Extensive genetic and biological characterization of infectious bronchitis virus strain D2860 of genotype GVIII.

Infectious bronchitis virus (IBV) strains of genotype GVIII have been emerging in Europe in the last decade, but no biological characterization has been reported so far. This paper reports the extensive genetic and biological characterization of IBV strain D2860 of genotype GVIII which was isolated from a Dutch layer flock that showed a drop in egg production. Whole genome sequencing showed that it has a high similarity (95%) to CK/DE/IB80/2016 (commonly known as IB80). Cross-neutralization tests with antigens and serotype-specific antisera of a panel of different non-GVIII genotypes consistently gave less than 2% antigenic cross-relationship with D2860. Five experiments using specified pathogen-free chickens of 0, 4, 29 and 63 weeks of age showed that D2860 was not able to cause clinical signs, drop in egg production, false layers or renal pathology. There was also a distinct lack of ciliostasis at both 5 and 8 days post-inoculation at any age, despite proof of infection by immunohistochemical (IHC) staining, RT-PCR and serology. IHC showed immunostaining between 5 and 8 days post inoculation in epithelial cells of sinuses and conchae, while only a few birds displayed immunostaining in the trachea. In vitro comparison of replication of D2860 and M41 in chicken embryo kidney cells at 37°C and at 41°C indicated that D2860 might have a degree of temperature sensitivity that might cause it to prefer the colder parts of the respiratory tract.

Genetic analysis of infectious bronchitis virus (IBV) in vaccinated poultry populations over a period of 10 years.

Infectious bronchitis virus (IBV) is an avian pathogen from the Coronavirus family causing major health issues in poultry flocks worldwide. Because of its negative impact on health, performance, and bird welfare, commercial poultry are routinely vaccinated by administering live attenuated virus. However, field strains are capable of rapid adaptation and may evade vaccine-induced immunity. We set out to describe dynamics within and between lineages and assess potential escape from vaccine-induced immunity. We investigated a large nucleotide sequence database of over 1700 partial sequences of the S1 spike protein gene collected from clinical samples of Dutch chickens submitted to the laboratory of Royal GD between 2011 and 2020. Relative frequencies of the two major lineages GI-13 (793B) and GI-19 (QX) did not change in the investigated period, but we found a succession of distinct GI-19 sublineages. Analysis of dN/dS ratio over all sequences demonstrated episodic diversifying selection acting on multiple sites, some of which overlap predicted N-glycosylation motifs. We assessed several measures that would indicate divergence from vaccine strains, both in the overall database and in the two major lineages. However, the frequency of vaccine-homologous lineages did not decrease, no increase in genetic variation with time was detected, and the sequences did not grow more divergent from vaccine sequences in the examined time window. Concluding, our results show sublineage turnover within the GI-19 lineage and we demonstrate episodic diversifying selection acting on the partial sequence, but we cannot confirm nor rule out escape from vaccine-induced immunity.RESEARCH HIGHLIGHTSSuccession of GI-19 IBV variants in broiler populations.IBV lineages overrepresented in either broiler, or layer production chickens.Ongoing episodic selection at the IBV S1 spike protein gene sequence.Several positively selected codons coincident with N-glycosylation motifs.

The inactivated infectious bronchitis virus (IBV) vaccine used as booster in layer hens influences the breadth of protection against challenge with IBV variants.

To achieve long term protection of laying and breeding hens against aberrant egg production caused by infectious bronchitis virus (IBV), a vaccination programme incorporating both live-attenuated and inactivated IBV vaccines is required. High quality IBV vaccines of both types are widely available, but the number of IBV variants of global importance continues to increase and it is not possible to develop vaccines against each one of them. Therefore, it is desirable to perform studies under controlled conditions to determine which IBV vaccine(s) provide the best protection for laying hens against different IBV challenges. Previous vaccination and challenge studies have shown that it is possible to obtain relevant data in a small number of laying hens housed under conditions of strict isolation. The present work extends this finding by investigating the efficacy, against challenge with five IBV strains of global importance, of an IBV vaccination programme including two live-attenuated IBV vaccines (Massachusetts and 793B types) and three different commercially available inactivated vaccines each containing antigen against at least one IBV strain. The results reported here confirm the importance of IBV vaccination for laying hens, show that efficient live priming makes a beneficial contribution to this protection and confirm that inactivated IBV vaccines contribute significantly to effective protection against at least the five IBV challenge strains used here. Furthermore, we provide data to support the "protectotype concept", long-established using different live-attenuated IBV vaccines in young chickens, is valid in broadening protection against IBV challenges in laying birds.RESEARCH HIGHLIGHTSIBV vaccination is essential as an aid in protecting laying hens against IBV infection.Live priming is a beneficial part of the IBV vaccination programme.IBV inactivated vaccine improves IBV protection.Heterologous IBV protection is confirmed in laying hens.

