Mass depopulation of laying hens in whole barns with liquid carbon dioxide: evaluation of welfare impact.

Appropriate emergency disaster preparedness is a key priority for agricultural agencies to allow effective response to serious avian disease outbreaks. There is a need to develop rapid, humane, and safe depopulation techniques for poultry that are widely applicable across a range of farm settings. Whole barn depopulation with carbon dioxide (CO(2)) has been investigated as a humane and efficient means of killing large numbers of birds in the event of a reportable disease outbreak. It has also been considered as a method for depopulating barns containing end-of-lay hens, particularly when there is limited local slaughter and rendering capacity. Determining the best method of humanely killing large flocks of birds remains problematic and is being investigated by a coordinated international effort. While whole barn depopulation using CO(2) inhalation has been explored, physiologic responses of chickens have not been characterized in field settings and assessment of animal welfare is hampered without this information. In this study, 12 cull laying hens were surgically instrumented with telemetry transmitters to record electroencephalographs, electrocardiographs, body temperature, and activity during 2 large-scale field CO(2) euthanasia trials of end-of-lay hens. The day following surgery, instrumented hens were placed in barns with other birds, barns were sealed, and animals were killed by CO(2) inhalation delivered via a specially designed liquid CO(2) manifold. Instrumented birds were monitored by infrared thermography, and ambient temperature, CO(2), and O(2) concentrations were recorded. Results from these studies indicate that instrumented hens lost consciousness within 2 min of CO(2) levels reaching 18 to 20%. Mild to moderate head shaking, gasping, and 1 to 2 clonic muscle contractions were noted in hens before unconsciousness; however, brain death followed rapidly (<5 min). Evaluation of welfare costs and benefits suggest clear advantages over catching and transporting cull hens for slaughter. The financial costs with this method are greater, however, than those estimated for traditional slaughter techniques. Results of these studies are being used to develop national protocols for whole barn depopulation of hens by CO(2) inhalation.

Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations.

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.

Cellular and cytokine responses in feathers of chickens vaccinated against Marek's disease.

In Marek's disease virus infection, feather follicle epithelium (FFE) constitutes the site of formation of infectious virus particles and virus shedding. The objective of this study was to characterize cellular and cytokine responses as indicators of cell-mediated immune response in FFE and associated feather pulp following immunization against Marek's disease. Analysis of feather tips collected between 4 and 28 days post-immunization (d.p.i.) from chickens vaccinated post-hatch with either CVI988/Rispens or herpesvirus of turkeys revealed that replication of these vaccine viruses started at 7d.p.i., peaked by 21d.p.i., and subsequently, showed a declining trend. This pattern of viral replication, which led to viral genome accumulation in feather tips, was associated with infiltration of T cell subsets particularly CD8+ T cells into the feather pulp area and the expression of cytokine genes such as interferon-gamma, which is an indication of elicitation of cell-mediated immune responses at the site of virus shedding.

Cellular and cytokine responses associated with dinitrofluorobenzene-induced contact hypersensitivity in the chicken.

The objective of the study was to determine the cellular and cytokine responses associated with dinitrofluorobenzene (DNFB)-induced skin contact hypersensitivity (SCH), as an indicator of cell-mediated immune response, in the chicken. The thickness of the DNFB-treated foot web was increased by 6h.p.i. (hours post-induction), peaked by 24h.p.i. and then declined gradually until the lowest measurements were observed at 72h.p.i. Infiltration of eosinophils was the highest at 6 and 12h.p.i. and gradually declined by 48h.p.i. The degree of infiltration of both CD8+ and CD4+ T cells varied with mild infiltration observed at 6h.p.i., moderate to heavy infiltration observed at 12h.p.i. that persisted through 24 and 48h.p.i. and declined by 72h.p.i. Infiltration of macrophages during the study period was prominent, yet less remarkable differences were recorded between observations. Expression of interleukin (IL)-4, IL-6, IL-10 and interferon (IFN)-gamma in skin tissue was at its highest at 6h.p.i. compared to other observed time points, yet only the expression of IFN-gamma and IL-10 genes turned out to be significantly higher at 6h.p.i. compared to all other time points. In conclusion, DNFB-induced SCH in chicken was associated with an early up-regulation of cytokine genes, and infiltration of eosinophils along with macrophages, CD8+, and CD4+ T cells at the site of induction.

