Effects of lighted incubation and foraging enrichment during rearing on individual fear behavior, corticosterone, and neuroplasticity in laying hen pullets.

Environmental conditions during incubation and rearing can influence stress responsivity of laying hen pullets throughout their lifespan, and therefore have important implications for their welfare. In this study, a 12:12h green LED light-dark cycle during incubation and larvae provisioning as enrichment during rearing were tested as strategies to optimize early-life conditions and thereby decrease stress responsivity in ISA Brown laying hens. A combination of parameters was measured to indicate neuronal, physiological, and behavioral changes that may affect fear and stress. The proteins calbindin D28k (calbindin1), doublecortin (DCX), and neuronal nuclein protein (NeuN) were quantified after hatch as a proxy for brain plasticity. Plasma and feather corticosterone levels were measured after hatch and at the end of the rearing phase, and fearfulness was investigated through a series of behavioral tests (i.e., voluntary approach, open field, tonic immobility, and manual restraint tests). No effects of light during incubation were found on calbindin1, DCX, or NeuN. Neither of the treatments affected corticosterone levels in blood plasma and feathers. Light-incubated pullets showed less fearfulness towards humans in the voluntary approach test, but not in the other behavioral tests reported in this study. Larvae provisioning had no effect on behavior. Our study showed minor effects of light during incubation and no effects of enrichment during rearing on stress responsivity of laying hen pullets. The small effects may be explained by the enriched rearing conditions for all birds in this experiment (low stocking density, natural daylight, and 24/7 classical music). Given the promising results of lighted incubation in other studies, which were mostly performed in broiler chickens, and evidence regarding the positive effects of enrichment during rearing, the potential of these strategies to improve laying hen welfare needs to be explored further.

Early-life interventions to prevent feather pecking and reduce fearfulness in laying hens.

Severe feather pecking, the pulling out of feathers of conspecifics, is a major welfare issue in laying hens. Possible underlying causes are fearfulness and lack of foraging opportunities. Because early life is a crucial stage in behavioral development, adapting the incubation and rearing environment to the birds' needs may reduce fearfulness and prevent the development of feather pecking. In a 2 × 2 factorial design study, we investigated whether a green light-dark cycle throughout incubation, which resembles natural incubation circumstances more than the standard dark incubation, and foraging enrichment with live larvae during rearing reduce fearfulness and feather pecking and increase foraging behavior of laying hen pullets from an early age onwards. In this 2-batch experiment, 1,100 ISA Brown eggs were incubated under either 0 h of light/24 h of darkness or 12 h of green LED light/12 h of darkness. After hatching, 400 female chicks (200 per batch) were housed in 44 pens (8-10 chicks per pen). During the entire rearing phase (0-17 wk of age), half of the pens received black soldier fly larvae in a food puzzle as foraging enrichment. We assessed fear of novel objects and humans, feather pecking, plumage condition, foraging behavior, and recovery time after a 3-fold vaccination (acute stressor). A slight increase in the number of foraging bouts was only seen with larvae provisioning (rate ratio 1.19, 95% CI 1.02-1.29, P = 0.008). Neither lighted incubation nor larvae provisioning affected fearfulness, feather pecking, plumage condition or recovery time after vaccination. In conclusion, the present study showed no effects of light during incubation and minor effects of foraging enrichment during rearing on the behavior of laying hen pullets. Further research is recommended on other welfare aspects.

Repeated cross-sectional sampling of pigs at slaughter indicates varying age of hepatitis E virus infection within and between pig farms.

