhibition assay using post-infection ferret sera. RESULTS: Nucleotide sequences of the haemaglutinin (HA) and neuraminidase (NA) genes of Brazilian isolate A/equine/Rio Grande do Sul/2012 were very similar to those of viruses belonging to Florida clade 1 and clustered with contemporary isolates from the USA. Comparison of their amino acid sequences against the OIE-recommended Florida clade 1 vaccine strain A/equine/South Africa/4/03 revealed five amino acid substitutions in HA and seven in NA. Changes in HA included one within antigenic site A and one within the 220-loop of the sialic acid receptor binding site. However, antigenic analysis by haemagglutination inhibition (HI) assay with ferret antisera raised against representatives of European, Kentucky and Florida sublineages failed to indicate any obvious differences in antigenicity. CONCLUSIONS: An extensive outbreak of equine influenza in South America during 2012 was caused by a virus belonging to Florida clade 1, closely related to strains circulating in the USA in 2011. Despite reports of vaccine breakdown with products containing the recommended strain South Africa/03, no evidence was found of significant antigenic drift. Other factors may have contributed to the rapid spread of this virus, including poor control of horse movement.

First Reported Circulation of Equine Influenza H3N8 Florida Clade 1 Virus in Horses in Italy.

BACKGROUND: Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance. METHODS: Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories. RESULTS: None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761). CONCLUSIONS: This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy.

Equine influenza outbreak in Eastern of Algeria in 2021: The first introduction of Florida Clade 1 to Maghreb area.

We have performed an equine influenza (EI) serological study of the equine population in Algeria by testing 298 serum samples collected between February and August 2021 from 5 provinces. The results were obtained performing an NP-ELISA. Our results revealed that 49.3% (147/298) samples positive for antibodies to EI (H3N8). During this study and after a gap of one decade an outbreak of EI was reported in Algeria in the first week of March 2021. The disease was confirmed by virus detection from the nasal swabs (n = 39) by qRT-PCR and by identifying 5 EI seroconversion. The virus sequences were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 1 of the Florida sublineage in the American lineage. This study indicate the first detection of FC1 strain of EIV in Maghreb area.

Highly pathogenic avian influenza H5N1 virus outbreak among Cape cormorants (Phalacrocorax capensis) in Namibia, 2022.

In January 2022, significant mortality was observed among Cape cormorants (Phalacrocorax capensis) on the west coast of Namibia. Samples collected were shown to be positive for H5N1 avian influenza by multiplex RT-qPCR. Full genome analysis and phylogenetic analysis identified the viruses as belonging to clade 2.3.4.4b and that it clustered with similar viruses identified in Lesotho and Botswana in 2021. This is the first genomic characterization of H5N1 viruses in Namibia and has important implications for poultry disease management and wildlife conservation in the region.

Pathological and Genomic Findings of Erysipelothrix rhusiopathiae Isolated From a Free-Ranging Rough-Toothed Dolphin Steno bredanensis (Cetacea: Delphinidae) Stranded in Korea.

Erysipelas, caused by Erysipelothrix rhusiopathiae, is considered one of the most serious infectious diseases of captive and free-ranging cetaceans worldwide, as these animals are known to be highly susceptible to the bacterial infections. The potential diversity between E. rhusiopathiae isolates from captive cetaceans has been previously described; however, the microbiological features of the free-ranging cetacean isolates remain unclear. Here, we describe a case of bacteremia in a rough-toothed dolphin (Steno bredanensis) caused by E. rhusiopathiae. Additionally, we present the first genomic features of the bacteria from free-ranging cetacean individuals. Histopathological and microbial examinations revealed that E. rhusiopathiae caused bacteremia and systemic infection in the dolphin. The genome of the isolated E. rhusiopathiae strain KC-Sb-R1, which was classified as Clade 1 possessing SpaB gene, was clearly differentiated from the other swine-isolated E. rhusiopathiae, and the comparison of its serovar-defining chromosomal region revealed that our isolate was greatly similar to those of other previously reported serovar 2/15 isolates, including the captive-dolphin isolate. Moreover, most of the potential virulence factors in the strain KC-Sb-R1 were similar to those in the strain Fujisawa. Further, a potential cytotoxicity of the isolate was confirmed, suggesting that marine mammal-isolated E. rhusiopathiae could possess strong pathogenic potential in other animals, including humans. These results would further increase our understanding on the risk factors for controlling zoonotic pathogens of emerging infectious diseases in captive or free-ranging cetaceans, and also provide important insight into the diversity of E. rhusiopathiae in animals.

Patient-Derived Antibody Data Yields Development of Broadly Cross-Protective Monoclonal Antibody against ST258 Carbapenem-Resistant Klebsiella pneumoniae.

