Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Cytopathic effect of Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptation & extensive multiplication of PPRV in Vero cells.

Outbreaks among Wild Birds and Domestic Poultry Caused by Reassorted Influenza A(H5N8) Clade 2.3.4.4 Viruses, Germany, 2016.

In November 2016, an influenza A(H5N8) outbreak caused deaths of wild birds and domestic poultry in Germany. Clade 2.3.4.4 virus was closely related to viruses detected at the Russia-Mongolia border in 2016 but had new polymerase acidic and nucleoprotein segments. These new strains may be more efficiently transmitted to and shed by birds.

Rope-based oral fluid sampling for early detection of classical swine fever in domestic pigs at group level.

BACKGROUND: Non-invasive sampling techniques based on the analysis of oral fluid specimen have gained substantial importance in the field of swine herd management. Methodological advances have a focus on endemic viral diseases in commercial pig production. More recently, these approaches have been adapted to non-invasive sampling of wild boar for transboundary animal disease detection for which these effective population level sampling methods have not been available. In this study, a rope-in-a-bait based oral fluid sampling technique was tested to detect classical swine fever virus nucleic acid shedding from experimentally infected domestic pigs. RESULTS: Separated in two groups treated identically, the course of the infection was slightly differing in terms of onset of the clinical signs and levels of viral ribonucleic acid detection in the blood and oral fluid. The technique was capable of detecting classical swine fever virus nucleic acid as of day 7 post infection coinciding with the first detection in conventional oropharyngeal swab samples from some individual animals. Except for day 7 post infection in the "slower onset group", the chances of classical swine fever virus nucleic acid detection in ropes were identical or higher as compared to the individual sampling. CONCLUSIONS: With the provided evidence, non-invasive oral fluid sampling at group level can be considered as additional cost-effective detection tool in classical swine fever prevention and control strategies. The proposed methodology is of particular use in production systems with reduced access to veterinary services such as backyard or scavenging pig production where it can be integrated in feeding or baiting practices.

Evaluation of Selective Culling as a Containment Strategy for African Swine Fever at a Vietnamese Sow Farm.

Selective culling, also known as the "tooth extraction approach", is a strategy for controlling African swine fever (ASF) by removing only sick and suspect animals instead of the entire herd in Vietnam. This method prioritizes preserving healthy animals, particularly valuable breeding pigs. Despite its implementation in various forms, no standardized protocol based on scientific principles has been established. Farms typically adapt this strategy based on their understanding, which can vary significantly. In implementing of selective culling that is not based on scientific principles, there is a significant risk of spreading the disease. The aim of this study is to evaluate the consequences of selective culling as currently implemented in Vietnam. Our analysis on a large sow farm revealed that current practices rely heavily on clinical observations without laboratory confirmations. This approach allows ASF-infected animals to remain on the farm longer, potentially exacerbating the spread of the virus. Thus, selective culling poses a substantial risk by potentially exacerbating the spread of disease. Our findings emphasize that early diagnosis of ASF and systematic removal of infected pigs are critical components for the effective implementation of selective culling strategies and that a high level of fragmentation to minimize contact between animals plays a key role. The optimal approach is to test conspicuous animals and separate them. Under no circumstances should suspect animals be left in the herd for several days before they become severely ill and succumb to the disease.

SARS-CoV and SARS-CoV -2 cross-reactive antibodies in domestic animals and wildlife in Nigeria suggest circulation of sarbecoviruses.

Anthropogenic exposure of domestic animals, as well as wildlife, can result in zoonotic transmission events with known and unknown pathogens including sarbecoviruses. During the COVID-19 pandemic, SARS-CoV-2 infections in animals, most likely resulting from spill-over from humans, have been documented worldwide. However, only limited information is available for Africa. The anthropozoonotic transmission from humans to animals, followed by further inter- and intraspecies propagation may contribute to viral evolution, and thereby subsequently alter the epidemiological patterns of transmission. To shed light on the possible role of domestic animals and wildlife in the ecology and epidemiology of sarbecoviruses in Nigeria, and to analyze the possible circulation of other, undiscovered, but potentially zoonotic sarbecoviruses in animals, we tested 504 serum samples from dogs, rabbits, bats, and pangolins collected between December 2020 and April 2022. The samples were analyzed using an indirect multi-species enzyme-linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of SARS-CoV and SARS-CoV -2, respectively. ELISA reactive sera were further analyzed by highly specific virus neutralization test and indirect immunofluorescence assay for confirmation of the presence of antibodies. In this study, we found SARS-CoV reactive antibodies in 16 (11.5%) dogs, 7 (2.97%) rabbits, 2 (7.7%) pangolins and SARS-CoV-2 reactive antibodies in 20 (13.4%) dogs, 6 (2.5%) rabbits and 2 (7.7%) pangolins, respectively. Interestingly, 2 (2.3%) bat samples were positive only for SARS-CoV RBD reactive antibodies. These serological findings of SARS-CoV and/or SARS-CoV-2 infections in both domestic animals and wildlife indicates exposure to sarbecoviruses and requires further One Health-oriented research on the potential reservoir role that different species might play in the ecology and epidemiology of coronaviruses at the human-animal interface.