Genetic diversity of Clostridium perfringens isolated from healthy broiler chickens at a commercial farm.

Clostridium perfringens is an important commensal and bacterial pathogen of many animal species. It has particular significance in poultry, where it may cause necrotic enteritis. Our objective was to characterize the population diversity of C. perfringens colonizing healthy birds, and to observe how diversity changed over time. Isolates were obtained from broiler chicken cecal samples in two barns on a single farm, on days 7, 14, 22, 27, 30 and 34 of a single 42-day rearing cycle. Bacitracin was used as a feed additive in one of the barns and withdrawn from the second barn for the duration of the experiment. Each isolate was typed using pulsed-field gel electrophoresis (PFGE) using SmaI restriction endonuclease. A total of 205 cecal isolates from 49 birds were typed, as well as 93 isolates from the barn environment (bedding, drinking water and feces). Eight major PFGE types and 17 subtypes were found in the 298 total isolates. The results show that an optimal sampling strategy would involve a large number of birds, with only a few isolates sampled per bird. The diversity of C. perfringens in this study appears to be low within a single bird, and increases as the bird matures. There was no significant difference in genetic diversity between the two barns. In addition, isolates from fresh fecal samples appear to represent the cecal C. perfringens population accurately, although this was not proven statistically. Antimicrobial susceptibility testing was performed on selected isolates (n=41) representing a cross-section of PFGE types. Based on minimum inhibitory concentration distributions, 95% of the isolates tested were deemed resistant to bacitracin, with a 16 microg/mL breakpoint. Three new cpb2 (beta2 toxin gene) variants were found in the study.

Ontogeny of cytokine gene expression in the chicken spleen.

Cytokine gene expression in the chicken spleen during embryogenesis and the early posthatch period was investigated in the present study. The constitutive expression of interleukin-4, interleukin-10, interleukin-18, and interferon-gamma genes was detectable as early as embryonic day 12. Expression of cytokine genes was higher in the spleen of posthatch chickens compared with chick embryos. There was a gradual increase in expression of all the cytokine genes in the spleen, which peaked by d 7 posthatch. This expression pattern coincided with the completion of T-cell colonization and structural development of the spleen during the early posthatch period. It is therefore possible that the cytokines examined in the present study are involved in the maturation of colonized T cells and in shaping the spleen microenvironment.

Avian hepatitis E virus in an outbreak of hepatitis--splenomegaly syndrome and fatty liver haemorrhage syndrome in two flaxseed-fed layer flocks in Ontario.

Two commercial layer chicken flocks that were fed a flax-based diet beginning at 28 weeks of age for the production of omega-3 fatty-acid-enriched eggs experienced increased mortality when the birds reached 37 weeks. The average weekly mortality was 0.34% over a 20-week period, with peak mortality of 0.9% for 1 week. Reduced feed consumption, reduced body weight gain and poor peak production were noticed prior to the onset of increased mortality. A total of 245 birds were necropsied and 78% of these had lesions in the liver and spleen, with 44% of those necropsied having changes consistent with hepatitis-splenomegaly syndrome, with lesions ranging from acute periportal lymphoplasmacytic hepatitis to chronic severe cholangiohepatitis with haemorrhage, vasculitis and amyloidosis. A total of 11% of the birds had lesions typical of fatty liver haemorrhagic syndrome, and 22% had lesions found in both hepatitis-splenomegaly syndrome and fatty liver haemorrhagic syndrome. No significant bacteria or viruses were recovered from samples of the liver/bile or spleen but 11 of 21 bile samples contained avian hepatitis E virus RNA detectable with a reverse transcriptase-polymerase chain reaction assay. Comparative sequence analysis found identities of 82 to 92% and 78 to 80% between the helicase and capsid protein genes, respectively, of the virus detected in this outbreak and those of other avian hepatitis E virus isolates, suggesting extensive genetic heterogeneity in avian hepatitis E viruses in Ontario flocks.