The most pressing challenge for the development of anti-capsular antibodies is maximizing coverage against the heterogenous capsular polysaccharide (CPS) of carbapenem-resistant Klebsiella pneumoniae (CR-Kp). So far, only CR-Kp with wzi154 CPS has been successfully targeted by antibodies. Here, we present murine antibody 24D11, which was developed by vaccinating mice with purified wzi50-type CPS. Cross-reactivity and protective efficacy of MAb 24D11 were confirmed against CR-Kp that express the 3 most prevalent CPS types (wzi29, wzi154, wzi50) using both in vitro and in vivo infection models. 24D11 induced complement-mediated and independent opsonophagocytosis in macrophages as well as killing of all CR-Kp strains in whole blood cells derived from healthy donors. In a murine intratracheal infection model, 24D11 reduced lung burden and dissemination of CR-Kp strains when administered 4 h pre- or postinfection. The protective efficacy of 24D11 remained effective in neutropenic mice. This is the first antibody which exhibits cross-protective efficacy against clade 1 and 2 ST258 CR-Kp strains. It overcomes a major barrier to successfully target wzi29, a major CPS expressed by ST258 CR-Kp. The finding that 24D11 also exhibits potent protective efficacy against wzi154 CR-Kp strains highlights its high potential as a lead agent for the development of broadly active immunotherapy. IMPORTANCE Here, we present in vitro and in vivo data for the wzi50 CPS-specific monoclonal antibody MAb 24D11, demonstrating its cross-protective efficacy against three prominent win types (wzi29, wzi154, and wzi50) of the carbapenem-resistant clonal group CG258. In a murine pulmonary infection model, MAb 24D11 reduced bacterial lung burden and dissemination to other organs even if administered 4 h postinfection. Its protective efficacy was also observed in neutropenic mice, which highlights its potential value in clinical settings where oncology patients with CG258 infections may also be neutropenic.

Antigenic differences between equine influenza virus vaccine strains and Florida sublineage clade 1 strains isolated in Europe in 2019.

From late 2018 to 2019, equine influenza virus (EIV) strains of Florida sublineage clade 1 (Fc1), which had until then been circulating mainly in the United States, suddenly spread across Europe causing many outbreaks, and Florida sublineage clade 2 (Fc2) strains, which had been circulating mainly in Europe, have not been detected in Europe since 2018. Since 2010, the World Organisation for Animal Health (OIE) has recommended that EIV vaccines contain an Fc1 strain that is like A/equine/South Africa/4/2003 or A/equine/Ohio/2003. Accordingly, Japanese vaccines contain A/equine/Ibaraki/1/2007 as the Fc1 strain. To evaluate the effectiveness of these vaccines against the Fc1 strains detected in Europe in 2019, we performed virus neutralization tests using horse antisera. Challenge viruses used were Irish strain A/equine/Tipperary/1/2019 and two recombinant viruses generated by reverse genetics. Recombinant viruses possessing hemagglutinin (HA) and neuraminidase (NA) derived from A/equine/Tipperary/1/2019 (rA/equine/Tipperary/1/2019) or British strain A/equine/Essex/1/2019 (rA/equine/Essex/1/2019) were generated. Equine antisera against A/equine/South Africa/2003 and A/equine/Ibaraki/2007 were produced by experimental infection. Antibody titers against A/equine/Tipperary/1/2019, rA/equine/Tipperary/1/2019, and rA/equine/Essex/1/2019 were 2.5- to 6.3-fold lower than those against the homologous vaccine strains A/equine/South Africa/4/2003 or A/equine/Ibaraki/2007. These results suggest that the ongoing evolution of the Fc1 viruses may impact on antigenicity and although antibodies against current vaccine strains neutralize the 2019 strains, ongoing surveillance is essential for optimum choice of candidate vaccine strains.

Multifocal outbreak of equine influenza in vaccinated horses in Argentina in 2018: Epidemiological aspects and molecular characterisation of the involved virus strains.

