Evaluation of the Agar-gel Immunodiffusion Test for the Detection of Precipitating Antibodies Against Progressive Pneumonia Virus of Sheep T. W. Molitor, 1. A. Schipper, D. L. Berryhill and M. R. Light* ABSTRACT
Various cell cultures were evaluated for their ability to support progressive pneumonia virus infection in vitro. Ovine trachea cells supported progressive pneumonia virus infection for an extended time, were extremely durable and could be passaged up until 30 passages. Progressive pneumonia virus infected ovine trachea cells were then used for the production of antigen for agar-gel immunodiffusion. A method for concentrating antigen, diafiltration, was compared to dialysis against polyethylene glycol. Using diafiltration, the concentrated virus was easily quantitated, less viscous (and therefore easier to apply) and only produced one precipitation line. Agar-gel immunodiffusion was used to survey 401 animals from two sheep flocks. One flock (96 sheep) was free of progressive pneumonia while the other flock had 111 of 305 total animals positive for precipitating antibodies. The incidence of precipitating antibodies in sheep ranged from 23% for yearling ewes to 80 % in ewes seven years old.
une longue periode; elles s'avererent aussi extremement durables, parce qu'elles permirent de realiser jusqu'a 30 passages successifs. On utilisa par consequent ce genre de culture cellulaire pour obtenir un antigene utilisable dans l'epreuve d'immunodiffusion sur gelose. On compara ensuite la diafiltration, un moyen de concentrer l'antigene, a la dialyse en presence de poly ethyllene glycol. L'antigene concentre 'a l'aide de la diafiltration se revela facile a quantifier, moins visqueux et par consequent plus facile a utiliser; il ne produisit en outre qu'une seule ligne de precipitation. L'epreuve d'immunodiffusion sur gelose servit 'a effectuer un releve serologique impliquant les 401 sujets de deux troupeaux. L'un d'eux, qui comptait 96 moutons, s'avera exempt de pneumonie progressive. On decela par ailleurs 111 reacteurs au sein de l'autre troupeau qui comptait 305 sujets. La proportion des moutons qui possedaient des anticorps precipitants varia de 23%, chez les sujets ages d'un an, 'a 80%, chez les brebis agees de sept ans.
INTRODUCTION
RASUME Cette experience visait 'a determiner l'aptitude de diverses cultures cellulaires a supporter une infection in vitro par le virus de la pneumonie progressive. Des cellules tracheales ovines supporterent une telle infection, pour Departments
of
Veterinary
Science
(Molitor
and
Schipper), Bacteriology (Berryhill) and Animal Science (Light), North Dakota State University, Fargo, North Dakota 58102.
The publication in part fulfills the requirements of M.S. degree, North Dakota State University, for the senior author.
Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Scientific Series No. 977.
Submitted November 21, 1978.
280
Progressive pneumonia of sheep is a chronic pulmonary disease, characterized by a slow, progressive loss of weight, increasingly severe respiratory distress and death (10,13). The etiological agent, progressive pneumonia virus (PPV), (2,10) is an enveloped, spherical, single-stranded RNA virus classified in the family Retroviridae, subfamily Lentivirinae (4). Various primary cell cultures have been reported to support the growth of Lentivirinae, including cells derived from ovine lung, liver, testes, choroid plexus and trachea (9). Bovine choroid plexus and trachea cells have been used for the cultivation of other slow viruses (7). Virus development of Lentivirinae is accompanied by cytopathic ef-
Con. J. comp. Med.
fects (CPE), culminating in eventual destruction of the entire monolayer (8). The changes include the formation of elongated fibroblastic cells with refractile cytoplasm, and large multinucleated giant cells (polykaryocytes) with stellate processes (12). Cytoplasmic inclusion bodies are absent. An extended time and numerous passages are often needed for the appearance of CPE, sometimes up to eight weeks. For this reason, a durable cell strain is needed to support PPV infections for extended periods. A number of diseases of sheep resembling progressive pneumonia occur in different parts of the world. Clinical, histopathological, biochemical and virological studies have disclosed that zwoegerziekte, maedi and progressive pneumonia are identical diseases (3,17,19). Visna, a demyelinating disease, resembles these diseases serologically but differs greatly clinically (5,15). Clinically, progressive pneumonia is characterized by an obdurate dry cough, afebrile state, increased loss of weight and increasingly severe respiratory distress. The clinical signs appear one to four years after the animal is exposed to the causative virus (13). The disease course lasts over a two to eight month period but may be enhanced if the animal is under stress (5). Diagnosis of progressive pneumonia can be made from clinical signs in later stages of the disease, (13,16) lesions upon necropsy (5,8) and/or the detection of precipitating antibodies (1). The relatively early appearance of precipitating antibodies in progressive pneumonia can result in the detection of diseased animals in which specific lesions have not sufficiently developed (18). Agar-gel immunodiffusion (AGID) is a reliable, simple serological procedure for the detection of precipitating antibodies against PPV(1). A problem with AGID is the production of a strong yet specific soluble antigen. In this study, various primary cell cultures were evaluated for their ability to support PPV infection in vitro. The cell culture which best supported PPV infection was used for the production of AGID antigen. Methods for concentrating PPV antigen were also examined and compared to the procedure previously reported (1). Improved techniques for production of virus and preparation of antigen provide additional information leading to the better understanding of the epidemiology and pathogenesis of this disease.
