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Horizontal Transmission of Turkey Herpesvirus to Chickens
Horizontal Transmission of Turkey Herpesvirus to Chickens 2. SOME FACTORS AFFECTING TRANSMISSION 1 B. R. CHO AND S. G. KENZY Department of Veterinary Science, Washington Agricultural Experiment Station; and Department of Veterinary Microbiology, Washington State University, Pullman, Washington 99163 (Received for publication July 3, 1972) ABSTRACT The likelihood of horizontal spread of turkey herpesvirus (HVT) increased as the dose of the cell-associated HVT given to donor chickens (8-week-old) was increased from 7,400 to 14,800 and to 74,000 plaque-forming-units (PFU). However, further increase in virus dose to 370,000 PFU per donor did not result in spread. Similar increase in the horizontal spread of HVT resulted with the cell-free HVT when the virus dose was increased from 1,400 to 11,280 PFU per donor bird. With further increase to 490,000 PFU per donor, the virus srpead poorly. Cell-associated HVT appeared to spread more effectively than cell-free virus. HVT grown in duck embryo fibroblast cell cultures spread readily when a small (5,800 PFU) or large dose (435,000 PFU) of the virus was given to donor birds, and seemed to spread more effectively than HVT grown in chicken embryo fibroblast cell cultures. Marek's disease virus maternal antibodies present in chicks (10-day-old) at the time of HVT inoculation had no suppressive effect on the horizontal transmission of HVT to contact cagemates. POULTRY SCIENCE 52: 608-613, 1973
INTRODUCTION
I
N preliminary studies (Cho et al., 1971), turkey herpesvirus (HVT) was found to spread readily to contact cagemates from chickens inoculated with HVT at 8 weeks of age. However, when younger birds (4 weeks old or younger) were inoculated with HVT, the virus failed to spread or spread poorly. This paper reports some of the factors affecting the horizontal transmission of HVT in chickens. MATERIALS AND METHODS
The source, hatching, and rearing of experimental chickens; methods of growing cell cultures and HVT; and agar gel precipitin (AGP) test have been described (Cho et ah, 1971). 1 Supported in part by funds from U. S. Public Health Service, National Cancer Institute Grant CA 07517,07; Project 11D-3073-5961, National Poultry Research Foundation; and Project 1773, Washington Agricultural Experiment Station, Scientific Paper No. 3895.
Fertile White Leghorn eggs from a commercial specific-pathogen-free (SPF) flock (H&N Inc.) and duck eggs from a flock maintained by the department served as a source for chick embryo fibroblast (CEF) and duck embryo fibroblast (DEF) cell cultures, respectively. HVT: In Trials 1 and 4, the cell-associated HVT (FC 126 strain) was employed for chicken inoculation. The virus material was a suspension of viable HVTinfected cells prepared from the 10th (Trial 1) and 14th (Trial 4) CEF-passage. The cells were removed from dishes with trypsin-versene, sedimented, and then resuspended in tissue culture medium. In Trial 2, donor chickens were inoculated with the cell-free HVT prepared by sonical treatment of HVT-infected CEF cell cultures (11th passage) by the method of Calnek et al. (1970). The sonicated material was centrifuged at 2000 X g for 20 minutes and the supernatant fluids were considered free of viable cells and used as cell-free HVT.
608
HERPESVIRUS TRANSMISSION
