Tularemia on the rise in Switzerland? A one health approach is needed!

In the last 10 years, an increase in tularemia cases has been observed in both humans and animals in Switzerland. In these, infection with Francisella tularensis, the causative agent of the zoonotic disease tularemia, can occur through arthropod vectors or contact to infected animals or exposure to contaminated environmental sources. Currently, we are only able to postulate potential aetiologies: (i) behavioral changes of humans with more exposure to endemic habitats of infected arthropod vectors; (ii) an increased rate of tularemia infected ticks; (iii) increasing number and geographical regions of tick biotopes; (iv) increasing and/or more diverse reservoir populations; (v) increasing presence of bacteria in the environment; (vi) raised awareness and increased testing among physicians; (vii) improved laboratory techniques including molecular testing. To approach these questions, a one-health strategy is necessary. A functioning collaboration between public health, human medicine, and diagnostic and veterinary units for the control of tularemia must be established. Furthermore, the public should be included within citizen-supported-science-projects.

Improvement of a Real-Time Reverse Transcription-Polymerase Chain Reaction Assay for the Sensitive Detection of the F Gene of Avian Orthoavulavirus-1 (AOAV-1).

Avian orthoavulavirus-1 (AOAV-1) is the causative agent of Newcastle disease in poultry. This highly infectious disease causes large economic losses annually and worldwide. AOAV-1 does not only infect poultry, but it has a very broad host range and has been detected in over 230 bird species to date. A distinct group of viral strains within AOAV-1 are pigeon-adapted strains, also named pigeon paramyxovirus-1 (PPMV-1). AOAV-1 is transmitted through the feces of infected birds and secretions from the nasal and oral cavities and eyes. It is worth mentioning that wild birds can transmit the virus to captive birds, especially feral pigeons to poultry. Therefore, early and sensitive detection of this virus-including the monitoring of pigeons-is of utmost importance. A variety of molecular methods for the detection of AOAV-1 already exist, but the detection of the F gene cleavage site of currently circulating PPMV-1 strains has not proven to be particularly sensitive or suitable. As presented here, by modifying the primers and probe of an already established real-time reverse-transcription PCR, the sensitivity could be increased, allowing for a more reliable detection of the AOAV-1 F gene cleavage site. Furthermore, it becomes clear how important it is to constantly monitor and, if necessary, adapt existing diagnostic procedures.

A Case Study of Zoonotic Chlamydia abortus Infection: Diagnostic Challenges From Clinical and Microbiological Perspectives.

Chlamydia abortus is the most common causative agent of abortion in small ruminants, but it is poorly recognized as a human pathogen. In most published case studies, diagnosis remained difficult and often resulted in delayed initiation of therapy. In this case study of severe C abortus infection in a pregnant farmer from Switzerland, we highlight the clinical and microbiological diagnostic challenges and provide evidence of a zoonotic epidemiological link.

Screening of Healthy Feral Pigeons (Columba livia domestica) in the City of Zurich Reveals Continuous Circulation of Pigeon Paramyxovirus-1 and a Serious Threat of Transmission to Domestic Poultry.

Pigeon paramyxovirus-1 (PPMV-1) is predominantly isolated from pigeons or doves and forms a separate group of viral strains within Avian Orthoavulavirus-1, the causative agent of Newcastle disease in poultry. Since the introduction of PPMV-1 into Europe in 1981, these strains have rapidly spread all over Europe, and are nowadays considered to be enzootic in feral and hobby pigeons (Columba livia domestica). Infections with PPMV-1 can range from asymptomatic to fatal. To assess whether PPMV-1 continuously circulates in healthy feral pigeons, 396 tissue samples of pigeons from the city of Zurich were tested by reverse transcriptase real-time PCR over the period of one year. PPMV-1-RNA was detected in 41 feral pigeons (10.35%), determined as the dominant European genotype VI.2.1.1.2.2. In 38 of the 41 pigeons where organ samples tested positive, PPMV-1-RNA was also detected in either choana or cloaca swabs. There were no significant differences in positivity rates between seasons, age, and sex. The current study shows that feral pigeons without clinical signs of disease can harbour and most likely excrete PPMV-1. Spill-over into free-range holdings of chickens are therefore possible, as observed in a recent outbreak of Newcastle disease in laying hens due to PPMV-1 genotype VI.2.1.1.2.2. in the canton of Zurich in January 2022.

Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris.

BACKGROUND: Species of Plasmodium (Haemosporida, Plasmodiidae) are remarkably diverse haemoparasites. Information on genetic diversity of avian malaria pathogens has been accumulating rapidly, however exo-erythrocytic development of these organisms remains insufficiently addressed. This is unfortunate because, contrary to Plasmodium species parasitizing mammals, the avian malaria parasites undergo several cycles of exo-erythrocytic development, often resulting in damage of various organs. Insufficient knowledge on the exo-erythrocytic development in most described Plasmodium species precludes the understanding of mechanisms of virulence during avian malaria. This study extends information on the exo-erythrocytic development of bird malaria parasites. METHODS: A roadkill fieldfare (Turdus pilaris) was sampled in Switzerland and examined using pathologic, cytologic, histologic, molecular and microbiologic methods. Avian malaria was diagnosed, and erythrocytic and exo-erythrocytic stages of the parasite were identified using morphologic characteristics and barcode DNA sequences of the cytochrome b gene. The species-specific characteristics were described, illustrated, and pathologic changes were reported. RESULTS: An infection with Plasmodium matutinum lineage pLINN1 was detected. Parasitaemia was relatively low (0.3%), with all erythrocytic stages (trophozoites, meronts and gametocytes) present in blood films. Most growing erythrocytic meronts were markedly vacuolated, which is a species-specific feature of this parasite's development. Phanerozoites at different stages of maturation were seen in leukocytes, macrophages, and capillary endothelial cells in most organs examined; they were particularly numerous in the brain. Like the erythrocytic meronts, growing phanerozoites were markedly vacuolated. Conspicuous exo-erythrocytic development and maturation in leucocytes suggests that this fieldfare was not adapted to the infection and the parasite was capable to escape from cellular immunity. CONCLUSIONS: This is the first report of exo-erythrocytic development of the malaria parasite lineage pLINN1 during single infection and the first report of this lineage in the fieldfare. The findings of multiple phanerozoites in brain, skeletal muscle, and eye tissue in combination with signs of vascular blockage and thrombus formation strongly suggest an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slowly moving car. Further studies on exo-erythrocytic stages of haemosporidian parasites are pivotal to understand the true level of populational damage of avian malaria in wild birds.

A new duplex real-time PCR for simultaneous detection and differentiation of Tetratrichomonas gallinarum and Trichomonas gallinae.

Tetratrichomonas gallinarum and Trichomonas gallinae are pathogenic avian parasites that infect a wide range of bird species. The pathologic potential of T. gallinarum is controversial, whereas T. gallinae causes disease in many avian species. Infections are often asymptomatic in doves and pigeons; thus, columbids are presumed to represent the natural hosts for trichomonads. The detection of T. gallinarum and T. gallinae is based on direct microscopic observation or a conventional PCR assay. Microscopy is not very sensitive, and identification of the trichomonads at the genus or species level is not possible. Conventional PCR assays have been developed primarily for phylogenetic studies, which detect a wide range of Trichomonas spp. but do not allow their differentiation. We developed a duplex real-time PCR (rtPCR) assay for the simultaneous detection and differentiation of T. gallinarum and T. gallinae. We found that the rtPCR assay detected 102 plasmid DNA copies of T. gallinarum and as few as 101 plasmid DNA copies of T. gallinae.

Survey of Salmonellae occurrence in meat-producing rabbitries in Switzerland.

