Success rates of inoculation of the various compartments of embryonated chicken eggs at different incubation days.

Inoculation of embryonated chicken eggs has been widely used during the past decades; however, inoculation success rates have not been investigated systematically. In this study named success rates were assessed in brown eggs incubated between 5 and 19 days, which were inoculated with 0.2 ml methylene blue per egg. Inoculations were performed in a simple and fully standardized way. Five embryonic compartments were targeted blindly (amniotic cavity, embryo, allantoic cavity, albumen and yolk) with needles of four different lengths; albumen and yolk were targeted with eggs in upside down position. Three compartments were inoculated within sight (air chamber, chorioallantoic membrane and blood vessel). Twenty embryos were used per incubation day, intended deposition site and needle length. Success rates were assessed by visual inspection after breaking the eggs. The inoculations targeting albumen, yolk, amniotic cavity and embryo yielded low scores. Magnetic resonance imaging was performed to elucidate the reason(s) for these low success rates: needles used were of appropriate length, but embryo and amniotic cavity had variable positions in the eggs, while albumen and yolk rapidly changed position after turning the eggs upside down. The latter led to adjustment of the inoculation method for albumen and yolk. Failures to inoculate compartments within sight were immediately visible; therefore, these eggs could be discarded. Except for the amniotic cavity, full scores (20/20) were obtained for all compartments although not always on every day of incubation. In conclusion, the present study may serve as a guide to more accurately inoculate the various chicken embryo compartments. RESEARCH HIGHLIGHTS Blind inoculation of embryonated egg compartments was successful, except for the amniotic cavity. MRI showed rapid position change of albumen and yolk after turning eggs upside down. In ovo vaccination against Marek's disease might be improved by using 38 mm needles.

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.

Success rates of intrauterine inoculations of layers via the vagina.

Intrauterine inoculation of layer hens has been documented previously in the literature; however, its efficiency has only been assessed on a very small scale. Attempts were therefore made to inoculate 14 experimental groups each consisting of 10 commercial table egg-producing hens intrauterinely with methylene blue. The effect of four variables-oviposition (natural, hormonally induced or not), position of the hen at inoculation (vertical/horizontal), technique to access the uterus (three methods) and inoculation device (four types)-on the success rate of intrauterine inoculation of layers of different breed and age was studied. Immediately after inoculation, hens were euthanized and the presence of methylene blue and perforations in the oviduct were examined. Successful inoculation rates ranged from 0/10 to 10/10. The 100% success rate was obtained in birds without an egg in the uterus, which were restrained in a horizontal position and inoculated with a hard catheter after exposure of the uterovaginal junction. The second best score was obtained in hens inoculated shortly after natural oviposition, maintained in a vertical position and using a corkscrew-shaped saliva ejector after exposure of the vaginal orifice (7/10). In all other groups the success rate was 5/10 or less. Vaginal perforation occurred in none to eight birds per group. It is concluded that intrauterine inoculation of laying hens is only reliable in birds without an egg in the uterus after exposure of the uterovaginal junction. All other methods proved unreliable and often caused vaginal perforations. However, intravaginal inoculation can be performed with 100% accuracy providing the vaginal orifice is exposed.

Broiler resilience to colibacillosis is affected by incubation temperature and post-hatch feeding strategy.

Colibacillosis is a poultry disease that negatively affects welfare and causes economic losses. Treatment with antibiotics raises concerns on antimicrobial resistance. Consequently, alternative approaches to enhance poultry resilience are needed. Access to feed and water directly after hatch (early feeding) may enhance resilience at later ages. Additionally, a high eggshell temperature (EST) during mid incubation may improve chick quality at hatch, supporting potential positive effects of early feeding. Effects of EST [37.8°C (control) or 38.9°C (higher)] during mid-incubation (embryo days 7-14) and feeding strategy (early feeding or 48 h delayed feeding) were tested in a 2 × 2 factorial arrangement. At hatch, Ì´ 1,800 broilers were divided over 36 pens and grown for 6 wk. At d 8 post hatch, avian pathogenic E. coli (APEC) was inoculated intratracheally as model to investigate broiler resilience against respiratory diseases. Incidence and severity of colibacillosis, local infection, and systemic infection were assessed at 6 moments between 3 h and 7 d postinoculation. Broilers were weighed daily during 13 d postinoculation and weekly thereafter. At higher EST, early feeding resulted in higher incidence of systemic infection compared to delayed feeding whereas at control EST, systemic infection was not different between feeding strategies. Regardless of EST, early compared to delayed feeding resulted in lower incidence of local infection, fewer BW deviations, and higher growth until d 35. In conclusion, early feeding could be considered as a strategy to enhance broiler resilience, but only when EST is not too high.