Development of cell culture based peste des petits ruminants (PPR) virus vaccine candidate from Bangladeshi isolates.

This study was conducted to develop a cell culture based PPR virus vaccine candidate using recent Bangladeshi strain of peste des petits ruminant's (PPR) virus. PPR virus was isolated from field outbreaks, confirmed by RT-PCR and used as viral inoculum for serial passaging in Vero cells for adaptation and attenuation. 60th serial passage had completed and RT-PCR and real time RT-PCR were done in every 5 passages for confirmation of PPR virus in tissue culture fluid (TCF). To assess the adaptation and attenuation cytopathology, virus titration, sequencing of both F and N genes and live animal experimentation were done. Different cellular alterations produced by PPR virus in infected Vero cells including syncytia formation, development of both intranuclear and intra cytoplasmic inclusion bodies and finally cell degradation are the indications of adaptation. The virus titre was found 2.5, 3.31, 3.55, 4.44, 4.71 and 6.5 Log10 TCID50/ml at 10th, 20th, 30th, 40th, 50th and 60th passages level respectively. In F gene sequence analysis it has been observed that few nucleotide (nt) and mino acid (aa) has been substituted as the effects of serial passaging of PPR virus in Vero cells. TCF at 60th passage level was found effective to produced protective antibody (Ab) titre in live animal experimentation. It is concluded that serially passaged and Vero cells adapted PPR virus TCF could be used as a vaccine candidate for further use to develop a potent & effective vaccine against PPR diseases.

Continuous surveillance of potentially zoonotic avian pathogens detects contemporaneous occurrence of highly pathogenic avian influenza viruses (HPAIV H5) and flaviviruses (USUV, WNV) in several wild and captive birds.

Three avian viral pathogens circulate in Germany with particular importance for animal disease surveillance due to their zoonotic potential, their impact on wild bird populations and/or poultry farms: Highly pathogenic (HP) avian influenza virus (AIV) of subtype H5 (HPAIV H5), Usutu virus (USUV), and West Nile virus (WNV). Whereas HPAIV H5 has been mainly related to epizootic outbreaks in winter, the arthropod-borne viruses USUV and WNV have been detected more frequently during summer months corresponding to peak mosquito activity. Since 2021, tendencies of a potentially year-round, i.e. enzootic, status of HPAIV in Germany have raised concerns that Orthomyxoviruses (AIV) and Flaviviruses (USUV, WNV) may not only circulate in the same region, but also at the same time and in the same avian host range. In search of a host species group suitable for a combined surveillance approach for all mentioned pathogens, we retrospectively screened and summarized case reports, mainly provided by the respective German National Reference Laboratories (NRLs) from 2006 to 2021. Our dataset revealed an overlap of reported infections among nine avian genera. We identified raptors as a particularly affected host group, as the genera Accipiter, Bubo, Buteo, Falco, and Strix represented five of the nine genera, and highlighted their role in passive surveillance. This study may provide a basis for broader, pan-European studies that could deepen our understanding of reservoir and vector species, as HPAIV, USUV, and WNV are expected to further become established and/or spread in Europe in the future and thus improved surveillance measures are of high importance.

Effect of swab matrix, storage time, and temperature on detection of avian influenza virus RNA in swab samples.

Virologic monitoring of avian influenza viruses (AIV) mainly relies on the collection of oropharyngeal, cloacal, or fecal swab samples. The quality of swab samples, therefore, contributes to limitations of the informative value of such monitoring, but the cost of sampling has a great impact on the feasibility of wild bird monitoring studies or poultry surveillance programs. Here, the effect of different swab material and storage conditions on quality and quantity of AIV RNA detection in swab samples by real-time reverse-transcription quantitative PCR has been studied. Two commercial swab products, a rayon-tipped and a flocked nylon type, were compared. Similar suitability of the two swab types, despite a huge price difference, was observed. Superior results by using both types of swab were gained provided that 1) swabs stayed immersed overnight in an appropriate viral transport medium (VTM), or that 2) swabs were vigorously shaken in VTM for at least 1 min and up to 1 hr to release as much trapped virus material as possible. Degradation of RNA over a period of 2 wk for virus-containing samples is negligible when using constant storage conditions at 4 C or 20 C; temperature shifts proved to be more harmful.

