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Horizontal Transmission of Turkey Herpesvirus to ChickensA
FEEDING POULTRY LITTER
suspensions of bovine rumen bacteria. J. Dairy Sci. 4 1 : 190-202. Merwin, R. T., 1956. The determination of drugs in medicated feeds. Symposium on Medicated Feeds. Medical Encyclopedia, Inc., New York, p. 83. Noland, P. R., B. F. Ford and M. L. Ray, 1955. The use of ground chicken litter as a source of nitrogen for gestating-lactating ewes and fattening steers. J. Animal Sci. 14: 860-865. Olson, J. C , Jr., H. Macy and H. O. Halvorson, 1952. Thermal death-time studies of coliform bacteria in milk. Minnesota Agr. Exp. Sta. Tech. Bull. 202. Schafer, M. L., K. A. Busch and J. E. Campbell, 1963. A rapid method for DDT in milk by use of gas chromatography. J. Dairy Sci. 46: 10251032. Southwell, B. L., O. M. Hale and W. C. McCormick, 1958. Poultry house litter as a protein supplement in steer fattening rations. Georgia Agr. Exp. Sta. Mimeo. Ser. N. S. 55.
Horizontal Transmission of Turkey Herpesvirus to ChickensA 1. PRELIMINARY OBSERVATION B. R. CHO, S. G. KENZY AND S. A. HAIDERB Department of Veterinary Science, Washington Agricultural Experiment Station; Department of Veterinary Microbiology, Washington State University, Pullman, Washington 99163 (Received for publication November 6, 1970) INTRODUCTION
I
SOLATION of a cell-associated herpesvirus from clinically normal turkeys has been reported (Kawamura et al., 1969; Witter etal., 1970). Turkey herpesvirus (HVT) is antigeniA
Supported in part by funds from U.S. Public Health Service, National Cancer Institute Grant CA 07517, 07; Project 13K-3073^1961, National Poultry Research Foundation; and Project 1773, Washington Agricultural Experiment Station, Scientific Paper No. 3565. B Present Address: Sterwin Laboratories, Inc., Millsboro, Delaware, 1966.
cally related to Marek's disease herpesvirus (MDHV), non-pathogenic for both turkeys and chickens and capable of spreading horizontally among turkeys (Witter et al, 1970). The virus, however, does not spread by contact between chickens (Okazaki et al., 1970) or spreads poorly, if at all (Witter et al, 1970; Purchase et al, 1970). This paper reports observations of horizontal transmission of HVT to chickens in contact with chickens that had been inoculated at 8 weeks of age with HVT which had been passaged in chick embryo fibroblast cell cultures.
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on February 6, 2015
sociation of Official Agricultural Chemists, Washington, D.C. Baker, C. J. L., 1946. A note on the estimation of the uric acid radical in avian excreta. Poultry Sci. 25: 593-596. Ballinger, D. G., R. J. Lishka and M. E. Gales, Jr., 1962. Application of silver diethyldithiocarbamate method to determination of arsenic. J. Am. Water Works Assoc. 54: 1424-1428. Belasco, I. J., 1954. New nitrogen feed compounds for ruminants—a laboratory evaluation. J. Animal Sci. 13: 601-610. Brugman, H. H., H. C. Dickey, B. E. Plummer and B. R. Poulton, 1964. Nutritive value of poultry litter. J. Animal Sci. 23: 869. Bose, S., 1944. An iodometric estimation of uric acid in poultry excreta. Poultry Sci. 23: 130134. Camp, A. A., 1959. Broiler-house litter as a livestock feed. Texas Agricultural Progress, 5: 17. Jurtshuk, P., Jr., R. M. Doetsch and J. C. Shaw, 1955. Anaerobic purine dissimilation by washed
881
882
B. R. CHO, S. G. KENZY AND S. A. HAIDER
MATERIALS AND METHODS
° Falcon Plastics, Los Angeles, California.