Volume 43
July, 1979
MATERIALS AND METHODS CELL CULTURE Ovine choroid plexus (OCP), ovine lung (OL) and bovine choroid plexus (BCP) cells were prepared by trypsin dispersion of choroid plexuses and lungs from exsanguinated fetal ovine or bovine animals as previously described (8). To prepare ovine trachea (OT) cells a trachea (from a Border-Leicester fetus) was attached to a ring stand and one end was clamped. Then 2.0-5.0 ml of trypsin was added to the distal end of the trachea. After ten minutes the trypsin was removed and fresh trypsin was added. This process was repeated for one hour. Trypsin released the cells lining the trachea and these cells were propagated in cell culture. Monolayers were grown in bicarbonate-buffered Eagle's minimal essential medium (MEM)I containing either 5% fetal bovine serum (FBS) or 5% fetal ovine serum, 0.25% lactalbumin and antibiotics (100 units of penicillin and 100 ,ug of streptomycin per ml). Cells were grown in plastic tissue culture flasks,2 ranging in size from 25 to 150 cm2, and incubated at 37°C. The OL, OCP and BCP cell strains prepared in this manner consisted of elongated fibroblastic cells which survived ten to 20 serial passages. The OT cell strains survived 20-30 passages.
VIRUS Progressive pneumonia virus was isolated from the lungs of a naturally infected sheep from the North Dakota State University (NDSU) flock. The isolate was verified as PPV in a personal communication from Dr. Terry Jackson at the National Animal Disease Centre (NADC), Agricultural Research Service, P.O. Box 70, Ames, Iowa and designated NDSU-1. This virus isolate was used for all further ex-
periments. ANTIGEN For virus production, OT, OL and OCP cells were infected with PPV. After the lGibco, Grand Island, New York. 2Corning Glass Works, Corning, New York or Falcon Plastics, Oxnard, California.
281
appearance of CPE, culture fluids were collected and stored at -20°C. The fluids were pooled, inactivated at 560C for 60 min and centrifuged (2,000 x g) for 20 min to remove cellular debris. Different methods for concentrating inactivated culture fluids were compared. Concentration of culture fluids from PPV infected OL cells by dialysis against polyethylene glycol was previously described (1). Progressive pneumonia virus antigen prepared by this method, antigen supplied by NADC3 and antigens prepared by concentration by diafiltration4 were all compared. In diafiltration, molecules can be separated according to molecular weight by varying the filter. Filters can separate molecules ranging in molecular weight from 300,000 (XM 300 filter) to 500 (UM 05 filter). The diafiltration unit is called an "Amicon cell". Three cells, containing volumes of 200, 50 and 10 ml, respectively, were available for this study. To concentrate the antigen a hundredfold, the 200 ml cell and then the 10 ml cell were most often used in succession. Concentrated antigen preparations were tested against known positive serums from sheep that died of naturally acquired progressive pneumonia. Antigens showing strong precipitation lines were used for further testing. AGAR-GEL IMMUNODIFFUSION
The AGID test was conducted in 15 x 100 mm plastic Petri dishes containing either 10, 15 or 20 ml of 1.5% Noble agar in 0.05 M tris buffer, pH 7.5. A series of tests was made to compare PPV precipitation reactions in different gel preparations. Salt concentration, pH and buffer were varied in these experiments. The salt concentration ranged from 0.85 to 10.0% and pH ranged from 7.0 to 10.0. On occasion, 0.15 M borate buffer or 25 mM barbital buffer was substituted for tris buffer. Agar was poured into plates and allowed to solidify at 25°C for one hour. A template with one central and six peripheral punches was used to make wells in the agar. Well sizes and locations were varied to determine optimal conditions. Wells varied from 4.0 to 8.0 mm in diameter and from 2.0 to 3.5 3National Animal Disease Center, Agricultural Research Service, P.O. Box 70, Ames, Iowa.
4Amicon, Lexington, Massachusetts.
282
mm apart in all directions. Antigen was placed in the center well while PPV positive serums, PPV negative serums and test serums were placed in the outer wells. The gels were examined over a narrow beam of light for precipitin lines at eight, 24 and
48 hours. ANIMALS
The Department of Animal Science, NDSU, manages two separate sheep flocks. One (PPV infected) is housed at the NDSU sheep barn, and the other (noninfected) is housed at the Animal Research Center (ARC). The NDSU sheep barn maintains a flock of 250-300 animals. The ARC flock consists of approximately 100 animals. A distance of 1.6 km separates the PPV infected and the noninfected flocks. Separation of sheep into two flocks was originally accomplished by removing lambs postpartum and formula feeding them. Serum samples from the sheep housed at the ARC have periodically been tested for the presence of PPV antibodies. Results have been negative. Histopathological examinations and lack of mortality due to PPV further substantiate the absence of progressive pneumonia in this flock. The NDSU sheepbarn flock is extensively infected with progressive pneumonia. This fact is documented by positive diagnoses at necropsy, histopathological results and the presence of PPV antibodies. Sheep vary in age from one to eight years. SERUM SAMPLES
Blood was collected from all sheep housed at the ARC and the NDSU sheep barn at various intervals. The blood was allowed to clot and the serum was extracted and stored at -20°C. Blood samples were also collected from all sheep brought to the NDSU Veterinary Diagnostic Laboratories which exhibited clinical signs associated with progressive pneumonia. All blood was collected from the jugular vein.
RESULTS CELL CULTURE
Four processed cell cultures were evalu-
Can. J. comp. Med.
ated for growth characteristics including the ability to support PPV infection, the number of serial passages possible and the length of time cells were able to adhere to the flask without disruption of confluent growth. The OT cells retained confluent monolayers up to 30 passages. Figure 1 depicts a confluent monolayer of OT cells before infection with PPV. The OT cells were capable of adherence to plastic flasks for extended periods of time (weeks to months) before passage without disruption of the monolayers. Since PPV may require one to two months to cause CPE, the longer the cell culture remains attached, the more suitable it is for PPV growth. The OCP, OL and BCP cells require passage after approximately one week of growth. Figure 2 depicts PPV infected, unstained ovine trachea cells six days following inoculation. Figure 3 shows a Giemsa-stained PPV infected multinucleated giant cell. The most important cell culture characteristic is the ability to support viral replication. Of the four cell strains tested, all ovine-derived cell strains supported PPV growth (CPE formation) but bovine cells did not. The OT cells were the only
cells that could be continuously passaged. After the appearance of CPE and destruction of a portion of the monolayer, fresh uninfected OT cells were added to the flask. These cells remained continuously infected and could be later passaged. Antigens prepared from concentrated PPV infected OL and OCP cells produced weaker precipitation lines with PPV positive serums than antigens prepared from PPV infected OT cells. For this reason, OT cells were used for the production of antigens used for AGID.
