Vaccination is routinely used in industrial poultry to control infectious diseases. Vaccines based on mRNA and self-amplifying RNA (saRNA) are approved for human use, but research on their application in poultry is limited. In this study the saRNA vaccine platform is evaluated in poultry. First, a luciferase-encoding saRNA (luc-saRNA) was tested as a model vaccine across different administration routes and doses in broilers. High luciferase expression, and anti-luciferase antibodies were observed after intramuscular (IM), subcutaneous (SC), and in ovo (IO) administration. After a second Luc-saRNA injection, seroconversion rates and antibody titers increased in the IM and SC group to almost 100%. Higher doses of Luc-saRNA increased luciferase production. However, they did not linearly increase antibody production, as all tested doses (0.20-5.0 µg) elicited an equipotent immune response. A vaccination experiment with saRNA encoding the hemagglutinin head-domain (HA-HD) of H5N1 avian influenza showed hemagglutinin inhibition (HI) titers that are indicative for protection after a single injection and these titers remained above the protective threshold during 6 weeks without boosting. When boosted, the HI titers increased four-fold. This study confirms effective protein translation and immune response induction in chickens with IM or SC administered saRNA-LNPs, even at the lowest dose of 0.20 µg.

De huisvesting van industrieel gehouden pluimvee is streng gereglementeerd. De huisvesting van hobbykippen valt echter buiten de wetgeving. De Vlaamse overheid heeft wel een richtlijn opgesteld met betrekking tot het houden en huisvesten van hobbypluimvee. Deze richtlijn is online beschikbaar. In het voorliggende onderzoek werd door middel van een veldstudie nagegaan of de hobbyhouder kennis heeft van deze richtlijn en in welke mate ze effectief wordt toegepast. Hiervoor werd een survey opgesteld en vijftig houders van hobbypluimvee werden erin opgenomen. De resultaten tonen aan dat verscheidene richtlijnen opgesteld door de Vlaamse overheid onvoldoende worden nageleefd. De tekortkomingen kunnen leiden tot welzijns- en gezondheidsproblemen bij de kip. Uit de studie blijkt ook een gebrek aan kennis van de richtlijn bij de kippenhouders. Op basis van de bevindingen worden de noodzaak van sensibilisering en het promoten van de huisvestingsrichtlijn benadrukt alvorens kippen worden aangeschaft.

Thermophilic Campylobacter species are the most common cause of bacterium-mediated diarrheal disease in humans globally. Poultry is considered the most important reservoir of human campylobacteriosis, but so far, no effective countermeasures are in place to prevent the bacterium from colonizing broiler flocks. This study investigated maternal antibodies’ potential to offer protection against Campylobacter in broiler chicks via a field trial and an immunization trial. In the field trial, breeder flocks with high and low anti-Campylobacter antibody levels in the yolk were selected based on serological screening. Offspring were subsequently monitored for maternal antibodies and Campylobacter prevalence during early life. Although maternal antibodies declined rapidly in the serum of broilers, offspring from flocks with lower anti-Campylobacter antibody levels seemed to be more susceptible to colonization. In the immunization trial, breeders from a seropositive breeder flock were vaccinated with an experimental bacterin or subunit vaccine. Immunization increased antibody levels in the yolk and consequently in the offspring. Elevated maternal antibody levels were significantly associated with reduced Campylobacter susceptibility in broilers at 2 weeks old but not at 1 and 3 weeks old. Overall, the protective effect of maternal immunity should be cautiously considered in the context of Campylobacter control in broilers. Immunization of breeders may enhance resistance but is not a comprehensive solution.

