Selection of vaccine-candidate peptides from Mycobacterium avium subsp. paratuberculosis by in silico prediction, in vitro T-cell line proliferation, and in vivo immunogenicity.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

A molecular epidemiological study on Escherichia coli in young chicks with colibacillosis identified two possible outbreaks across farms.

Avian pathogenic Escherichia coli (APEC) is the cause of colibacillosis outbreaks in young poultry chicks, resulting in acute to peracute death. The high morbidity and mortality caused by colibacillosis results in poor animal welfare, reduced sustainability and economical loss worldwide. To advance the understanding of the molecular epidemiology, genomic relatedness and virulence traits of APEC, we performed systematic sampling from 45 confirmed colibacillosis broiler flocks with high first week mortality (FWM) during 2018-2021. From these flocks, 219 APEC isolates were whole genome sequenced (WGS) and bioinformatic analyses were performed. The bioinformatic analyses included sequence typing (ST), serotyping, detection of virulence-associated genes (VAGs) and phylogenetic analysis. Our results showed a high prevalence of ST23, ST429 and ST95 among APEC isolates from Norwegian broiler flocks, and identified ST23, ST429, ST117 and ST371 to cause disease more often alone, compared to ST95, ST69 and ST10. Phylogenetic analyses, together with associated metadata, identified two distinct outbreaks of colibacillosis across farms caused by ST429 and ST23 and gave insight into expected SNP distances within and between flocks identified with the same ST. Further, our results highlighted the need for combining two typing methods, such as serotyping and sequence typing, to better discriminate strains of APEC. Ultimately, systematic sampling of APEC from multiple birds in a flock, together with WGS as a diagnostic tool is important to identify the disease-causing APEC within a flock and to detect outbreaks of colibacillosis across farms.

High sequence similarity between avian pathogenic E. coli isolates from individual birds and within broiler chicken flocks during colibacillosis outbreaks.

Avian pathogenic E. coli (APEC) cause high first week mortality (FWM) in broiler chickens worldwide. In order to investigate the epidemiologic aspects of colibacillosis in broiler flocks it is important to develop reliable and cost-effective sampling guidelines. In this context, it is particularly important to define the minimum number of samples required to reliably identify the causative APEC clone during outbreaks of colibacillosis. This study describes the diversity of E. coli isolates between and within three flocks with high FWM due to colibacillosis. Each flock was represented by five animals, showing typical lesions of colibacillosis, and spleen, liver and one other organ from each animal was sampled for APEC. A total of 47 E. coli isolates, one per organ, and approximately 15 isolates per flock were whole genome sequenced and compared by multilocus sequence typing (MLST), serotyping and phylogenetic analysis to deduce their relationship. The results revealed that within individual birds there was little or no sequence type (ST) or serotype diversity between APEC isolates from different organs. Based on phylogenetic analysis, isolates belonging to the same ST and serotype showed a low number of single nucleotide polymorphisms (SNPs) across more than 95 % of the genome. Isolates from the liver always represented the major disease-causing APEC in individual birds, even when more than one ST was detected within an individual bird and flock. This study guides us towards an economically efficient way of sampling for future epidemiological studies on colibacillosis, by determining the causative APEC-clone at flock level.

An Official Outbreak Investigation of Acute Haemorrhagic Diarrhoea in Dogs in Norway Points to Providencia alcalifaciens as a Likely Cause.