For Trial 3, two different HVT preparations (cell-free) were employed, one made with the virus serially passaged 15 times in DEF cell cultures (referred to as HVT/DEF) and the other with the virus passaged 13 times in CEF cell cultures (referred to as HVT/CEF). Assay of HVT: In all trials, in vitro assay of cell-associated and cell-free HVT inocula was essentially that described by Calnek el al. (1972). Procedures: Trial 1. This trial was designed to determine if the dose of HVT (10th CEF-passage) given to donor chickens had any effect on the horizontal spread of the virus. Eight-week-old and 3-day-old birds from the same parent flock were allocated into Groups A through E. Each group, except for Group E, consisted of 2 chickens (8-week-old) as inoculated donors and 15 chicks (3-day-old) as contact cagemates. Group E had 6 untreated chicks which served as chick controls. Each of 2 donor birds from Groups A through D was inoculated subcutaneously with 7,400, 14,800, 74,000, and 370,000 plaque-forming-units (PFU) of the same cell-associated HVT preparation, respectively. Each group was then housed separately in a Horsfall-Bauer type (HB) isolator. Half of the contact cagemates of Groups A through D was examined for HVT infection at 4 weeks, and the rest, including donor birds of each group and chick controls (Group E), at 8 weeks after treatment. Horizontal transmission of HVT was determined by examining heparinized blood for HVT viremia and serum samples for precipitins to HVT antigens (Cho etal., 1971). Trial 2. This trial was similar to Trial 1 except for the employment of the cell-free
609
HVT. Each of 2 donor chickens from Groups A through C was inoculated subcutaneously with 1,400, 11,280, and 490,000 PFU of the cell-free HVT respectively. Six untreated hatchmates were placed in Group D as controls. Trial 3. This trial was designed to determine if HVT serially passaged in DEF cell cultures (HVT/DEF) spread similarly to HVT/CEF. Group A consisted of 2 donor chickens (8-week-old) inoculated subcutaneously with 5,800 PFU of HVT/DEF and 16 chicks (2-day-old) as contact cagemates. Group B was treated like Group A except that each of the 2 donor chickens received 435,000 PFU of HVT/DEF. Group C consisted of 2 donors (8-week-old) inoculated with 23,200 PFU of HVT/CEF and 22 contact cagemates (2-day-old). Housing and examination of the birds were done as in Trial 1. Trial 4. This trial was conducted similarly to other trials but designed to determine the effect of Marek's disease virus (MDV) maternal antibodies in donor chicks on the horizontal transmission of HVT. Group A consisted of 3 inoculated donor chicks and 19 contact cagemates. The three donor chicks were positive for MDV maternal antibodies when tested by AGP test at the time of HVT inoculation (10-day-old). Five hatchmates of the donor chicks were placed as controls into Group D. Group B was the same as Group A except that the three donor chicks were negative for MDV maternal antibodies. Group C had two 8-week-old chickens as inoculated donors and 20 contact cagemates. The two 8-week-old birds were negative for precipitins to MDV antigens at the time of HVT inoculation. Two hatchmates of the donors in Group C were also placed in Group D as controls. Five untreated hatchmates of
610
B. R. CHO AND S. G. KENZY TABLE 1.—Horizontal transmission of turkey herpesvirus {HVT) with varying doses of the cell-associated and cell-free virus {Trials 1 and 2) HVT viremia and/or antibodies demonstrated
Exposure to HVT
Trial
Group
Post-inoculation or contact 4 weeks 8 weeks
Exposure age
Exposure method
Viremia
l4
1
2
Antibodies
Viremia 3
Antibodies
NT 0/7
2/2 3/7
NT 0/7
NT 0/7
2/2 5/8
2/2 4/8
8wks 3 days
NT 0/7
NT 0/7
2/2 7/8
2/2 7/8
8wks 3 days
NT 0/7
NT 0/7
2/2 0/7
2/2 0/7
NT
NT
0/6
0/6
8wks 2 days
NT 0/7
NT 0/7
1/2 0/8
2/2 0/8
Inoculated (11,280 PFU) Contact cagemates
8wks 2 days
NT 1/9
NT 0/9
2/2 3/9
2/2 3/9
C
Inoculated (490,000 PFU) Contact cagemates
8wks 2 days
NT 1/10
NT 0/10
0/2 2/10
2/2 1/10
D
Chick controls
NT
NT
0/6
0/6
A
Inoculated (7,400 PFU) Contact cagemates6
8wks 3 days
NT 0/7
B
Inoculated (14,800 PFU) Contact cagemates
8wks 3 days
C
Inoculated (74,000 PFU) Contact cagemates
D
Inoculated (370,000 PFU) Contact cagemates6
E
Chick controls
A
Inoculated (1,400 PFU) Contact cagemates6
B
2/2 2/7
1
Plaque-forming-units per donor bird. Not tested. Number of birds positive over number of birds examined. 4 Employed cell-associated HVT. 6 A few birds died of smothering within a week. 6 Employed cell-free HVT.