ANTIGEN Soluble PPV antigens, concentrated either by diafiltration or by dialysis against polyethylene glycol, gave strong precipitation lines when reacted against positive serums of naturally infected sheep. Infected cell culture fluids were collected, pooled, subjected to one cycle of freezing and thawing and then concentrated. Concentrated cellular debris from PPV infected OT cells produced no precipitation lines. Concentration by dialysis required three
Fig. 1. Uninfected ovine trachea cells. X200.
Volume 43 - July, 1979
283
Fig. 2. PPV infected ovine trachea cells six days following inoculation. X200.
molecular weight, best retained the antigen used for AGID. Diafiltration required one to two days to concentrate antigen one hundredfold. This method was readily quantitated and little antigen was lost in transferring it to a tube. Because smaller molecular weight molecules were removed, the diafiltered antigen was less viscous and easier to apply than that prepared by dialysis. The diafiltered antigen yielded one distinct line of precipitation on every occasion. Fig. 3. PPV infected multinucleated giant cell. Giemsa stain. X470.
to four days, was difficult to quantitate, was difficult to perform due to the viscosity of the material and antigen was usually lost in the process of transferring it to a tube. The dialyzed antigen sometimes yielded two lines of precipitation in AGID and sometimes none at all. The Amicon XM100 diafiltration filter, which filters molecules less than 100,000 in
284
AGAR-GEL IMMUNODIFFUSION
Semisolid medium containing 1.5% Noble agar, 8.5% NaCl and 0.05 M tris buffer allowed the production of distinct precipitation lines by various antigens and positive serums. Tris buffer was superior to either borate or barbital buffer with regard to the sharpness of precipitation lines. Although the pH of the medium had little effect on the development of precipitation lines in the range of 7.0-9.0, pH 7.5 tris buffer was consistently better relative to the sharpness of precipitation lines. The
Can. J. comp. Med.
Fig. 4. AGID test place with PPV antigen in the center well, positive control serums in wells 1, 3 and 5 and test serums in wells 2, 4 and 6. The serum in well 2 forms a distinct line of identity with positive control serums.
concentration of NaCl in the medium had a significant effect on the development of precipitation lines. No lines were observed when NaCl was absent but as the NaCl concentration was increased to 8.5% they appeared and increased in intensity. Examples of AGID reactions between PPV antigen and negative or positive serum samples are shown in Fig. 4. One line of precipitation was produced by serums from naturally infected sheep. For all testing, PPV positive control serums were placed in alternate wells and the test serums were placed between positive controls. Serums were recorded as negative if the precipitation lines from positive control serums migrated into the well, slightly positive if the precipitation lines from the positive control serums curved into the well and strongly positive if a line of identity formed with those of the positive control serums.
Volume 43
-
July, 1979
FLOCK SURVEY
The entire sheep population at NDSU was surveyed for precipitating antibodies against PPV by AGID. All 96 serums from sheep at the ARC were negative. Of the 305 serums collected from sheep at the NDSU sheep barn 111 were positive and 194 were negative. These results include animals from seven different breeds which ranged from one to seven years in age (Table I). Newborn lambs were not tested. Of the 401 total samples, 100 serums were tested against PPV antigen supplied by NADC. Results were in complete agreement with results from the same serums reacting against the NDSU-1 antigen. In general, the proportion of sheep with positive serums increased with the age of the sheep. Only 23% of the yearlings had positive serums, whereas 80% of the seven year olds were positive.
TABLE I. Agar-Gel Immunodiffusion Analysis of Serum Samples According to Age of the Sheep Age (years) 1 2 3 4 5 6 7 Total
Proportion of Positive Percent Serums 25/107 23%
11/52
19/50 11/21 17/30 12/25
16/20
111/305
21% 38% 52% 57% 48%
80% 36%
DISCUSSION The initial task in these experiments was to determine the cell culture which best supported the propagation of PPV in vitro. Diagnosis of progressive pneumonia has been primarily based on lesions but a serological test (AGID) is now available to detect precipitating antibodies against PPV (1). An improved method for preparing PPV antigen for AGID was discovered. The AGID test was then used to survey two sheep flocks for the incidence of pro-
gressive pneumonia. CELL CULTURE
Whether or not a particular virus may infect and replicate in vitro depends a great deal upon the type of cell culture used. In particular, PPV is a difficult virus to isolate and propagate in cell culture due to the length of time for appearance of CPE, the sensitivity of PPV to various conditions and the specificity of the virus for one host, sheep. Cell cultures derived from numerous ovine organs have been reported to support RNA slow virus growth (8). In this study, OT cells supported PPV growth for an extended time, were extremely durable and could be passaged up until 30 passages. Infected OT cell cultures readily developed CPE characteristics of PPV infection (Figs. 1-3). Bovine-derived cell cultures did not support PPV growth.
