Implementing wildlife disease surveillance in the Netherlands, a One Health approach.

The surveillance of (emerging) wildlife diseases can provide important, objective evidence of the circulation of pathogens of interest for veterinary and/or public health. The involvement of multiple research institutions in wildlife disease surveillance can ensure the best use of existing knowledge and expertise, but can also complicate or add challenges to the integration of wildlife disease surveillance components into a national programme. Documenting the existing efforts in a country's surveillance of wildlife diseases, including the institutes in which it takes place, provides a basis for policy-makers and authorities to identify gaps and priorities in their current surveillance programmes. This paper describes the wildlife disease surveillance activities taking place in the Netherlands. The authors recommend that, in addition to funding these current activities, surveillance resources should be allocated with the flexibility to allow for additional targeted surveillance, to detect and adequately respond to newly introduced or emerging pathogens. Similar structured overviews of wildlife disease surveillance in other countries would be very useful to facilitate international collaboration.

La surveillance exercée sur les maladies (émergentes) de la faune sauvage permet de réunir des données déterminantes, objectives et probantes sur la présence d'agents pathogènes importants pour la santé animale et/ou publique. La participation de plusieurs instituts de recherche dans les activités de surveillance des maladies de la faune sauvage permet de tirer le meilleur parti des connaissances et de l'expertise disponibles mais, dans certains cas, elle peut aussi se traduire par une complexité ou des difficultés supplémentaires qui compromettent l'intégration des composantes axées sur les maladies de la faune sauvage dans les programmes nationaux de surveillance. La collecte d'informations sur les efforts déployés au niveau national pour surveiller les maladies des animaux sauvages ainsi que sur les institutions chargées de cette surveillance constitue une première étape essentielle pour que les responsables des politiques sanitaires et les autorités puissent identifier les lacunes et les priorités des programmes de surveillance en vigueur. Les auteurs décrivent les activités de surveillance des maladies de la faune sauvage conduites actuellement aux Pays-Bas. Ils recommandent que parallèlement au financement des activités en cours, les ressources destinées à la surveillance soient allouées de manière plus souple afin de couvrir de nouvelles activités ciblées, de détecter les agents pathogènes émergents ou d'introduction récente et de préparer une réponse adéquate. Ils préconisent de réaliser dans d'autres pays des études structurées similaires sur la surveillance des maladies de la faune sauvage afin de faciliter la collaboration internationale.

La vigilancia de enfermedades (emergentes) de la fauna silvestre puede proporcionar importantes elementos de prueba objetivos sobre la circulación de patógenos de interés para la salud pública y/o veterinaria. La participación de numerosos establecimientos de investigación en estas actividades de vigilancia puede garantizar que se haga un uso idóneo de los conocimientos teóricos y técnicos existentes, pero a veces también complica o dificulta la integración en un programa nacional de las tareas de vigilancia de las enfermedades de la fauna silvestre. El hecho de repertoriar las actividades en la materia que se llevan a cabo en un país, incluidos los establecimientos donde tienen lugar, sienta las bases para que las autoridades e instancias de planificación de políticas puedan determinar las carencias y prioridades de los programas de vigilancia que ya tengan en marcha. Tras describir las actividades de vigilancia sanitaria de la fauna silvestre que se llevan a cabo en los Países Bajos, los autores recomiendan que los recursos para fines de vigilancia se asignen de manera flexible para que, además de costear las actividades ya en curso, sirvan para financiar otras labores de vigilancia selectiva que permitan detectar patógenos emergentes o recién introducidos en el país y responder debidamente a ellos. Para facilitar la colaboración internacional sería muy útil contar con estudios estructurados similares, que ofrezcan una visión de conjunto de la vigilancia sanitaria de la fauna silvestre en otros países.

Wild bird surveillance around outbreaks of highly pathogenic avian influenza A(H5N8) virus in the Netherlands, 2014, within the context of global flyways.

Highly pathogenic avian influenza (HPAI) A(H5N8) viruses that emerged in poultry in east Asia since 2010 spread to Europe and North America by late 2014. Despite detections in migrating birds, the role of free-living wild birds in the global dispersal of H5N8 virus is unclear. Here, wild bird sampling activities in response to the H5N8 virus outbreaks in poultry in the Netherlands are summarised along with a review on ring recoveries. HPAI H5N8 virus was detected exclusively in two samples from ducks of the Eurasian wigeon species, among 4,018 birds sampled within a three months period from mid-November 2014. The H5N8 viruses isolated from wild birds in the Netherlands were genetically closely related to and had the same gene constellation as H5N8 viruses detected elsewhere in Europe, in Asia and in North America, suggesting a common origin. Ring recoveries of migratory duck species from which H5N8 viruses have been isolated overall provide evidence for indirect migratory connections between East Asia and Western Europe and between East Asia and North America. This study is useful for better understanding the role of wild birds in the global epidemiology of H5N8 viruses. The need for sampling large numbers of wild birds for the detection of H5N8 virus and H5N8-virus-specific antibodies in a variety of species globally is highlighted, with specific emphasis in north-eastern Europe, Russia and northern China.

