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July 2001, Volume 25, Number 7, Pages 990-996
Table of contents    Previous  Article  Next   [PDF]
Paper
Transmissibility of adenovirus-induced adiposity in a chicken model
N V Dhurandhar1, B A Israel2, J M Kolesar3, G Mayhew4, M E Cook5 and R L Atkinson6

1Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan, USA

2Department of Pathobiological Sciences, University of Wisconsin, Madison, Wisconsin, USA

3School of Pharmacy, University of Wisconsin, Madison, Wisconsin, USA

4Department of Genetics, University of Wisconsin, Madison, Wisconsin, USA

5Department of Poultry Science, University of Wisconsin, Madison, Wisconsin, USA

6Department of Medicine and Nutritional Sciences, University of Wisconsin, Madison, Wisconsin, USA

Correspondence to: N V Dhurandhar, Department of Nutrition and Food Science, Wayne State University, 3009 Science Hall, Detroit, MI 48202, USA. Email: ndhurand@sun.science.wayne.edu

Abstract

BACKGROUND: We previously reported that human adenovirus Ad-36 induces adiposity and paradoxically lower levels of serum cholesterol (CHOL) and triglycerides (TG) in animals.

OBJECTIVE: To evaluate the transmissibility of Ad-36 and Ad-36 induced adiposity using a chicken model.

DESIGN: Experiment 1¾four chickens were housed (two per cage) and one from each cage was inoculated with Ad-36. Duration of presence of Ad-36 DNA in the blood of all chickens was monitored. Experiment 2¾two groups of chickens were intranasally inoculated with Ad-36 (infected donors, I-D) or media (control donors, C-D). Blood drawn 36 h later from I-D and C-D groups was inoculated into wing veins of recipient chickens (infected receivers, I-R, and control receivers, C-R, respectively). On sacrifice, 5 weeks post-inoculation, blood was drawn, body weight noted and visceral fat was separated and weighed.

RESULTS: Experiment 1¾Ad-36 DNA appeared in the blood of the inoculated chickens and that of uninoculated chickens (cage mates) within 12 h of inoculation and the viral DNA persisted up to 25 days in the blood. Experiment 2¾compared with C-D, visceral and total body fat were significantly greater and CHOL significantly lower for the I-D and I-R. TG were significantly lower for the I-D. Ad-36 was isolated from 12 out of 16 blood samples of the I-D that were used for inoculating I-R chickens. Ad-36 DNA was present in the blood and the adipose tissue of the I-D and I-R but not in the skeletal muscles of animals selected randomly for testing.

CONCLUSION: As seen in experiment 1, Ad-36 infection can be transmitted horizontally from an infected chicken to another chicken sharing the cage. Additionally, experiment 2 demonstrated blood-borne transmission of Ad-36-induced adiposity in chickens. Transmissibility of Ad-36-induced adiposity in chicken model raises serious concerns about such a possibility in humans that needs further investigation.

International Journal of Obesity (2001) 25, 990-996

Keywords

cholesterol; triglycerides; body fat; visceral fat; Ad-36; obesity

Introduction

Obesity has been called the number one public health problem in America.1 Although obesity is recognized as a disease of multiple etiologies, a virus infection as an etiological factor has been ignored until now. Five different viruses have been shown to cause obesity in animal models,2,3,4,5,6,7,8 but until recently there was no evidence to suspect that viruses might play a role in the etiology of human obesity. We have shown that Ad-36, a human adenovirus, produces adiposity and paradoxically low levels of serum cholesterol (CHOL) and triglycerides (TG) in animal models,8 and that a unique syndrome consisting of low serum CHOL and TG levels is present in about 30% of obese humans subjects from three different states (Wisconsin, Florida and New York) who have antibodies to Ad-36.9,10 Only 5% of the lean humans tested to date from the three states had antibodies to Ad-36, suggesting that infection with this virus carries a high probability of association with obesity.10 The possible link between a virus and obesity in humans warrants serious investigation of the adiposity-promoting effect of this virus. For ethical reasons, we cannot infect humans to study the virus-induced adiposity and, therefore, we have used animal models to understand the characteristics of Ad-36 induced adiposity. Chickens develop Ad-36-induced adiposity in as little as 3 weeks8 and therefore we have used a chicken model in this experiment.

