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.

Fluorochrome-labeled deoxyribonuclease: specific stain for cell nuclei.

Nuclei of cells in tissue culture may be stained with deoxyribonuclease labeled with fluoresceitn isothiocyatnate; this is a simple, rapid, and specific process which is useful in localizing DNA in situ.

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.

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.

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.

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.

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 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.

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.

Endoscopic gross anatomy findings of the thoracic air sac of the turkey.

The gross anatomy of the cranial thoracic air sacs was studied in the live turkey with a fiber-optic endoscope fitted with a video system. Thirty-five out of 39 (89.7%) of the turkeys had paired single thoracic air sacs with two ostia. The other 10.3% of the turkeys had paired cranial and caudal thoracic air sacs, each with a single ostium. The air flow pattern through the common thoracic air sac with two ostia is unknown. Furthermore, 42.3% of the turkeys with a single thoracic air sac had an invagination arising from the floor of the air sac that partially divided the single thoracic air sac creating a blind-ended sac. The blind sac formed by the invagination may trap small inhaled particulate matter.

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.

Effect of exercise on the cardiovascular and respiratory systems of heavy turkeys and relevance to sudden death syndrome.

The cardiorespiratory response to walking exercise was studied in 14-to-18-week-old male and female heavy turkeys. Blood pressure, electrocardiograms, respiratory rate, body temperature, blood gases, and electrolytes were measured at rest and after exercise at two different room temperatures. After exercise, systolic, diastolic, and mean blood pressures decreased, and heart rate and body temperature increased, while a severe lactic acidosis developed, in both sexes and at both room temperatures. Fifty-four percent of the turkeys developed arrhythmias, mostly a transient post-exercise sinus arrhythmia of no clinical significance. Changes in electrolytes were clinically insignificant. Blood gases remained within normal limits, demonstrating adequate gas-exchange and pulmonary capability. Within the limits of this study, the cardiac capacity of the heavy turkey in response to exercise appeared very limited. The pathogenesis of sudden death syndrome of turkeys is still unknown, but cardiovascular changes occurring after exercise could be severe enough to cause death.

Effect of exercise on cardiac output and other cardiovascular parameters of heavy turkeys and relevance to the sudden death syndrome.

The cardiovascular response to treadmill exercise was studied in 14-to-18-week-old male and female heavy turkeys. Arterial blood pressure, cardiac output, body temperature, and heart rate were measured before and during each period of exercise. Total peripheral resistance, stroke volume, and stroke index were calculated from those parameters. Systolic, diastolic, and mean blood pressures significantly decreased during exercise. Cardiac output increased during exercise in males and females by 64% and 23%, respectively, while total peripheral resistance fell by approximately 40% in both sexes. Exercise had no significant effect on stroke volume and stroke index. The cardiac functional capacity of the heavy turkey in response to mild exercise appears very limited. This failure of the cardiovascular system to adequately respond to increased metabolic needs suggests that hemodynamic failure is a major component in the pathogenesis of sudden death syndrome of turkeys.

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.

Cardiac muscle mass distribution in the domestic turkey and relationship to electrocardiogram.

Ventricular weights were measured in 20-week-old male and female heavy turkeys, and electrocardiograms (ECGs) were recorded in 10- and 20-week-old male and female heavy turkeys. Twenty-week-old males had heavier relative left ventricular and total ventricular weights than females of the same age. Left-to-right ventricular weight ratios were 4.4:1 in males and 4.5:1 in females. Heart rate was faster in young birds and in females, and that influenced the duration of the intervals of the ECG. Wave amplitudes were smaller in 10-week-old turkeys than in 20-week-old turkeys. There was no correlation between electrocardiographic parameters and ventricular weights of male and female turkeys. Two-dimensional ECGs were therefore found to be of minimal value for detecting turkeys with heavier ventricles.

Use of equine chorionic gonadotrophin in female mink.

This study was comprised of three trials to determine the effects of equine chrionic gonadotrophin (eCG) on induction of sexual receptivity in female mink that had failed to mate by late in the breeding season. In the first trial one ovary was removed from unmated mink, which were then injected with 100 IU eCG. This treatment induced ovarian activity, including ovulation in the remaining ovary. In the second experiment, mink that had not been observed to mate were treated with 100 IU eCG or saline, resulting in mating of 10 11 of the eCG-treated animals, compared to 5 11 controls. Litter sizes were larger in mink in the control group, suggesting that eCG interfered with some phase of the reproductive process. In the third trial, 226 mink that had failed to mate until late in the breeding season were treated with 100 IU eCG. Of the 191 that subsequently mated, 99 produced litters, but litter sizes were reduced slightly from those observed in the remainder of the herd that bred without hormone treatment prior to March 20. Neonatal kit loss per female whelping was greater in mink treated with eCG. It is concluded that eCG treatment will induce mating in mink that refuse to mate, but this treatment results in reduced whelping success and greater neonatal kit loss. Its utility may be restricted to salvage situations where large numbers of mink fail to mate.

Campylobacter colitis in ranch mink in Ontario.

Outbreaks of colitis, where Campylobacter jejuni and Campylobacter coli were the only pathogens isolated occurred in weanling mink (Mustella vision) on two commercial mink ranches in Ontario. Lesions were restricted to the proximal colon and were characterized by multiple 1 mm focal or 1 mm linear erosions/ulcers in the region 2 cm distal to the ileal-colonic junction. Histological changes included thickening of the colonic mucosa, inflammatory cell infiltrate in the lamina propria and submucosa, cellular debris and inflammatory exudate within cryptal lumens and multiple areas of mucosal erosion/ulceration. Four C. jejuni negative mink were challenged with 5.1 X 10(9) colony forming units of C. jejuni by oral inoculation. Three of four experimentally infected mink developed diarrhea by day 4 postinfection with lesions grossly and microscopically similar to mink in the naturally occurring outbreak. Examination of lesions by transmission electron microscope failed to show evidence of C. jejuni invasion of intestinal epithelium. Feeding uncooked slaughterhouse chicken offal was the likely source of C. jejuni in the naturally occurring outbreaks.

Mortality in fledgling great horned owls from black fly hematophaga and leucocytozoonosis.

Black fly feeding alone and in concert with Leucocytozoon spp. infection caused mortality in fledgling great horned owls (Bubo virginianus) in the Yukon, Canada 1990 to 1991. These mortalities occurred during a year of food shortage corresponding with a decline in the population of snowshoe hare (Lepus americanus), the main prey for great horned owls. We hypothesize an interaction between food availability and the consequences of host-parasite interactions.

Tyzzer's disease in muskrats: occurrence in free-living animals.

Tyzzer's disease was diagnosed in four muskrats (Ondatra zibethica) found dead over a 2 month period in a single feed-house in a Saskatchewan marsh. No dead animals were found elsewhere in the marsh, although several hundred apparently healthy animals were trapped during this period. Similarities in the pathology and epizootiology of Tyzzer's and Errington's diseases of muskrats support an hypothesis that these diseases are a single entity.