Is dolphin morbillivirus virulent for white-beaked dolphins (Lagenorhynchus albirostris)?

The virulence of morbilliviruses for toothed whales (odontocetes) appears to differ according to host species. In 4 species of odontocetes, morbilliviruses are highly virulent, causing large-scale epizootics with high mortality. In 8 other species of odontocetes, including white-beaked dolphins (Lagenorhynchus albirostris), morbilliviruses have been found as an incidental infection. In these species, the virulence of morbilliviruses is not clear. Therefore, the admission of 2 white-beaked dolphins with morbillivirus infection into a rehabilitation center provided a unique opportunity to investigate the virulence of morbillivirus in this species. By phylogenetic analysis, the morbilliviruses in both animals were identified as a dolphin morbillivirus (DMV) most closely related to that detected in a white-beaked dolphin in Germany in 2007. Both animals were examined clinically and pathologically. Case No. 1 had a chronic neural DMV infection, characterized by polioencephalitis in the cerebrum and morbillivirus antigen expression limited to neurons and glial cells. Surprisingly, no nervous signs were observed in this animal during the 6 months before death. Case No. 2 had a subacute systemic DMV infection, characterized by interstitial pneumonia, leucopenia, lymphoid depletion, and DMV antigen expression in mononuclear cells and syncytia in the lung and in mononuclear cells in multiple lymphoid organs. Cause of death was not attributed to DMV infection in either animal. DMV was not detected in 2 contemporaneously stranded white-beaked dolphins. Stranding rate did not increase in the region. These results suggest that DMV is not highly virulent for white-beaked dolphins.

Replication of 2 subtypes of low-pathogenicity avian influenza virus of duck and gull origins in experimentally infected Mallard ducks.

Many subtypes of low-pathogenicity avian influenza (LPAI) virus circulate in wild bird reservoirs, but their prevalence may vary among species. We aimed to compare by real-time reverse-transcriptase polymerase chain reaction, virus isolation, histology, and immunohistochemistry the distribution and pathogenicity of 2 such subtypes of markedly different origins in Mallard ducks (Anas platyrhynchos): H2N3 isolated from a Mallard duck and H13N6 isolated from a Ring-billed Gull (Larus delawarensis). Following intratracheal and intraesophageal inoculation, neither virus caused detectable clinical signs, although H2N3 virus infection was associated with a significantly decreased body weight gain during the period of virus shedding. Both viruses replicated in the lungs and air sacs until approximately day 3 after inoculation and were associated with a locally extensive interstitial, exudative, and proliferative pneumonia. Subtype H2N3, but not subtype H13N6, went on to infect the epithelia of the intestinal mucosa and cloacal bursa, where it replicated without causing lesions until approximately day 5 after inoculation. Larger quantities of subtype H2N3 virus were detected in cloacal swabs than in pharyngeal swabs. The possible clinical significance of LPAI virus-associated pulmonary lesions and intestinal tract infection in ducks deserves further evaluation.

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets.

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.

Modification of the ferret model for pneumonia from seasonal human influenza A virus infection.

The primary complication of seasonal influenza in humans is viral pneumonia. A conventional animal model--intranasal inoculation of ferrets with 10(6) median tissue culture infectious dose of virus--results in disease that is neither consistent nor comparable with severe viral pneumonia in humans. Therefore, the authors modified the experimental procedures by increasing the median tissue culture infectious dose to 10(9) and by inoculating via the intratracheal route, testing these procedures with H1N1 strains (A/Bilthoven/3075/1978 and A/Netherlands/26/2007) and H3N2 strains (A/Bilthoven/16190/1968 and A/Netherlands/177/2008) of seasonal influenza virus. The ferrets of all groups (n = 3 per virus strain) had clinical signs, increased body temperature, virus excretion from day 1, loss of body weight, and increased relative lung weight at 4 days postinoculation. All ferrets had severe pulmonary consolidation, and histologic examination revealed moderate to severe necrotizing bronchointerstitial pneumonia with severe edema, necrosis of alveolar epithelium, inflammatory infiltrates in alveolar septa and lumina, epithelial regeneration, and perivascular and peribronchiolar inflammatory infiltrates. The lesions were associated with the presence of influenza virus antigen in respiratory epithelium by immunohistochemistry. Although all 4 virus strains caused pulmonary lesions of comparable severity, virus isolation in the lungs, trachea, nasal concha, and tonsils showed higher mean virus titers in the H1/07 and H3/68 groups than in the H1/78 and H3/08 groups. In conclusion, the above H1N1 and H3N2 strains cause severe pneumonia in ferrets by use of the modified experimental procedures and provide a good model for pneumonia caused by seasonal influenza A virus infection in humans.