Highly pathogenic avian influenza virus incursions of subtype H5N8, H5N5, H5N1, H5N4, and H5N3 in Germany during 2020-21.

From October 2020 to July 2021, five different subtypes (H5N8, H5N5, H5N1, H5N4, and H5N3) and seven genotypes of highly pathogenic avian influenza viruses (HPAIV) belonging to clade 2.3.4.4b were detected in a broad array of avian hosts in Germany. Initial incursion by wild birds with an unprecedented involvement of charadriiforme species at the Wadden Sea coast only carrying subtype H5N3, lateral spread between poultry with detection of novel reassortants and mixed infections in poultry holdings, suspected spillback of HPAIV from poultry to wild birds, and detection of HPAIV-infected wild birds during the following summer in 2021 were hallmarks of this epizootic. Local reassortment events with low pathogenic AIV strains were detected by phylogenetic analyses, with a dominating HP H5N8 and later HP H5N1 strain responsible for most cases. In addition, the first-ever described HPAIV strain of subtype H5N4 could be genetically characterized.

Investigation into a Superspreading Event of the German 2020-2021 Avian Influenza Epidemic.

Between November 2020 and May 2021, Germany faced the largest highly pathogenic avian influenza (HPAI) epidemic recorded so far with 245 outbreaks in poultry and captive birds and more than 1000 diagnosed cases in wild birds. In March 2021, an HPAI outbreak of subtype H5N8 was confirmed in a holding rearing laying hens for sales. Disease introduction probably occurred via indirect contact with infected wild birds. Since the index farm sold chicken to customers including many smallholders, partly in travel trade, the primary outbreak triggered 105 known secondary outbreaks in five German federal states. An outbreak investigation was carried out with links between the involved farms retrieved from the German Animal Disease Notification System used for network analysis. In some cases, links were confirmed through sequence-based molecular analysis. Special emphasis was put on the estimation of the flock incubation period as a prerequisite of sound contact tracing. The unique circumstances of an outbreak farm with frequent direct trade contacts prior to disease suspicion enabled an assessment of the flock incubation period based on the consequences of contacts, further supported by molecular analysis and modeling of disease spread. In this case, the flock incubation period was at least 14 days.

Longitudinal Study on Extended-Spectrum Beta-Lactamase-E. coli in Sentinel Mallard Ducks in an Important Baltic Stop-Over Site for Migratory Ducks in Germany.

Antimicrobial resistance (AMR) is a serious global health threat with extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales as the most critical ones. Studies on AMR in wild birds imply a possible dissemination function and indicate their potential role as sentinel animals. This study aimed to gain a deeper insight into the AMR burden of wild waterfowl by sampling semi-wild mallard ducks used as sentinels and to identify if AMR bacteria could be recommended to be added to the pathogens of public health risks to be screened for. In total, 376 cloacal and pooled fecal samples were collected from the sentinel plant over a period of two years. Samples were screened for ESBL-carrying E. coli and isolates found further analyzed using antimicrobial susceptibility testing and whole-genome sequencing. Over the sampling period, 4.26% (16/376) of the samples were positive for ESBL-producing E. coli. BlaCTX-M-1 and blaCTX-M-32 were the most abundant CTX-M types. Although none of the top global sequence types (ST) could be detected, poultry-derived ST115 and non-poultry-related STs were found and could be followed over time. The current study revealed low cases of ESBL-producing E. coli in semi-wild mallard ducks, which proves the suitability of sentinel surveillance for AMR detection in water-associated wildlife.

A practical guide for strategic and efficient sampling in African swine fever-affected pig farms.