2
3
the contact exposed chicks in Groups A, B, and C were also placed in Groups D as controls. Each of the donors in Groups A, B, and C was inoculated subcutaneously with 7,650 PFU of the cell-associated HVT. Donor chicks with MDV maternal antibodies were the progeny from a group of chickens experimentally exposed to the Id-1 isolate of MDV (Kenzy et al., 1969). All birds, except for 8-week-olds, were treated at 10 days of age. RESULTS
Trial 1: The results of Trials 1 and 2 are summarized in Table 1. When half of the contact cagemates was examined
at 4 weeks after contact, none was either viremic with HVT or positive for precipitins to HVT antigens. At 8 weeks after contact, however, infection of cagemates with HVT was detected. In Group A with 2 donor chickens inoculated with 7,400 PFU of HVT, 3 of the 7 cagemates had HVT viremia, 2 of which were also positive for precipitins. As the virus dose given to donor birds was increased to 14,800 PFU in Group B and 74,000 PFU in Group C, more contact cagemates were found to be infected with HVT and positive for precipitins (Table 1). However, in Group D, of which 2 donor birds were inoculated with 370,000 PFU of HVT, none of the cagemates was
611
HERPESVIRUS TRANSMISSION
either viremic or positive for precipitins through 8 weeks after contact exposure. All inoculated donor chickens of Groups A through D had an HVT viremia as well as precipitins when examined 8 weeks after virus inoculation. Six untreated control birds (Group E) were free of HVT viremia and precipitins. Trial 2: The results of Trial 2, in which varying doses of cell-free HVT (1,400 to 490,000 PFU) were employed for inoculation of donor chickens, were similar to those of Trial 1 except for Group A (Table 1). In Group A, of which 2 donor birds were inoculated with 1,400 PFU of HVT per bird, there was no evidence of contact infection among cagemates through 8 weeks after contact, although the two donor chickens were infected with HVT as indicated by HVT viremia and/or precipitins in their blood. When a larger dose of the same virus material (11,280 PFU) was given to donor chickens, the virus did spread horizontally (Group B). Further increase in the virus dose per donor bird to 490,000 PFU, however, did not result in a greater incidence of contact infection among cagemates (Group The two donor chickens of Group B,
when examined 8 weeks after virus inoculation, carried both the virus and precipitins in their blood, while in the 2 donors of Group C only precipitins were noted. Six untreated control birds were free of any cytopathic agents in their blood and were negative for precipitins to HVT antigens. Trial 3: HVT/DEF spread readily from inoculated chickens to contact cagemates (Table 2). Similar horizontal spread of HVT resulted in both Groups A and B when donor birds were inoculated with 5,800 and 435,000 PFU of H V T / DEF respectively. In Group C, where donor birds were each inoculated with 23,200 PFU of HVT/CEF, the virus also spread horizontally but less effectively than HVT/DEF in Groups A and B. Trial 4: The results are summarized in Table 3. When HVT was inoculated into 10-day-old chicks either positive (Group A) or negative (Group B) for MDV maternal antibodies, none of the cagemates from Groups A and B was infected with HVT at 4 weeks. Two of the 6 cagemates of Group A and none from Group B had an HVT viremia at 8 week after contact. All three inoculated donors from each of Groups A and B were viremic when examined 8 weeks after virus
T A B L E 2.—Horizontal transmission in chickens of turkey herpesvirus grown in duck embryo fibroblast cell cultures {Trial 3) Exposure to HVT
{HVT)
HVT viremia/antibodies demonstrated Age exposed
Post-inoculation or contact 4 weeks 8 weeks
Group
Exposure method
A
Inoculated Contact cagemates5
DEF-passaged1 (5,800)2 None
8wks 2 days
NT» 0/4
NT 0/4
0/2< 5/8
1/2 2/8
B
Inoculated Contact cagemates
DEF-passaged (435,000) None
8 wks 2 days
NT 0/6
NT 0/6
2/2 5/10
2/2 3/10
C
Inoculated Contact cagemates
CEF-passaged« (23,200) None
8 wks 2 days
NT 0/10
NT 0/10
2/2 3/12
2/2 1/12
1 2
HVT used
HVT passaged 15 times in duck embryo fibroblast cell cultures. Virus dose in plaque-forming-units per donor chicken. »Not tested. *6 Number of birds positive over number of birds examined. Four chicks died of smothering within a week. • HVT passaged 13 times in chicken embryo fibroblast cell cultures.
Viremia Antibodies
Viremia Antibodies
612
B. R. CHO AND S. G. KENZY T A B L E S.^~Effects of Marek's disease maternal antibodies in the inoculated chicks upon transmission of turkey herpesvirus (HVT) to contact cagemates {Trial 4)
Group
MD maternal abyi in HVTinoculated donor chicks
Exposure to HVT
horizontal
HVT viremia and/or antibodies demonstrated
Exposure method
Age exposed
Post-inoculation or contact 4 weeks 8 weeks Viremia
Antibodies
A
Positive
Inoculated (7,650)*5 Contact cagemates
10 days 10 days
NT 5 0/10
NT 0/10
3/3* 2/6
3/3 1/6
B
Negative
Inoculated (7,650)6 Contact cagemates
10 days 10 days
NT 0/10
NT 0/10
3/3 0/7
3/3 0/7
C
Negative
Inoculated (7,560) 5 Contact cagemates
8 wks 10 days
NT 2/10
NT 0/10
1/2 4/9
1/2 3/9
NT
NT
0/12
0/12
D
None (chick controls)
Viremia Antibodies
1 Marek's disease virus maternal antibodies as determined by agar gel precipitin test. 3 HVT dose in plaque-forming-units per bird. «4 Not tested. Number of birds positive over number of birds examined. 6 A few chicks died of smothering within a week.
inoculation. In Group C, of which 2 donor chickens were inoculated at 8 weeks of age with the same HVT, 2 of the 10 cagemates and 4 of the 9 cagemates at 4 and 8 weeks respectively post-contact were viremic with HVT. None of the contact cagemates had precipitins to HVT antigens at 4 weeks, but more than half of the viremic cagemates as well as most of the inoculated donors were positive at 8 weeks. Untreated control birds were free of any demonstable cytopathic agents in their blood and were also negative for precipitins to HVT antigens. In Trials 1 through 4, contact cagemates which developed precipitins were invariably also viremic and the antibodies were not detected in any contact cagemates without a detectable HVT viremia. Inoculated birds and their contact cagemates in all trials remained clinically normal and free of any gross pathologic lesions throughout the course of the experiments. DISCUSSION
This study further demonstrated that HVT could spread from inoculated chickens to cagemates through contact.