Mechanisms of strontium and uranium removal from high-level radioactive waste simulant solutions by the sorbent monosodium titanate.

High-level waste (HLW) is a waste associated with the dissolution of spent nuclear fuel for the recovery of weapons-grade material. It is the priority problem for the U.S. Department of Energy's Environmental Management Program. Current HLW treatment processes at the Savannah River Site (Aiken, SC) include the use of monosodium titanate (MST, with a similar stoichiometry to NaTi2O5 x xH2O) to concentrate strontium (Sr) and actinides. The high affinity of MST for Sr and actinides in HLW solutions rich in Na+ is poorly understood. Mechanistic information about the nature of radionuclide uptake will provide insight about MST treatment reliability. Our study characterized the morphology of MST and the chemistry of sorbed Sr2+ and uranium [U(VI)] as uranyl ion, UO2(2+), on MST, which were added (individually) from stock solutions of Sr and 238U(VI) with spectroscopic and transmission electron microscopic techniques. The local structure of sorbed U varied with loading, but the local structure of Sr did not vary with loading. Sorbed Sr exhibited specific adsorption as partially hydrated species whereas sorbed U exhibited specific adsorption as monomeric and dimeric U(VI)-carbonate complexes. Sorption proved site specific. These differences in site specificity and sorption mechanism may account forthe difficulties associated with predicting Sr and U loading and removal kinetics using MST.

Retrospective evidence that the MHC (B haplotype) of chickens influences genetic resistance to attenuated infectious bronchitis vaccine strains in chickens.

Infectious bronchitis is a respiratory disease of chickens that is caused by the coronavirus infectious bronchitis virus (IBV). Virtually all broiler and layer breeder flocks are routinely vaccinated against IBV. Two hatches of 1-day-old chicks from four lines were mistakenly vaccinated for infectious bronchitis using a moderately attenuated vaccine designed for chicks of an older age. The vaccination resulted in high mortality, and chicks from three of four lines died with signs typical of infectious bronchitis. The mortality that occurred using this less-attenuated vaccine was significantly influenced by the genetic line, and the MHC (B) haplotype in chickens of three B congenic lines. B congenic chickens possessing the B*15 haplotype were resistant in contrast to chickens possessing the B*13 or B*21 haplotypes. Chicks from two further hatches of the four lines were vaccinated appropriately with a more attenuated IBV vaccine, and only limited chick mortality was seen. These retrospective data from two repeated hatches confirm earlier data indicating chicken genes influence resistance to IBV, and indicate for the first time that genes tightly linked to the B haplotype are relevant in resistance to IBV. Due to extenuating circumstances it was not possible to verify results with chicks from F2 matings. Factors that may enhance definition of the role of the B haplotype in immune response to IBV, and the desirability for further analysis of a B haplotype-linked influence on immunity to IBV are discussed.

Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens.

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.

Safety, immunogenicity, and efficacy of two Escherichia coli cya crp mutants as vaccines for broilers.