Assessment of the virulence for chickens of Newcastle Disease virus with an engineered multi-basic cleavage site in the fusion protein and disrupted V protein gene.

Newcastle Disease virus (NDV) has shown promise as an oncolytic virus for treatment of a wide range of tumours. NDV with a multi-basic cleavage site (MBCS) in the fusion (F) protein (NDV F3aa) has increased oncolytic efficacy in several tumour models, but also increased virulence in chickens compared to non-virulent NDV F0, raising potential environmental safety issues. Previously, we generated a variant of NDV F3aa with a disrupted V protein gene and a substitution of phenylalanine to serine at position 117 of the F protein (NDV F3aa-S-STOPV). Compared to NDV F3aa this virus had decreased virulence in embryonated chicken eggs. In this study, the virulence of the virus was evaluated upon inoculation of six-week-old chickens through a natural infection route and by determination of the intracerebral pathogenicity index (ICPI). Based on these data NDV F3aa-S-STOPV classified as a non-virulent virus. Although NDV F3aa was classified as a virulent virus based on the ICPI, the virus was also less pathogenic than NDV F0 upon inoculation of six-week-old chickens. These data indicate that NDV with a MBCS is not necessarily pathogenic in chickens. In addition, these data show that F3aa-S-STOPV is safe to use in viro-immunotherapies without posing a threat for chickens upon accidental exposure.

Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021.

Highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage (GsGd), which threaten the health of poultry, wildlife and humans, are spreading across Asia, Europe, Africa and North America but are currently absent from South America and Oceania. In December 2021, H5N1 HPAI viruses were detected in poultry and a free-living gull in St. John's, Newfoundland and Labrador, Canada. Our phylogenetic analysis showed that these viruses were most closely related to HPAI GsGd viruses circulating in northwestern Europe in spring 2021. Our analysis of wild bird migration suggested that these viruses may have been carried across the Atlantic via Iceland, Greenland/Arctic or pelagic routes. The here documented incursion of HPAI GsGd viruses into North America raises concern for further virus spread across the Americas by wild bird migration.

A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets.

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 microg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.

A newly discovered human pneumovirus isolated from young children with respiratory tract disease.

From 28 young children in the Netherlands, we isolated a paramyxovirus that was identified as a tentative new member of the Metapneumovirus genus based on virological data, sequence homology and gene constellation. Previously, avian pneumovirus was the sole member of this recently assigned genus, hence the provisional name for the newly discovered virus: human metapneumovirus. The clinical symptoms of the children from whom the virus was isolated were similar to those caused by human respiratory syncytial virus infection, ranging from upper respiratory tract disease to severe bronchiolitis and pneumonia. Serological studies showed that by the age of five years, virtually all children in the Netherlands have been exposed to human metapneumovirus and that the virus has been circulating in humans for at least 50 years.

Establishing a European network for wildlife health surveillance.

Surveillance of wildlife health in Europe remains informal and reporting wildlife diseases is not yet coordinated among countries. At a meeting in Brussels on 15 October 2009, delegates from 25 countries provided an overview of the current status of wildlife health surveillance in Europe. This showed that every year in Europe over 18,000 wild animals are examined as part of general surveillance programmes and over 50,000 wild animals are examined in the course of targeted surveillance. The participants at the Brussels meeting agreed to set up a European network for wildlife health surveillance. The goals of this network, which was established in February 2010, are to improve procedures for the rapid exchange of information, harmonise procedures for investigation and diagnosis of wildlife diseases, share relevant expertise, and provide training opportunities for wildlife health surveillance.

Comparative pathology of select agent influenza a virus infections.