Decrease of Mycoplasma gallisepticum seroprevalence and introduction of new genotypes in Dutch commercial poultry during the years 2001-2018.

Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

In ovo application of a live infectious bursal disease vaccine to commercial broilers confers proper immunity.

Infectious bursal disease (IBD) is an economically important disease of young chickens caused by the Avibirnavirus infectious bursal disease virus (IBDV). Besides biosecurity, vaccination is the most important measure for IBDV control. Sufficient levels of maternally derived antibodies (MDA) protect against early challenge and also interfere with the take of live conventional vaccines. Recently, the field surveys conducted in four countries, published by Ashash, U., Noach, C., Perelman, B., Costello, C., Sansalone, P., Brazil, T. & Raviv, Z. [(2019). In ovo and day of hatch application of a live infectious bursal disease virus vaccine to commercial broilers. Avian Diseases, 63, 713-720] using the MB-1 vaccine strain by in ovo application or sub-cutaneous route at the day of hatch seem to conflict with the rule that very early application of a conventional live vaccine in birds with significant levels of MDA has very little chance of a successful immune response. An in ovo vaccination-challenge controlled experiment with MB-1 vaccine was performed using commercial broilers with high levels of MDA against IBDV and a vvIBDV challenge at 22 or 36 days of age. Clinical signs, bursa-bodyweight ratios, histology, serology, RT-PCR, Sanger- and deep sequencing were used to study the efficacy and safety of the in ovo-applied MB1 vaccine in comparison to an established immuno-complex vaccine. The study findings confirmed that the in ovo application of the live MB-1 vaccine in commercial broilers was successful and induced full protection against a vvIBDV challenge at 22 and 36 days of age, demonstrated by the bursa lesion score and qPCR and IBDV genotyping. Comparable to the field studies, a delayed viral replication of 2-3 weeks, following the in ovo administration of the MB1 vaccine, was observed.

Relative contribution of vertical, within-farm and between-farm transmission of Mycoplasma synoviae in layer pullet flocks.

In this study, the relative contribution of vertical transmission, within-farm transmission and between-farm transmission of Mycoplasma synoviae in layer pullet flocks was quantified using logistic regression analysis. Data from 311 Dutch pullet flocks, of which 172 (55%) were positive for M. synoviae, were included in the study. Also the M. synoviae status of the parent stock of these flocks was included. The M. synoviae status was determined with the M. synoviae rapid plate agglutination test. Data analysis showed that vertical transmission was the most important transmission route for M. synoviae in layers as is demonstrated by an odds ratio of 5.8 (P = 0.000). A positive association with M. synoviae infections was found for layer pullet flocks on a multi-house farm where at least one other flock was M. synoviae-positive compared to single-house farms (odds ratio 3.1, P = 0.022), while a negative association was found when no other M. synoviae-positive flocks were present (odds ratio = 0.2, P = 0.003). No association was found between M. synoviae status of pullet flocks and poultry farm density. Odds ratios were 0.54 (P = 0.288) and 0.34 (P = 0.073), respectively, for medium and highest poultry farm density compared to lowest poultry farm density. This is the first time that the relative contribution of horizontal and vertical transmission of M. synoviae has been quantified. These results can be extrapolated to M. synoviae control in general, and emphasize the importance of M. synoviae control in parent stock and practical channelling.

Spotlight on avian coronaviruses.

Coronaviruses (CoVs) mainly cause enteric and/or respiratory signs. Mammalian CoVs including COVID-19 (now officially named SARS-CoV-2) belong to either the Alphacoronavirus or Betacoronavirus genera. In birds, the majority of the known CoVs belong to the Gammacoronavirus genus, whilst a small number are classified as Deltacoronaviruses. Gammacoronaviruses continue to be reported in an increasing number of avian species, generally by detection of viral RNA. Apart from infectious bronchitis virus in chickens, the only avian species in which CoV has been definitively associated with disease are the turkey, pheasant and guinea fowl. Whilst there is strong evidence for recombination between gammacoronaviruses of different avian species, and between betacoronaviruses in different mammals, evidence of recombination between coronaviruses of different genera is lacking. Furthermore, the recombination of an alpha or betacoronavirus with a gammacoronavirus is extremely unlikely. For recombination to happen, the two viruses would need to be present in the same cell of the same animal at the same time, a highly unlikely scenario as they cannot replicate in the same host!