BACKGROUND: Equine influenza is an important cause of respiratory disease of horses worldwide. The equine influenza virus (EIV) undergoes antigenic drift through the accumulation of amino acid substitutions in the viral proteins, which may lead to vaccine breakdown. OBJECTIVES: To describe the epidemiological findings and the molecular characteristics of the EIV detected during the multifocal outbreak that occurred in Argentina between March and July 2018 and evidence a vaccine breakdown. STUDY DESIGN: Observational, descriptive study. METHODS: Virus was detected in nasopharyngeal swabs using real-time reverse transcriptase PCR (RT-PCR). Nucleotide and deduced amino acid sequences of the haemagglutinin (HA) and neuraminidase (NA) genes were obtained from EIV positive nasopharyngeal swabs, and phylogenetic analysis was undertaken. Amino acid sequences were compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 1 vaccine strain and strain components of vaccines used in Argentina. Serum samples were tested using haemagglutination inhibition test. RESULTS: Equine influenza virus infection was confirmed using real-time RT-PCR and serological testing. The phylogenetic analysis of the HA and NA genes revealed that all the EIV identified during the outbreak belong to the H3N8 subtype, Florida clade 1. Multiple amino acid changes, some of them at antigenic sites, were observed in the circulating virus when compared with the strains included in the most commonly used vaccine in Argentina. Seventy-six percent of the affected horses had been vaccinated with this vaccine, suggesting the occurrence of vaccine breakdown. MAIN LIMITATIONS: The study does not include antigenic characterisation and full genome sequencing of Argentinian strains, that could provide additional information. CONCLUSIONS: The occurrence of this multifocal equine influenza outbreak in regularly vaccinated horses is a field evidence of vaccine breakdown, reinforcing the necessity of keeping vaccine strains updated according to OIE recommendations. It also underlines the importance of the implementation of appropriate quarantine measures and restriction of horse movement in the face of disease.

Fatal multiple outbreaks of equine influenza H3N8 in Nigeria, 2019: The first introduction of Florida clade 1 to West Africa.

In December 2018, suspected outbreaks of equine influenza (EI) were observed in donkeys in Sokoto State, in the extreme northwest of Nigeria bordering the Republic of the Niger. Equine influenza virus (EIV) subtype H3N8 was the etiologic agent identified in the outbreaks using real-time RT-qPCR and sequencing of both the partial haemagglutinin (HA) gene and the complete genome. Since then the H3N8 virus spread to 7 of the 19 northern states of Nigeria, where it affected both donkeys and horses. Phylogenetic analysis of the partial and complete HA gene revealed the closest nucleotide similarity (99.7%) with EIVs belonging to the Florida clade 1 (Fc-1) of the American lineage isolated in 2018 from Argentina and Chile. In total, 80 amino acid substitutions were observed in the viral proteins when compared to the OIE-recommended Fc-1 vaccine strains. The HA and neuraminidase proteins respectively had 13 and 16 amino acid substitutions. This study represents the first reported outbreak of EI caused by an Fc-1 virus in Nigeria and in the West Africa sub-region. Based on this report, extensive disease surveillance in equids is required to establish the circulating lineages and design an effective control strategy to protect the considerable population of horses and donkeys in the country.

Whole Genome Sequencing of the First H3N8 Equine Influenza Virus Identified in Malaysia.

In August 2015, Malaysia experienced an outbreak of acute respiratory disease in racehorses. Clinical signs observed were consistent with equine influenza (EI) infection. The index cases were horses recently imported from New Zealand. Rapid control measures, including temporary cancellation of racing, were implemented to minimize the impact of the outbreak. By November, the disease outbreak was resolved, and movement restrictions were lifted. The aim of this study was to confirm the clinical diagnosis and characterize the causal virus. A pan-reactive influenza type A real-time RT-PCR was used for confirmatory diagnosis. Antigenic characterization by haemagglutinin inhibition using a panel of specific ferret antisera indicated that the causal virus belonged to clade 1 of the H3N8 Florida sub-lineage. The genetic characterization was achieved by the whole genome sequencing of positive nasal swabs from clinically affected animals. Pylogenetic analysis of the haemagglutinin (HA) and neuraminidase (NA) genes demonstrated ≥99% homology with several EI strains that had recently circulated in the USA and Japan. The antigenic and genetic characterization did not indicate that the current World Organisation for Animal Health (OIE) recommendations for EI vaccine composition required modification.

Differences in virulence gene expression between human blood and stool Campylobacter coli clade 1 ST828CC isolates.

BACKGROUND: Campylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates. RESULTS: We assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two- to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins. CONCLUSIONS: This study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream.

Success and Limitation of Equine Influenza Vaccination: The First Incursion in a Decade of a Florida Clade 1 Equine Influenza Virus that Shakes Protection Despite High Vaccine Coverage.