ANTIGEN A problem with AGID is the production of a strong but specific antigen. In a previous report (1), soluble PPV antigen was
286
produced by concentrating PPV infected ovine lung cells by dialysis against polyethylene glycol. This method produced a strong antigen but quantitation was difficult and, due to viscosity, the antigen was difficult to apply to the wells. The production of antigen by diafiltration of PPV infected OT cells circumvented many of the problems of dialysis. Using diafiltration, the concentrated virus was easily quantitated and, because the filters selectively removed molecules with smaller weights, concentrated antigen was less viscous and only produced one precipitation line. All serums that produced precipitation lines against the NADC dialysis antigen also produced lines against the NDSU-1 diafiltered antigen. A complete line of identity was formed between the NADC antigen and the NDSU-1 antigen. AGAR-GEL IMMUNODIFFUSION
Sodium chloride at a concentration of 8.5 % was found to be essential for the production of precipitation bands. These results are similar to those for a microimmunodiffusiorn test for infectious bovine rhinotracheitis virus (11). An explanation of the requirement for a high NaCl concentration has been suggested (11). Some antigen-antibody complexes are too hydrophilic to precipitate, become trapped in the agar and fail to appear as a precipitation band in a hypotonic saline environment. A high salt concentration reduces the solubility of the hydrophilic complex and this results in the formation of a visible precipitin line. The AGID test was a satisfactory and uncomplicated technique for detecting pre-
cipitating antibodies against PPV. Compared to other tests (6,18), AGID is simple and economical and diagnostic results are obtained sooner. FLOCK SURVEY
One hundred and eleven of 305 animals tested were positive for precipitating antibodies against PPV (Table I). Of the sheep housed at the NDSU sheep barn, 36% were positive by AGID. Incidence according to age varied from 23% for yearling ewes to 80% in ewes seven years old. Age must play an important role in the incidence of progressive pneumonia. A high
Can. J. comp. Med.
percentage of three year old sheep was positive by the AGID test (Table I). The older the animal and the longer it remains in the flock the greater the chance for exposure to PPV. Very few one and two year old sheep were positive by AGID. Sheep do not normally succumb to progressive pneumonia before two years of age because of the long disease course (13,14). REFERENCES 1. CUTLIP, R. C., T. A. JACKSON and G. A. LAIRD. Immunodiffusion test for ovine progressive pneumonia. Am. J. vet. Res. 38: 1081-1084. 1977. R. C. and G. A. LAIRD. Isolation and CUTLIP, 2. characterization of a virus associated with progressive pneumonia (maedi) of sheep. Am. J. vet. Res. 38: 1377-1382. 1976. 3. DEBOER, G. F. Zwoegerziekte virus, the causative agent for progressive interstitial pneumonia (maedi) and leucoencephalitis (visna) in sheep. Res. vet. Sci. 18: 15-25. 1975. 4. FENNER, F. Classification and nomenclature of viruses. Intervirology 3: 61-64. 1977. 5. GEORGSSON, G., N. NATHANSON, P. A. PALSSON and G. PETURSSON. The pathology of visna and maedi in sheep. In The Slow Virus Diseases of Animals and Man. Edited by R. H. Kimberlin. New York: American Elsevier. 1976. 6. GUDNADOTTIR, M. and K. KRISTENSDOTTIR. Complement fixing antibodies in sera of sheep affected with visna and maedi. J. Immun. 98: 663-667. 1967. 7. HANSON, R. P., R. J. ECKROADE, R. MARSH, G. M. ZUREINE, C. L. KANITZ and D. P. GUSTAFSON. Susceptibility of mink to sheep scrapie. Science 172: 859-861. 1971.
Volume 43 - July, 1979
8. HARTER, D. H. and J. E. COWARD. Sheep progressive pneumonia viruses. "Slow" cytolytic agents with tumor virus properties: a review. Tex. Rep. Biol. Med. 32: 649-664. 1974. 9. HARTER, D. H., K. C. HSU and H. M. ROSE. Multiplication of visna virus in bovine and porcine cell lines. Proc. Soc. exp. Biol. Med. 129: 295-300. 1968. 10. KENNEDY R. C., E. M. EKLUND, C. LOPEZ and W. J. HADLOW. Isolation of a vir-us from the lungs of Montana shep afflicted with progressive pneumonia. Virology 35: 483-484. 1968. 11. LEJEUNE, J. M., L. T. HART, A. D. LARSON and C. L. SEGER. Microimmunodiffusion test for detection of antibodies to infectious bovine rhinotracheitis vir-us in bovine serum. Am. J. vet. Res. 38: 459-463. 1977. 12. LOPEZ, C., C. E. EKLUND and W. HADLOW. Tissue culture studies of progressive pneumonia, a slow infectious disease of sheep. J. infect. Dis. 138: 1035-1040. 1971. 13. MARSH. H. Progressive pneumonia in sheep. J. Am. vet. med. Ass. 15: 458-473. 1923. 14. SIGURDORDOTTIR, B. H. and H. THORMAR. Isolation of a viral agent from the lung of sheep infected with maedi. J. infeet. Dis. 114: 55-60. 1964. 15. SIGURDSSON, B. Maedi, a slow progressive pneumonia of sheep: an epizoological and a pathological study. Br. vet. J. 110: 225-270. 1954. 16. SIGURDSSON, B. and P. A. PALSSON. Visna of sheep: A slow demyelinating infection. Br. J. exp. Path. 39: 519-528. 1958. 17. TAKEMOTO, K. K., C. F. MATTERN, L. B. STONE, J. E. COE and G. LAVELLE. Antigenic and morphological similarities of progressive pneu6monia virus, a recently isolated "slow virus" of sheep, to visna and maedi viruses. J. Virol. 7: 301308. 1971. 18. TERPESTRA, C. and G. F. DEBOER. Precipitating antibodies against maedi-visna virus in experimentally infected sheep. Arch. ges. Virusforsch. 43: 53-62. 1973. 19. WEISS, M. J., E. P. ZEELON, R. W. SWEET, D. H. HARTER and S. SPIELGMAN. Immunological cross reaction of the major internal protein component from "slow" viruses of sheep. Virology 76: 851-854. 1977.