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on February 6, 2015
Experimental chicks and eggs: Chicks for this experiment came from breeder flocks of White Leghorn Crosses (F 2 and F 3 Cornell S X Line 7). For cell cultures {chick embryo fibroblast) embryonated eggs from Cornell S Line breeder flock were used. These breeder flocks maintained by this department were free of RIF (resistance inducing factor) and RVNA (Rous virus neutralizing antibodies) when tested with subgroups A and B pseudotypes of Bryan high-titer strain of Rous sarcoma virus. The progeny from these breeders were hatched as described previously (Cho et •al., 1968) and reared in Horsfall-Bauer type cages in an isolation building. Cell cultures: Chick embryo fibroblast (CEF) cell cultures were prepared as described by Witter et al. (1969) and grown in tissue culture petri dishes (60 X 15 mm) 0 which were incubated in a humidified C0 2 incubator (approximately 3% C0 2 ). CEF cultures were used for growing and assaying HVT throughout this study by the method of Witter et al. (1970). Turkey herpesvirus (HVT): Turkey herpesvirus (FC 126 strain) was obtained as the 11th duck embryo fibroblast (DEF) culture passage from the Regional Poultry Research Laboratory, East Lansing, Michigan, through the courtesy of Dr. W. Okazaki. The virus was further passaged at this laboratory in CEF cultures. Cultured CEF cells infected with the FC 126 strain of HVT (7-10th passage in CEF), dispersed with trypsin-versene (Madin and Darby, 1958), and suspended in growth medium, were used as the source of HVT (FC126). Another strain of HVT referred to as
WY70, isolated in this laboratory from kidney cell cultures of apparently normal turkey poults, was also used. Strain WY70 was identified as HVT on the basis of growth characteristics and cytopathology in cell cultures, type of nucleic acid (DNA virus), cell association of infectivity, absence of pathogenicity for chicks, and antigenic similarity with the FC 126 strain of HVT as determined by agar gel precipitin tests. Agar gel precipitin test (AGP): This test was employed for detecting antibodies against HVT. The procedures described by Chubb and Churchill (1968) for detecting antibodies associated with MD were employed. HVT antigens were prepared from the second passage in CEF cultures exhibiting nearly confluent plaques following infection with HVT (FC126). Experimental designs: Experimental designs are shown in Table 1. Eight trials were conducted in a similar manner but varying in age of donors when HVT was inoculated and varying in route and dose of HVT inoculated. In Trials 1 through 4 donor chicks (1 to 4 days old) were inoculated intraabdominally or subcutaneously with 4.3 X 103 to 106 pfu (plaque forming units) of HVT per chick. In other trials donor birds were inoculated subcutaneously with 9.3 X 104 to 2 X 105 pfu of HVT per bird at 4 weeks (Trial 7) or 8 weeks of age (Trials 5, 6 and 8). The FC 126 strain was employed for all experiments except Trial 6 in which the WY70 strain was used. In Trials 1 through 4 siblings of the same hatch were used as contacts, while in Trials 5 through 8 siblings of the same and different hatches were used as contacts. In Trials 7 and 8 which were conducted simultaneously, siblings of the same hatches and the same HVT preparation were employed. In each respective trial, donors and con-
TURKEY
883
HERPESVIRUS
TABLE 1.—Experimental designs of Trials {1-8 Donors (Inoculates)-4 Trial
Aw incited
1
10
4 days
2
10
3
Dose
(P fu ) o f HVT(CEF passage level)
Observation period (wk.)
Route
No. of birds
Exposure Age
4.3X10 3 (7th)
IA B
10
4 days
6
4 days
3.1X10" (7th)
IA
10
4 days
6
10
2 days
4.8X10" (9th)
SQC
20
2 days
6 and 8
4
20
1 day
106 (7th)
SQ
50
1 day
6 and 8
5
7
8 weeks
9.3X10" (9th)
SQ
3
8 weeks
8
5
2 weeks
6
6
D
8 weeks
10.3X10 (14th)
4
8 weeks
SQ
2 weeks 2 7
5
4 weeks
5
2X10 (10th)
SQ
7 20
8 weeks
A B 0 D
6
2X10 (10th)
SQ
10 days 4 weeks
7 4 and 8
3 days
3
8 weeks
10
4 weeks
10
2 weeks
20
3 days
4 and
Donors and Pen-mates from respective trials were housed together. Intraabdominal. Subcutaneous. WY70 strain of HVT was used.
tacts were housed together in a HorsfallBauer type cage. Contact transmission of HVT from the inoculated donors to contact exposed pen mates was determined by examining heparinized blood and serum samples for the presence of HVT and precipitin antibodies from all or representative samples of birds at a specified observation period. Heparinized blood (0.1 ml.) from each bird was inoculated into each of two dishes of CEF
monolayers which were fed with 5 ml. of maintenance medium. The medium was replaced after 24 hours and then at 2- to 3day intervals. Control cultures, not inoculated with blood, were treated similarly. All cultures were examined daily and a total plaque count determined on the 7th day post-inoculation. Serum samples from each bird were tested by AGP for precipitins against HVT. The criteria for establishing HVT infec-
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No. of birds
Pen-mates (Contacts) A
884
B. R. CHO, S. G. KENZY AND S. A. HAIDER
tion included plaque formation with cytopathological changes, characteristics of HVT in CEF cultures inoculated with blood and the presence of precipitins against HVT in the serum of the chickens. RESULTS
The presence of precipitins for HVT in the inoculated donors closely paralleled the presence of viremia. None of the contact pen mates in Trials 1 to 3 was positive for precipitins. However, in Trial 4 several of the contact pen mates were positive for antibodies, although none were demonstrated to be viremic. On histological examination of the contact pen mates in Trial 4, peripheral nerve lesions typical of MD (Payne and Biggs, 1967) were noted in one of 8 and in 7 of 12 birds examined at 6 and 8 weeks after contact, respectively. Young chicks in Trial 7 which were inoculated at 4 weeks of age with 2 X 105 pfu of HVT occasionally transmitted the virus to their pen mates. Only one of the 10 birds •which were contact exposed at 3 days of age developed detectable virus in blood when examined 4 weeks later, but none of the 10 T)irds examined 8 weeks later carried HVT
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The results of Trials 1 though 8 are summarized in Table 2. As shown in Trials 1 through 4 there was no evidence that HVT spread from the inoculates to pen mates during contact for 68 weeks, when day-old to 4-day-old chicks were inoculated with 4.3 X 10s to 106 pfu of HVT. None of the pen mates in these four trials when examined at 6 to 8 weeks after contact carried the virus in their blood, although the virus could be reisolated from most of the inoculated donor birds. The number of HVT plaques induced in CEF cultures per 0.1 ml. of blood from donor birds varied from 1 to 116 plaques, and was apparently independent of the dose given the donors.