Attenuated derivatives (delta cya delta crp mutants) of an O2 and an O78 avian septicemic Escherichia coli strain were used to immunize broiler chickens by spray to determine the safety, immunogenicity, and efficacy of the derivatives in single- and double-dose regimens. In the safety and immunogenicity studies, groups of 10 chickens were vaccinated by spray (droplet size approximately 20 microm) with the parent E. coli, the mutant organisms, or phosphate-buffered saline (PBS) at 14 days of age and euthanatised 21 days later. There was no deaths or gross pathologic finding in any of the chickens immunized with the vaccine strains. Compared with the levels in chickens exposed to PBS, there were significantly higher levels of immunoglobulin (Ig) G antibody in serum and air sac washings and of IgA antibody in air sac washings in response to the virulent parent strains than to the vaccine strains. In efficacy studies, chickens were immunized with the O2 or the O78 vaccine strain or PBS at day 14 and with the O2 vaccine strain or PBS at days 10 and 14 and challenged with the parent strain 10 days after the last vaccination. There was no significant difference in local IgA and IgG and serum IgG responses between vaccinated and control groups. Chickens vaccinated with the O2 strain, but not the O78 strain, had significantly lower air sac lesion scores compared with those of the unvaccinated groups in both single- and double-dose regimens. We conclude that the mutant O2 strain provided moderate protection against airsacculitis.

Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2.

Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus). We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E. coli O2. Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E. coli O2 isolate. A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose. An analogous protein in air sac fluid proteins bound to intact E. coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients. However, endogenous amino acid sequences were unrelated to other known proteins. LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins. These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E. coli responsible for respiratory disease.

Comparison of two micro-analytical methods for detecting the spatial distribution of sorbed Pu on geologic materials.

Subsurface transport of groundwater contaminants is greatly influenced by chemical speciation, precipitation and sorption processes at the mineral-water interface. The retardation of contaminants is often greatest at boundaries between minerals and in fractures and pore spaces. The investigation of the spatial distribution of sorbed contaminants along these boundaries requires micro-analytical techniques. The sorption of dissolved Pu(V) on a natural zeolitic tuff from Yucca Mountain (NV, USA) was examined using microautoradiography (MAR), X-ray diffraction (XRD), electron microprobe (EM) techniques, and synchrotron-based micro-X-ray fluorescence (micro-SXRF). The tuff contained a heterogeneous distribution of zeolites and trace quantities of smectites, Fe oxides (hematite), and Mn oxides (rancieite), which are present as fracture fill and pore space materials. Micro-SXRF studies showed that Pu is mostly associated with bodies of smectite plus Mn oxides, which were typically elevated in Ce, Ga, Nb, Pb, Y, Ca, Ti, and Zn. Sorbed Pu was not associated with Fe-rich bodies, which were enriched in Cl and Rb. Results of the MAR studies were complementary to that of the micro-SXRF studies in that Pu was associated with similar elements in the tuff. Indirect detection of Pu by EM or micro-SXRF (by analyzing Ag developed on the MAR photoemulsion) was a more sensitive method for detecting lower levels of sorbed Pu than the direct detection of sorbed Pu via micro-SXRF in the absence of the photoemulsion.

Hygiene at winter bird feeders in a southwestern Ontario city.

To further understand the source of the epidemic of salmonellosis in some species of birds using bird feeders in southern Ontario in the winter of 1997-1998, 124 bird feeder stations were examined for their state of hygiene and for Salmonella on 5 occasions during the winter of 1999 in a city of 100,000 people in southwestern Ontario. No Salmonella were isolated from feed contaminated with feces recovered from the feeders. Squirrel-proof feeders were significantly less contaminated with feces than were other feeder types (hopper, platform, silo), which did not differ significantly in their hygiene scores. Contamination of squirrel-proof feeders increased significantly through the course of the study, but other feeder types showed no significant change. Hygiene was poorer if feeders were maintained equally by both male and female household members, particularly as they grew older, but no age or gender effect was observed if only one person was largely responsible for maintaining the feeders. We concluded that winter bird feeder stations in a southern Ontario city were not contaminated with Salmonella but that bird feeder stations could be designed better to reduce fecal contamination of feed.

Direct measurement of aluminum uptake and distribution in single cells of Chara corallina.