This study explores upright versus inverted catching and crating of spent laying hens. Both catching methods were compared using a cost-benefit analysis that focused on animal welfare, ergonomic, and financial considerations. Data were collected on seven commercial farms (one floor system and six aviary systems) during depopulation of approximately 3,000 hens per method per flock. Parameters such as wing flapping frequency, catcher bird interaction, incidence of catching damage and hens dead on arrival (DOA) were measured and compared between catching methods. Ergonomic evaluations were performed via catcher surveys and expert assessment of video recordings. The wing flapping frequency was lower (3.1 ± 0.6 vs. 4.0 ± 0.5, P < 0.001) and handling was gentler (1.9 ± 0.5 vs. 4.4 ± 0.5, P < 0.001), both on a 7-point Likert scale, for upright versus inverted catching. However, more person-hours per 1000 hens were required for upright than inverted catching (8.2 ± 3.2 h vs. 4.8 ± 2.0 h, P = 0.011), with only wing bruises being significantly less common for upright than inverted catching (1.1 ± 0.6 % vs. 1.7 ± 0.7%, P = 0.04). Upright catching was 1.8 times more expensive than inverted catching; compensation for this cost would require a premium price of approximately €0.0005 extra per egg. Ergonomically, both catching methods were considered demanding, although catchers (n = 29) preferred inverted catching. In conclusion, this study showed animal welfare benefits of upright vs. inverted catching. Industry adoption of upright catching will depend on compensation of the additional labor costs, adjustments to labor conditions and shorter loading times.

The pre-transport phase induces stress, fear, and injury in poultry, but management choices greatly influence this. Pre-transport practices for spent hens and broilers in Flanders (Belgium) were studied. Poultry farmers (31 of 156 layers and 48 of 203 broiler farmers completed the survey) were surveyed on the selection of unfit chickens, catching and crating, and farmer opinion. A minority of farmers made a specific selection of chickens unfit for transport prior to catching (layers 25%: 5.1 ± 5.9 h, broilers 39%: 6.8 ± 7.0 h). More layer (69%) than broiler farmers (19%) withdrew feed too early (EU regulations stipulate max. 12 h before expected slaughter time). Layer farmers withdrew water earlier than broiler farmers (47.9 ± 51.1 min vs. 20.6 ± 23.3 min). More broiler than layer farmers believed that the container type affects the birds’ welfare (48% vs. 27%; p < 0.05). On broiler farms, mechanical catching was preferred for catchers’ well-being, while upright catching was considered better for animal welfare than catching more than three chickens by one/two legs, wings, or mechanically. Poultry farmers should be sensitized about the need for additional selection before catching, including clear guidelines about judging which birds are fit for transport.

Pre-transport management activities can cause stress, injuries, and mortality in poultry. This study aimed to gather information about the current pre-transport practices (selection of unfit chickens, catching preparations, catching, and crating) for spent hens and broilers by questioning Flemish poultry farmers. The results showed that catching preparations such as an extra selection of chickens unfit for transport was performed by a minority of the poultry farmers, and that layer farmers were less in line with the EU-legislation for water and feed withdrawal than broiler farmers. All birds were caught inverted except for one broiler farmer who used mechanical catching (not yet common in Flanders). Additionally, mechanical catching may involve extra costs, increased biosecurity risks, and specific recommendations for the stable (height and width). The broiler farmers preferred mechanical catching for broiler catchers' well-being, while upright catching was considered better for animal welfare than catching more than three chickens by one/two legs, mechanically, or by wings. Awareness of the need to perform an extra selection by the poultry farmers before catching is required. Preparations like closing areas under the aviary system and removing litter (layers) can further streamline the process and reduce animal suffering.