The virus dose given to donor chickens appeared to influence the horizontal spread of HVT. The likelihood of horizontal transmission increased when the virus dose given to donor chickens was increased from 7,400 to 74,000 PFU of the cell-associated and from 1,400 to 11,280 PFU of the cell-free HVT. There was little or no spread when donor birds were each inoculated with an extremely large dose of the cell-associated (370,000 PFU) or the cell-free HVT (490,000 PFU) which had been grown in CEF cell cultures. However, HVT which had been grown in DEF cell cultures spread readily when a similarly large dose (435,000 PFU) of the virus was given to donor birds. The reason for this discrepancy was not determined but may be due to differences in growing the virus (i.e., growing in CEF versus DEF cell cultures) and/or "auto-interference" as observed with influenza virus when a large amount of the virus was inoculated into embryonated eggs (Ziegler et al., 1944). It appeared that cell-associated HVT spread more readily (Trial 1) than cellfree virus (Trial 2), and so did HVT grown in DEF cell cultures (Trial 3) than the virus grown in CEF cell cultures.
HERPESVIRUS TRANSMISSION
MDV maternal antibodies present in the donor chickens at the time of HVT inoculation did not inhibit the horizontal spread of HVT (Trial 4). It was reported that MDV maternal antibodies were of little or no influence on infection of chicks with HVT (Patrascu et al., 1972). The results of Trial 4 rather seemed to confirm the preliminary observations (Cho et al., 1971) which described age effect of donors on the horizontal spread of HVT. Since donor and contact chickens in each trial were siblings of respective hatches and the HB isolators were identical in design, the differences in horizontal spread of HVT were not likely due to genetic or husbandry factors. ACKNOWLEDGMENT
We gratefully acknowledge the fine technical assistance of Mr. Wayne Bryson.
613
REFERENCES Calnek, B. W., S. B. Hitchner and H. K. Adldinger, 1970. Lyophilization of cell-free Marek's disease herpesvirus and a herpesvirus from turkeys. Appl. Microbiol. 20: 723-726. Calnek, B. W., G. Garrido, W. Okazaki and I. V. Patrascu, 1972. In vitro methods for assay of turkey herpesvirus. Avian Dis. 16: 52-56. Cho, B. R., S. G. Kenzy and S. A. Haider, 1971. Horizontal transmission of turkey herpesvirus to chickens. 1. Preliminary observation. Poultry Sci. 50: 881-887. Kenzy, S. G., R. F. Lapen and J. M. Sharma, 1969. Transmission of cutaneous Marek's disease (skin leukosis). Presented at the 106th Annual AVMA Meeting, July 13-17, Minneapolis, Minnesota. Patrascu, I. V., B. W. Calnek and N. W. Smith, 1972. Vaccination with lyophilized turkey herpesvirus (HVT): Minimum infective and protective doses. Avian Dis. 16: 86-93. Ziegler, J. E., Jr., G. I. Lavin and F. L. Horsfall, Jr., 1944. Interference between the influenza viruses. II. The effect of virus rendered non-infective by ultraviolet radiation upon the multiplication of influenza viruses in the chick embryo. J. Exp. Med. 79:379-400.