Selective breeding for high natural antibody level increases resistance to avian pathogenic Escherichia coli (APEC) in chickens.

Keyhole limpet hemocyanin (KLH)-binding natural antibody (NAb) titers in chickens are heritable, and higher levels have previously been associated with a higher survival. This suggests that selective breeding for higher NAb levels might increase survival by means of improved general disease resistance. Chickens were divergently selected and bred for total NAb levels binding KLH at 16 weeks of age for six generations, resulting in a High NAb selection line and a Low NAb selection line. To for test differences in disease resistance, chickens were challenged with avian pathogenic Escherichia coli (APEC) in two separate experiments. Chickens at 8 days of age received one of four intratracheal inoculations of 0.2 mL phosphate buffered saline (PBS): 1) mock inoculate, 2) with 0.2 mL PBS containing 108.20 colony-forming units (CFU)/mL APEC, 3) with 0.2 mL PBS containing 106.64 CFU/mL APEC, and 4) with 0.2 mL PBS containing 107.55 CFU/mL APEC. Mortality was recorded during 7 days post inoculation. Overall, 50-60% reduced mortality was observed in the High line compared to the Low line for all APEC doses. In addition, morbidity was determined of the surviving chickens at 15 days of age. The High line had lower morbidity scores compared to the Low line. We conclude that selective breeding for high KLH-binding NAb levels at 16 weeks of age increase APEC resistance in early life. This study and previous studies support the hypothesis that KLH-binding NAb might be used as an indicator trait for to selective breed for general disease resistance in an antigen non-specific fashion.

Transmissibility of infectious bronchitis virus H120 vaccine strain among broilers under experimental conditions.

The aim of this study was to quantify transmission of infectious bronchitis virus (IBV) H120 vaccine strain among broilers, and to assess whether birds that have been exposed to vaccine strain-shedding birds were protected against clinical signs after infection with a virulent strain of the same serotype. A transmission experiment and a replicate were carried out, each with six groups of commercial broilers. At day of hatch (n = 30) or at 15 days of age (n = 20), half of each group was inoculated with either IBV H120 vaccine (H120 group), virulent IBV M41 (M41 group), or were mock-infected, thereby contact-exposing the other half of each group. Nasal discharge was recorded, and antibody response and virus shedding were measured. To measure clinical protection, four weeks after inoculation all birds, in all groups, were challenged with IBV M41. The reproduction ratio (R; the average number of contact infections caused by one infectious bird) was determined to quantify virus transmission. All contact-exposed birds, except for one in an H120 group, became infected with either IBV H120 or IBV M41. Almost all birds contact-infected with IBV H120 or IBV M41 were subsequently protected against clinical signs after challenge with IBV M41. The lower limits of the 95% confidence interval (CI) of the R of IBV H120 vaccine, and of IBV M41, were significantly <1. For both IBV H120 and IBV M41, the 95% CI was [2.1-infinity] following inoculation at day of hatch and [1.8-infinity] after inoculation at 15 days of age. This finding demonstrates that IBV H120 vaccine is able to spread extensively among broilers. This implies that this vaccine strain might be able to become endemically present in the poultry population. It also implies that, even if not all birds received vaccine during spray application, due to the ability of the vaccine to spread in the flock, they will most likely be protected against clinical signs after a subsequent field virus infection.

Signs Indicating Imminent Death in Escherichia coli-Infected Broilers.