Not much is known about the transmissibility of Ad-36 and Ad-36 induced adiposity. Blood is one of the possible routes of viral transmission. This paper describes two experiments which address the issue of Ad-36 transmission in a chicken model.

Methods

Experiment 1

The purpose of this experiment was to investigate whether Ad-36 inoculation is followed by appearance of the viral titers in the blood and to determine if exposure by close contact with Ad-36-infected animals produces infection. Four specific pathogen free (SPF) male chickens (1 day old) were obtained from Specific Pathogen-Free Avian Supply (SPAFAS, Roanoke, IL). At 3 weeks of age they were housed two per cage and one chicken from each cage was inoculated intranasally (i.n.) with Ad-36 (4´106 plaque forming units, PFU). The other chicken from each cage was not inoculated and served as a chicken 'in-contact' with the Ad-36 inoculated chicken. Blood was drawn from all the chickens before inoculation and 12 h, 36 h, 4 days, 11 days, 18 days and 25 days post inoculation. DNA obtained from the blood was screened for the presence of Ad-36 DNA using capillary electrophoresis technique described below.

Experiment 2

This experiment tested the possibility of blood-borne transmission of infection and of Ad-36-associated adiposity. One-day-old male SPF white leghorn broiler chickens (n=64) were obtained from SPAFAS and were housed under Biosafety level 2 conditions, with negative air pressure in each room. Food (Purina Starter Grow) and water were provided ad libitum throughout the study period and food consumption, corrected for spillage, was recorded for individual cages. For the first 3 weeks, the chicks were housed in a brooder with a 12 h light cycle and a temperature of 95°F that was reduced gradually to 70°F at the end of 3 weeks. At 3 weeks of age, the chickens were removed from the brooder and maintained at 70±2°F thereafter. Chickens were weighed at 1 week, 3 weeks, and then every week until the termination of the study at 8 weeks.

After 3 weeks, the chickens were divided into four weight-matched groups of 16 each. Blood was drawn from a wing vein for measurement of adenovirus antibodies to insure that the chickens had not been previously exposed to Ad-36. These assays were repeated at the time of sacrifice. At 4 weeks of age one group (infected donors, I-D) was inoculated i.n. with 200 mul of Ad-36 virus suspension (1.8´106 PFU). A second group (control donors, C-D) was inoculated i.n. with 200 mul sterile media. After 36 h, blood was drawn from the wing veins of the chickens from I-D and C-D groups and 200mul blood was injected into the wing veins of I-R (infected receivers) and C-R (control receivers) groups, respectively. About 400 mul blood from I-D and I-R groups was also used to isolate the virus. Blood was drawn 10 days post inoculation to screen for Ad-36 antibodies.

Five weeks after inoculation the animals were fasted overnight and sacrificed. The omental-mesenteric (visceral) fat from each bird was carefully dissected and weighed. Body weights were measured and blood was drawn to determine CHOL and TG. About 1 g each of visceral fat and skeletal muscle from the right side of the keel bone (breast muscle) were flash frozen in liquid nitrogen. Total fat content of the carcasses of 11 chickens from each group was determined using the Folch extraction method.11 DNA obtained from blood, visceral adipose tissue and skeletal muscle from three chickens each from the four groups was assayed for Ad-36 DNA by capillary electrophoresis assay.

Statistical analysis

In experiment 1, log (number of Ad-36 DNA copies per ml of blood) was plotted against the time points. In experiment 2, student's t-test followed by Bonferroni adjustments were used to analyze the differences in the means. Chi square test was used to analyze the difference in the prevalence of obesity in the four groups.

Techniques and assays

Utilization of the virus

Media used for tissue culture: Minimum Essential Media Eagle (MEM) (catalog no. M-0643, Sigma Chemicals) with non-essential amino acids, Earle's salts and l-glutamine was used for growing A549 cells for tissue culture. MEM with 10% fetal bovine serum (FBS), 2.9% NaHCO3 (v/v) pH 7.4, was used for growing A549 cells, and for harvesting or isolating the virus.

Preparation of plaque-purified Ad-36 (Ad-36P): Human adenovirus-36 (Ad-36, ATCC no. VR-913) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and the work stock was grown on A549 cells, a human bronchial carcinoma tissue culture line. This virus was plaque purified as described below to obtain genetic homogeneity.