Influenza A virus infections may spread rapidly in human populations and cause variable mortality. Two of these influenza viruses have been designated as select agents: 1918 H1N1 virus and highly pathogenic avian influenza (HPAI) virus. Knowledge of the pathology of these virus infections in humans, other naturally infected species, and experimental animals is important to understand the pathogenesis of influenza, to design appropriate models for evaluation of medical countermeasures, and to make correct diagnoses. The most important complication of influenza in humans is viral pneumonia, which often occurs with or is followed by bacterial pneumonia. Viremia and extrarespiratory disease are uncommon. HPAI viruses, including HPAI H5N1 virus, cause severe systemic disease in galliform species as well as in anseriform species and bird species of other orders. HPAI H5N1 virus infection also causes severe disease in humans and several species of carnivores. Experimental animals are used to model different aspects of influenza in humans, including uncomplicated influenza, pneumonia, and virus transmission. The most commonly used experimental animal species are laboratory mouse, domestic ferret, and cynomolgus macaque. Experimental influenza virus infections are performed in various other species, including domestic pig, guinea pig, and domestic cat. Each of these species has advantages and disadvantages that need to be assessed before choosing the most appropriate model to reach a particular goal. Such animal models may be applied for the development of more effective antiviral drugs and vaccines to protect humans from the threat of these virus infections.

Pandemic 2009 H1N1 influenza virus causes diffuse alveolar damage in cynomolgus macaques.

The pathogenesis of lower respiratory tract disease from the pandemic 2009 H1N1 (H1N1v) influenza A virus is poorly understood. Therefore, either H1N1v virus or a seasonal human H1N1 influenza A virus was inoculated into cynomolgus macaques as a nonhuman primate model of influenza pneumonia, and virological, pathological, and microarray analyses were performed. Macaques in the H1N1v group had virus-associated diffuse alveolar damage involving both type I and type II alveolar epithelial cells and affecting an average of 16% of the lung area. In comparison, macaques in the seasonal H1N1 group had milder pulmonary lesions. H1N1v virus tended to be reisolated from more locations in the respiratory tract and at higher titers than seasonal H1N1 virus. In contrast, differential expression of messenger RNA transcripts between H1N1v and seasonal H1N1 groups did not show significant differences. The most upregulated genes in H1N1v lung samples with lesions belonged to the innate immune response and proinflammatory pathways and correlated with histopathological results. Our results demonstrate that the H1N1v virus infects alveolar epithelial cells and causes diffuse alveolar damage in a nonhuman primate model. Its higher pathogenicity compared with a seasonal H1N1 virus may be explained in part by higher replication in the lower respiratory tract.

Pathology and virus distribution in chickens naturally infected with highly pathogenic avian influenza A virus (H7N7) During the 2003 outbreak in The Netherlands.

The largest recorded outbreak of highly pathogenic avian influenza virus of the subtype H7N7 occurred in The Netherlands in 2003. We describe the immunohistochemical and histopathologic findings of 3 chickens naturally infected during this outbreak. Influenza virus antigen occurred in endothelial cells and mononuclear cells of all tissues examined and occurred in parenchymal cells of heart, lung, kidney, pancreas, and trachea, often associated with multifocal inflammation and necrosis. These findings are consistent with the acute stage of highly pathogenic avian influenza from other subtypes. In the severely edematous wattle skin, most endothelial cells contained virus antigen, while in all other tissues virus antigen was only detected in a few endothelial cells. Virus histochemistry showed that this H7N7 virus attached to more endothelial cells in wattle skin than in other vascular beds. This might explain, at least partly, the tropism of the virus and the associated severity of lesions in this tissue.

Avian influenza viruses in mammals.

Highly pathogenic avian influenza viruses of subtype H5N1 are remarkable because of their expanding non-avian host range and wide tissue tropism. They have caused severe or fatal respiratory and extra-respiratory disease in seven naturally infected species of carnivore. However, they are not unique in their ability to cross the species barrier, to cause clinical disease and mortality, or to replicate in extra-respiratory organs. Low pathogenic avian influenza viruses have crossed from birds to swine, horses, harbour seals, whales and mink; have resulted in severe respiratory disease and mortality; and may have spread beyond the respiratory tract in some of these species. They are also transmitted from mammal to mammal in most species, and have become endemic in swine and horse populations, demonstrating their ability to adapt to and become sustained in mammals. Until now, highly pathogenic avian influenza viruses H5N1 have not acquired this ability, but there are concerns that they may adapt to mammalian species and, thus, could spark an influenza pandemic in humans.

Neurological signs in juvenile harbour seals (Phoca vitulina) with fatal phocine distemper.