Quantitative information on the uptake and distribution of Al at the cellular level is required to understand mechanisms of Al toxicity, but direct measurement of uptake across the plasma membrane has remained elusive. We measured rates of Al transport across membranes in single cells of Chara corallina using the rare (26)Al isotope, an emerging technology (accelerator mass spectrometry), and a surgical technique for isolating subcellular compartments. Accumulation of Al in the cell wall dominated total uptake (71-318 microgram m(-2) min(-1)), although transport across the plasma membrane was detectable (71-540 ng m(-2) min(-1)) within 30 min of exposure. Transport across the tonoplast was initially negligible, but accelerated to rates approximating uptake across the plasma membrane. The avacuolate protoplasm showed signs of saturation after 60 min, but continued movement across the plasma membrane was supported by sequestration in the vacuole. Saturation of all compartments was observed after 12 to 24 h. Accumulation of Al in the cell wall reflected variation in [Al(3+)] induced by changes in Al supply or complexing ligands, but was unaffected by pH. In contrast, transport across the plasma membrane peaked at pH 4.3 and increased when [Al(3+)] was reduced by complexing ligands. Cold temperature (4 degrees C) reduced accumulation in the cell wall and protoplasm, whereas 2,4-dinitrophenol and m-chlorocarbonylcyanidephenyl hydrazone increased membrane transport by 12- to 13-fold. Our data suggest that the cell wall is the major site of Al accumulation. Nonetheless, membrane transport occurs within minutes of exposure and is supported by subsequent sequestration in the vacuole. The rapid delivery of Al to the protoplasm suggests that intracellular lesions may be possible.

Observations on pigeon circovirus infection in Ontario.

Subclinical pigeon circovirus infection was diagnosed in 1-day-old to 6-week-old birds from a loft with no history of clinical disease. Pigeons from other lofts presented with various illnesses and were found at necropsy to be concurrently infected with pigeon circovirus.

Evaluation of different procedures for processing avian air sac lavage fluid.

Air sac lavage has become a routine diagnostic tool used in pet birds; however, cytologic techniques for collection and handling of avian cells have not been reported. In this study, directed endoscopy was used to obtain the air sac washes. Different factors were compared that might affect final preservation of cell morphology, such as centrifugation speed, anticoagulant, storage temperature, time to processing, addition of glutaraldehyde to the sample, and addition of protein. The goal was to find the processing technique best suited for the evaluation of cells from air sac washes. The use of an anticoagulant did not influence cell quality. Optimal cell morphology was achieved when the sample was centrifuged at a speed of 1000 rpm (89.4 X g) and when prepared in less than an hour from the time of collection. Low temperature (5 C) tended to preserve cell quality better. The addition of protein (fetal bovine serum) helped to preserve cell quality and was of value if the sample storage time was greater than 30 min. The addition of glutaraldehyde resulted in a significant reduction in cell quality.

Development of the thoracic air sacs of turkeys with age and rearing conditions.

Cytology and structure of the thoracic air sac of turkeys were investigated at four different ages (26-day embryo, 1 day, 2 wk, and 10 wk old) and two rearing conditions (isolation and commercial). Cytology was performed by guided fiberoptic endoscopy on the left thoracic air sac of each bird. The right thoracic air sac was sampled for light and electron microscopy. Heterophils were the most common nonepithelial cell found in air sac fluid. followed by macrophages and lymphocytes. Macrophages were most abundant in 1-day-old turkeys and turkeys raised in commercial conditions. The epithelium of the air sac consisted of squamous and cuboidal cells, with a few ciliated columnar and nonciliated columnar cells. Cuboidal cells had similar characteristics to type II pneumocytes. The mucociliary system was organized in tracts extended from the ostium to the posterior parts of the air sac. The number of ciliated tracts decreased with age, and the air sacs of commercial turkeys had a larger proportion of ciliated epithelium than did those of isolation birds. The epithelium may protect against disease by a structured mucociliary transport system, the production of surfactant, and phagocytosis of foreign particles. Differences in cytology and structure may reflect the maturation of the immune system and/or response to environment.