Two hundred microliters of the 10-4 dilution of the virus work stock was inoculated in two wells of a six-well plate. Two wells of the plate were inoculated with 200 mul of media as negative controls. Plates were overlaid with 2 ml of 1% agar in media in each well, and the plates were inverted and incubated at 37°C till the plaques appeared. Under a microscope cells from a single plaque were aspirated using a micropipette, placed in 200 mul of media, and diluted 100-fold. This was considered the first passage. The diluted virus solution was once again grown on A549 cells in a six-well plate and the procedure to select a single plaque was repeated. A total of three such passages were done and the resultant purified virus work stock was grown and was used for all the experiments. This purified Ad-36 tested positive with an enzyme immuno-assay (Adenoclone EIA, Meridian Diagnostics, Cincinnati, OH) that detects human adenoviruses. A PFU assay was done to determine the viral dose in the harvested material.

Preparation of the viral suspension: The plaque-purified virus was grown on A549 cells in 75 cm2 tissue culture flasks at 0.1 multiplicity of infection and 90% destruction of the cells was observed due to cytopathic effect (CPE) after about 10 days. At this time, the flasks were frozen at -80°C and later thawed to lyse the cells and release the virus. The cell suspension was then centrifuged to pellet the cell debris and the clear supernatant was used as the viral suspension for inoculating the animals.

Plaque forming units assay: Titers of Ad-36 virus were determined using A549 cells by this assay. Starting with 100 mul of virus suspension and 900 mul of media, serial 10-fold dilutions were made. A549 cells were grown to confluence in six-well plates and three wells were used for each dilution. Three wells were used as the blank control and were not infected with the virus suspension. Media was removed from each well and 100 mul of the serially diluted virus suspension were pipetted out in the wells. The plates were incubated at 37°C, shaking gently every 15 min. After 1 h of incubation, the viral suspension from the wells was removed and discarded. The wells were overlaid with about 3 ml of 1% agar in media per well, with 1´antibiotic-antimycotic solution. The plates were inverted and incubated at 37°C for 8 days until plaques appear. After 8 days, wells were stained overnight with about 1 ml of crystal violet per well. The next day, number of plaques formed was counted after removing the agar. The number of plaques formed´dilution of viral suspension used gave PFU/100 mul of inoculum used. This was multiplied by 10 to express PFU/ml.

Tissue culture infectivity dose: The titer of the work stocks that caused a CPE in 50% of the wells containing A549 cells were calculated and expressed as tissue culture infectivity dose (TCID-50) units per ml. TCID-50 of the work stocks were determined using serial 10-fold dilutions of the virus work stock. TCID50 was calculated by serially diluting the virus stock solution and inoculating cells with the dilutions to find out the reciprocal of the highest dilution of virus which causes CPE in 50% of the cells inoculated. Titers were calculated after the cells inoculated with the virus dilutions were incubated at 37°C for 8 days.

Virus isolation

A549 cells in tissue culture were used for isolation of Ad-36 from the blood samples drawn 36 h post-inoculation of I-D and C-D groups. Flasks containing A549 cells were inoculated with about 200 mul of whole blood samples and were incubated at 37°C in MEM media containing 2´antibiotic-antimycotic solution and 10% FBS. Culture media from the flasks was collected after 8 days and transferred to cells in new set of flasks. This was repeated for a total of three passages. Infection was confirmed by observing whether viral CPE occurred in the respective cells 8 days after inoculation of the flask. The isolated virus was confirmed to be a human adenovirus by using a human adenovirus specific enzyme immuno-assay kit (Adenoclone EIA, Meridian Diagnostics, Cincinnati, OH).

Capillary electrophoresis assay

This assay is used to directly detect Ad-36 DNA in the blood.12,13 Briefly, the assay is divided into three parts namely, probe synthesis, DNA extraction and hybridization, and CE-LIF (capillary electrophoresis-laser-induced fluorescence) analysis.

Probe synthesis: We have the entire Ad-36 genome sequenced. To ensure specificity of the DNA detection only a sequence unique to Ad-36 from the fiber protein region of the viral genome was probed. Uniqueness of the sequence was verified by a gene bank search. A 5'fluorscein phosphoramidite labeled DNA probe for Ad-36 adenovirus (5' AGT TGA AAC AGC AAG AGA CTC AAA G) was synthesized by IDT Laboratories (Coralville, IA).