NEWS AND NOTES (Continuedfrom page 603) analysis of lipids, 15—Precipitation of low density lipoproteins with polyanions, 16—Electrophoretic methods for lipoprotein separation, 17—New techniques for the study of lipoproteins, and 18—Automated methods of gas analysis. Registration fees for the Conference are $110 for A.O.C.S. members and $140 for nonmembers. More detailed information is available from James Lyon, Executive Director, American Oil Chemists' Society, 508 South Sixth Street, Champaign, Illinois 61820. Telephone (217) 359-2344. The American Oil Chemists' Society is a not-forprofit association of chemists, other scientists and technologists. The Society's work is directed to the advancement of technology in oils, other lipids, and associated materials; to the improvement of the technical competence of its members and others in the field; and to education and training at all levels in these and related fields of science and technology. ANNUAL A.I.B.S. MEETING The 24th Annual Meeting of the American Insti-
tute of Biological Sciences will be held at the University of Massachusetts, Amherst, June 17-22. The Plenary Session entitled "Manpower in the Biological Sciences" will be held on Monday, June 18, at 8 p.m. Betty Vetter of the Scientific Manpower Commission, who will be the keynote speaker, will discuss the national manpower needs brought to light by the A.I.B.S. Manpower Survey. The following day's symposium "Manpower: Supply and Demand" under the joint sponsorship of the A.I.B.S., F.A.S.E.B., and Scientific Manpower Commission, will continue on this most important subject. In addition the A.I.B.S. Education Division has planned several symposia in the mornings, including: Graduate Education, Research in Biological Education, Environmental Education, and Environmental Regulation. Informal sessions for open discussion will be held in the afternoons. A number of Workshops are also scheduled, as well as Contributed Paper Sessions. Other Symposia sponsored by A.I.B.S. and participating societies are: Physiological Adaptation to
(Continued on page 647)
INTRODUCTION
I
N preliminary studies (Cho et al., 1971), turkey herpesvirus (HVT) was found to spread readily to contact cagemates from chickens inoculated with HVT at 8 weeks of age. However, when younger birds (4 weeks old or younger) were inoculated with HVT, the virus failed to spread or spread poorly. This paper reports some of the factors affecting the horizontal transmission of HVT in chickens. MATERIALS AND METHODS
The source, hatching, and rearing of experimental chickens; methods of growing cell cultures and HVT; and agar gel precipitin (AGP) test have been described (Cho et ah, 1971). 1 Supported in part by funds from U. S. Public Health Service, National Cancer Institute Grant CA 07517,07; Project 11D-3073-5961, National Poultry Research Foundation; and Project 1773, Washington Agricultural Experiment Station, Scientific Paper No. 3895.
Fertile White Leghorn eggs from a commercial specific-pathogen-free (SPF) flock (H&N Inc.) and duck eggs from a flock maintained by the department served as a source for chick embryo fibroblast (CEF) and duck embryo fibroblast (DEF) cell cultures, respectively. HVT: In Trials 1 and 4, the cell-associated HVT (FC 126 strain) was employed for chicken inoculation. The virus material was a suspension of viable HVTinfected cells prepared from the 10th (Trial 1) and 14th (Trial 4) CEF-passage. The cells were removed from dishes with trypsin-versene, sedimented, and then resuspended in tissue culture medium. In Trial 2, donor chickens were inoculated with the cell-free HVT prepared by sonical treatment of HVT-infected CEF cell cultures (11th passage) by the method of Calnek et al. (1970). The sonicated material was centrifuged at 2000 X g for 20 minutes and the supernatant fluids were considered free of viable cells and used as cell-free HVT.
608
HERPESVIRUS TRANSMISSION
For Trial 3, two different HVT preparations (cell-free) were employed, one made with the virus serially passaged 15 times in DEF cell cultures (referred to as HVT/DEF) and the other with the virus passaged 13 times in CEF cell cultures (referred to as HVT/CEF). Assay of HVT: In all trials, in vitro assay of cell-associated and cell-free HVT inocula was essentially that described by Calnek el al. (1972). Procedures: Trial 1. This trial was designed to determine if the dose of HVT (10th CEF-passage) given to donor chickens had any effect on the horizontal spread of the virus. Eight-week-old and 3-day-old birds from the same parent flock were allocated into Groups A through E. Each group, except for Group E, consisted of 2 chickens (8-week-old) as inoculated donors and 15 chicks (3-day-old) as contact cagemates. Group E had 6 untreated chicks which served as chick controls. Each of 2 donor birds from Groups A through D was inoculated subcutaneously with 7,400, 14,800, 74,000, and 370,000 plaque-forming-units (PFU) of the same cell-associated HVT preparation, respectively. Each group was then housed separately in a Horsfall-Bauer type (HB) isolator. Half of the contact cagemates of Groups A through D was examined for HVT infection at 4 weeks, and the rest, including donor birds of each group and chick controls (Group E), at 8 weeks after treatment. Horizontal transmission of HVT was determined by examining heparinized blood for HVT viremia and serum samples for precipitins to HVT antigens (Cho etal., 1971). Trial 2. This trial was similar to Trial 1 except for the employment of the cell-free