BACKGROUND: An outbreak of salmonellosis due to Salmonella Typhimurium was detected coincidentally in a Swiss meat rabbitry, given that surveillance of Salmonella in rabbits is not mandatory in Switzerland. METHODS: To assess the extent of potentially subclinical Salmonella carriage in meat rabbits, faecal pool samples of 50 farms (90% of Swiss commercial rabbitries) with ground covering litter and group housing were bacteriologically tested. Additionally, 236 rabbits showing clinical signs compatible with intestinal diseases, such as salmonellosis, were examined postmortem and analysed bacteriologically. Salmonella isolates were serotyped and analysed by whole genome sequencing (WGS). RESULTS: Salmonella Typhimurium was detected in three commercial farms (6.0% of all tested farms). The affected farms were directly linked to the animal trade and Salmonella isolates were shown to be identical by WGS. CONCLUSION: There is no increased hazard for Salmonella carriage in the animal welfare-friendly Swiss husbandry systems in general, despite risk factors such as ground covering litter.

Zoonotic Chlamydiae as rare causes of severe pneumonia.

Zoonotic species of the Chlamydiaceae family should be considered as rare pathogenic agents of severe atypical pneumonia. A fatal case of a severe pneumonia due to Chlamydia psittaci was traced back to pet birds, and pneumonia in a pregnant woman was attributed to abortions in a sheep and goat flock, being the source of Chlamydia abortus. The two SARS­CoV­2-negative pneumonia cases presented here were investigated in an inter-disciplinary approach involving physicians and veterinarians. State-of-art molecular methods allowed the identification and genotyping of zoonotic Chlamydiae.

Falcons From the United Arab Emirates Infected With Chlamydia psittaci/C abortus Intermediates Specified as Chlamydia buteonis by Polymerase Chain Reaction.

Chlamydiaceae are obligate intracellular bacteria with a broad host range. Several studies have found chlamydial species that are genetically intermediate between Chlamydia psittaci and Chlamydia abortus in various avian species. One of these intermediate Chlamydia species, found in a red-shouldered hawk (Buteo lineatus), was recently classified as a new species Chlamydia buteonis. This newly described Chlamydia species has, so far, only been reported in hawks exhibiting clinical signs of conjunctivitis, dyspnea, and diarrhea. In the present study, fecal samples of 5 gyrfalcons (Falco rusticolus), 3 gyr/peregrine falcon hybrids (Falco rusticolus × Falco peregrinus), and 15 falcons of unknown species presented to falcon clinics on the Arabian Peninsula were shipped to the Vetsuisse Faculty, University of Zurich (Zurich, Switzerland), for examination for the presence of Chlamydiaceae. A step-wise diagnostic approach was performed to identify the chlamydial species involved. Chlamydiaceae were detected in 21/23 falcons by a family-specific real-time quantitative PCR (qPCR). Further identification with a 23S ribosomal RNA-based microarray assay and 16S conventional PCR and sequencing yielded inconclusive results, indicating the presence of an intermediate Chlamydia species. Because none of the falcons tested positive for Chlamydia psittaci by specific qPCR, all 23 samples were subjected to a Chlamydia buteonis-specific qPCR, which was positive in 16/23 samples. Detailed information regarding clinical history was available for 8 falcons admitted to a falcon clinic in Dubai, United Arab Emirates. Six of those birds that were presented to the clinic because of loss of performance and poor general condition, including vomiting and diarrhea, were positive for C buteonis. In 2 birds without clinical disease signs admitted for a routine health examination, 1 was positive for C buteonis, and 1 was negative. It is yet unknown whether Chlamydia buteonis causes disease in birds, but the findings in this study indicate that Chlamydia buteonis may be an infectious pathogen in falcon species.

A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses.

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

Low occurrence of Salmonella spp. in wild birds from a Swiss rehabilitation centre.