or precipitins. All pen mates contact exposed at 4 weeks of age were free of HVT and precipitins when examined 4 and 8 weeks later, respectively. However, when donor birds were inoculated at 8 weeks of age with 9.3 X 104 to 2 X 105 pfu of HVT, the HVT spread laterally as indicated by the presence of viremia and precipitins in pen mates (Trials 5, 6 and 8). In Trial 5 when 8-week-old donor chickens were inoculated with HVT (9.3 X 10* pfu), all three contact pen mates of the same age became viremic and two of them developed precipitins when tested 8 weeks later. However, only one of three birds contact exposed at 2 weeks of age was found to be viremic when examined 6 weeks later, and no precipitins for HVT were demonstrated. All three inoculated donors carried both HVT and precipitins in their blood after 8 weeks of observation. Similar lateral transmission was also demonstrated in Trial 6 where the WY70 strain of HVT was employed. In Trial 8 HVT again spread from the donor birds which were inoculated at 8 weeks of age with 2 X 105 pfu of HVT. A few contact pen mates (20-40%) when examined 4 weeks later were viremic. The number of HVT plaques per 0.1 ml. of blood varied from one to 23. Two donor birds examined at this time carried both HVT and precipitins. At 8 weeks post-contact, more contact pen mates (50-80%) were found to have HVT in their blood and some of these viremic pen mates were also positive for precipitins, although none of the contact infected were positive for precipitins at 4 weeks post-contact. The spread of HVT from inoculated chickens to their pen mates occurred regardless of the age of pen mates when they were first exposed. In this trial pen mates were contact exposed when 3 days, 2 weeks, 4 weeks, and 8 weeks of age, respectively.
885
TURKEY HERPESVIRUS
TABLE 2.—Horizontal transmission of turkey herpesvirus {HVT) from inoculated chickens to pen males Exposure to HVT Trial 1
2
4
5
6
7
(pfu)*
Exposure age
c D E
8/10 D
—
4.3X10 3
4 days
6
Pen mates
Contact
None
4 days
6
0/9
—
0/9 D
Donors
IA
3.1X10"
4 days
6
9/10
8/9
1/1
Pen mates
Contact
None
4 days
6
0/9
—
0/9
Donors
SQE
4.8X10"
2 days
6 8
5/5 3/4
4/5 3/3
1/1
Pen mates
Contact
None
2 days
6 8
0/10 0/9
—
0/10 0/9
Donors
SQ
10»
1 day
6 8
8/8 2/2
3/8 2/2
—
Pen mates
Contact
None
1 day
6 8
0/8 0/12
—
2/8 1/12
Donors
SQ
9.3X10"
8 weeks
8
3/3
3/3
—
Pen mates
Contact
None
8 weeks 2 weeks
8 6
3/3 1/3
2/3 0/1
0/2
8 weeks
8
3/3
3/3
—
8 weeks 2 weeks 10 days
8 6 7
2/2 1/3 2/2
2/2 1/1 1/2
0/2
4 weeks
4 8
2/2 1/3
2/2 1/1
Donors
SQ
10.3X10"
Penmates
Contact
None
Donors
Donors
SQ Contact
SQ Contact
2X10 5 None
2X10 5 None
1/2
4 weeks
0/3 0/4
3 days
1/10 0/10
0/1
8 weeks
2/2 3/4
2/2 3/3
1/1
0/3 0/4 0/9 0/10
8 weeks
8
2/3
1/2
0/1
4 weeks
4 8
1/5 4/5
0/1 4/4
0/4 0/1
2 weeks
4 8
2/5 4/5
0/2 1/4
0/3 0/1
3/10 3/6
0/3 2/3
0/7 0/3
3 days
B
10/10 c
Nonviremic
IA B
Pen mates
A
Viremic
Donors
Pen mates
8
Route
Precipitins demonstrated in chickens
HVT viremia demonstrated
Plaque forming units of HVT. Intra-abdominal. No. of birds with HVT in their blood/no. of birds examined. No. of birds with precipitin antibodies/no. of birds viremic or non-viremic. Subcutaneous.
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3
Group
Observation P er iod (wk.)
886
B. R. CHO, S. G. KENZY AND S. A. HAIDER
Similar horizontal transmission of HVT among chickens was not observed by other workers (Witter et al, 1970; Okazaki et al., 1970; Purchase et al., 1970), possibly due to the much younger age of chicks used as donors. In their observations donor chicks were inoculated as day-olds. It remains to be seen whether horizontal
transmission of HVT also occurs when the virus has been passaged in only DEF cell cultures. Both FC 126 and WY70 strains of HVT used for this study were passaged in CEF cultures. The FC 126 strain had been passaged 11 times in DEF cultures prior to passage in CEF cultures at this laboratory. It was not determined if the contact-infected pen mates served as secondary sources of HVT. SUMMARY When 8-week-old chickens were inoculated subcutaneously with 9.3 X 104 to 2 X 105 pfu of turkey herpesvirus (HVT), the virus spread within 8 weeks post-contact to most of the pen mates exposed at 3 days, 2 weeks, 4 weeks and 8 weeks of age, respectively. There was no horizontal transmission of HVT to pen mates when contact exposed for 6-8 weeks to chicks inoculated at one day to 4 days of age with 4.3 X 103 to 106 pfu of HVT. The virus spread to pen mates occasionally when donor birds were inoculated at 4 weeks of age with 2 X 105 pfu of HVT. ACKNOWLEDGMENT The technical assistance of Mr. W. Bryson is acknowledged. REFERENCES Cho, G. R., S. G. Kenzy and U. H. Kim, 1968. Atypical cells in the peripheral blood of chickens exposed to Marek's disease agent. Canad. J. Comp. Med. 32: 562-567. Chubb, R. C , and A. E. Churchill, 1968. Precipitating antibodies associated with Marek's disease. Vet. Rec. 83: 4-7. Kawamura, H., D. J. King and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13: 853863. Madin, S. H., and N. B. Darby, Jr., 1958. Established kidney cell lines of normal adult bovine and ovine origin. Proc. Soc. Exptl. Biol. Med. 98: 574-576.