Humans can become infected with hepatitis E virus (HEV) by consumption of undercooked pork. To reduce the burden of HEV in humans, mitigation on pig farms is needed. HEV is found on most pig farms globally, yet within-farm seroprevalence estimates vary considerably. Understanding of the underlying variation in infection dynamics within and between farms currently lacks. Therefore, we investigated HEV infection dynamics by sampling 1711 batches of slaughter pigs from 208 Dutch farms over an 8-month period. Four farm types, conventional, organic, and two types with strict focus on biosecurity, were included. Sera were tested individually with an anti-HEV antibody ELISA and pooled per batch with PCR. All farms delivered seropositive pigs to slaughter, yet batches (resembling farm compartments) had varying results. By combining PCR and ELISA results, infection moment and extent per batch could be classified as low transmission, early, intermediate or late. Cluster analysis of batch infection moments per farm resulted in four clusters with distinct infection patterns. Cluster 1 farms delivered almost exclusively PCR negative, ELISA positive batches to slaughter (PCR-ELISA+), indicating relatively early age of HEV infection. Cluster 2 and 3 farms delivered 0.3 and 0.7 of batches with intermediate infection moment (PCR+ELISA+) respectively and only few batches with early infection. Cluster 4 farms delivered low transmission (PCR-ELISA-) and late infection (PCR+ELISA-) batches, demonstrating that those farms can prevent or delay HEV transmission to farm compartments. Farm type partly coincided with cluster assignment, indicating that biosecurity and management are related to age of HEV infection.

Prevalence and Demographic Risk Factors of Mycobacterium tuberculosis Infections in Captive Asian Elephants (Elephas maximus) Based on Serological Assays.

To address putative TB statuses of elephants and to identify and quantify potential demographic risk factors for TB, three ELISAs specific for different mycobacterial antigens (ESAT6, CFP10, MPB83) and the TB Stat-Pak assay were used as surrogate serological markers for TB infection in elephants. In view of the low number of animals of which the infected status could be confirmed (4 out of 708) Latent Class Analyses of TB serology test outcomes was used to predict the putative TB status of each of 708 elephants as positive (17.3%), inconclusive (48.7%), or negative (34%) when assessed on a population basis. Correlation between test performance of the individual assays was high between the ELISAs, but low with that of the TB Stat-Pak assay. Risk factors, assessed based on cut off values for each of the ELISAs determined by ROC analysis, included sex, BCS, age, working time, feed type, management system, camp size and region. Old age elephants were more likely to show a positive TB serology test outcome, than younger ones. Elephants working 7 h per day and the ones in good condition BCS (7-11) were less likely to be positive in TB serology testing. In addition, fewer animals in the large camp size (31-50 elephants) were found to be positive in ELISA tests, compared to elephants in the other camp sizes. In this study, the North region had the lowest percentages of elephants with positive TB test outcome, the West region and to a lesser extend the other regions showed clearly higher percentages of positive animals. Even though assays used in the present study have not been validated yet, results obtained showed promise as diagnostic or screening tests. For the diagnosis of animals suspected to be infected, the ELISA tests, once further optimized for the individual antigens, can be used in parallel. For screening of complete camps for presence or absence of infection, a single optimized ELISA test can be utilized.

Seroprevalence of porcine reproductive and respiratory syndrome virus on swine farms in a tropical country of the Middle Americas: the case of Costa Rica.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Little is known regarding the epidemiology of this infection in tropical countries. To address this problem in Costa Rica, a seroepidemiological study was carried out in two phases. In the first phase, a pilot study was conducted in nine farms with the clinical diagnosis of PRRSV. In total, 265 pig serum samples were collected from animals ranging in age from 1 to 15 weeks of age. This study aimed to establish the duration of maternal immunity in piglets, to identify the period of viremia, and to determine when seroconversion occurs. In the second phase, a cross-sectional serology study was performed on a representative sample of the Costa Rican national herds in the second phase. The twenty-five selected farms represent all provinces and were classified according to herd size (100 to 2000 sows). In each farm, pigs aged 8, 10, and 12 weeks were sampled, as well as gilts based on the pilot study. In total 1281 pigs were sampled across all 25 farms. The aim of the cross-sectional study was to quantify the seroprevalence of PRRSV in Costa Rican pig farms and to describe its geographical distribution in this tropical country. The prevalence of positive farms was 44% (11/25), and these farms were located in six of the seven provinces of Costa Rica. Overall, 58% (344/596) of the pigs were seropositive to PRRSV. The age of the pigs and the ecozone where farms were located were significantly related with PRRSV seroprevalence in animals and herds, respectively.