Catching, carrying, and loading of broilers before transport to the slaughterhouse causes stress. In this study three catching methods (two manual (inverted, upright) and one mechanical) were compared using a cost-benefit analysis of animal welfare, ergonomics and economic analysis. Depopulation of approximately 5,000 broilers per catching method per flock (upright vs. inverted vs. mechanical: n=3; upright vs. inverted: n=9; inverted vs. mechanical: n=3 flocks) was analyzed on 15 commercial farms. Economic considerations (person-hours per 1,000 chickens), ergonomics (catcher survey, ergonomic assessment of simulated catching), and animal welfare on-farm (wing flapping frequency, catcher-bird interaction) and at the slaughterhouse (catch damage and DOA prevalence) were considered. Wing flapping frequency was lower (2.0 ± 0.1 vs. 5.4 ± 0.1, P < 0.001), and catcher-bird interaction was better (3.7 ± 0.2 vs. 4.4 ± 0.2, P < 0.01) for upright catching compared to inverted catching based on a 7-point Likert scale. Prevalence of catch damage was lower for upright versus mechanical catching (15.5 ± 1.3% vs. 17.7 ± 1.4%, P = 0.046). More person-hours per 1,000 broilers were required for upright versus inverted (1.6 ± 0.1 h vs. 1.0 ± 0.1 h) and mechanical catching (0.6 ± 0.3 h) (P < 0.001). Upright catching was 1.5 and 1.2 times more expensive than inverted and mechanical catching based on 20,000 broilers. Compared to inverted catching, fair compensation would increase by €0.012 (upright) and €0.006 (mechanical) per kg of live weight. An ergonomics expert rated manual catching as very demanding, but catchers (n = 16) disliked upright catching (more labor-intensive). This study revealed animal welfare benefits of upright versus inverted (less wing flapping, better catcher-bird interaction) and mechanical catching (less catch damage), whereas mechanical catching provided the best labor conditions. Widespread application of upright catching would require testing of entire flocks and collaboration with the poultry sector to determine fair compensation, improve labor conditions and identify strategies to minimize catch and load duration.

Campylobacter is the main cause of human gastroenteritis worldwide, with 50 to 80% of the cases related to consumption of poultry products. Maternal antibodies (MAB) from commercial breeder flocks may protect their progeny against infection during the first few weeks of life. We here studied the prevalence of Campylobacter antibody titers in broiler breeder flocks and to which extent immunization of broiler breeders increases maternal anti-Campylobacter titers in their progeny and protects the offspring against Campylobacter colonization. Two vaccines were used: a bacterin mix of 13 Campylobacter strains and a subunit vaccine comprising 6 immunodominant Campylobacter antigens. All sampled on-farm breeder flocks were positive for anti-Campylobacter antibodies, yet in some breeder flocks only very low titers were detected. Vaccination of SPF broiler breeder flocks with both subunit and bacterin vaccines resulted in a prolonged presence of anti-Campylobacter antibodies in the serum and intestinal mucus of chicks. These bacterin- or subunit vaccine-induced MAB conferred protection against Campylobacter colonization in chicks until 7 and 21 d of age, respectively, but only at a low challenge dose (102.5 cfu). The concentration of MAB in the mucus is probably too low to sufficiently capture Campylobacter when higher challenge doses are used. In conclusion, vaccinating broiler breeders protects their offspring against Campylobacter colonization under low pathogen exposure conditions.

In order to minimize animal loss and economical loss, industrial poultry is heavily vaccinated against infectious agents. mRNA vaccination is an effective vaccination platform, yet little to no comprehensive, comparative studies in avians can be found. Nevertheless, poultry mRNA vaccination could prove to be very interesting due to the relatively low production cost, especially true when using self-amplifying mRNA (saRNA), and their extreme adaptability to new pathogens. The latter could be particularly useful when new pathogens join the stage or new variants arise. As a first step toward the investigation of saRNA vaccines in poultry, this study evaluates a luciferase-encoding saRNA in avian tracheal explants, conjunctival explants, primary chicken cecal cells and 18-day embryonated eggs. Naked saRNA in combination with RNase inhibitor and 2 different lipid-based formulations, that is, ionizable lipid nanoparticles (LNPs) and Lipofectamine Messenger Max, were evaluated. The saRNA-LNP formulation led to the highest bioluminescent signal in the tracheal explants, conjunctival explants and cecal cell cultures. A dose-response experiment with these saRNA-LNPs (33-900 ng/well) in these avian organoids and cells showed a nonlinear dose-response relationship. After in ovo administration, the highest dose of the saRNA-LNPs (5 µg) resulted in a visual expression as a weak bioluminescence signal could be seen. The other delivery approaches did not lead to a visual saRNA expression in the embryos. In conclusion, effective entry of saRNA encapsulated in LNPs followed by successful saRNA translation in poultry was established. Hence, mRNA vaccination in poultry could be possible, but further in vivo testing is needed.