In the case of African swine fever (ASF) outbreaks in pig farms, EU legislation requires a thorough epidemiological investigation to determine, among other tasks, the extent of infection in the affected farm. The main aim of this study was to implement a reliable sampling strategy to quickly obtain an overview of the extent of ASF virus spread in an affected pig farm. We developed and tested a three-step approach: (i) identification of sub-units within the affected farm, (ii) categorization of sub-units, and (iii) targeted selection of animals for testing. We used commercially available lateral flow devices (LFDs) to detect ASF antigen and antibodies under field conditions and compared them with routinely performed laboratory tests (qPCR, ELISA, IPT). The study was conducted in three commercial farms in Latvia that were affected by ASF in July 2020. One of the affected farms was relatively small with only 31 pigs, whereas the other two were large with 1800 and 9800 animals, respectively. The approach proved to be helpful and practical for efficient and reliably assess the ASF situation on the farm and to identify sub-units within a farm where infected animals are present and sub-units which might (still) be free of infection. This important epidemiological information helps to better estimate the high-risk period and to track the potential spread of infection outside the farm. It allows also to prioritize culling and, if appropriate, to pursue a partial culling strategy taking into account the absence of clinical signs, implemented biosecurity measures, quarantine and negative test results, among others. This might be of interest for large commercial farms where the infection was identified very early and has not yet spread widely. Due to its limited sensitivity, the antigen LFD test is useful for testing animals showing signs of disease.

SARS-CoV-2 at the Human-Animal Interface: Implication for Global Public Health from an African Perspective.

The coronavirus disease 2019 (COVID-19) pandemic has become the most far-reaching public health crisis of modern times. Several efforts are underway to unravel its root cause as well as to proffer adequate preventive or inhibitive measures. Zoonotic spillover of the causative virus from an animal reservoir to the human population is being studied as the most likely event leading to the pandemic. Consequently, it is important to consider viral evolution and the process of spread within zoonotic anthropogenic transmission cycles as a global public health impact. The diverse routes of interspecies transmission of SARS-CoV-2 offer great potential for a future reservoir of pandemic viruses evolving from the current SARS-CoV-2 pandemic circulation. To mitigate possible future infectious disease outbreaks in Africa and elsewhere, there is an urgent need for adequate global surveillance, prevention, and control measures that must include a focus on known and novel emerging zoonotic pathogens through a one health approach. Human immunization efforts should be approached equally through the transfer of cutting-edge technology for vaccine manufacturing throughout the world to ensure global public health and one health.

Clinical Epidemiology, Pathology, and Molecular Investigation of Lumpy Skin Disease Outbreaks in Bangladesh during 2020-2021 Indicate the Re-Emergence of an Old African Strain.

Lumpy skin disease (LSD) emerged in Bangladesh in mid-2019, leading to great economic losses for cattle farmers. This study describes the recent occurrence of the LSDV in Bangladesh and examines the clinical manifestation of the disease in local cattle breeds, characteristic epidemiological features, and pathological findings in affected animals. In addition, a full-genome sequencing of two local LSDV isolates was carried out. A total of 565 animals from 88 households were investigated, and 165 samples (skin lesions, saliva, nasal discharge, feces, and milk) were collected for virus detection. Pathology and immunohistochemistry were performed on nodule biopsies. Fever, nodular skin lesions, and swelling of the joints were the most common clinical manifestations. Skin lesions had a higher concentration of viral DNA compared to other sample types and were therefore selected for virus isolation and characterization. Pathology of the LSD skin nodules comprised a granulomatous reaction in the dermis and hypodermis that extended to the surrounding tissues. Development of the skin lesions started with swelling of keratinocytes with cytoplasmic vacuolation, vasculitis, panniculitis, thrombosis, and infarction. Altogether, the LSDV produced transmural, hemorrhagic, necrotizing, proliferative and ulcerative dermatitis. The LSD viral antigen was detected occasionally in the macrophages, epithelial cells, and vascular smooth muscle cells. The complete genome sequence analysis revealed that the two Bangladeshi field strains (BD-V392.1 and BD-V395.1) were distinct from the contemporary field strains and were closely related to the ancestral African Neethling strain. The findings of this study will improve the diagnosis, monitoring, and control of LSD in Bangladesh.

African Swine Fever Outbreak Investigations-The Significance of Disease-Related Anecdotal Information Coming from Laypersons.

Veterinarians who have conducted numerous investigations of African swine fever outbreaks in pig farms in various European countries over the years shared their experiences during a workshop in Germany in early 2020. One focus was on the so-called "anecdotal information" obtained from farmers, farm workers or other lay people during the outbreak investigations. Discussions revolved around how to correctly interpret and classify such information and how the subjective character of the statements can influence follow-up examinations. The statements of the lay persons were grouped into three categories according to their plausibility: (i) statements that were plausible and prompted further investigation, (ii) statements that were not plausible and could therefore be ignored, and (iii) statements that were rather implausible but should not be ignored completely. The easiest to deal with were statements that could be classified without doubt as important and very plausible and statements that were not plausible at all. Particularly difficult to assess were statements that had a certain plausibility and could not be immediately dismissed out of hand. We aim to show that during outbreak investigations, one is confronted with human subjective stories that are difficult to interpret but still important to understand the overall picture. Here, we present and briefly discuss an arbitrary selection of reports made by lay persons during outbreak investigations.