BACKGROUND: Salmonella are bacteria of the family Enterobacteriaceae with a wide host range. Infection in birds causes subclinical disease to mass mortality events. Wild birds may act as healthy carriers posing a hazard to livestock and humans. The present study investigated the occurrence of Salmonella in wild birds admitted to a rehabilitation centre in order to assess the exposure of the staff to this zoonotic pathogen. METHODS: Faecal swabs of 552 avian patients (68 species) were collected over the course of 12 months. Each sample was propagated in enrichment broth and subsequently incubated on a RAPID'Salmonella plate. Salmonella isolates were serotyped, and antimicrobial susceptibility testing was performed. RESULTS: Six Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and 1 S. Schleissheim were detected; all were pansusceptible to the antibiotics tested. CONCLUSION: Despite the low positive rate in the tested population, the authors recommend applying protective equipment and hygiene measures when handling wild birds.

Feedborne Salmonella enterica Serovar Jerusalem Outbreak in Different Organic Poultry Flocks in Switzerland and Italy Linked to Soya Expeller.

Poultry feed is a leading source of Salmonella infection in poultry. In Switzerland, heat-treated feed is used to reduce Salmonella incursions into flocks in conventional poultry production. By contrast, organic feed is only treated with organic acids. In 2019, the Swiss National Reference Center for Enteropathogenic Bacteria identified the rare serovar S. Jerusalem from samples of organic soya feed. Further, in July 2020, the European Union's Rapid Alert System for Food and Feed published a notification of the detection of S. Jerusalem in soya expeller from Italy. During 2020, seven S. Jerusalem isolates from seven different poultry productions distributed over six cantons in Switzerland were reported, providing further evidence of a possible outbreak. Using whole-genome sequencing (WGS), S. Jerusalem isolates from feed and from animals in Switzerland were further characterized and compared to S. Jerusalem from organic poultry farm environments in Italy. WGS results showed that feed isolates and isolates from Swiss and Italian poultry flocks belonged to the sequence type (ST)1028, grouped in a very tight cluster, and were closely related. This outbreak highlights the risk of spreading Salmonella by feed and emphasizes the need for a heat-treatment process for feed, also in organic poultry production.

Alternatives to robenidine to control gastrointestinal disorders of weaner rabbits in the field.

Gastrointestinal disorders due to Eimeria sp. and E. coli overgrowth cause high mortality in weaner rabbits and the interest in alternatives to coccidiostats is high. This study aimed to investigate the superiority of natural feed additives towards robenidine preserving gastrointestinal health in the field. Rabbits were divided into four groups, Control Group (CG) exclusively supplemented with robenidine, Sainfoin Group (SG) was supplemented with a combination of robenidine and sainfoin, and two additional groups were respectively supplemented with Herb-All COCC-X (garlic; conessi tree) (HG: Herbal Group) and by a combination of Herb-All COCC-X and Klinofeed (clinoptilolite) (MG: Mineral Group). Eimeria sp. (98,40%) and E. coli overgrowth (73.60%) could be confirmed as the main causes for losses. High mortality rates (SG: 30.00% - MG: 47.50%), also in the groups receiving robenidine (SG: 30.00%; CG: 45.00%), reinforced the importance of alternatives in the field. The natural additives of groups SG, HG and MG did not have a significant influence on the weight gains and the oocyst counts in the jejunum/ileum and caecum of slaughter rabbits at the end of the trial, compared to group CG. Significantly higher oocyst shedding in SG (p = 1.4E-03) and HG (p = 1.4E-05) during the trial may be explained by a higher surviving rate of diseased rabbits in those groups, fostered by beneficial effects of the additives, which should be investigated further.

Erysipelothrix rhusiopathiae infection by geese to human transmission.