287
Various cell cultures were evaluated for their ability to support progressive pneumonia virus infection in vitro. Ovine trachea cells supported progressive pneumonia virus infection for an extended time, were extremely durable and could be passaged up until 30 passages. Progressive pneumonia virus infected ovine trachea cells were then used for the production of antigen for agar-gel immunodiffusion. A method for concentrating antigen, diafiltration, was compared to dialysis against polyethylene glycol. Using diafiltration, the concentrated virus was easily quantitated, less viscous (and therefore easier to apply) and only produced one precipitation line. Agar-gel immunodiffusion was used to survey 401 animals from two sheep flocks. One flock (96 sheep) was free of progressive pneumonia while the other flock had 111 of 305 total animals positive for precipitating antibodies. The incidence of precipitating antibodies in sheep ranged from 23% for yearling ewes to 80 % in ewes seven years old.
une longue periode; elles s'avererent aussi extremement durables, parce qu'elles permirent de realiser jusqu'a 30 passages successifs. On utilisa par consequent ce genre de culture cellulaire pour obtenir un antigene utilisable dans l'epreuve d'immunodiffusion sur gelose. On compara ensuite la diafiltration, un moyen de concentrer l'antigene, a la dialyse en presence de poly ethyllene glycol. L'antigene concentre 'a l'aide de la diafiltration se revela facile a quantifier, moins visqueux et par consequent plus facile a utiliser; il ne produisit en outre qu'une seule ligne de precipitation. L'epreuve d'immunodiffusion sur gelose servit 'a effectuer un releve serologique impliquant les 401 sujets de deux troupeaux. L'un d'eux, qui comptait 96 moutons, s'avera exempt de pneumonie progressive. On decela par ailleurs 111 reacteurs au sein de l'autre troupeau qui comptait 305 sujets. La proportion des moutons qui possedaient des anticorps precipitants varia de 23%, chez les sujets ages d'un an, 'a 80%, chez les brebis agees de sept ans.
INTRODUCTION
RASUME Cette experience visait 'a determiner l'aptitude de diverses cultures cellulaires a supporter une infection in vitro par le virus de la pneumonie progressive. Des cellules tracheales ovines supporterent une telle infection, pour Departments
of
Veterinary
Science
(Molitor
and
Schipper), Bacteriology (Berryhill) and Animal Science (Light), North Dakota State University, Fargo, North Dakota 58102.
The publication in part fulfills the requirements of M.S. degree, North Dakota State University, for the senior author.
Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Scientific Series No. 977.
Submitted November 21, 1978.
280
Progressive pneumonia of sheep is a chronic pulmonary disease, characterized by a slow, progressive loss of weight, increasingly severe respiratory distress and death (10,13). The etiological agent, progressive pneumonia virus (PPV), (2,10) is an enveloped, spherical, single-stranded RNA virus classified in the family Retroviridae, subfamily Lentivirinae (4). Various primary cell cultures have been reported to support the growth of Lentivirinae, including cells derived from ovine lung, liver, testes, choroid plexus and trachea (9). Bovine choroid plexus and trachea cells have been used for the cultivation of other slow viruses (7). Virus development of Lentivirinae is accompanied by cytopathic ef-
Con. J. comp. Med.
fects (CPE), culminating in eventual destruction of the entire monolayer (8). The changes include the formation of elongated fibroblastic cells with refractile cytoplasm, and large multinucleated giant cells (polykaryocytes) with stellate processes (12). Cytoplasmic inclusion bodies are absent. An extended time and numerous passages are often needed for the appearance of CPE, sometimes up to eight weeks. For this reason, a durable cell strain is needed to support PPV infections for extended periods. A number of diseases of sheep resembling progressive pneumonia occur in different parts of the world. Clinical, histopathological, biochemical and virological studies have disclosed that zwoegerziekte, maedi and progressive pneumonia are identical diseases (3,17,19). Visna, a demyelinating disease, resembles these diseases serologically but differs greatly clinically (5,15). Clinically, progressive pneumonia is characterized by an obdurate dry cough, afebrile state, increased loss of weight and increasingly severe respiratory distress. The clinical signs appear one to four years after the animal is exposed to the causative virus (13). The disease course lasts over a two to eight month period but may be enhanced if the animal is under stress (5). Diagnosis of progressive pneumonia can be made from clinical signs in later stages of the disease, (13,16) lesions upon necropsy (5,8) and/or the detection of precipitating antibodies (1). The relatively early appearance of precipitating antibodies in progressive pneumonia can result in the detection of diseased animals in which specific lesions have not sufficiently developed (18). Agar-gel immunodiffusion (AGID) is a reliable, simple serological procedure for the detection of precipitating antibodies against PPV(1). A problem with AGID is the production of a strong yet specific soluble antigen. In this study, various primary cell cultures were evaluated for their ability to support PPV infection in vitro. The cell culture which best supported PPV infection was used for the production of AGID antigen. Methods for concentrating PPV antigen were also examined and compared to the procedure previously reported (1). Improved techniques for production of virus and preparation of antigen provide additional information leading to the better understanding of the epidemiology and pathogenesis of this disease.