Co-infections: Simultaneous detections of West Nile virus and Usutu virus in birds from Germany.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in Europe resulted in significant outbreaks leading to avifauna mortality and human infections. Both viruses have overlapping geographical, host and vector ranges, and are often co-circulating in Europe. In Germany, a nationwide bird surveillance network was established to monitor these zoonotic arthropod-borne viruses in migratory and resident birds. In this framework, co-infections with WNV and USUV were detected in six dead birds collected in 2018 and 2019. Genomic sequencing and phylogenetic analyses classified the detected WNV strains as lineage 2 and the USUV strains as lineages Africa 2 (n = 2), Africa 3 (n = 3) and Europe 2 (n = 1). Preliminary attempts to co-propagate both viruses in vitro failed. However, we successfully cultivated WNV from two animals. Further evidence for WNV-USUV co-infection was obtained by sampling live birds in four zoological gardens with confirmed WNV cases. Three snowy owls had high neutralizing antibody titres against both WNV and USUV, of which two were also positive for USUV-RNA. In conclusion, further reports of co-infections in animals as well as in humans are expected in the future, particularly in areas where both viruses are present in the vector population.

Spread of West Nile Virus and Usutu Virus in the German Bird Population, 2019-2020.

West Nile virus (WNV) and Usutu virus (USUV) are important flaviviruses circulating in Germany. While USUV was first reported more than 10 years ago, WNV has only reached the country in 2018. Wild birds are important amplifying hosts for both viruses. Therefore, we have been monitoring the bird population in different regions of Germany by a previously established network for many years. This report summarizes the results of molecular and/or serological methods of 2345 blood samples from birds of 22 different orders and over 2900 bird carcasses from 2019 and 2020. USUV RNA circulation was found in different regions of Germany, with emphasis on USUV lineages Europe 3 and Africa 3. Increased evidence of USUV lineage Europe 2 was detected in eastern Germany. WNV RNA was found only in birds from the eastern part of the country. The seroprevalence for USUV was between 3.11% and 7.20% in all three regions investigated, whereas the WNV seroprevalence spanned from 14.77% to 16.15% in eastern Germany, with a noticeable tendency for a westward and southward expansion in both years. Thus, wild bird monitoring for WNV and USUV can serve as an early warning system for a human exposure risk.

Evidence-Based African Swine Fever Policies: Do We Address Virus and Host Adequately?

African swine fever (ASF) is one of the most threatening diseases for the pig farming sector worldwide. Prevention, control and eradication remain a challenge, especially in the absence of an effective vaccine or cure and despite the relatively low contagiousness of this pathogen in contrast to Classical Swine Fever or Foot and Mouth disease, for example. Usually lethal in pigs and wild boar, this viral transboundary animal disease has the potential to significantly disrupt global trade and threaten food security. This paper outlines the importance of a disease-specific legal framework, based on the latest scientific evidence in order to improve ASF control. It compares the legal basis for ASF control in a number of pig-producing regions globally, considering diverse production systems, taking into account current scientific evidence in relation to ASF spread and control. We argue that blanket policies that do not take into account disease-relevant characteristics of a biological agent, nor the specifics under which the host species are kept, can hamper disease control efforts and may prove disproportionate.

The course of African swine fever in Romanian backyard holdings - A case report.

African swine fever (ASF) was diagnosed for the first time in Romania in a backyard holding in Satu-Mare County in July 2017. Since then, more than 3800 outbreaks occurred in the entire country. Disease control strategies in the backyard sector rely almost exclusively on reactive measures implemented upon appearance of clinical signs and laboratory confirmation of ASF. In our descriptive study, infection course and outbreak investigation data of 56 affected backyard holdings in Satu-Mare County has been investigated. Early disease detection based on clinical signs appeared to be efficient. In the majority of outbreaks, ASF was detected within the first 2 weeks after the estimated virus introduction. A clinical phase of 2-8 days was observed before pigs either succumbed to the disease or control measures were implemented on affected farms. A moderate on-farm transmissibility of ASF virus between pigs was observed. Four clusters of outbreaks were identified indicating virus perpetuation and transmission from farm to farm. To suspend infection chains, rapid intervention by isolating affected farms combined with effective biosecurity measures is required. However, due to the backyard peculiarities, quick and effective implementation of control measures has shown to be rather difficult.