Erysipelothrix rhusiopathiae transmission to human is often occupation-related, but in most cases, a detailed case history is missing. This case report is based on an interdisciplinary approach and includes a thorough medical record. A 58-year-old laboratory technician working on geese necropsy cut open her glove at a rib fragment of a goose and subsequently noticed a slowly progressive, reddish skin alteration in the particular region of the hand. Bacteriological investigations on the geese revealed septicaemia due to E. rhusiopathiae and therefore substantiated the diagnosis of the patient. The infectious agent could not be cultured from the patient; however, antibiotic susceptibility testing was performed using the goose isolate. An entire follow-up until full recovery of the patient was conducted. Zoonotic infections possibly have a significant impact on certain occupations. This case report analyses a rare but important zoonotic infection to create awareness of this in physicians caring for human patients.

Detection of Chlamydiaceae in Swiss wild birds sampled at a bird rehabilitation centre.

BACKGROUND: Annually, 800-1500 wild birds are admitted to the rehabilitation centre of the Swiss Ornithological Institute, Sempach, Lucerne, Switzerland. The workers of the centre come in close contact with the avian patients and might therefore be exposed to zoonotic agents shed by these birds, such as Chlamydia psittaci. METHODS: In the present study, 91 choanal, 91 cloacal and 267 faecal swabs from 339 wild birds of 42 species were investigated using a stepwise diagnostic approach. RESULTS: Chlamydiaceae were detected in 0.9 per cent (0.3-2.6 per cent) of birds (n=3), all of them members of the Columbidae family. The Chlamydiaceae species of two of these birds (one Eurasian collared dove, one fancy pigeon) were identified as C psittaci types B and E by PCR and outer membrane protein A genotyping. CONCLUSION: The findings of the current study suggest that zoonotic transmission of Chlamydiaceae is very unlikely for songbird and waterfowl species tested herein, while pigeons might pose a risk to workers at rehabilitation centres.

Occurrence of Chlamydiaceae in Raptors and Crows in Switzerland.

Bacteria of the family Chlamydiaceae are globally disseminated and able to infect many bird species. So far, 11 species of Chlamydia have been detected in wild birds, and several studies found chlamydial strains classified as genetically intermediate between Chlamydia (C.) psittaci and C.abortus. Recently, a group of these intermediate strains was shown to form a separate species, i.e., C.buteonis. In the present study, 1128 samples from 341 raptors of 16 bird species and 253 corvids representing six species were examined using a stepwise diagnostic approach. Chlamydiaceae DNA was detected in 23.7% of the corvids and 5.9% of the raptors. In corvids, the most frequently detected Chlamydia species was C.psittaci of outer membrane protein A (ompA) genotype 1V, which is known to have a host preference for corvids. The most frequently detected ompA genotype in raptors was M56. Furthermore, one of the raptors harbored C.psittaci 1V, and two others carried genotype A. C.buteonis was not detected in the bird population investigated, so it remains unknown whether this species occurs in Switzerland. The infection rate of Chlamydiaceae in corvids was high compared to rates reported in other wild bird species, but neither Chlamydiaceae-positive corvids nor raptors showed overt signs of disease. Since the Chlamydiaceae of both, raptors and crows were identified as C.psittaci and all C.psittaci genotypes are considered to be zoonotic, it can be suggested that raptors and crows pose a potential hazard to the health of their handlers.

Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Chlamydiaceae in wild, feral and domestic pigeons in Switzerland and insight into population dynamics by Chlamydia psittaci multilocus sequence typing.