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DISCUSSION These studies indicate that HVT mayspread horizontally to 50% or more of the pen mates when contact exposed to chickens inoculated at 8 weeks of age with HVT (9.3 X 104 to 2 X 105 pfu). Birds infected through contact developed viremia which was often accompanied by presence of precipitins against HVT. The age of donor birds when HVT was inoculated appeared to be an important factor determining horizontal transmission of HVT among chickens, since baby chicks (1 day to 4 days old) inoculated with even 106 pfu of HVT did not spread the virus to their pen mates after 8 weeks of observation. Chickens inoculated at 4 weeks of age with 2 X 105 pfu of HVT occasionally spread the virus. A few pen mates contact exposed to day-old chicks inoculated with 10e pfu of HVT (Trial 4) had precipitins when tested with HVT antigens, but HVT could not be detected from any of these pen mates. In those trials (Trials 5, 6 and 8) in which horizontal transmission was observed, pen mates which developed precipitins through contact infection were invariably also viremic. However, precipitins were not detected in the pen mates without viremia. In Trial 4, it appeared that precipitins in the pen mates resulted from an inadvertent infection with MDHV. The HVT and MDHV are antigenically related and cross react in agar gel precipitin test (Witter et al., 1970). Presence of histologic lesions in these birds typical of MD further indicated possible MD infection in these birds (Trial 4).
TURKEY HERPESVIRUS
1968. Preliminary studies on cell cultures infected with Marek's disease agent. Avian Dis. 12: 169-18S. Witter, R. L., J. J. Solomon and G. H. Burgoyne, 1969. Cell culture techniques for primary isolation of Marek's disease associated-herpesvirus. Avian Dis. 13: 101-118. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 3 1 : 525-538.
Identification of P-Alanyl-Lysine in Chick Muscle1 M. W. STUTZ,2 J. E. SAVAGE AND B. L. O'DELL Departments of Agricultural Chemistry, and Poultry Husbandry, University of Missouri, Columbia, Missouri 65201 (Received for publication November 7, 1970)
W
HILE studying the metabolism of lysine in growing chicks fed a diet containing 35% casein, an unidentified ninhydrin-positive peak was observed in acid extracts of muscle analyzed for free amino acids. The compound was hydrolyzable and was eventually identified as a peptide, composed of ^-alanine and lysine. There is little information in the literature concerning such a peptide and no details concerning its presence in avian species. Other related peptides, camosine (^-alanylhistidine) and anserine ([3-alanyl1-methylhistidine) have been recognized as constituents of muscle for many years. Kalyankar and Meister (1959) have described the enzymatic synthesis of camosine, anserine and several related peptides, including (3-alanyl-lysine. They used a cell1 Contribution from the Missouri Agricultural Experiment Journal series No. 607S. 2 Present address: Animal Physiology Laboratories, Merck Institute for Therapeutic Research, Rahway, N. J.
free system obtained from chick pectoral muscle. Meister (1965) referred to an unpublished observation that a peptide with the properties of p-alanyl-lysine occurs in the muscle of chicks fed diets containing 2% lysine. Recently a- ((3-alanyl) -lysine has been isolated from rabbit muscle (Matsuoka et al., 1969). EXPERIMENTAL PROCEDURES AND RESULTS
Previous experiments had shown that extracts of muscle from chicks fed casein based diets contained an unidentified compound which eluted at the same position as the internal standard, a-amino-f-guanidobutyric acid. To isolate the compound, a chick, fed a practical corn-soybean meal diet for 16 days, was fasted for 4 hours and fed a 35% casein diet (O'Dell and Savage, 1966) for 24 hours. Lysine-X4C (uniformly labeled) was injected intraperitoneally at a dose of 15 (*Ci. per 100 grams of body weight (44.6 [j-Ci. total). Immediately after
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on February 6, 2015
Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14: 413^29. Payne, L. N., and P. M. Biggs, 1967. Studies on Marek's disease. II. Pathogenesis. J. Natl. Cancer Inst. 39: 281-302. Purchase, H. G., W. Okazaki and B. R. Burmester, 1970. Control of Marek's disease by vaccination with a herpesvirus of turkeys. Presented at the Poultry Section, 107th Annual A.V.M.A. Meeting, June 23-26, 1970, Las Vegas, Nevada. Witter, R. L., G. H. Burgoyne and J. J. Solomon,
887
suspensions of bovine rumen bacteria. J. Dairy Sci. 4 1 : 190-202. Merwin, R. T., 1956. The determination of drugs in medicated feeds. Symposium on Medicated Feeds. Medical Encyclopedia, Inc., New York, p. 83. Noland, P. R., B. F. Ford and M. L. Ray, 1955. The use of ground chicken litter as a source of nitrogen for gestating-lactating ewes and fattening steers. J. Animal Sci. 14: 860-865. Olson, J. C , Jr., H. Macy and H. O. Halvorson, 1952. Thermal death-time studies of coliform bacteria in milk. Minnesota Agr. Exp. Sta. Tech. Bull. 202. Schafer, M. L., K. A. Busch and J. E. Campbell, 1963. A rapid method for DDT in milk by use of gas chromatography. J. Dairy Sci. 46: 10251032. Southwell, B. L., O. M. Hale and W. C. McCormick, 1958. Poultry house litter as a protein supplement in steer fattening rations. Georgia Agr. Exp. Sta. Mimeo. Ser. N. S. 55.