Characterization of infectious bronchitis virus D181, a new serotype (GII-2).

This paper describes the characterization of a new infectious bronchitis virus (IBV) strain D181, that rapidly evolved from a low-level incidental finding in 2017 to become the second most isolated IBV strain in Dutch layers and breeders in 2018, as well as being found in samples from Germany and Belgium. Based on the sequence of the S gene and the results of cross-neutralization tests, D181 can be considered as a new serotype and the second lineage within genotype II (GII-2). The experimental infection of SPF hens confirmed the ability of D181 to cause a drop in egg production, and immunohistochemistry showed presence of the virus in the trachea, lung and conjunctiva at 5 days post inoculation and in the caecal tonsils at 5 and 8 days post inoculation. In silico analysis of several widely used PCR primers indicated that primer sets adapted for GII might be needed to detect D181, as many general S1 primers might miss it.

Effect of IBV D1466 on egg production and egg quality and the effect of heterologous priming to increase the efficacy of an inactivated IBV vaccine.

To protect layers, breeders and grandparents against damage by infectious bronchitis virus infections during the laying period, vaccination using live priming followed by a boost with inactivated IB vaccine is commonly used. For many IB variants, homologous live vaccines are not available for priming. Very little is known about the efficacy of priming with heterologous live IB vaccines (or combination of live IB vaccines) to induce broad IB protection in long-living chickens. In this study, the protection levels induced by vaccination programmes with only heterologous live priming by a Massachusetts vaccine and a 4/91 vaccine, only a multivalent inactivated vaccine that contained D1466 antigen and a combination of both, against a D1466 challenge were compared. The infection with infectious bronchitis virus D1466, a genotype II, lineage 1 virus, was able to cause serious damage to the unvaccinated laying hens resulting in respiratory signs, a long-lasting drop in egg production and loss of egg quality. All three vaccination programmes induced significant levels of protection against challenge with a pathogenic D1466 strain. Overall, the vaccination programme using the broad heterologous live priming and the inactivated vaccine provided high protection against the combination of egg drop and loss of egg quality. The results showed that this combination of heterologous live vaccines was able to increase the efficacy of the inactivated infectious bronchitis virus vaccine despite the very low antigenic relationship of both live vaccines with the challenge strain.

Major difference in clinical outcome and replication of a H3N1 avian influenza strain in young pullets and adult layers.

In this study, we investigated the pathogenicity, replication and tropism of the low pathogenic avian influenza (LPAI) strain A/chicken/Belgium/460/2019(H3N1) in adult SPF layers and young SPF males. The inoculated hens showed 58% mortality and a 100% drop in egg production in the second week post inoculation. The high viral loads in the cloacal samples coincided with the period of the positive immunohistochemistry of the oviduct, acute peritonitis and time of mortality, suggesting that the replication of H3N1 in the oviduct was a major component of the onset of clinical disease and increased level of excretion of the virus. In the inoculated young birds, the clinical signs were very mild with the exception of one bird. The results suggest that the time of replication of the virus was much shorter than in the adult layers; some of the young males did not show any proof of being infected at all. To conclude, the results of the study in young birds confirmed the intravenous pathogenicity test results but also showed that the clinical signs in adult layers were very severe. Based on the mortality without a bacterial component, complete drop of egg production and post mortem findings, this H3N1 strain is a moderately virulent strain, the highest category for LPAI strains. It is important to realize that if HPAI did not exist, this moderately virulent H3N1 virus would most likely to be considered as a very virulent virus.

Spotlight on avian pathology: infectious bronchitis virus.

Infectious bronchitis is a highly infectious disease of the domestic chicken of all ages and type, affecting the respiratory, renal and reproductive systems. Secondary bacterial infections are common and have a serious economic and welfare impact. Many genotypic and serotypic variants of infectious bronchitis virus (IBV) exist worldwide, making diagnosis difficult, and challenging control strategies. Vaccination, requiring the use of both live-attenuated and inactivated vaccines, is needed to control IBV infections; to date, attempts to develop vectored vaccines as effective as the traditional vaccines have been unsuccessful.

Induction of IBV strain-specific neutralizing antibodies and broad spectrum protection in layer pullets primed with IBV Massachusetts (Mass) and 793B vaccines prior to injection of inactivated vaccine containing Mass antigen.