609
HVT. Each of 2 donor chickens from Groups A through C was inoculated subcutaneously with 1,400, 11,280, and 490,000 PFU of the cell-free HVT respectively. Six untreated hatchmates were placed in Group D as controls. Trial 3. This trial was designed to determine if HVT serially passaged in DEF cell cultures (HVT/DEF) spread similarly to HVT/CEF. Group A consisted of 2 donor chickens (8-week-old) inoculated subcutaneously with 5,800 PFU of HVT/DEF and 16 chicks (2-day-old) as contact cagemates. Group B was treated like Group A except that each of the 2 donor chickens received 435,000 PFU of HVT/DEF. Group C consisted of 2 donors (8-week-old) inoculated with 23,200 PFU of HVT/CEF and 22 contact cagemates (2-day-old). Housing and examination of the birds were done as in Trial 1. Trial 4. This trial was conducted similarly to other trials but designed to determine the effect of Marek's disease virus (MDV) maternal antibodies in donor chicks on the horizontal transmission of HVT. Group A consisted of 3 inoculated donor chicks and 19 contact cagemates. The three donor chicks were positive for MDV maternal antibodies when tested by AGP test at the time of HVT inoculation (10-day-old). Five hatchmates of the donor chicks were placed as controls into Group D. Group B was the same as Group A except that the three donor chicks were negative for MDV maternal antibodies. Group C had two 8-week-old chickens as inoculated donors and 20 contact cagemates. The two 8-week-old birds were negative for precipitins to MDV antigens at the time of HVT inoculation. Two hatchmates of the donors in Group C were also placed in Group D as controls. Five untreated hatchmates of
610
B. R. CHO AND S. G. KENZY TABLE 1.—Horizontal transmission of turkey herpesvirus {HVT) with varying doses of the cell-associated and cell-free virus {Trials 1 and 2) HVT viremia and/or antibodies demonstrated
Exposure to HVT
Trial
Group
Post-inoculation or contact 4 weeks 8 weeks
Exposure age
Exposure method
Viremia
l4
1
2
Antibodies
Viremia 3
Antibodies
NT 0/7
2/2 3/7
NT 0/7
NT 0/7
2/2 5/8
2/2 4/8
8wks 3 days
NT 0/7
NT 0/7
2/2 7/8
2/2 7/8
8wks 3 days
NT 0/7
NT 0/7
2/2 0/7
2/2 0/7
NT
NT
0/6
0/6
8wks 2 days
NT 0/7
NT 0/7
1/2 0/8
2/2 0/8
Inoculated (11,280 PFU) Contact cagemates
8wks 2 days
NT 1/9
NT 0/9
2/2 3/9
2/2 3/9
C
Inoculated (490,000 PFU) Contact cagemates
8wks 2 days
NT 1/10
NT 0/10
0/2 2/10
2/2 1/10
D
Chick controls
NT
NT
0/6
0/6
A
Inoculated (7,400 PFU) Contact cagemates6
8wks 3 days
NT 0/7
B
Inoculated (14,800 PFU) Contact cagemates
8wks 3 days
C
Inoculated (74,000 PFU) Contact cagemates
D
Inoculated (370,000 PFU) Contact cagemates6
E
Chick controls
A
Inoculated (1,400 PFU) Contact cagemates6
B
2/2 2/7
1
Plaque-forming-units per donor bird. Not tested. Number of birds positive over number of birds examined. 4 Employed cell-associated HVT. 6 A few birds died of smothering within a week. 6 Employed cell-free HVT.
2
3
the contact exposed chicks in Groups A, B, and C were also placed in Groups D as controls. Each of the donors in Groups A, B, and C was inoculated subcutaneously with 7,650 PFU of the cell-associated HVT. Donor chicks with MDV maternal antibodies were the progeny from a group of chickens experimentally exposed to the Id-1 isolate of MDV (Kenzy et al., 1969). All birds, except for 8-week-olds, were treated at 10 days of age. RESULTS
Trial 1: The results of Trials 1 and 2 are summarized in Table 1. When half of the contact cagemates was examined
at 4 weeks after contact, none was either viremic with HVT or positive for precipitins to HVT antigens. At 8 weeks after contact, however, infection of cagemates with HVT was detected. In Group A with 2 donor chickens inoculated with 7,400 PFU of HVT, 3 of the 7 cagemates had HVT viremia, 2 of which were also positive for precipitins. As the virus dose given to donor birds was increased to 14,800 PFU in Group B and 74,000 PFU in Group C, more contact cagemates were found to be infected with HVT and positive for precipitins (Table 1). However, in Group D, of which 2 donor birds were inoculated with 370,000 PFU of HVT, none of the cagemates was
611
HERPESVIRUS TRANSMISSION