Horizontal Transmission of Turkey Herpesvirus to ChickensA 1. PRELIMINARY OBSERVATION B. R. CHO, S. G. KENZY AND S. A. HAIDERB Department of Veterinary Science, Washington Agricultural Experiment Station; Department of Veterinary Microbiology, Washington State University, Pullman, Washington 99163 (Received for publication November 6, 1970) INTRODUCTION
I
SOLATION of a cell-associated herpesvirus from clinically normal turkeys has been reported (Kawamura et al., 1969; Witter etal., 1970). Turkey herpesvirus (HVT) is antigeniA
Supported in part by funds from U.S. Public Health Service, National Cancer Institute Grant CA 07517, 07; Project 13K-3073^1961, National Poultry Research Foundation; and Project 1773, Washington Agricultural Experiment Station, Scientific Paper No. 3565. B Present Address: Sterwin Laboratories, Inc., Millsboro, Delaware, 1966.
cally related to Marek's disease herpesvirus (MDHV), non-pathogenic for both turkeys and chickens and capable of spreading horizontally among turkeys (Witter et al, 1970). The virus, however, does not spread by contact between chickens (Okazaki et al., 1970) or spreads poorly, if at all (Witter et al, 1970; Purchase et al, 1970). This paper reports observations of horizontal transmission of HVT to chickens in contact with chickens that had been inoculated at 8 weeks of age with HVT which had been passaged in chick embryo fibroblast cell cultures.
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on February 6, 2015
sociation of Official Agricultural Chemists, Washington, D.C. Baker, C. J. L., 1946. A note on the estimation of the uric acid radical in avian excreta. Poultry Sci. 25: 593-596. Ballinger, D. G., R. J. Lishka and M. E. Gales, Jr., 1962. Application of silver diethyldithiocarbamate method to determination of arsenic. J. Am. Water Works Assoc. 54: 1424-1428. Belasco, I. J., 1954. New nitrogen feed compounds for ruminants—a laboratory evaluation. J. Animal Sci. 13: 601-610. Brugman, H. H., H. C. Dickey, B. E. Plummer and B. R. Poulton, 1964. Nutritive value of poultry litter. J. Animal Sci. 23: 869. Bose, S., 1944. An iodometric estimation of uric acid in poultry excreta. Poultry Sci. 23: 130134. Camp, A. A., 1959. Broiler-house litter as a livestock feed. Texas Agricultural Progress, 5: 17. Jurtshuk, P., Jr., R. M. Doetsch and J. C. Shaw, 1955. Anaerobic purine dissimilation by washed
881
882
B. R. CHO, S. G. KENZY AND S. A. HAIDER
MATERIALS AND METHODS
° Falcon Plastics, Los Angeles, California.