An outbreak investigation was initiated in September 2019, following a notification to the Norwegian Food Safety Authority (NFSA) of an unusually high number of dogs with acute haemorrhagic diarrhoea (AHD) in Oslo. Diagnostic testing by reporting veterinarians had not detected a cause. The official investigation sought to identify a possible common cause, the extent of the outbreak and prevent spread. Epidemiological data were collected through a survey to veterinarians and interviews with dog owners. Diagnostic investigations included necropsies and microbiological, parasitological and toxicological analysis of faecal samples and food. In total, 511 dogs with acute haemorrhagic diarrhoea were registered between 1 August and 1 October. Results indicated a common point source for affected dogs, but were inconclusive with regard to common exposures. A notable finding was that 134 of 325 faecal samples (41%) cultured positive for Providencia alcalifaciens. Whole genome sequencing (WGS) of 75 P. alcalifaciens isolates from 73 dogs revealed that strains from 51 dogs belonged to the same WGS clone. Findings point to P. alcalifaciens as implicated in the outbreak, but investigations are needed to reveal the pathogenic potential of P. alcalifaciens in dogs and its epidemiology.

Bovine respiratory syncytial virus in experimentally exposed and rechallenged calves; viral shedding related to clinical signs and the potential for transmission.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen worldwide, detrimentally affecting the economy and animal welfare. To prevent and control BRSV infection, further knowledge on virus shedding and transmission potential in individual animals is required. This study aimed to detect viral RNA and infective virions during BRSV infection to evaluate duration of the transmission period and correlation with clinical signs of disease. The outcome of BRSV re-exposure on calves, their housing environment and effect of introduction of sentinel calves was also investigated. A live animal experiment including 10 calves was conducted over 61 days. Initially, two calves were inoculated with a non-passaged BRSV field isolate. Two days later, six naïve calves (EG: Exposed group) were introduced for commingling and four weeks later, another two naïve calves (SG: Sentinel group) were introduced. Seven weeks after commingling, EG animals were re-inoculated. Clinical examination was performed daily. Nasal swabs were collected regularly and analysed for viral RNA by RT-ddPCR, while virus isolation was performed in cell culture. BRSV serology was performed with ELISA. RESULTS: All the EG calves seroconverted and showed clinical signs of respiratory disease. Viral RNA was detected from days 1-27 after exposure, while the infective virus was isolated on day 6 and 13. On day 19, all animals were seropositive and virus could not be isolated. Total clinical score for respiratory signs corresponded well with the shedding of viral RNA. The SG animals, introduced 27 days after exposure, remained negative for BRSV RNA and stayed seronegative throughout the study. Inoculation of the EG calves seven weeks after primary infection did not lead to new shedding of viral RNA or clinical signs of disease. CONCLUSION: Viral RNA was detected in nasal swabs from the calves up to four weeks after exposure. The detection and amount of viral RNA corresponded well with the degree of respiratory signs. The calves were shedding infective virions for a considerable shorter period, and naïve calves introduced after four weeks were not infected. Infected calves were protected from reinfection for at least seven weeks. This knowledge is useful to prevent spread of BRSV.

Human to animal transmission of influenza A(H1N1)pdm09 in a turkey breeder flock in Norway.

Introduction: Routine surveillance samples disclosed seropositivity to influenza A virus (IAV) in a Norwegian turkey breeder flock. Simultaneous reports of influenza-like symptoms in farm workers and a laboratory confirmed influenza A(H1N1)pdm09 (H1N1pdm09) infection in one person led to the suspicion of a H1N1pdm09 infection in the turkeys. Animals and methods: H1N1pdm09 infection was confirmed by a positive haemaggutinin inhibition test using H1N1pdm09 antigens, and detection of H1N1pdm09 nucleic acid in reproductive organs of turkey hens. The flock showed no clinical signs except for a temporary drop in egg production. Previous reports of H1N1pdm09 infection in turkeys suggested human-to-turkey transmission (anthroponosis) during artificial insemination. Results and discussion: The flock remained seropositive to IAV and the homologous H1N1pdm09 antigen throughout the following 106 days, with decreasing seroprevalence over time. IAV was not detected in fertilised eggs or in turkey poults from the farm, however, maternally derived antibodies against H1N1pdm09 were found in egg yolks and in day-old poults. Genetic analyses of haemagglutinin gene sequences from one of the infected farm workers and turkeys revealed a close phylogenetic relationship, and confirmed human-to-turkey virus transmission.