DNA extraction and hybridization: The DNA from the blood, adipose tissue and muscle samples of chickens was extracted with the Qiagen QIAmp Blood or tissue kit and quantitated spectrophotometrically. The genomic DNA was then digested with MboI (restriction enzyme from bacteria Moraxella Bovis) by standard procedures to generate smaller DNA fragments for hybridization, and treated with RNAse One to remove any RNA contamination. This DNA was then hybridized with the DNA probe (1.0125 mug) in a buffer volume of 30 mul containing 10 mM Tris-HCl (pH 7.2), 1 mM EDTA (pH 8.0) and 50 nM NaCl. The mixture was heated at 85°C for 10 min, and then incubated at 42°C for 4 h. Following the incubation, samples were flash frozen at -80°C.

CE-LIF analysis

Separations were performed on a P/ACE 2050 system using LIF in the reversed-polarity mode (anode at the detector side) at excitation of 488 nm and emission of 520 nm. Samples were introduced hydrodynamically by 10 s injections at 0.34 Pa pressure across a 65 cm´100 muM coated eCAP double stranded DNA (dsDNA) capillary filled with replaced linear polyacrylamide. The capillary was conditioned with eCAP dsDNA 1000 gel buffer. Separations were performed under constant voltage at 7.0 kV for 15-30 min. The intra-day and inter-day migration time precision was 0.18% (n=9) and 0.22% (n=6), respectively. The intra-day peak area precision was 7.3% (n=6) and the inter-day peak area precision was 11% (n=9). The minimal detectable level was 36 ag (signal to noise ratio 3:1).

Data are obtained as relative fluorescence units (RFU), which are converted to picogram DNA by comparing the RFU obtained from a standard (pGEM Molecular Markers, catalog no. G174, Promega, Madison, WI) of known concentration picogram DNA. Viral loads in the blood samples were determined by comparison to a standard curve as previously described12 and by expressing picogram DNA as number of copies of the virus. Briefly, one copy of Ad-36 DNA contains 33 068 base pairs. One picogram of DNA contains 9´108 bp, making 1 pg equal to approximately 27 216 copies of Ad-36 DNA. Results of the capillary electrophoresis assay (picogram of viral DNA) were expressed as the number of copies of Ad-36 per ml of the blood samples obtained from the two inoculated and two in-contact chickens before and after the viral inoculation (Figure 1).

DNA from A549 cells without virus, and DNA from A549 cells infected with Ad-36 were used as negative and positive controls, respectively. Also, DNA from A549 cells infected with other human adenoviruses Ad-2, Ad-31 and Ad-37 were used as negative controls.

Serum neutralization assay

This assay was used to screen the serum for Ad-36 antibodies. Serum was heat inactivated for 30 min at 56°C. The assay was carried out using 96-well microtiter plates. Serial 2-fold dilutions (1:2 to 1:1024) of serum were made with media and 50 mul of each dilution were added per well in duplicate. Fifty microliters of Ad-36 virus (100 TCID50) were added to each dilution. The plates were incubated at 37°C for 1 h. One hundred microliters of cell suspension containing about 20 000 cells were added to each well and the plates were incubated at 37°C for 12 days. Crystal violet-ethanol was added to each well to fix and stain the cells and the plates examined for CPE. The highest serum dilution with no CPE was considered the titer. Controls included wells containing no virus and/or wells with virus but no serum. A back titration to confirm the use of the appropriate virus dilutions was included. Presence of CPE with the virus and none in the presence of serum was considered an indication of effective neutralization of the virus with antibody in serum and the serum was considered to have antibody against the virus. A titer lower than 1:8 was considered to be non-specific neutralization and a titer of 1:8 was considered positive.

Assays for serum cholesterol and triglycerides

Serum CHOL was assayed using the CHOL oxidase-peroxidase method. Colorimetric determinations were made using Sigma kits (catalog no. 352) and the absorbance read at 500 nm. Cholesterol calibrator (Sigma catalog no. C 7921) and Cardiolipid control (Sigma catalog no. C 4571) were used.

TG were determined using the glycerol phosphate-peroxidase method. Colorimetric determinations were made using Sigma kits (catalog no. 339-50) and the absorbance was read at 540 nm. Glycerol (Sigma catalog no. 339-11) was used as a standard.