ESBL-Producing, Carbapenem- and Ciprofloxacin-Resistant Escherichia coli in Belgian and Dutch Broiler and Pig Farms: A Cross-Sectional and Cross-Border Study.

BACKGROUND: The use of antibiotics in food production selects for resistant bacteria and may cause a threat to human and animal health. Belgium and the Netherlands have one of the highest densities of broilers and pigs in Europe, making active monitoring of antibiotic use and resistance in this region vital. OBJECTIVES: This study aimed to quantify ESBL-producing (ESBL-E. coli), carbapenem- and ciprofloxacin-resistant (CiproR) Escherichia coli in animal feces on broiler and pig farms with a history of high antibiotic use in Belgium and the Netherlands. METHODS: A total of 779 broiler and 817 pig fecal samples, collected from 29 conventional broiler and 31 multiplier pig farms in the cross-border region of Belgium and the Netherlands, were screened for the presence of antibiotic-resistant E. coli using selective culturing. RESULTS: Carbapenem-resistant E. coli were not detected. ESBL-E. coli were remarkably more prevalent in samples from Belgian than Dutch farms. However, CiproR-E. coli were highly prevalent in broilers of both countries. The percentage of samples with ESBL- and CiproR-E. coli was lower in pig compared to poultry farms and varied between farms. No clear association with the on-farm antibiotic use in the year preceding sampling was observed. Multidrug resistance was frequently observed in samples from both countries, but ESBL-production in combination with ciprofloxacin resistance was higher in samples from Belgium. CONCLUSIONS: This study demonstrated marked differences in antibiotic resistance between countries, farms and within farms. The observed variation cannot be explained straightforward by prior quantity of antibiotic use suggesting that it results from more complex interactions that warrant further investigation.

The role of different Culex mosquito species in the transmission of West Nile virus and avian malaria parasites in Mediterranean areas.

Vector-borne diseases, especially those transmitted by mosquitoes, have severe impacts on public health and economy. West Nile virus (WNV) and avian malaria parasites of the genus Plasmodium are mosquito-borne pathogens that may produce severe disease and illness in humans and birds, respectively, and circulate in an endemic form in southern Europe. Here, we used field-collected data to identify the impact of Culex pipiens, Cx. perexiguus and Cx. modestus, on the circulation of both WNV and Plasmodium in Andalusia (SW Spain) using mathematical modelling of the basic reproduction number (R0 ). Models were calibrated with field-collected data on WNV seroprevalence and Plasmodium infection in wild house sparrows, presence of WNV and Plasmodium in mosquito pools, and mosquito blood-feeding patterns. This approach allowed us to determine the contribution of each vector species to pathogen amplification. Overall, 0.7% and 29.6% of house sparrows were positive to WNV antibodies and Plasmodium infection, respectively. In addition, the prevalence of Plasmodium was higher in Cx. pipiens (2.0%), followed by Cx. perexiguus (1.8%) and Cx. modestus (0.7%). Three pools of Cx. perexiguus were positive to WVN. Models identified Cx. perexiguus as the most important species contributing to the amplification of WNV in southern Spain. For Plasmodium models, R0 values were higher when Cx. pipiens was present in the population, either alone or in combination with the other mosquito species. These results suggest that the transmission of these vector-borne pathogens depends on different Culex species, and consequently, their transmission niches will present different spatial and temporal patterns. For WNV, targeted surveillance and control of Cx. perexiguus populations appear as the most effective measure to reduce WNV amplification. Also, preventing Culex populations near human settlements, or reducing the abundance of these species, are potential strategies to reduce WNV spillover into human populations in southern Spain.