either viremic or positive for precipitins through 8 weeks after contact exposure. All inoculated donor chickens of Groups A through D had an HVT viremia as well as precipitins when examined 8 weeks after virus inoculation. Six untreated control birds (Group E) were free of HVT viremia and precipitins. Trial 2: The results of Trial 2, in which varying doses of cell-free HVT (1,400 to 490,000 PFU) were employed for inoculation of donor chickens, were similar to those of Trial 1 except for Group A (Table 1). In Group A, of which 2 donor birds were inoculated with 1,400 PFU of HVT per bird, there was no evidence of contact infection among cagemates through 8 weeks after contact, although the two donor chickens were infected with HVT as indicated by HVT viremia and/or precipitins in their blood. When a larger dose of the same virus material (11,280 PFU) was given to donor chickens, the virus did spread horizontally (Group B). Further increase in the virus dose per donor bird to 490,000 PFU, however, did not result in a greater incidence of contact infection among cagemates (Group The two donor chickens of Group B,
when examined 8 weeks after virus inoculation, carried both the virus and precipitins in their blood, while in the 2 donors of Group C only precipitins were noted. Six untreated control birds were free of any cytopathic agents in their blood and were negative for precipitins to HVT antigens. Trial 3: HVT/DEF spread readily from inoculated chickens to contact cagemates (Table 2). Similar horizontal spread of HVT resulted in both Groups A and B when donor birds were inoculated with 5,800 and 435,000 PFU of H V T / DEF respectively. In Group C, where donor birds were each inoculated with 23,200 PFU of HVT/CEF, the virus also spread horizontally but less effectively than HVT/DEF in Groups A and B. Trial 4: The results are summarized in Table 3. When HVT was inoculated into 10-day-old chicks either positive (Group A) or negative (Group B) for MDV maternal antibodies, none of the cagemates from Groups A and B was infected with HVT at 4 weeks. Two of the 6 cagemates of Group A and none from Group B had an HVT viremia at 8 week after contact. All three inoculated donors from each of Groups A and B were viremic when examined 8 weeks after virus
T A B L E 2.—Horizontal transmission in chickens of turkey herpesvirus grown in duck embryo fibroblast cell cultures {Trial 3) Exposure to HVT
{HVT)
HVT viremia/antibodies demonstrated Age exposed
Post-inoculation or contact 4 weeks 8 weeks
Group
Exposure method
A
Inoculated Contact cagemates5
DEF-passaged1 (5,800)2 None
8wks 2 days
NT» 0/4
NT 0/4
0/2< 5/8
1/2 2/8
B
Inoculated Contact cagemates
DEF-passaged (435,000) None
8 wks 2 days
NT 0/6
NT 0/6
2/2 5/10
2/2 3/10
C
Inoculated Contact cagemates
CEF-passaged« (23,200) None
8 wks 2 days
NT 0/10
NT 0/10
2/2 3/12
2/2 1/12
1 2
HVT used
HVT passaged 15 times in duck embryo fibroblast cell cultures. Virus dose in plaque-forming-units per donor chicken. »Not tested. *6 Number of birds positive over number of birds examined. Four chicks died of smothering within a week. • HVT passaged 13 times in chicken embryo fibroblast cell cultures.
Viremia Antibodies
Viremia Antibodies
612
B. R. CHO AND S. G. KENZY T A B L E S.^~Effects of Marek's disease maternal antibodies in the inoculated chicks upon transmission of turkey herpesvirus (HVT) to contact cagemates {Trial 4)
Group
MD maternal abyi in HVTinoculated donor chicks
Exposure to HVT
horizontal
HVT viremia and/or antibodies demonstrated
Exposure method
Age exposed
Post-inoculation or contact 4 weeks 8 weeks Viremia
Antibodies
A
Positive
Inoculated (7,650)*5 Contact cagemates
10 days 10 days
NT 5 0/10
NT 0/10
3/3* 2/6
3/3 1/6
B
Negative
Inoculated (7,650)6 Contact cagemates
10 days 10 days
NT 0/10
NT 0/10
3/3 0/7
3/3 0/7
C
Negative
Inoculated (7,560) 5 Contact cagemates
8 wks 10 days
NT 2/10
NT 0/10
1/2 4/9
1/2 3/9
NT
NT
0/12
0/12
D
None (chick controls)
Viremia Antibodies
1 Marek's disease virus maternal antibodies as determined by agar gel precipitin test. 3 HVT dose in plaque-forming-units per bird. «4 Not tested. Number of birds positive over number of birds examined. 6 A few chicks died of smothering within a week.
inoculation. In Group C, of which 2 donor chickens were inoculated at 8 weeks of age with the same HVT, 2 of the 10 cagemates and 4 of the 9 cagemates at 4 and 8 weeks respectively post-contact were viremic with HVT. None of the contact cagemates had precipitins to HVT antigens at 4 weeks, but more than half of the viremic cagemates as well as most of the inoculated donors were positive at 8 weeks. Untreated control birds were free of any demonstable cytopathic agents in their blood and were also negative for precipitins to HVT antigens. In Trials 1 through 4, contact cagemates which developed precipitins were invariably also viremic and the antibodies were not detected in any contact cagemates without a detectable HVT viremia. Inoculated birds and their contact cagemates in all trials remained clinically normal and free of any gross pathologic lesions throughout the course of the experiments. DISCUSSION
This study further demonstrated that HVT could spread from inoculated chickens to cagemates through contact.