Feral pigeons, common wood pigeons and Eurasian collared doves are the most common representatives of the Columbidae family in Switzerland and are mostly present in highly populated, urban areas. Pigeons may carry various members of the obligate intracellular Chlamydiaceae family, particularly Chlamydia (C.) psittaci, a known zoonotic agent, and C. avium. The objective of the study was to identify the infection rates of common free-roaming pigeons for different Chlamydia species with the overall aim to assess the risk pigeons pose to public health. In this study, 431 pigeons (323 feral pigeons, 34 domestic pigeons, 39 Eurasian collared doves, 35 common wood pigeons) from several geographic locations in Switzerland were investigated for the presence of Chlamydiaceae. Samples consisted of pooled choanal-cloacal swabs (n = 174), liver samples (n = 52), and paired swab and liver samples from 205 pigeons (n = 410). All 636 samples were screened using a Chlamydiaceae family-specific 23S rRNA real-time PCR (qPCR). Subsequent species identification was performed by DNA-microarray assay, sequencing of a 16S rRNA gene fragment and a C. psittaci specific qPCR. In total, 73 of the 431 pigeons tested positive for Chlamydiaceae, of which 68 were positive for C. psittaci, four were C. avium-positive and one pigeon was co-infected with C. avium and C. psittaci. The highest infection rates were detected in feral (64/323) and domestic pigeons (5/34). Common wood pigeons (2/35) and Eurasian collared doves (2/39) revealed lower infection rates. Additionally, multilocus sequence typing of twelve selected C. psittaci-positive samples revealed closely related sequence types (ST) between and within different Swiss cities. Furthermore, liver and corresponding swab samples from the same bird were colonized by the same ST. Considering the high infection rates of C. psittaci in domestic and feral pigeons, close or frequent contact to these birds poses a human health risk.

Survey on Chlamydiaceae in cloacal swabs from Swiss turkeys demonstrates absence of Chlamydia psittaci and low occurrence of Chlamydia gallinacean.

In Switzerland, domestic turkey meat is a niche product. Turkeys are fattened on mixed family-based farms scattered across the country, with most providing access to an uncovered outdoor pasture for the birds. Swiss fattening turkeys may therefore get infected with Chlamydiaceae via wild birds or their faeces, potentially shedding these bacteria at a later stage. The aim of the present study was to acquire baseline data about the shedding of Chlamydiaceae in clinically unremarkable Swiss fattening turkeys at slaughter, potentially exposing slaughterhouse workers to infection. In this large-scale study, 1008 cloacal swabs of Swiss turkeys out of 53 flocks from 28 different grow-out farms with uncovered outdoor pasture were collected over the course of 14 months and examined for the occurrence of Chlamydiaceae by a family-specific 23S-rRNA real-time PCR. Positive samples were further analyzed by Chlamydia psittaci (C. psittaci)-specific real-time PCR and the Arraymate DNA Microarray for species identification. All samples were negative for C. psittaci, but seven swabs out of one flock were tested positive for Chlamydia gallinacea (0.7%). Although turkeys with access to pasture may have contact with Chlamydiaceae-harbouring wild birds or their faeces, the infection rate in Swiss turkeys was shown to be low.

Virus-like particles in a new vaccination approach against infectious laryngotracheitis.

Gallid alphaherpesvirus 1 (syn. infectious laryngotracheitis virus; ILTV) is the causative agent of infectious laryngotracheitis, a respiratory disease of chickens causing substantial economic losses in the poultry industry every year. Currently, the most efficient way to achieve protection against infection is immunization with live-attenuated vaccines. However, this vaccination strategy entails the risk of generating new pathogenic viruses resulting from spontaneous mutations or from recombination with field strains. This work presents a new approach based on virus-like particles (VLPs) displaying ILTV glycoproteins B (gB) or G (gG) on their surface. The main focus of this pilot study was to determine the tolerability of VLPs delivered in ovo and intramuscularly (i.m.) into chickens and to investigate the nature of the immune response elicited. The study revealed that the new vaccines were well tolerated in hybrid layer chicks independent of the administration method (in ovo or i.m.). Upon in ovo injection, vaccination with VLP-gG led to an antibody response, while a cellular immune response in VLP-gB-immunized chickens was hardly detectable. Since the administration of VLPs had no visible side effects in vivo and was shown to elicit an antibody-based immune response, we anticipate that VLPs will become a valuable platform for the development of new safe vaccines for poultry.