Equine cervical intervertebral disc degeneration is associated with location and MRI features.

Morphology of the equine cervical intervertebral disc is different from that in humans and small companion animals and published imaging data are scarcely available. The objectives of this exploratory, methods comparison study were (a) to describe MRI features of macroscopically nondegenerated and degenerated intervertebral discs (b) to test associations between spinal location and macroscopic degeneration or MRI-detected annular protrusion and between MRI-detected annular protrusion and macroscopic degeneration, and (c) to define MRI sequences for characterizing equine cervical intervertebral disc degeneration. Ex vivo MRI of intervertebral discs was performed in 11 horses with clinical signs related to the cervical region prior to macroscopic assessment. Mixed-effect logistic regression modeling included spinal location, MRI-detected annular protrusion, and presence of macroscopic degeneration with "horse" as random effect. Odds ratio and 95% confidence interval were determined. Reduced signal intensity in proton density turbo SE represented intervertebral disc degeneration. Signal voids due to presence of gas and/or hemorrhage were seen in gradient echo sequences. Presence of macroscopic intervertebral disc degeneration was significantly associated with spinal location with odds being higher in the caudal (C5 to T1) versus cranial (C2 to C5) part of the cervical vertebral column. Intervertebral discs with MRI-detected annular protrusion grades 2-4 did have higher odds than with grade 1 to have macroscopic degeneration. It was concluded that MRI findings corresponded well with gross macroscopic data. Magnetic resonance imaging of the equine cervical intervertebral disc seems to be a promising technique, but its potential clinical value for live horses needs to be explored further in a larger and more diverse population of horses.

Evidence of high EEHV antibody seroprevalence and spatial variation among captive Asian elephants (Elephas maximus) in Thailand.

BACKGROUND: Elephant endotheliotropic herpesviruses (EEHV) can cause an acute highly fatal hemorrhagic disease in young Asian elephants (Elephas maximus), both ex situ and in situ. Amongst eight EEHV types described so far, type 1 (subtype 1A and 1B) is the predominant disease-associated type. Little is known about routes of infection and pathogenesis of EEHV, and knowledge of disease prevalence, especially in range countries, is limited. METHODS: A large cross-sectional serological survey was conducted in captive elephants (n = 994) throughout Thailand using an EEHV-1A glycoprotein B protein antigen specific antibody ELISA. RESULTS: Antibody seroprevalence was 42.3%, with 420 of 994 elephants testing positive. Associations between seropositivity and potential risk factors for EEHV infection were assessed and included: elephant age, sex, camp cluster size, management type (extensive versus intensive), sampling period (wet vs. dry season) and location of camp (region). Univariable regression analysis identified management system and region as risk factors for the presence of EEHV antibodies in elephants, with region being significant in the final multivariable regression model. Prevalence was highest in the North region of the country (49.4%). CONCLUSIONS: This study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.

Exploring contacts facilitating transmission of influenza A(H5N1) virus between poultry farms in West Java, Indonesia: A major role for backyard farms?

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been reported in Asia, including Indonesia since 2003. Although several risk factors related to the HPAIV outbreaks in poultry in Indonesia have been identified, little is known of the contact structure of farms of different poultry production types (backyard chickens, broilers, layers, and ducks). This study aims to quantify the contact rates associated with the movement of people, and movements of live birds and products and equipment that affect the risk of HPAIV H5N1 transmission between poultry farms in Indonesia. On 124 poultry farms in 6 districts in West Java, logbooks were distributed to record the movements of farmers/staff and visitors and their poultry contacts. Most movements in backyard chicken, commercial native chicken, broiler and duck farms were visits to and from other poultry farms, whilst in layer farms visits to and from poultry companies, visits to egg collection houses and visit from other poultry farms were most frequent. Over 75% of persons visiting backyard chicken and duck farms had previously visited other poultry farms on the same day. Visitors of backyard chicken farms had the highest average contact rate, either direct contact with poultry on other farms before the visits (1.35 contact/day) or contact during their visits in the farms (10.03 contact/day). These results suggest that backyard chicken farms are most at risk for transmission of HPAIV compared to farms of the other poultry production types. Since visits of farm-to-farm were high, backyard farms could also a potential source for HPAIV transmission to commercial poultry farms.