Body composition analysis

Digestive tracts of the carcasses were cleaned and returned to the carcasses. After autoclaving and homogenization of the carcasses, aliquots were used for water, ash and fat contents. All measurements were performed in triplicate. Water content was determined by heating samples to a constant weight in a drying oven overnight at 90°C. Ash content was determined by incinerating the sample in a furnace at 600°C for 4 h. The Folch extraction method11 was used for body fat determination. Fat was extracted with methanol-chloroform.

Results

Experiment 1

Onset and duration of presence of Ad-36 DNA in the blood: Ad-36 DNA was absent in the blood of all four chickens drawn prior to the viral inoculation (0 time, Figure 1). Ad-36 DNA appeared in the blood 12-36 h post-inoculation in the chickens receiving Ad-36 inoculation and was detectable up to 25 days when the last blood sample was obtained (Figure 1). Ad-36 DNA was detectable in 12 h in both the 'in-contact' chickens. One of the 'in-contact' chickens had Ad-36 DNA up to 25 days and the DNA was undetectable at 11 days in the other 'in-contact' chicken.

Experiment 2

Transmission of Ad-36-induced adiposity by transfusion of blood obtained from Ad-36 infected animals. Detection of Ad-36: The virus could be isolated from 12 out of 16 blood samples of the I-D group that were used for inoculating I-R chickens, confirming active viremia at transfusion. The Adenoclone EIA kit confirmed that the isolated virus was a human adenovirus. No virus was isolated from the blood from the C-D group. Three animals from each of the four groups were randomly selected to test for Ad-36 DNA in the blood, adipose tissue and the skeletal muscle. Ad-36 DNA was detected in the DNA obtained from the blood and the adipose tissue of the I-D and I-R groups but not in the skeletal muscle DNA from these groups (Table 1). Ad-36 DNA could not be detected in the blood, adipose tissue or skeletal muscle of the control groups (C-D and C-R).

Antibodies to Ad-36: Ad-36 antibodies were absent in all chickens at the start of the experiment. Ad-36 neutralizing antibodies were detected in all chickens from the infected groups (I-D and I-R) 10 days after the viral inoculation and at sacrifice. Ad-36 antibodies were not detected in the control groups (C-D and C-R).

Body composition: Total cumulative food intake per chicken after the first inoculation was not different for any of the groups (mean±s.d.; C-D, 5401.6±268.1 g; C-R, 5585.5±560.0 g; I-D, 5234.6±338.3 g; I-R, 5316.2±687.7 g, P=0.55 by ANOVA). Mean body weights, visceral fat, total body fat (%), CHOL and TG levels or the prevalence of obesity were not different for the two control groups (C-D and C-R, Table 2). Compared to the C-D, final mean body weights of the I-D and I-R groups were not significantly different. Compared to the C-D group, visceral fat was greater by 142% and 80% for I-D (P<0.002) and I-R (P<0.01) groups, respectively (Table 2).

Compared to the C-D group, total carcass fat was greater by 35% for the I-D group (P<0.01) and by 44% for the I-R group (P<0.01). Using the definition of obesity as a total body fat greater than the 85th percentile of the range of the control groups (C-D and C-R), seven I-D chickens (64%) and eight I-R chickens (72%) were obese (P<0.02 and<0.004, respectively). Only four chickens (18%) were obese from the uninfected control groups together (C-D and C-R).

Serum lipids: Serum CHOL and TG for the I-D group and serum CHOL for the I-R group were significantly lower compared to the C-D group (Table 2).

Discussion

Whether Ad-36 causes viremia in the infected animals was previously unknown. Blood-borne spread of Ad-36 would be a possibility if Ad-36 does cause viremia. The aim of experiment 1 was limited to observing viral DNA titers in the blood. Therefore, only four animals were used for the experiment. The data from experiment 1 demonstrate that Ad-36 DNA appears in the blood after animals have been inoculated with Ad-36. The onset of Ad-36 DNA appearance in the blood was as soon as 12 h following the inoculation. Although the titers were gradually declining, Ad-36 DNA showed a prolonged and continued presence in the blood, which raises the possibility of a long carrier state for infected individuals. Substantially high titers for the viral DNA were present 36 h post-inoculation. Rise and fall of Ad-36 DNA titers in the blood suggested viral replication and clearance phases in the body. Appearance of Ad-36 DNA and the rapidity of the onset of viremia in the blood of chickens sharing cages with Ad-36 inoculated animals demonstrated the ease of transmission of Ad-36 infection in chickens. It appears that Ad-36 virus quickly spreads from the nasal passages of a chicken, circulates in the blood of the infected chickens, and is also very quickly spread horizontally to uninfected chickens kept in contact with the infected chickens. The exact route of infection of the 'in-contact' chickens is not clear. The results suggest a high degree of infectivity from either nasal-oral secretions and/or by fecal excretion and contamination. Both chickens in a cage were males and too young to be sexually active, which rules out sexual transmission of the infection.