Broilers were observed during 9 days for clinical signs after intratracheal inoculation at 8 days of age with 107 E. coli 506. It was determined if these signs were predictive for imminent death. Hourly observations were made daily from a distance of 1-2 m and nightly by camera observation, with respect to the following parameters: level of attention, locomotory activity, posture and appearance, interaction, and impairment of respiration. For deviations of the normal state for these five parameters (i.e., typical clinical signs of disease), scores were defined in up to four classes. The periods of time elapsing from attaining a score for the first time to death were registered per bird for each score for each parameter. Of 114 birds, 85 did not present typical signs of illness as described, and 29 presented the following clinical history: 25 died after presenting signs of illness, 2 died without previous signs, 1 fell ill but survived, and 1 fell ill and recovered. Extended clinical examination was performed in birds presenting clinical signs; temperature, heart rate, respiratory rate, and subcutaneous capillary refill time were measured. The level of attention, and posture and appearance were affected most often in ill birds; 25% of these birds died within 5 and 4 hr, respectively; 50% died within 12 hr; and 75% died within 20 and 19 hr, respectively. Any of these typical signs of illness visible from 1-2 m indicated imminent death, with 75% of the birds dying within 20 hr. Measurements resulting from extended clinical examination proved of lesser predictive value. From these observations, a protocol for intervention to prevent animal suffering may be designed.

Outbreak of avian influenza H7N3 on a turkey farm in the Netherlands.

This case report describes the course of an outbreak of avian influenza on a Dutch turkey farm. When clinical signs were observed their cause remained unclear. However, serum samples taken for the monitoring campaign launched during the epidemic of highly pathogenic avian influenza in 2003, showed that all the remaining turkeys were seropositive against an H7 strain of avian influenza virus, and the virus was subsequently isolated from stored carcases. The results of a reverse-transcriptase pcr showed that a H7N3 strain was involved, and it was characterised as of low pathogenicity. However, its intravenous pathogenicity index was 2.4, characterising it as of high pathogenicity, suggesting that a mixture of strains of low and high pathogenicity may have been present in the isolate. The outbreak remained limited to three farms.

Effect of IBV-H120 vaccination in broilers on colibacillosis susceptibility after infection with a virulent Massachusetts-type IBV strain.

Vaccination against infectious bronchitis (IB) is aimed to protect against clinical IB. The question is, however, whether vaccinated birds are also protected against predisposure for colibacillosis after a subsequent IBV infection. We examined this research question in four experiments. One-day-old commercial broilers, housed in isolators, were vaccinated with IB vaccine strain H120 by coarse spray or ocularly. Twenty-eight days after vaccination, broilers were challenged with the virulent IBV strain M41. Five days later, broilers were inoculated with Escherichia coli strain 506. Body weight uniformity, severity of E. coli airsacculitis, and systemic E. coli infection at 7 days following E. coli inoculation were used as parameters for colibacillosis. IBV vaccination reduced both the number of broilers with E. coli airsacculitis as well as the severity of airsacculitis significantly after challenge with IBV-M41 and E. coli 506. However, in spray-vaccinated groups, no significant reduction of the number of birds with systemic colibacillosis or the severity of this infection was obtained, and body weight uniformity was not significantly improved compared with nonvaccinated, IBV-M41, and E. coli 506-challenged groups. Eye-drop vaccination resulted in conflicting results.

Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41.

Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1 dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-ß mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4 dpi. In contrast, IFN-γ production in blood was highest at 1 dpi and decreased over time down to levels comparable to uninfected birds at 4 dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-ß compared to uninfected birds.

Effect of anti-inflammatory drugs on colibacillosis lesions in broilers after Infectious Bronchitis Virus and subsequent Escherichia coli infection.