In an initial study in specified pathogen free (SPF) chickens, a heterologous virus neutralizing (VN) antibody response to IBV variants Q1, Variant 2 (Var 2), D388/QX (D388), D274 and Arkansas (DPI) was observed using a vaccination programme incorporating two different live-attenuated IBV vaccines, followed by boosting with an inactivated vaccine containing IBV Massachusetts (Mass) antigen. Therefore, a more detailed study was undertaken in SPF layer-type chickens primed with IBV Mass and 793B vaccines. The efficacy of single or repeated vaccination with a multivalent inactivated vaccine containing IBV antigen was determined against challenge with five virulent IBVs: Mass (M41), 793B (4/91), D388, Q1 and Var 2. The parameters assessed were serological response, respiratory signs, egg production, post mortem abnormalities in the reproductive organs and abdomen, and incidence of IBV antigen in kidneys. Increased VN titres were recorded against the five IBV challenge strains, with a significantly higher level of protection against drops in egg production following challenge. The difference between one or two vaccinations with inactivated vaccine was not significant in terms of egg production. However, a significantly increased level of protection was seen in the lower percentage of hens with free yolk in the abdomen and/or peritonitis post challenge with IBV variants, D388, Q1 and Var 2 not included in the vaccination programme. A lower incidence of acute, degenerated ovaries was found in groups given one injection of inactivated vaccine following live priming, and this was significantly lower than in groups given only live priming.

Detection of different genotypes of infectious bronchitis virus and of infectious bursal disease virus in European broilers during an epidemiological study in 2013 and the consequences for the diagnostic approach.

To gather recent data regarding the infectious bronchitis (IB) and infectious bursal disease (IBD) situation in Europe, a large-scale field epidemiological survey using diagnostic samples has been implemented in 2013 for about six months in several European countries: France, Germany, Greece, Italy, the Netherlands, Poland, Portugal, the Republic of Ireland, Spain and the UK. In 234 flocks that were sampled, strains from 10 different IBV genotypes were detected: the 793B genotype was detected most frequently, followed by QX, Massachusetts (Mass) and the Xindadi-like strains. Strains belonging to the Q1, Ark, D274, D1466, Italy-02 and B1648 genotypes were detected as well, although less frequently. The separate sampling of tracheas and kidneys for IBV detection using reverse transcriptase PCR was very useful, as different genotypes or significant differences in sequences of the same genotype were detected between both organs. The data of this survey also provided valuable information about the replication of IBD vaccines and subsequent infectious bursal disease virus (IBDV) antibody responses under field conditions. The detection of five non-vvIBDV field strains of two different genotypes shows the presence of non-vvIBDV non-vaccine strains, which can easily be undetected in Europe due to the focus on sampling of clinically ill birds. Detection of vaccine virus in the bursa and antibody response to the IBD vaccination in flocks that had been vaccinated by the drinking water with a live attenuated vaccine compared to a vaccination in the hatchery using an immune-complex vaccine showed a delayed replication of the vaccines that had been applied by the drinking water, indicating mistakes in the timing and/or application of the vaccines.

Field and laboratory findings following the large-scale use of intermediate type infectious bursal disease vaccines in Denmark.

Following a period of clinical outbreaks of very virulent infectious bursal disease virus (vvIBDV) in Denmark, the histological bursal lesion score (HBLS) was used on a national scale to screen broiler flocks vaccinated with intermediate IBD vaccines for lesions indicative of IBDV challenge. High lesion scores were detected in a high percentage of healthy and well performing flocks despite the lack of other indications of the presence of vvIBDV. RT-PCR and subsequent sequencing showed the frequent presence of H253Q and H253N IBDV strains that were genetically close to the sequence of the intermediate vaccines with a relative risk ratio of 13.0 (P < 0.0001) in intermediate vaccine A or B vaccinated flocks compared to unvaccinated flocks. The relevance of these H253Q and H253N strains was tested under experimental conditions using a protocol derived from the European Pharmacopoeia for safety of live IBD vaccines. The results confirmed the higher pathogenicity for the bursa of these strains compared to intermediate vaccines as well as the negative effect on antibody response to a Newcastle disease (ND) vaccination performed at the peak of the bursa damage. The efficacy of the ND vaccination was still 100% showing that the H253N and H253Q IBDV strains would be considered as safe vaccine viruses. In conclusion, the use of the HBLS to screen commercial broiler flocks vaccinated with intermediate IBD vaccines for the presence of vvIBDV does not seem to be a reliable method due to the frequent occurrence of H253N and H253Q strains in those flocks. For screening of IBD vaccinated flocks for the presence of vvIBDV or other field strains, the RT-PCR with subsequent sequencing seems to be most suitable.