Intervertebral Disc Degeneration in Warmblood Horses: Morphology, Grading, and Distribution of Lesions.

Equine intervertebral disc degeneration is thought to be rare and of limited clinical relevance, although research is lacking. To objectively assess pathological changes of the equine intervertebral disc and their clinical relevance, description of the normal morphology and a practical, biologically credible grading scheme are needed. The objectives of this study are to describe the gross and histological appearance of the equine intervertebral discs and to propose a grading scheme for macroscopic degeneration. Spinal units from 33 warmblood horses were grossly analyzed and scored. Of the 286 intervertebral discs analyzed, 107 (37%) were assigned grade 1 and grade 2 (considered normal) and were analyzed histologically. A nucleus pulposus and an annulus fibrosus could be identified macroscopically and histologically. Histologically, the nucleus pulposus was composed of a cartilaginous matrix and the annulus fibrosus of parallel collagenous bands. A transition zone was also histologically visible. Intra- and inter-observer reliability scores were high for all observers. Higher grades were associated with greater age. Gross changes associated with equine intervertebral disc degeneration (grades 3-5)-that is, yellow discoloration, cleft formation (tearing), and changes in consistency of the nucleus pulposus-were largely similar to those in humans and dogs and were most prevalent in the caudal cervical spine. Equine intervertebral disc degeneration was not associated with osteophyte formation. Changes of the vertebral bone were most common in the thoracolumbar spine but were not correlated with higher grades of intervertebral disc degeneration. Thus, changes of the vertebral bone should be excluded from grading for equine intervertebral disc degeneration.

Effect of Simultaneous Exposure of Pigs to Streptococcus suis Serotypes 2 and 9 on Their Colonization and Transmission, and on Mortality.

The distribution of Streptococcus suis serotypes isolated from clinically infected pigs differs between geographical areas, and varies over time. In several European countries, predomination of serotype 2 has changed to serotype 9. We hypothesize a relation, with one serotype affecting the other in colonization and invasion. The aim of this study was to evaluate whether simultaneous exposure of pigs to serotypes 2 and 9 affects colonization and transmission of each type, and mortality. Thirty-six caesarean-derived/colostrum-deprived piglets were randomly assigned to three groups, and there housed pair-wise. At six weeks old, one pig per pair was inoculated with either one (serotype 2 or 9; mono-group) or two serotypes simultaneously (dual-group); the other pig was contact-exposed. Tonsillar and nasal samples were collected within three weeks post inoculation. Bacterial loads in samples were quantified using multiplex real-time polymerase chain reaction (PCR). Transmission rates of the serotypes among pigs were estimated using a mathematical Susceptible-Infectious (SI) model. Bacterial loads and transmission rates did not differ significantly between serotypes. Compared to the mono-group, in the dual-group the average serotype 2 load in tonsillar samples from contact pigs was reduced on days 1 to 4 and on day 6. Simultaneous exposure to the serotypes reduced the mortality hazard 6.3 times (95% C.I.: 2.0-19.8) compared to exposure to serotype 2 only, and increased it 6.6 times (95% C.I.: 1.4-30.9) compared to exposure to serotype 9 only. This study indicates that serotype 2 load and mortality were affected in pigs exposed to these two serotypes.

Quantifying antimicrobial resistance at veal calf farms.