Experiment 2 is the first report describing transmission of adiposity due to blood transfusions. We have previously shown that chickens and mice inoculated with Ad-36 develop adiposity and paradoxically low levels of serum CHOL and TG compared to uninfected controls.8 In the present experiment, this adiposity syndrome was observed in the two infected groups. Relative to the total body fat increase, the infected chickens showed preferential increases in visceral adipose tissue. Presence of virus in the blood transfused to I-R chickens was demonstrated by isolating the virus and by DNA detection using CE. Also, the more than 4-fold rise in antibody titers, and the presence of Ad-36 DNA in adipose tissue and blood of the infected groups (I-D and I-R) confirm the presence of infection in these animals.

Selective presence of Ad-36 DNA in the visceral adipose tissue compared to the skeletal muscle despite its presence in the blood is an important finding and may indicate a viral affinity for the adipose tissue.

The results of the two experiments taken together suggest that Ad-36 can be transmitted from an experimentally infected chicken to a cage-mate through close physical contact, and that viral DNA may persist in blood for at least 3.5 weeks following acute infection. Not only the virus itself, but also the adiposity induced by the virus can be transmitted between chickens via blood.

One of the most significant findings of this project is the fulfilment of the Koch's postulate. Experiment 2 demonstrated that Ad-36 infection induced adiposity in animals. The same organism (Ad-36) could be recovered from the blood of these animals and on inoculation produced adiposity once again in a separate set of animals. These data along with our previous data that showed that adiposity induced by Ad-36 is not a non-specific effect of a viral infection8 provide stronger evidence for a causative role for Ad-36 in inducing adiposity in animals under experimental conditions.

Ad-36 is a human virus and it was first isolated from a fecal sample of a diabetic girl with enteritis.14 Almost nothing is known about its pathogenicity in humans. Serum neutralizing antibody assays show a widespread presence of Ad-36 antibodies in the population.10 Therefore, Ad-36 pathogenicity has high relevance to human health. Obviously, we cannot demonstrate Ad-36 induced adiposity in humans by deliberate transfusion of blood contaminated with the virus. Therefore, we have used an animal model to study the transmissibility of Ad-36 infection and Ad-36 induced adiposity.

Conclusion

In conclusion, this study demonstrated that Ad-36 infection as well as Ad-36-induced adiposity could be transmitted in a chicken model. Further investigation is needed to investigate the potential for transmission of virus-induced adiposity in humans.

Acknowledgements

We gratefully acknowledge Drs Geoffrey Letchworth and Lisa Krugner-Higby for advice on virological aspects and Sharon Gathright and Kathleen Taylor for laboratory assistance. This work was supported by funds from the Wisconsin Alumni Research Foundation's Beers-Murphy Clinical Nutrition Center.

References

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Figures

Figure 1 Ad-36 DNA titers in the blood of chickens inoculated with Ad-36 as well as the chickens kept in contact with the inoculated chickens. Time 0 denotes blood sample obtained before the viral inoculation.

Tables

Table 1 Experiment 2: capillary electrophoretic detection of Ad-36 DNA in chicken blood and tissue obtained 5 weeks post-inoculation from control donors (C-D), control receivers (C-R), infected donors (I-D) and infected receivers (I-R)

Table 2 Effect of blood transfusion from Ad-36 inoculated chickens (I-D) and chicken inoculated with media (C-D) to infected receivers (I-R) and control receivers (C-R), respectively (Experiment 2)

Received 14 July 2000; revised 22 January 2001; accepted 12 February 2001
July 2001, Volume 25, Number 7, Pages 990-996
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