Every year, several epizooties of equine influenza (EI) are reported worldwide. However, no EI case has been identified in France between 2015 and late 2018, despite an effective field surveillance of the pathogen and the disease. Vaccination against equine influenza virus (EIV) remains to this day one of the most effective methods to prevent or limit EI outbreaks and the lack of detection of the pathogen could be linked to vaccination coverage. The aim of this study was to evaluate EI immunity and vaccine coverage in France through a large-scale serological study. A total of 3004 archived surplus serums from French horses of all ages, breeds and sexes were selected from four different geographical regions and categories (i.e., sanitary check prior to exportation, sale, breeding protocol or illness diagnosis). EIV-specific antibody response was measured by single radial hemolysis (SRH) and an EIV-nucleoprotein (NP) ELISA (used as a DIVA test). Overall immunity coverage against EIV infection (i.e., titers induced by vaccination and/or natural infection above the clinical protection threshold) reached 87.6%. The EIV NP ELISA results showed that 83% of SRH positive serum samples from young horses (≤3 years old) did not have NP antibodies, which indicates that the SRH antibody response was likely induced by EI vaccination alone (the HA recombinant canarypoxvirus-based EI vaccine is mostly used in France) and supports the absence of EIV circulation in French horse populations between 2015 and late 2018, as reported by the French equine infectious diseases surveillance network (RESPE). Results from this study confirm a strong EI immunity in a large cohort of French horses, which provides an explanation to the lack of clinical EI in France in recent years and highlights the success of vaccination against this disease. However, such EI protection has been challenged since late 2018 by the incursion in the EU of a Florida Clade 1 sub-lineage EIV (undetected in France since 2009), which is also reported here.

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin-Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012.

BACKGROUND: In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. OBJECTIVE: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. METHODS: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alignments were constructed and virus was antigenically characterised with specific ferret antisera. Paired serum samples were tested by haemagglutination inhibition and single radial haemolysis. RESULTS: The diagnosis of EIV was confirmed by real-time RT-PCR, virus isolation and serological testing. The phylogenetic analysis of HA1 gene sequences of 18 EI viruses indicated that all of them belong to clade 1 of the Florida sublineage of the American lineage and are closely related to viruses isolated in the United States in 2012. The HA1 of viruses identified in horses in racing facilities in Maroñas, Uruguay, and in Palermo, Argentina, displayed 100% amino acid sequence identity and were identical to that of a virus isolated in Dubai in 2012, from vaccinated endurance horses recently imported from Uruguay. CONCLUSIONS: The surveillance data reported illustrate the international spread of EI viruses and support the recommendations of the OIE expert surveillance panel to include viruses of the Florida sublineage in vaccines.

The first reported Florida clade 1 virus in the Nordic countries, isolated from a Swedish outbreak of equine influenza in 2011.

Equine Influenza Virus (EIV) is a major cause of respiratory disease in horses and the virus constantly undergoes antigenic drift. Here we characterize and describe the HA1 and the NA genes of H3N8 within samples obtained from outbreaks in Sweden during November-December 2011. Both clade 1 and clade 2 viruses of the Florida sublineage were identified. The index case of clade 2 was transported to Sweden from Spain through the Netherlands, whereas the clade 1 had its origin from a Swedish stud farm. The clade 1 virus was efficiently spread between training yards by unvaccinated young horses, but vaccinated horses were also presented with clinical signs of respiratory disease. No virus of the Eurasian lineage was isolated during this outbreak. Clade 1 has previously been described in outbreaks in numerous of other countries, but this is the first time it has been detected in Sweden. The results from this study shows the importance of including both clade 1 and clade 2 of the Florida sublineage in equine influenza vaccines, supporting the ESP and OIE recommendations.

Structure of the monofunctional heme catalase DR1998 from Deinococcus radiodurans.

UNLABELLED: Deinococcus radiodurans is an aerobic organism with the ability to survive under conditions of high radiation doses or desiccation. As part of its protection system against oxidative stress, this bacterium encodes three monofunctional catalases. The DR1998 catalase belongs to clade 1, and is present at high levels under normal growth conditions. The crystals of DR1998 diffracted very weakly, and the merged diffraction data showed an R sym of 0.308. Its crystal structure was determined and refined to 2.6 Å. The four molecules present in the asymmetric unit form, by crystallographic symmetry, two homotetramers with 222 point-group symmetry. The overall structure of DR1998 is similar to that of other monofunctional catalases, showing higher structural homology with the catalase structures of clade 1. Each monomer shows the typical catalase fold, and contains one heme b in the active site. The heme is coordinated by the proximal ligand Tyr369, and on the heme distal side the essential His81 and Asn159 are hydrogen-bonded to a water molecule. A 25-Å-long channel is the main channel connecting the active site to the external surface. This channel starts with a hydrophobic region from the catalytic heme site, which is followed by a hydrophilic region that begins on Asp139 and expands up to the protein surface. Apart from this channel, an alternative channel, also near the heme active site, is presented and discussed. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank in Europe under accession code 4CAB.