Behandeling van chronische eileg bij een grijze roodstaartpapegaai

There is a trend towards extended periods of lay in the laying hen industry. Extended cycles without a moulting stage gives the opportunity to obtain more eggs from a single hen. However, appropriate management and care for older laying hens is needed. In this trial we assessed the prevalence of conditions in old laying hens with a focus on neoplastic diseases. In total 150 ISA Brown and 150 Dekalb white laying hens were selected at 86 weeks of age. Of each hen line, 75 hens were necropsied at 86 weeks of age; the other half were monitored for 44 weeks after which they were necropsied. At week 86, 15.3% of the hens suffered from a neoplasm, ISA Brown being the most affected. During the follow up period, 50 birds died because of a natural cause of which 20 hens showed signs of a neoplasms. At the end of the follow up period, 43% of the hens were affected by a neoplasm. Adenocarcinoma was the most prevalent neoplasm and equally distributed among both hen lines. Leiomyomas were most frequently observed in ISA brown hens. Among causes of death, 19.05% of ISA brown and 20.69% of Dekalb White was attributed to a neoplasm. Furthermore, link with ovarian activity and other pathologies were made with significant correlations between adenocarcinomas and inactive ovaries. In conclusion, this study shows that the prevalence of adenocarcinoma and leiomyoma is a factor to be considered in longer laying cycles with 1/5th of the mortality caused by these processes.RESEARCH HIGHLIGHTSAt 86 weeks of age, the prevalence of neoplasms was 15.3%, mainly in brown hens.At 130 weeks of age, 43% of the hens were affected by a neoplasm.Adenocarcinoma was the most prevalent neoplasm equally distributed among hen lines.Leiomyoma was the second most prevalent neoplasm, mainly found in brown hens.

Marek’s disease (MD) has a high economic impact in poultry production worldwide. MD consists of several pathologic syndromes of which the most commonly encountered are lymphoproliferative syndromes such as acute MD. Sporadically, MD leads to central nervous system syndromes characterized by non-neoplastic brain pathology. In this case report, an onset of neurological signs on two different broiler farms, housing ‘slow-growing’ Hubbard JA 757 broilers, is described. On the first farm, clinical signs were observed from 42 days of age onwards and on the second farm, respectively from 42 (stable 1) and 30 days (stable 2) of age onwards. The neurological symptoms consisted of acute central nervous system signs, which started with flaccid neck paralysis. Histopathological examination revealed multifocal perivascular cuffing of CD3 positive mononuclear cells in the brain tissues. These findings pointed towards the development of transient paralysis. qPCR analysis confirmed the diagnosis. In broiler flocks, which only have a limited life span, vaccination is not common practice. In flocks of slow-growing breeds, outbreaks of MD might become more important again. Therefore, vaccination of slow-growing broilers against MD seems recommended.

Sinds enkele jaren is er een toename van het aantal uitbraken van aviaire influenza in industriële pluimveehouderijen en hobbyhouderijen in Europa. Ook duren de epizoötieën langer. De voorbije jaren bleef het virus ook tijdens de zomer in de Belgische wildevogelpopulatie circuleren. Dierenartsen met vogels in hun patiëntenbestand worden daarom aangespoord om gedurende het hele jaar waakzaam te blijven voor deze aangifteplichtige ziekte. In dit artikel wordt een overzicht gegeven van de huidige epidemiologische situatie, de mogelijke ziektebeelden, de te ondernemen acties in het geval van verdenking van een uitbraak en de bestrijdingsmaatregelen van overheidswege.

vIn recent years, there has been an increase in the number of outbreaks of avian influenza in indus-trial poultry flocks and backyard poultry in Europe. Also, the period of seasonal outbreaks seems to expand. In recent years, the virus has been circulating in the wild-bird population in Belgium, during summer time. Veterinarians are therefore urged to be watchful for this notifiable disease. In this review, an overview is given of the current epidemiological situation, the possible disease features, actions to be taken in case of suspicion of an outbreak and governmental control measures.