Volume 43
July, 1979
MATERIALS AND METHODS CELL CULTURE Ovine choroid plexus (OCP), ovine lung (OL) and bovine choroid plexus (BCP) cells were prepared by trypsin dispersion of choroid plexuses and lungs from exsanguinated fetal ovine or bovine animals as previously described (8). To prepare ovine trachea (OT) cells a trachea (from a Border-Leicester fetus) was attached to a ring stand and one end was clamped. Then 2.0-5.0 ml of trypsin was added to the distal end of the trachea. After ten minutes the trypsin was removed and fresh trypsin was added. This process was repeated for one hour. Trypsin released the cells lining the trachea and these cells were propagated in cell culture. Monolayers were grown in bicarbonate-buffered Eagle's minimal essential medium (MEM)I containing either 5% fetal bovine serum (FBS) or 5% fetal ovine serum, 0.25% lactalbumin and antibiotics (100 units of penicillin and 100 ,ug of streptomycin per ml). Cells were grown in plastic tissue culture flasks,2 ranging in size from 25 to 150 cm2, and incubated at 37°C. The OL, OCP and BCP cell strains prepared in this manner consisted of elongated fibroblastic cells which survived ten to 20 serial passages. The OT cell strains survived 20-30 passages.
VIRUS Progressive pneumonia virus was isolated from the lungs of a naturally infected sheep from the North Dakota State University (NDSU) flock. The isolate was verified as PPV in a personal communication from Dr. Terry Jackson at the National Animal Disease Centre (NADC), Agricultural Research Service, P.O. Box 70, Ames, Iowa and designated NDSU-1. This virus isolate was used for all further ex-
periments. ANTIGEN For virus production, OT, OL and OCP cells were infected with PPV. After the lGibco, Grand Island, New York. 2Corning Glass Works, Corning, New York or Falcon Plastics, Oxnard, California.
281
appearance of CPE, culture fluids were collected and stored at -20°C. The fluids were pooled, inactivated at 560C for 60 min and centrifuged (2,000 x g) for 20 min to remove cellular debris. Different methods for concentrating inactivated culture fluids were compared. Concentration of culture fluids from PPV infected OL cells by dialysis against polyethylene glycol was previously described (1). Progressive pneumonia virus antigen prepared by this method, antigen supplied by NADC3 and antigens prepared by concentration by diafiltration4 were all compared. In diafiltration, molecules can be separated according to molecular weight by varying the filter. Filters can separate molecules ranging in molecular weight from 300,000 (XM 300 filter) to 500 (UM 05 filter). The diafiltration unit is called an "Amicon cell". Three cells, containing volumes of 200, 50 and 10 ml, respectively, were available for this study. To concentrate the antigen a hundredfold, the 200 ml cell and then the 10 ml cell were most often used in succession. Concentrated antigen preparations were tested against known positive serums from sheep that died of naturally acquired progressive pneumonia. Antigens showing strong precipitation lines were used for further testing. AGAR-GEL IMMUNODIFFUSION
The AGID test was conducted in 15 x 100 mm plastic Petri dishes containing either 10, 15 or 20 ml of 1.5% Noble agar in 0.05 M tris buffer, pH 7.5. A series of tests was made to compare PPV precipitation reactions in different gel preparations. Salt concentration, pH and buffer were varied in these experiments. The salt concentration ranged from 0.85 to 10.0% and pH ranged from 7.0 to 10.0. On occasion, 0.15 M borate buffer or 25 mM barbital buffer was substituted for tris buffer. Agar was poured into plates and allowed to solidify at 25°C for one hour. A template with one central and six peripheral punches was used to make wells in the agar. Well sizes and locations were varied to determine optimal conditions. Wells varied from 4.0 to 8.0 mm in diameter and from 2.0 to 3.5 3National Animal Disease Center, Agricultural Research Service, P.O. Box 70, Ames, Iowa.
4Amicon, Lexington, Massachusetts.
282
mm apart in all directions. Antigen was placed in the center well while PPV positive serums, PPV negative serums and test serums were placed in the outer wells. The gels were examined over a narrow beam of light for precipitin lines at eight, 24 and
48 hours. ANIMALS
The Department of Animal Science, NDSU, manages two separate sheep flocks. One (PPV infected) is housed at the NDSU sheep barn, and the other (noninfected) is housed at the Animal Research Center (ARC). The NDSU sheep barn maintains a flock of 250-300 animals. The ARC flock consists of approximately 100 animals. A distance of 1.6 km separates the PPV infected and the noninfected flocks. Separation of sheep into two flocks was originally accomplished by removing lambs postpartum and formula feeding them. Serum samples from the sheep housed at the ARC have periodically been tested for the presence of PPV antibodies. Results have been negative. Histopathological examinations and lack of mortality due to PPV further substantiate the absence of progressive pneumonia in this flock. The NDSU sheepbarn flock is extensively infected with progressive pneumonia. This fact is documented by positive diagnoses at necropsy, histopathological results and the presence of PPV antibodies. Sheep vary in age from one to eight years. SERUM SAMPLES
Blood was collected from all sheep housed at the ARC and the NDSU sheep barn at various intervals. The blood was allowed to clot and the serum was extracted and stored at -20°C. Blood samples were also collected from all sheep brought to the NDSU Veterinary Diagnostic Laboratories which exhibited clinical signs associated with progressive pneumonia. All blood was collected from the jugular vein.
RESULTS CELL CULTURE
Four processed cell cultures were evalu-
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ated for growth characteristics including the ability to support PPV infection, the number of serial passages possible and the length of time cells were able to adhere to the flask without disruption of confluent growth. The OT cells retained confluent monolayers up to 30 passages. Figure 1 depicts a confluent monolayer of OT cells before infection with PPV. The OT cells were capable of adherence to plastic flasks for extended periods of time (weeks to months) before passage without disruption of the monolayers. Since PPV may require one to two months to cause CPE, the longer the cell culture remains attached, the more suitable it is for PPV growth. The OCP, OL and BCP cells require passage after approximately one week of growth. Figure 2 depicts PPV infected, unstained ovine trachea cells six days following inoculation. Figure 3 shows a Giemsa-stained PPV infected multinucleated giant cell. The most important cell culture characteristic is the ability to support viral replication. Of the four cell strains tested, all ovine-derived cell strains supported PPV growth (CPE formation) but bovine cells did not. The OT cells were the only
cells that could be continuously passaged. After the appearance of CPE and destruction of a portion of the monolayer, fresh uninfected OT cells were added to the flask. These cells remained continuously infected and could be later passaged. Antigens prepared from concentrated PPV infected OL and OCP cells produced weaker precipitation lines with PPV positive serums than antigens prepared from PPV infected OT cells. For this reason, OT cells were used for the production of antigens used for AGID.