The virus dose given to donor chickens appeared to influence the horizontal spread of HVT. The likelihood of horizontal transmission increased when the virus dose given to donor chickens was increased from 7,400 to 74,000 PFU of the cell-associated and from 1,400 to 11,280 PFU of the cell-free HVT. There was little or no spread when donor birds were each inoculated with an extremely large dose of the cell-associated (370,000 PFU) or the cell-free HVT (490,000 PFU) which had been grown in CEF cell cultures. However, HVT which had been grown in DEF cell cultures spread readily when a similarly large dose (435,000 PFU) of the virus was given to donor birds. The reason for this discrepancy was not determined but may be due to differences in growing the virus (i.e., growing in CEF versus DEF cell cultures) and/or "auto-interference" as observed with influenza virus when a large amount of the virus was inoculated into embryonated eggs (Ziegler et al., 1944). It appeared that cell-associated HVT spread more readily (Trial 1) than cellfree virus (Trial 2), and so did HVT grown in DEF cell cultures (Trial 3) than the virus grown in CEF cell cultures.
HERPESVIRUS TRANSMISSION
MDV maternal antibodies present in the donor chickens at the time of HVT inoculation did not inhibit the horizontal spread of HVT (Trial 4). It was reported that MDV maternal antibodies were of little or no influence on infection of chicks with HVT (Patrascu et al., 1972). The results of Trial 4 rather seemed to confirm the preliminary observations (Cho et al., 1971) which described age effect of donors on the horizontal spread of HVT. Since donor and contact chickens in each trial were siblings of respective hatches and the HB isolators were identical in design, the differences in horizontal spread of HVT were not likely due to genetic or husbandry factors. ACKNOWLEDGMENT
We gratefully acknowledge the fine technical assistance of Mr. Wayne Bryson.
613
REFERENCES Calnek, B. W., S. B. Hitchner and H. K. Adldinger, 1970. Lyophilization of cell-free Marek's disease herpesvirus and a herpesvirus from turkeys. Appl. Microbiol. 20: 723-726. Calnek, B. W., G. Garrido, W. Okazaki and I. V. Patrascu, 1972. In vitro methods for assay of turkey herpesvirus. Avian Dis. 16: 52-56. Cho, B. R., S. G. Kenzy and S. A. Haider, 1971. Horizontal transmission of turkey herpesvirus to chickens. 1. Preliminary observation. Poultry Sci. 50: 881-887. Kenzy, S. G., R. F. Lapen and J. M. Sharma, 1969. Transmission of cutaneous Marek's disease (skin leukosis). Presented at the 106th Annual AVMA Meeting, July 13-17, Minneapolis, Minnesota. Patrascu, I. V., B. W. Calnek and N. W. Smith, 1972. Vaccination with lyophilized turkey herpesvirus (HVT): Minimum infective and protective doses. Avian Dis. 16: 86-93. Ziegler, J. E., Jr., G. I. Lavin and F. L. Horsfall, Jr., 1944. Interference between the influenza viruses. II. The effect of virus rendered non-infective by ultraviolet radiation upon the multiplication of influenza viruses in the chick embryo. J. Exp. Med. 79:379-400.
NEWS AND NOTES (Continuedfrom page 603) analysis of lipids, 15—Precipitation of low density lipoproteins with polyanions, 16—Electrophoretic methods for lipoprotein separation, 17—New techniques for the study of lipoproteins, and 18—Automated methods of gas analysis. Registration fees for the Conference are $110 for A.O.C.S. members and $140 for nonmembers. More detailed information is available from James Lyon, Executive Director, American Oil Chemists' Society, 508 South Sixth Street, Champaign, Illinois 61820. Telephone (217) 359-2344. The American Oil Chemists' Society is a not-forprofit association of chemists, other scientists and technologists. The Society's work is directed to the advancement of technology in oils, other lipids, and associated materials; to the improvement of the technical competence of its members and others in the field; and to education and training at all levels in these and related fields of science and technology. ANNUAL A.I.B.S. MEETING The 24th Annual Meeting of the American Insti-
tute of Biological Sciences will be held at the University of Massachusetts, Amherst, June 17-22. The Plenary Session entitled "Manpower in the Biological Sciences" will be held on Monday, June 18, at 8 p.m. Betty Vetter of the Scientific Manpower Commission, who will be the keynote speaker, will discuss the national manpower needs brought to light by the A.I.B.S. Manpower Survey. The following day's symposium "Manpower: Supply and Demand" under the joint sponsorship of the A.I.B.S., F.A.S.E.B., and Scientific Manpower Commission, will continue on this most important subject. In addition the A.I.B.S. Education Division has planned several symposia in the mornings, including: Graduate Education, Research in Biological Education, Environmental Education, and Environmental Regulation. Informal sessions for open discussion will be held in the afternoons. A number of Workshops are also scheduled, as well as Contributed Paper Sessions. Other Symposia sponsored by A.I.B.S. and participating societies are: Physiological Adaptation to
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