Fluoroquinolone agents are considered critical for human medicine by the World Health Organization (WHO). However, they are often used for the treatment of avian colibacillosis in poultry production, creating considerable concern regarding the potential spread of fluoroquinolone resistance genes from commensals to pathogens. Therefore, there is a need to understand the impact of fluoroquinolone application on the reservoir of ARGs in poultry gut and devise means to circumvent potential resistome expansion. Building upon a recent dose optimization effort, we used shotgun metagenomics to investigate the time-course change in the cecal microbiome and resistome of broiler chickens receiving an optimized dosage [12.5 mg/kg body weight (bw)/day], with or without synbiotic supplementation (PoultryStar®, BIOMIN GmbH), and a high dosage of enrofloxacin (50 mg/kg bw/day). Compared to the high dose treatment, the low (optimized) dose of enrofloxacin caused the most significant perturbations in the cecal microbiota and resistome of the broiler chickens, demonstrated by a lower cecal microbiota diversity while substantially increasing the antibiotic resistance genes (ARGs) resistome diversity. Withdrawal of antibiotics resulted in a pronounced reduction in ARG diversity. Chickens receiving the synbiotic treatment had the lowest diversity and number of enriched ARGs, suggesting an alleviating impact on the burden of the gut resistome. Some Proteobacteria were significantly increased in the cecal metagenome of chickens receiving enrofloxacin and showed a positive association with increased ARG burden. Differential abundance (DA) analysis revealed a significant increase in the abundance of ARGs encoding resistance to macrolides-lincosamides-streptogramins (MLS), aminoglycosides, and tetracyclines over the period of enrofloxacin application, with the optimized dosage application resulting in a twofold higher number of affected ARG compared to high dosage application. Our results provide novel insights into the dose-dependent effects of clinically important enrofloxacin application in shaping the broiler gut resistome, which was mitigated by a synbiotic application. The contribution to ameliorating the adverse effects of antimicrobial agents, that is, lowering the spread of antimicrobial resistance genes, on the poultry and potentially other livestock gastrointestinal microbiomes and resistomes merits further study.

The humane killing of individual broiler chickens on-farm requires a minimum of suffering. In this regard, rapid and irreversible loss of consciousness are important determinants. This can be verified by cerebral and spinal reflexes. Also, on-farm feasibility determines whether producers will apply the method. The aim of the study was to compare the effectiveness and animal welfare impact of two different methods for killing individual broilers of varying ages (2, 4, and 6 weeks): manual cervical dislocation (CD) and captive bolt (CB). The evaluation of CD and CB was based on effectiveness and on time to onset (convulsions) or cessation (pain response, pupillary light reflex, convulsions, heartbeat) of non-invasive indicators. In addition, a pilot study was conducted on-farm to assess the feasibility of two alternative methods, CB and nitrogen gasification (N2), and to survey farmers’ opinions on them. The onset of convulsions was almost immediate for both methods in the first study. No differences between CD and CB were observed for the cessation of pain response for chickens at age of 2 weeks (5.0 and 7.5 s, respectively) and 6 weeks (14.0 and 14.1 s, respectively). However, at 4 weeks a longer pain response was measured after CD (11.3 s) than after CB (4.7 s). For the three age categories, the pupillary light reflex disappeared later after CD (54.9 - 80.7 s) compared to CB (8.3 - 13.7 s). The same was observed for cessation of convulsions in 2- and 6-week-old chickens (185.3 and 172.0 s for CD and 79.0 and 82.9 s for CB). This suggests that brain death occurred faster after CB compared to CD. No difference between the methods was found for the cessation of the heartbeat. After the pilot study, the producers preferred N2 over CB in terms of animal-friendliness, time-efficiency, ease of use, and effectiveness. However, both methods were found rather expensive and required some experience. CB and N2 are good killing alternatives to CD due to rapid and irreversible insensibility. However, more information and support for chicken producers will be needed for these to become routine killing methods.