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on February 6, 2015
Experimental chicks and eggs: Chicks for this experiment came from breeder flocks of White Leghorn Crosses (F 2 and F 3 Cornell S X Line 7). For cell cultures {chick embryo fibroblast) embryonated eggs from Cornell S Line breeder flock were used. These breeder flocks maintained by this department were free of RIF (resistance inducing factor) and RVNA (Rous virus neutralizing antibodies) when tested with subgroups A and B pseudotypes of Bryan high-titer strain of Rous sarcoma virus. The progeny from these breeders were hatched as described previously (Cho et •al., 1968) and reared in Horsfall-Bauer type cages in an isolation building. Cell cultures: Chick embryo fibroblast (CEF) cell cultures were prepared as described by Witter et al. (1969) and grown in tissue culture petri dishes (60 X 15 mm) 0 which were incubated in a humidified C0 2 incubator (approximately 3% C0 2 ). CEF cultures were used for growing and assaying HVT throughout this study by the method of Witter et al. (1970). Turkey herpesvirus (HVT): Turkey herpesvirus (FC 126 strain) was obtained as the 11th duck embryo fibroblast (DEF) culture passage from the Regional Poultry Research Laboratory, East Lansing, Michigan, through the courtesy of Dr. W. Okazaki. The virus was further passaged at this laboratory in CEF cultures. Cultured CEF cells infected with the FC 126 strain of HVT (7-10th passage in CEF), dispersed with trypsin-versene (Madin and Darby, 1958), and suspended in growth medium, were used as the source of HVT (FC126). Another strain of HVT referred to as
WY70, isolated in this laboratory from kidney cell cultures of apparently normal turkey poults, was also used. Strain WY70 was identified as HVT on the basis of growth characteristics and cytopathology in cell cultures, type of nucleic acid (DNA virus), cell association of infectivity, absence of pathogenicity for chicks, and antigenic similarity with the FC 126 strain of HVT as determined by agar gel precipitin tests. Agar gel precipitin test (AGP): This test was employed for detecting antibodies against HVT. The procedures described by Chubb and Churchill (1968) for detecting antibodies associated with MD were employed. HVT antigens were prepared from the second passage in CEF cultures exhibiting nearly confluent plaques following infection with HVT (FC126). Experimental designs: Experimental designs are shown in Table 1. Eight trials were conducted in a similar manner but varying in age of donors when HVT was inoculated and varying in route and dose of HVT inoculated. In Trials 1 through 4 donor chicks (1 to 4 days old) were inoculated intraabdominally or subcutaneously with 4.3 X 103 to 106 pfu (plaque forming units) of HVT per chick. In other trials donor birds were inoculated subcutaneously with 9.3 X 104 to 2 X 105 pfu of HVT per bird at 4 weeks (Trial 7) or 8 weeks of age (Trials 5, 6 and 8). The FC 126 strain was employed for all experiments except Trial 6 in which the WY70 strain was used. In Trials 1 through 4 siblings of the same hatch were used as contacts, while in Trials 5 through 8 siblings of the same and different hatches were used as contacts. In Trials 7 and 8 which were conducted simultaneously, siblings of the same hatches and the same HVT preparation were employed. In each respective trial, donors and con-
TURKEY
883
HERPESVIRUS
TABLE 1.—Experimental designs of Trials {1-8 Donors (Inoculates)-4 Trial
Aw incited
1
10
4 days
2
10
3
Dose
(P fu ) o f HVT(CEF passage level)
Observation period (wk.)
Route
No. of birds
Exposure Age
4.3X10 3 (7th)
IA B
10
4 days
6
4 days
3.1X10" (7th)
IA
10
4 days
6
10
2 days
4.8X10" (9th)
SQC
20
2 days
6 and 8
4
20
1 day
106 (7th)
SQ
50
1 day
6 and 8
5
7
8 weeks
9.3X10" (9th)
SQ
3
8 weeks
8
5
2 weeks
6
6
D
8 weeks
10.3X10 (14th)
4
8 weeks
SQ
2 weeks 2 7
5
4 weeks
5
2X10 (10th)
SQ
7 20
8 weeks
A B 0 D
6
2X10 (10th)
SQ
10 days 4 weeks
7 4 and 8
3 days
3
8 weeks
10
4 weeks
10
2 weeks
20
3 days
4 and
Donors and Pen-mates from respective trials were housed together. Intraabdominal. Subcutaneous. WY70 strain of HVT was used.
tacts were housed together in a HorsfallBauer type cage. Contact transmission of HVT from the inoculated donors to contact exposed pen mates was determined by examining heparinized blood and serum samples for the presence of HVT and precipitin antibodies from all or representative samples of birds at a specified observation period. Heparinized blood (0.1 ml.) from each bird was inoculated into each of two dishes of CEF
monolayers which were fed with 5 ml. of maintenance medium. The medium was replaced after 24 hours and then at 2- to 3day intervals. Control cultures, not inoculated with blood, were treated similarly. All cultures were examined daily and a total plaque count determined on the 7th day post-inoculation. Serum samples from each bird were tested by AGP for precipitins against HVT. The criteria for establishing HVT infec-
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No. of birds
Pen-mates (Contacts) A
884
B. R. CHO, S. G. KENZY AND S. A. HAIDER
tion included plaque formation with cytopathological changes, characteristics of HVT in CEF cultures inoculated with blood and the presence of precipitins against HVT in the serum of the chickens. RESULTS
The presence of precipitins for HVT in the inoculated donors closely paralleled the presence of viremia. None of the contact pen mates in Trials 1 to 3 was positive for precipitins. However, in Trial 4 several of the contact pen mates were positive for antibodies, although none were demonstrated to be viremic. On histological examination of the contact pen mates in Trial 4, peripheral nerve lesions typical of MD (Payne and Biggs, 1967) were noted in one of 8 and in 7 of 12 birds examined at 6 and 8 weeks after contact, respectively. Young chicks in Trial 7 which were inoculated at 4 weeks of age with 2 X 105 pfu of HVT occasionally transmitted the virus to their pen mates. Only one of the 10 birds •which were contact exposed at 3 days of age developed detectable virus in blood when examined 4 weeks later, but none of the 10 T)irds examined 8 weeks later carried HVT
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The results of Trials 1 though 8 are summarized in Table 2. As shown in Trials 1 through 4 there was no evidence that HVT spread from the inoculates to pen mates during contact for 68 weeks, when day-old to 4-day-old chicks were inoculated with 4.3 X 10s to 106 pfu of HVT. None of the pen mates in these four trials when examined at 6 to 8 weeks after contact carried the virus in their blood, although the virus could be reisolated from most of the inoculated donor birds. The number of HVT plaques induced in CEF cultures per 0.1 ml. of blood from donor birds varied from 1 to 116 plaques, and was apparently independent of the dose given the donors.