Prevalence of histopathological intestinal lesions and enteric pathogens in Dutch commercial broilers with time.

Intestinal disease has a major impact on the broiler industry due to economic and welfare reasons. Intestinal disease might occur due to a large number of reasons varying from well-defined pathogens to non-specific enteritis and complex syndromes. However, knowledge about the nature of intestinal disease and presence of enteric viruses in the Dutch broiler industry is largely absent. Therefore, a large-scale field study, in which 98 broiler flocks from 86 farms were sampled weekly, was started to assess the prevalence of histopathological lesions in the jejunum, a number of enterotropic viruses by real-time quantitative reverse transcriptase PCR (RT-qPCR) and coccidia by lesion scoring. Histopathological lesions indicative of intestinal disease were found in all flocks examined. The pathogens investigated were chicken astrovirus (99% of flocks positive), avian nephritis virus 3 (100%), rotavirus A (95%), rotavirus D (52%), reovirus (100%), Eimeria acervulina (94%), E. maxima (49%) and E. tenella (40%). The enteric viruses were more prevalent in the first weeks of the growing period, while coccidiosis was more frequently found at 4 and 5 weeks of age. The abundant presence of the enteric viruses and enteric disorders stresses the need to elucidate the role of these viruses in intestinal disease. Furthermore, the high prevalence of coccidiosis despite the use of anticoccidials shows that the current coccidial management programmes might be insufficient in controlling this disease.

Variability in biological behaviour, pathogenicity, protectotype and induction of virus neutralizing antibodies by different vaccination programmes to infectious bronchitis virus genotype Q1 strains from Chile.

In the period from July 2008 to 2010, a disease episode resulting in serious economic losses in the major production area of the Chilean poultry industry was reported. These losses were associated with respiratory problems, increase of condemnations, drops in egg production and nephritis in breeders, laying hens and broilers due to infections with infectious bronchitis virus (IBV). Twenty-five IBV isolates were genotyped and four strains were selected for further testing by pathotyping and protectotyping. Twenty-four IBV isolates were of the Q1 genotype. The experiments also included comparing the ability of six vaccination programmes to induce virus neutralizing antibodies (VNA) in layers against four selected Chilean strains. Despite the high genetic homology in the S1 gene between the four strains, the heterogeneity in biological behaviour of these different Q1 strains was substantial. These differences were seen in embryonated eggs, in cell culture, in pathogenicity and in level of cross-protection by IBV Massachusetts (Mass) vaccination. This variability underlines the importance of testing more than one strain per serotype or genotype to determine the characteristics of a certain serotype of genotype. The combination of Mass and 793B vaccine provided a high level of protection to the respiratory tract and the kidney for each strain tested in the young birds. The combination of broad live priming using Mass and 793B vaccines and boosting with multiple inactivated IBV antigens induced the highest level of VNA against Q1 strains, which might be indicative for higher levels of protection against Q1 challenge in laying birds.

Increased level of protection of respiratory tract and kidney by combining different infectious bronchitis virus vaccines against challenge with nephropathogenic Brazilian genotype subcluster 4 strains.

Genotyping of seven infectious bronchitis virus (IBV) strains isolated in Brazil showed that all belonged to the common Brazilian genotype and that these strains were closest to the subcluster of strain IBV/Brazil/2007/USP-19. Pathotyping of four selected Brazilian strains showed that they all caused a considerable level of ciliostasis in the trachea but at a somewhat lower level than did M41 and Brazilian strains 50/96, 57/96, 62/96 and 64/96 representing four different serotypes that had been reported earlier. In contrast to the M41 challenge strain, all Brazilian isolates replicated in kidney tissue in a high percentage of non-vaccinated challenged birds, clearly showing that they are nephropathogenic. As for the tracheal protection, the results using Massachusetts (Mass) vaccination against the recent strains seemed to show protection higher on average than for the strains reported earlier. A single or twofold vaccination with a Mass vaccine resulted in a mean tracheal protection level against the four challenge strains of 92% and 90%, respectively, whereas a single and twofold vaccination with a Mass vaccine halved the percentage of infected kidneys (14% and 13%, respectively, P < .05) compared to that of the unvaccinated birds (27%). The combination of the Mass and the 793B vaccine provided on average a tracheal protection of 99% and a reduction of the percentage of infected kidneys to a mean of 2%. This was a significantly (P < .05) higher protection than that achieved by a single or twofold Mass vaccination, showing the added value of the 793B vaccination following priming with a vaccine of the Mass type.