BACKGROUND: In case of persistent and sterile inflammation, anti-inflammatory drugs should be considered as first choice treatment instead of antibiotics. OBJECTIVE: To assess the effect of anti-inflammatory drugs on lesions due to colibacillosis. ANIMALS & METHODS: Five groups of day-old broilers of 15 birds each were housed in isolators and were inoculated at 29 days of age with Infectious Bronchitis Virus strain M41 by the oculo-nasal and IT (intratracheal) route (10(5.4) EID(50) (egg infectious dosis 50)/broiler) and at 33 days of age with Escherichia coli strain 506 by the IT route (10(7.6) colony forming units/broiler). Broilers of four groups were treated from day 28 up to and including day 39 orally on a daily basis with either carbasalate calcium (4 × 12.5 mg), meloxicam (2 × 0.5 mg), acetaminophen (4 × 2.5 mg), or dexamethasone (1 × 1.0 mg). The fifth group was placebo-medicated. At 40 days of age, the experiment was ended and at post-mortem examination, colibacillosis lesions were assessed. RESULTS: All broilers in the dexamethasone group died. This mortality exceeded significantly (p < 0.05) that of the other groups in which mortality ranged from 2 to 5. Mean lesion score of surviving broilers of medicated groups ranged from 5.3 to 5.8 compared to 3.9 in the placebo group and did not differ significantly between groups. CONCLUSION: None of the anti-inflammatory drugs had a positive effect on colibacillosis lesions. CLINICAL IMPORTANCE: Anti-inflammatory drugs cannot be considered as an alternative for antibiotic treatment.

Deposition of differently sized airborne microspheres in the respiratory tract of chickens.

As a part of the development of an efficient dry powder aerosol vaccine for poultry, the objective of this study was to accurately determine the deposition pattern of nebulized microspheres in the airways of unanaesthetized chickens of different ages (1 day, 2 weeks and 4 weeks old). In the first part of the study, the aerosol administration method was characterized: the influence of different nebulizers and nebulizing protocols on the relative humidity in the exposure chamber, the particle size distributions, the microsphere output and single microsphere percentage were determined. In the second part, birds were exposed to nebulized fluorescently labelled polystyrene microspheres (1 to 20 microm). Respiratory and gastro-intestinal tract tissue samples were collected and the number of fluorescent microspheres per sample was determined. In 2-week-old and 4-week-old chickens, microspheres of 5 and 10 microm, respectively, were too large for deposition in the lungs and air sacs as less than 5% of these microspheres penetrated into the lower airways. The larger size of microspheres reaching the lower airways of 4-week-old birds was explained by increasing airway dimensions with age. For 1-day-old chickens, deposition in the lungs decreased from 17 to 3% with increasing particle size (1 to 20 microm), but increased in the air sacs from 6 to 20%. Consequently, the total deposition percentage in the lower airways was independent of microsphere size and even 20 microm particles were able to penetrate into the lower airways, which was attributed to mouth breathing of the 1-day-old chickens.

Ability of Massachusetts-type infectious bronchitis virus to increase colibacillosis susceptibility in commercial broilers: a comparison between vaccine and virulent field virus.

The abilities of Massachusetts-type vaccine virus and virulent infectious bronchitis (IB) field virus to increase colibacillosis susceptibility were compared. In four experiments, 29-day-old female commercial broilers housed in isolators, were infected intratracheally and oculonasally with IB vaccine strains (H120 and H52) or virulent IB field strains (D387 and M41) (4.8 or 6.8 log(10) median embryo infective dose, per broiler). Five days later, Escherichia coli 506 strain was given intratracheally (5.6 to 8.8 log(10) colony forming units/broiler). The incidence of nasal discharge at 3 and 5 days after IB virus infection was used to assess the clinical effect of the IB infection, while mortality, body weight uniformity and E. coli lesions at 7 days following E. coli inoculation were used as parameters for colibacillosis. Nasal discharge was observed in 6/117 (5%), 26/119 (22%), 35/119 (29%) and 115/120 (96%) of broilers infected with H120, H52, D387 and M41 virus, respectively. Apart from H52 and D387, differences between IBV strains were significant. IB vaccine and virulent IB viruses did not generally differ significantly in their ability to induce colibacillosis susceptibility. Mean colibacillosis lesion scores of H52-infected birds even significantly exceeded those of birds infected with the other IB viruses. The ability of H120 virus to induce colibacillosis susceptibility tended to be the weakest. The practical consequences of these findings are discussed.