or precipitins. All pen mates contact exposed at 4 weeks of age were free of HVT and precipitins when examined 4 and 8 weeks later, respectively. However, when donor birds were inoculated at 8 weeks of age with 9.3 X 104 to 2 X 105 pfu of HVT, the HVT spread laterally as indicated by the presence of viremia and precipitins in pen mates (Trials 5, 6 and 8). In Trial 5 when 8-week-old donor chickens were inoculated with HVT (9.3 X 10* pfu), all three contact pen mates of the same age became viremic and two of them developed precipitins when tested 8 weeks later. However, only one of three birds contact exposed at 2 weeks of age was found to be viremic when examined 6 weeks later, and no precipitins for HVT were demonstrated. All three inoculated donors carried both HVT and precipitins in their blood after 8 weeks of observation. Similar lateral transmission was also demonstrated in Trial 6 where the WY70 strain of HVT was employed. In Trial 8 HVT again spread from the donor birds which were inoculated at 8 weeks of age with 2 X 105 pfu of HVT. A few contact pen mates (20-40%) when examined 4 weeks later were viremic. The number of HVT plaques per 0.1 ml. of blood varied from one to 23. Two donor birds examined at this time carried both HVT and precipitins. At 8 weeks post-contact, more contact pen mates (50-80%) were found to have HVT in their blood and some of these viremic pen mates were also positive for precipitins, although none of the contact infected were positive for precipitins at 4 weeks post-contact. The spread of HVT from inoculated chickens to their pen mates occurred regardless of the age of pen mates when they were first exposed. In this trial pen mates were contact exposed when 3 days, 2 weeks, 4 weeks, and 8 weeks of age, respectively.
885
TURKEY HERPESVIRUS
TABLE 2.—Horizontal transmission of turkey herpesvirus {HVT) from inoculated chickens to pen males Exposure to HVT Trial 1
2
4
5
6
7
(pfu)*
Exposure age
c D E
8/10 D
—
4.3X10 3
4 days
6
Pen mates
Contact
None
4 days
6
0/9
—
0/9 D
Donors
IA
3.1X10"
4 days
6
9/10
8/9
1/1
Pen mates
Contact
None
4 days
6
0/9
—
0/9
Donors
SQE
4.8X10"
2 days
6 8
5/5 3/4
4/5 3/3
1/1
Pen mates
Contact
None
2 days
6 8
0/10 0/9
—
0/10 0/9
Donors
SQ
10»
1 day
6 8
8/8 2/2
3/8 2/2
—
Pen mates
Contact
None
1 day
6 8
0/8 0/12
—
2/8 1/12
Donors
SQ
9.3X10"
8 weeks
8
3/3
3/3
—
Pen mates
Contact
None
8 weeks 2 weeks
8 6
3/3 1/3
2/3 0/1
0/2
8 weeks
8
3/3
3/3
—
8 weeks 2 weeks 10 days
8 6 7
2/2 1/3 2/2
2/2 1/1 1/2
0/2
4 weeks
4 8
2/2 1/3
2/2 1/1
Donors
SQ
10.3X10"
Penmates
Contact
None
Donors
Donors
SQ Contact
SQ Contact
2X10 5 None
2X10 5 None
1/2
4 weeks
0/3 0/4
3 days
1/10 0/10
0/1
8 weeks
2/2 3/4
2/2 3/3
1/1
0/3 0/4 0/9 0/10
8 weeks
8
2/3
1/2
0/1
4 weeks
4 8
1/5 4/5
0/1 4/4
0/4 0/1
2 weeks
4 8
2/5 4/5
0/2 1/4
0/3 0/1
3/10 3/6
0/3 2/3
0/7 0/3
3 days
B
10/10 c
Nonviremic
IA B
Pen mates
A
Viremic
Donors
Pen mates
8
Route
Precipitins demonstrated in chickens
HVT viremia demonstrated
Plaque forming units of HVT. Intra-abdominal. No. of birds with HVT in their blood/no. of birds examined. No. of birds with precipitin antibodies/no. of birds viremic or non-viremic. Subcutaneous.
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3
Group
Observation P er iod (wk.)
886
B. R. CHO, S. G. KENZY AND S. A. HAIDER
Similar horizontal transmission of HVT among chickens was not observed by other workers (Witter et al, 1970; Okazaki et al., 1970; Purchase et al., 1970), possibly due to the much younger age of chicks used as donors. In their observations donor chicks were inoculated as day-olds. It remains to be seen whether horizontal
transmission of HVT also occurs when the virus has been passaged in only DEF cell cultures. Both FC 126 and WY70 strains of HVT used for this study were passaged in CEF cultures. The FC 126 strain had been passaged 11 times in DEF cultures prior to passage in CEF cultures at this laboratory. It was not determined if the contact-infected pen mates served as secondary sources of HVT. SUMMARY When 8-week-old chickens were inoculated subcutaneously with 9.3 X 104 to 2 X 105 pfu of turkey herpesvirus (HVT), the virus spread within 8 weeks post-contact to most of the pen mates exposed at 3 days, 2 weeks, 4 weeks and 8 weeks of age, respectively. There was no horizontal transmission of HVT to pen mates when contact exposed for 6-8 weeks to chicks inoculated at one day to 4 days of age with 4.3 X 103 to 106 pfu of HVT. The virus spread to pen mates occasionally when donor birds were inoculated at 4 weeks of age with 2 X 105 pfu of HVT. ACKNOWLEDGMENT The technical assistance of Mr. W. Bryson is acknowledged. REFERENCES Cho, G. R., S. G. Kenzy and U. H. Kim, 1968. Atypical cells in the peripheral blood of chickens exposed to Marek's disease agent. Canad. J. Comp. Med. 32: 562-567. Chubb, R. C , and A. E. Churchill, 1968. Precipitating antibodies associated with Marek's disease. Vet. Rec. 83: 4-7. Kawamura, H., D. J. King and D. P. Anderson, 1969. A herpesvirus isolated from kidney cell culture of normal turkeys. Avian Dis. 13: 853863. Madin, S. H., and N. B. Darby, Jr., 1958. Established kidney cell lines of normal adult bovine and ovine origin. Proc. Soc. Exptl. Biol. Med. 98: 574-576.