ANTIGEN Soluble PPV antigens, concentrated either by diafiltration or by dialysis against polyethylene glycol, gave strong precipitation lines when reacted against positive serums of naturally infected sheep. Infected cell culture fluids were collected, pooled, subjected to one cycle of freezing and thawing and then concentrated. Concentrated cellular debris from PPV infected OT cells produced no precipitation lines. Concentration by dialysis required three
Fig. 1. Uninfected ovine trachea cells. X200.
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Fig. 2. PPV infected ovine trachea cells six days following inoculation. X200.
molecular weight, best retained the antigen used for AGID. Diafiltration required one to two days to concentrate antigen one hundredfold. This method was readily quantitated and little antigen was lost in transferring it to a tube. Because smaller molecular weight molecules were removed, the diafiltered antigen was less viscous and easier to apply than that prepared by dialysis. The diafiltered antigen yielded one distinct line of precipitation on every occasion. Fig. 3. PPV infected multinucleated giant cell. Giemsa stain. X470.
to four days, was difficult to quantitate, was difficult to perform due to the viscosity of the material and antigen was usually lost in the process of transferring it to a tube. The dialyzed antigen sometimes yielded two lines of precipitation in AGID and sometimes none at all. The Amicon XM100 diafiltration filter, which filters molecules less than 100,000 in
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AGAR-GEL IMMUNODIFFUSION
Semisolid medium containing 1.5% Noble agar, 8.5% NaCl and 0.05 M tris buffer allowed the production of distinct precipitation lines by various antigens and positive serums. Tris buffer was superior to either borate or barbital buffer with regard to the sharpness of precipitation lines. Although the pH of the medium had little effect on the development of precipitation lines in the range of 7.0-9.0, pH 7.5 tris buffer was consistently better relative to the sharpness of precipitation lines. The
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Fig. 4. AGID test place with PPV antigen in the center well, positive control serums in wells 1, 3 and 5 and test serums in wells 2, 4 and 6. The serum in well 2 forms a distinct line of identity with positive control serums.
concentration of NaCl in the medium had a significant effect on the development of precipitation lines. No lines were observed when NaCl was absent but as the NaCl concentration was increased to 8.5% they appeared and increased in intensity. Examples of AGID reactions between PPV antigen and negative or positive serum samples are shown in Fig. 4. One line of precipitation was produced by serums from naturally infected sheep. For all testing, PPV positive control serums were placed in alternate wells and the test serums were placed between positive controls. Serums were recorded as negative if the precipitation lines from positive control serums migrated into the well, slightly positive if the precipitation lines from the positive control serums curved into the well and strongly positive if a line of identity formed with those of the positive control serums.
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FLOCK SURVEY
The entire sheep population at NDSU was surveyed for precipitating antibodies against PPV by AGID. All 96 serums from sheep at the ARC were negative. Of the 305 serums collected from sheep at the NDSU sheep barn 111 were positive and 194 were negative. These results include animals from seven different breeds which ranged from one to seven years in age (Table I). Newborn lambs were not tested. Of the 401 total samples, 100 serums were tested against PPV antigen supplied by NADC. Results were in complete agreement with results from the same serums reacting against the NDSU-1 antigen. In general, the proportion of sheep with positive serums increased with the age of the sheep. Only 23% of the yearlings had positive serums, whereas 80% of the seven year olds were positive.
TABLE I. Agar-Gel Immunodiffusion Analysis of Serum Samples According to Age of the Sheep Age (years) 1 2 3 4 5 6 7 Total
Proportion of Positive Percent Serums 25/107 23%
11/52
19/50 11/21 17/30 12/25
16/20
111/305
21% 38% 52% 57% 48%
80% 36%
DISCUSSION The initial task in these experiments was to determine the cell culture which best supported the propagation of PPV in vitro. Diagnosis of progressive pneumonia has been primarily based on lesions but a serological test (AGID) is now available to detect precipitating antibodies against PPV (1). An improved method for preparing PPV antigen for AGID was discovered. The AGID test was then used to survey two sheep flocks for the incidence of pro-
gressive pneumonia. CELL CULTURE
Whether or not a particular virus may infect and replicate in vitro depends a great deal upon the type of cell culture used. In particular, PPV is a difficult virus to isolate and propagate in cell culture due to the length of time for appearance of CPE, the sensitivity of PPV to various conditions and the specificity of the virus for one host, sheep. Cell cultures derived from numerous ovine organs have been reported to support RNA slow virus growth (8). In this study, OT cells supported PPV growth for an extended time, were extremely durable and could be passaged up until 30 passages. Infected OT cell cultures readily developed CPE characteristics of PPV infection (Figs. 1-3). Bovine-derived cell cultures did not support PPV growth.