Factors influencing the outcome of infectious bronchitis vaccination and challenge experiments.

The factors influencing the outcome of infectious bronchitis vaccination and challenge experiments regarding the respiratory and renal systems are reviewed. Advantages and disadvantages of the available techniques for measuring protection against an infectious bronchitis virus challenge are discussed, including the definition of protection itself. Suggestions are made regarding some ways in which progress towards standardization of a recognized protocol for performing experimental challenge studies can be made and areas where more work is needed are indicated.

Broiler flocks in production systems with slower-growing breeds and reduced stocking density receive fewer antibiotic treatments and have lower mortality.

In the Netherlands, the number of broiler production systems with higher welfare standards, using slower-growing broilers and decreased stocking densities, has increased over the last decade. This study aimed to investigate the effect of this change on antibiotic treatments, mortality, and footpad lesions. Data from national monitoring databases from 2013 to 2021 were used, resulting in 113,380 included flocks from 917 farms. Flocks were divided into conventional (CONV), medium-growing (MED), and slow-growing (SLOW), based on breed and slaughter age (median age: CONV 42 d; MED 50 d; SLOW 56 d). Generalized mixed-effect models were created to compare antibiotic treatments in and after the first week, total on-farm mortality, and footpad lesion scores between these 3 production systems. Year, quarter, flock size, thinning, number of houses, and regional density of poultry farms were included as fixed effects. Random effects were farm and veterinary practice in all models, with an additional random slaughterhouse effect to describe footpad lesions. Probability of treatment in the first week of age in CONV flocks overall years (7.2%, 95% CI [5.9, 8.7]) was higher than in MED (2.0%, 95% CI [1.6, 2.5]) and SLOW flocks (1.3%, 95% CI [1.0, 1.7]). Treatment probability after the first week was similarly higher in CONV flocks (14.7%, 95% CI [12.1, 17.6]) than in MED (3.2%, 95% CI [2.5, 4.0]) and SLOW flocks (2.2%, 95% CI [1.7, 2.9]). CONV flocks had a higher mean mortality (3.2%, 95% CI [3.0, 3.4]) than MED (2.0%, 95% CI [1.9, 2.1]) and SLOW flocks (1.9%, 95% CI [1.8, 2.0]). Regarding footpad lesions, CONV flocks had the highest mean scores (range 0-200) over all years, whereas SLOW flocks had the lowest scores (CONV: 46.1, 95% CI [42.1, 50.6]; MED: 21.3, 95% CI [18.9, 24.0]; SLOW: 13.2, 95% CI [11.5, 15.1]). This analysis of data from flocks over a 9-yr period indicates that switching from conventional to alternative production systems with higher welfare standards could positively affect broiler health and antibiotic use.

The required sample size in vaccination-challenge experiments with infectious bronchitis virus, a meta-analysis.

For statistical, animal welfare and financial reasons the choice of the number of chickens per group in experiments is important. This estimation, together with the number of tracheal organ cultures (TOCs) that need to be examined from each chicken in order to assess protection, should be based on the difference in level of protection that one would like to be able to detect (effect size), the expected variability of the results between and within the chickens, the desired confidence level and the power of the study. To obtain data that would facilitate this estimation, a meta-analysis was performed on the data from 18 infectious bronchitis virus (IBV) vaccination-challenge experiments performed at the Dutch Animal Health Service Deventer, the Netherlands (GD) in order to determine and quantify the source of variation in the mean level of protection of different groups. For the calculations, 137 groups of chickens were subdivided into 10 clusters based on age (young or adult), vaccination (none, homologous or heterologous), challenge (IBV or mock infected) and location of vaccination (isolator at GD or in the field). The results were used to estimate the required number of chickens per group for the different clusters using 2, 5 or 10 TOCs per chicken to be able to detect effect sizes of 6.25%, 12.5%, 25% and 50% between groups of chickens with 95% confidence (P<0.05) and 80% power. The number of chickens that was required for the mentioned effect sizes varied greatly from 2 to 650. This meta-analysis provided data that allow research workers to estimate the number of chickens that should be included in each group in order to obtain reliable results based on particular combinations of infectious bronchitis vaccination and challenge strains as defined by the presented clusters.