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DISCUSSION These studies indicate that HVT mayspread horizontally to 50% or more of the pen mates when contact exposed to chickens inoculated at 8 weeks of age with HVT (9.3 X 104 to 2 X 105 pfu). Birds infected through contact developed viremia which was often accompanied by presence of precipitins against HVT. The age of donor birds when HVT was inoculated appeared to be an important factor determining horizontal transmission of HVT among chickens, since baby chicks (1 day to 4 days old) inoculated with even 106 pfu of HVT did not spread the virus to their pen mates after 8 weeks of observation. Chickens inoculated at 4 weeks of age with 2 X 105 pfu of HVT occasionally spread the virus. A few pen mates contact exposed to day-old chicks inoculated with 10e pfu of HVT (Trial 4) had precipitins when tested with HVT antigens, but HVT could not be detected from any of these pen mates. In those trials (Trials 5, 6 and 8) in which horizontal transmission was observed, pen mates which developed precipitins through contact infection were invariably also viremic. However, precipitins were not detected in the pen mates without viremia. In Trial 4, it appeared that precipitins in the pen mates resulted from an inadvertent infection with MDHV. The HVT and MDHV are antigenically related and cross react in agar gel precipitin test (Witter et al., 1970). Presence of histologic lesions in these birds typical of MD further indicated possible MD infection in these birds (Trial 4).
TURKEY HERPESVIRUS
1968. Preliminary studies on cell cultures infected with Marek's disease agent. Avian Dis. 12: 169-18S. Witter, R. L., J. J. Solomon and G. H. Burgoyne, 1969. Cell culture techniques for primary isolation of Marek's disease associated-herpesvirus. Avian Dis. 13: 101-118. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 3 1 : 525-538.
Identification of P-Alanyl-Lysine in Chick Muscle1 M. W. STUTZ,2 J. E. SAVAGE AND B. L. O'DELL Departments of Agricultural Chemistry, and Poultry Husbandry, University of Missouri, Columbia, Missouri 65201 (Received for publication November 7, 1970)
W
HILE studying the metabolism of lysine in growing chicks fed a diet containing 35% casein, an unidentified ninhydrin-positive peak was observed in acid extracts of muscle analyzed for free amino acids. The compound was hydrolyzable and was eventually identified as a peptide, composed of ^-alanine and lysine. There is little information in the literature concerning such a peptide and no details concerning its presence in avian species. Other related peptides, camosine (^-alanylhistidine) and anserine ([3-alanyl1-methylhistidine) have been recognized as constituents of muscle for many years. Kalyankar and Meister (1959) have described the enzymatic synthesis of camosine, anserine and several related peptides, including (3-alanyl-lysine. They used a cell1 Contribution from the Missouri Agricultural Experiment Journal series No. 607S. 2 Present address: Animal Physiology Laboratories, Merck Institute for Therapeutic Research, Rahway, N. J.
free system obtained from chick pectoral muscle. Meister (1965) referred to an unpublished observation that a peptide with the properties of p-alanyl-lysine occurs in the muscle of chicks fed diets containing 2% lysine. Recently a- ((3-alanyl) -lysine has been isolated from rabbit muscle (Matsuoka et al., 1969). EXPERIMENTAL PROCEDURES AND RESULTS
Previous experiments had shown that extracts of muscle from chicks fed casein based diets contained an unidentified compound which eluted at the same position as the internal standard, a-amino-f-guanidobutyric acid. To isolate the compound, a chick, fed a practical corn-soybean meal diet for 16 days, was fasted for 4 hours and fed a 35% casein diet (O'Dell and Savage, 1966) for 24 hours. Lysine-X4C (uniformly labeled) was injected intraperitoneally at a dose of 15 (*Ci. per 100 grams of body weight (44.6 [j-Ci. total). Immediately after
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Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14: 413^29. Payne, L. N., and P. M. Biggs, 1967. Studies on Marek's disease. II. Pathogenesis. J. Natl. Cancer Inst. 39: 281-302. Purchase, H. G., W. Okazaki and B. R. Burmester, 1970. Control of Marek's disease by vaccination with a herpesvirus of turkeys. Presented at the Poultry Section, 107th Annual A.V.M.A. Meeting, June 23-26, 1970, Las Vegas, Nevada. Witter, R. L., G. H. Burgoyne and J. J. Solomon,
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