ANTIGEN A problem with AGID is the production of a strong but specific antigen. In a previous report (1), soluble PPV antigen was
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produced by concentrating PPV infected ovine lung cells by dialysis against polyethylene glycol. This method produced a strong antigen but quantitation was difficult and, due to viscosity, the antigen was difficult to apply to the wells. The production of antigen by diafiltration of PPV infected OT cells circumvented many of the problems of dialysis. Using diafiltration, the concentrated virus was easily quantitated and, because the filters selectively removed molecules with smaller weights, concentrated antigen was less viscous and only produced one precipitation line. All serums that produced precipitation lines against the NADC dialysis antigen also produced lines against the NDSU-1 diafiltered antigen. A complete line of identity was formed between the NADC antigen and the NDSU-1 antigen. AGAR-GEL IMMUNODIFFUSION
Sodium chloride at a concentration of 8.5 % was found to be essential for the production of precipitation bands. These results are similar to those for a microimmunodiffusiorn test for infectious bovine rhinotracheitis virus (11). An explanation of the requirement for a high NaCl concentration has been suggested (11). Some antigen-antibody complexes are too hydrophilic to precipitate, become trapped in the agar and fail to appear as a precipitation band in a hypotonic saline environment. A high salt concentration reduces the solubility of the hydrophilic complex and this results in the formation of a visible precipitin line. The AGID test was a satisfactory and uncomplicated technique for detecting pre-
cipitating antibodies against PPV. Compared to other tests (6,18), AGID is simple and economical and diagnostic results are obtained sooner. FLOCK SURVEY
One hundred and eleven of 305 animals tested were positive for precipitating antibodies against PPV (Table I). Of the sheep housed at the NDSU sheep barn, 36% were positive by AGID. Incidence according to age varied from 23% for yearling ewes to 80% in ewes seven years old. Age must play an important role in the incidence of progressive pneumonia. A high
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percentage of three year old sheep was positive by the AGID test (Table I). The older the animal and the longer it remains in the flock the greater the chance for exposure to PPV. Very few one and two year old sheep were positive by AGID. Sheep do not normally succumb to progressive pneumonia before two years of age because of the long disease course (13,14). REFERENCES 1. CUTLIP, R. C., T. A. JACKSON and G. A. LAIRD. Immunodiffusion test for ovine progressive pneumonia. Am. J. vet. Res. 38: 1081-1084. 1977. R. C. and G. A. LAIRD. Isolation and CUTLIP, 2. characterization of a virus associated with progressive pneumonia (maedi) of sheep. Am. J. vet. Res. 38: 1377-1382. 1976. 3. DEBOER, G. F. Zwoegerziekte virus, the causative agent for progressive interstitial pneumonia (maedi) and leucoencephalitis (visna) in sheep. Res. vet. Sci. 18: 15-25. 1975. 4. FENNER, F. Classification and nomenclature of viruses. Intervirology 3: 61-64. 1977. 5. GEORGSSON, G., N. NATHANSON, P. A. PALSSON and G. PETURSSON. The pathology of visna and maedi in sheep. In The Slow Virus Diseases of Animals and Man. Edited by R. H. Kimberlin. New York: American Elsevier. 1976. 6. GUDNADOTTIR, M. and K. KRISTENSDOTTIR. Complement fixing antibodies in sera of sheep affected with visna and maedi. J. Immun. 98: 663-667. 1967. 7. HANSON, R. P., R. J. ECKROADE, R. MARSH, G. M. ZUREINE, C. L. KANITZ and D. P. GUSTAFSON. Susceptibility of mink to sheep scrapie. Science 172: 859-861. 1971.
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8. HARTER, D. H. and J. E. COWARD. Sheep progressive pneumonia viruses. "Slow" cytolytic agents with tumor virus properties: a review. Tex. Rep. Biol. Med. 32: 649-664. 1974. 9. HARTER, D. H., K. C. HSU and H. M. ROSE. Multiplication of visna virus in bovine and porcine cell lines. Proc. Soc. exp. Biol. Med. 129: 295-300. 1968. 10. KENNEDY R. C., E. M. EKLUND, C. LOPEZ and W. J. HADLOW. Isolation of a vir-us from the lungs of Montana shep afflicted with progressive pneumonia. Virology 35: 483-484. 1968. 11. LEJEUNE, J. M., L. T. HART, A. D. LARSON and C. L. SEGER. Microimmunodiffusion test for detection of antibodies to infectious bovine rhinotracheitis vir-us in bovine serum. Am. J. vet. Res. 38: 459-463. 1977. 12. LOPEZ, C., C. E. EKLUND and W. HADLOW. Tissue culture studies of progressive pneumonia, a slow infectious disease of sheep. J. infect. Dis. 138: 1035-1040. 1971. 13. MARSH. H. Progressive pneumonia in sheep. J. Am. vet. med. Ass. 15: 458-473. 1923. 14. SIGURDORDOTTIR, B. H. and H. THORMAR. Isolation of a viral agent from the lung of sheep infected with maedi. J. infeet. Dis. 114: 55-60. 1964. 15. SIGURDSSON, B. Maedi, a slow progressive pneumonia of sheep: an epizoological and a pathological study. Br. vet. J. 110: 225-270. 1954. 16. SIGURDSSON, B. and P. A. PALSSON. Visna of sheep: A slow demyelinating infection. Br. J. exp. Path. 39: 519-528. 1958. 17. TAKEMOTO, K. K., C. F. MATTERN, L. B. STONE, J. E. COE and G. LAVELLE. Antigenic and morphological similarities of progressive pneu6monia virus, a recently isolated "slow virus" of sheep, to visna and maedi viruses. J. Virol. 7: 301308. 1971. 18. TERPESTRA, C. and G. F. DEBOER. Precipitating antibodies against maedi-visna virus in experimentally infected sheep. Arch. ges. Virusforsch. 43: 53-62. 1973. 19. WEISS, M. J., E. P. ZEELON, R. W. SWEET, D. H. HARTER and S. SPIELGMAN. Immunological cross reaction of the major internal protein component from "slow" viruses of sheep. Virology 76: 851-854. 1977.
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