In 2002, the northern European harbour seal (Phoca vitulina) population experienced an epidemic of phocine distemper virus (PDV) in which 22,000 seals died. Clinical signs were recorded in 20 harbour seal pups admitted to the Seal Rehabilitation and Research Centre with clinical disease, and they were diagnosed PDV infection-positive by RT-PCR postmortem. All 20 had respiratory signs, 14 had conjunctivitis and 10 had neurological signs. Severe neurological signs were one of the criteria for euthanasia during the epidemic, and many pups that were euthanased were not included in this study owing to the lack of complete datasets. Neurological signs were therefore among the most prevalent signs of fatal PDV infection in harbour seal pups. The lymphoid depletion reported in dead seals during the epidemic was not reflected in the total mononuclear leucocyte count of the seal pups, but they had an absolute granulocytosis, thrombocytosis, anaemia, and high total white blood cell counts. When first examined, 11 of the pups had a positive serum IgG titre, and four had a positive serum IgM titre. High levels of PDV-specific serum IgG antibodies were not correlated with an absence of clinical signs or longer survival.

Heterozygosity and lungworm burden in harbour seals (Phoca vitulina).

In several studies, heterozygosity measured at around 10 microsatellite markers correlates with parasite load. Usually the effect size is small, but while this may reflect reality, it may also be possible that too few markers are used or the measure of fitness contains too much error to reveal what is actually a much stronger underlying effect. Here, we analysed over 200 stranded harbour seals (Phoca vitulina) for an association between lungworm burden and heterozygosity, conducting thorough necropsies on the seals and genotyping the samples obtained for 27 microsatellites. We found that homozygosity predicts higher worm burdens, but only in young animals, where the worms have the greatest impact on fitness. Testing each locus separately, we found that a significant majority reveal a weak but similar trend for heterozygosity to be protective against high lungworm burden, suggesting a genome-wide effect, that is, inbreeding. This conclusion is supported by the fact that heterozygosity is correlated among markers in young animals but not in otherwise equivalent older ones. Taken as a whole, our results support the notion that homozygosity increases susceptibility to parasitic infection and suggest that parasites can be effective in removing inbred individuals from the population.

Severe influenza resembling hemorrhagic shock and encephalopathy syndrome.

Influenza-associated encephalopathy is a clinically diverse syndrome and severe cases are not well documented outside Japan. Clinical, pathological and molecular aspects are described of two fatal cases presenting during 2004 and 2005 winter seasons in The Netherlands. Results showed that severe influenza can resemble hemorrhagic shock and encephalopathy syndrome, and proper testing for influenza virus should be considered in similar cases. The failure to detect viral replication in non-pulmonary organs including the brain would support the pathogenesis of this syndrome is based on proinflammatory cytokine responses.

Fatal enterocolitis in harbour seals (Phoca vitulina) caused by infection with Eimeria phocae.

Coccidiosis due to Eimeria phocae infection has been described in harbour seals (Phoca vitulina) from the western Atlantic population, but not in any detail in seals from the eastern Atlantic population. This paper describes fatal enterocolitis due to E phocae infection in three juvenile harbour seals at a rehabilitation centre in the Netherlands in July 2003. The clinical signs were lethargy, bloody faeces, and intermittent convulsions and muscle tremors just before they died; the nervous signs resembled those of nervous coccidiosis in calves. The main pathological finding was severe, diffuse, haemorrhagic enterocolitis; there were diffuse inflammatory changes in the lamina propria of the jejunal, ileal, caecal and colonic mucosa that were associated with the presence of the sexual stages and oocysts of a coccidian species identified as E phocae. A retrospective microscopical examination of intestinal tissues from 113 harbour seals that had died between 1999 and 2004 revealed one seal that was positive for E phocae.

Acute fatal sarcocystosis hepatitis in an Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Hong Kong.

Unlike most species in the genus Sarcocystis, Sarcocystis canis has a broad intermediate host range. Its life cycle is incompletely known and most reports are from the USA. Here we report fatal hepatitis in a 4year old male Indo-Pacific bottlenose dolphin (Tursiops aduncus) from Hong Kong associated with a S. canis-like infection. Diagnosis was made based on clinical presentation, histopathology, transmission electron microscopy (TEM), and molecular characterization. Microscopically, S. canis-like like infection was confined to the liver. Immature and mature schizonts were found in hepatocytes and the parasite was associated with generalized hepatic necrosis. By TEM, schizonts divided by endopolygeny, and merozoites lacked rhoptries. Molecular characterization of parasites present in liver and brain tissues at the cox1 gene showed a high degree of identity (97-98%) and clustered together with Sarcocystis canis, S. lutrae, S. arctica, S. speeri, S. turdusi, and S. rileyi in a phylogenetic study. This is the first report of S. canis-like infection from Asia.