This study was performed to determine a sampling strategy to quantify the prevalence of antimicrobial resistance on veal calf farms, based on the variation in antimicrobial resistance within and between calves on five farms. Faecal samples from 50 healthy calves (10 calves/farm) were collected. From each individual sample and one pooled faecal sample per farm, 90 selected Escherichia coli isolates were tested for their resistance against 25 mg/L amoxicillin, 25 mg/L tetracycline, 0.5 mg/L cefotaxime, 0.125 mg/L ciprofloxacin and 8/152 mg/L trimethoprim/sulfamethoxazole (tmp/s) by replica plating. From each faecal sample another 10 selected E. coli isolates were tested for their resistance by broth microdilution as a reference. Logistic regression analysis was performed to compare the odds of testing an isolate resistant between both test methods (replica plating vs. broth microdilution) and to evaluate the effect of pooling faecal samples. Bootstrap analysis was used to investigate the precision of the estimated prevalence of resistance to each antimicrobial obtained by several simulated sampling strategies. Replica plating showed similar odds of E. coli isolates tested resistant compared to broth microdilution, except for ciprofloxacin (OR 0.29, p ≤ 0.05). Pooled samples showed in general lower odds of an isolate being resistant compared to individual samples, although these differences were not significant. Bootstrap analysis showed that within each antimicrobial the various compositions of a pooled sample provided consistent estimates for the mean proportion of resistant isolates. Sampling strategies should be based on the variation in resistance among isolates within faecal samples and between faecal samples, which may vary by antimicrobial. In our study, the optimal sampling strategy from the perspective of precision of the estimated levels of resistance and practicality consists of a pooled faecal sample from 20 individual animals, of which 90 isolates are tested for their susceptibility by replica plating.

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration.

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte-like cells (CLCs). Wnt/ß-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non-chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non-chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/ß-catenin signaling in the healthy, NC-rich NP and early degenerated, CLC-rich NP of both breed types by immunohistochemistry of ß-catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c-myc. Both NCs and CLCs showed nuclear and cytoplasmic ß-catenin protein expression and axin2 gene expression, but ß-catenin signal intensity and Wnt target gene expression were higher in the CLC-rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/ß-catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c-myc gene expression. In conclusion, Wnt/ß-catenin signaling activity in the NC within the NC-rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/ß-catenin signaling activity in early IVD degeneration may be a reflection of its dual role.

Rate of foot-and-mouth disease virus transmission by carriers quantified from experimental data.

Upon infection with foot-and-mouth disease virus (FMDV) a considerable number of animals become carriers of the virus. These carriers are considered to be a risk for new outbreaks, but the rate at which these animals can transmit the infection has not been quantified. An analysis was carried out using data from previously published experiments in order to quantify the transmission rate parameter beta of FMDV infection from carriers to susceptible animals. The parameter beta was estimated at 0.0256 (likelihood-based confidence interval: 0.008-0.059) infections per carrier per month. Moreover, analysis of published experimental data indicates that the proportion of FMDV carriers decreases at a rate of 0.115 per month. Both parameters obtained from this study are useful for quantitative risk analyses of the trade of animals from FMDV-infected areas or the lifting of vaccination programs.

Transmission of the highly pathogenic avian influenza virus H5N1 within flocks during the 2004 epidemic in Thailand.

This present study is the first to quantify the transmission of avian influenza virus H5N1 within flocks during the 2004 epidemic in Thailand. It uses the flock-level mortality data to estimate the transmission-rate parameter ( beta ) and the basic reproduction number (R(0)). The point estimates of beta varied from 2.26/day (95% confidence interval [CI], 2.01-2.55) for a 1-day infectious period to 0.66/day (95% CI, 0.50-0.87) for a 4-day infectious period, whereas the accompanying R(0) varied from 2.26 (95% CI, 2.01-2.55) to 2.64 (95% CI, 2.02-3.47). Although the point estimates of beta of backyard chickens and fighting cocks raised together were lower than those of laying hens and broiler chickens, this difference was not statistically significant. These results will enable us to assess the control measures in simulation studies. They also indicate that, for the elimination of the virus, a critical proportion of the susceptible poultry population in a flock (i.e., 80% of the population) needs to be vaccinated.