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry worldwide [1]. In recent years, there has been a renewed interest in the therapeutic use of bacteriophages (phages) as alternative or supportive to existing antibiotics [2]. However, despite promising in vitro data successful in vivo therapeutic activity is not always guaranteed [3]. Therefore, a better understanding of the phage-bacterium interactions and the underlying mechanisms is essential for the successful development of a phage therapy. Here, we investigated the interactions of the APEC O1:H7 ST95 strain, AM621, and the well-characterized lytic Myoviridae phage, Escherichia phage vB_EcoM-P10, at different multiplicity of infection (MOI). These interactions led to 109 spontaneous in vitro generated phage-resistant mutants of the APEC strain. These mutants were obtained using the agar plate or the secondary culture method [4]. Resistance mechanisms that occurred in the bacterial mutants were investigated through analysis of whole genome sequencing (WGS) data. The analysis revealed 41 mutants with single-nucleotide polymorphisms (SNPs) in their core genome. In 32 of the mutants a single SNP was detected while two SNPs were identified in nine mutants. In total, 34 unique SNPs were detected. The SNPs resulted in nonsense (n=5), missense (n=19), or synonymous mutation (n=6). The remaining four SNPs were in non-coding regions. Affected genes included outer membrane protein (omp) A, lipopolysaccharide (LPS)-related genes, phosphate acetyltransferases,

Enrofloxacin is frequently administered via drinking water for the treatment of colibacillosis in broiler chickens. However, the EMA/CVMP has urged to re-evaluate historically approved doses, especially for antimicrobials administered via drinking water. In response, the objectives of this study were two-fold. First, to evaluate the pharmacokinetics (PK) of enrofloxacin following IV, PO and drinking water administration. Second, to predict the efficacy of a range of doses in the drinking water for the treatment of APEC infections. For the first objective, PK parameters were estimated by fitting a one-compartmental model with a zero-order IV infusion and an oral absorption lag function to the simultaneously modelled IV and PO data. After fixing these parameter values, a drinking behaviour pharmacokinetic (DBPK) model was developed for the description and prediction of drinking water PK profiles by adding three model improvements (different diurnal and nocturnal drinking rates, inter-animal variability in water consumption and taking account of dose non-proportionality). The subsequent simulations and probability of target attainment (PTA) analysis predicted that a dose of 12.5 mg/kg/24 h is efficacious in treating colibacillosis with an MIC up to 0.125 μg/mL (ECOFF), whereas the currently registered dose (10 mg/kg/24 h) reaches a PTA of 66% at ECOFF.

The therapeutic use of bacteriophages (phage therapy) represents a promising alternative to antibiotics to control bacterial pathogens. However, the understanding of the phage–bacterium interactions and population dynamics seems essential for successful phage therapy implementation. Here, we investigated the effect of three factors: phage species (18 lytic E. coli-infecting phages); bacterial strain (10 APEC strains); and multiplicity of infection (MOI) (MOI 10, 1, and 0.1) on the bacterial growth dynamics. All factors had a significant effect, but the phage appeared to be the most important. The results showed seven distinct growth patterns. The first pattern corresponded to the normal bacterial growth pattern in the absence of a phage. The second pattern was complete bacterial killing. The remaining patterns were in-between, characterised by delayed growth and/or variable killing of the bacterial cells. In conclusion, this study demonstrates that the phage–host dynamics is an important factor in the capacity of a phage to eliminate bacteria. The classified patterns show that this is an essential factor to consider when developing a phage therapy. This methodology can be used to rapidly screen for novel phage candidates for phage therapy. Accordingly, the most promising candidates were phages found in Group 2, characterised by growth dynamics with high bacterial killing.