”THS mill/1WI/l/I/l/l/II/l/lll/fl/l/Z/fll/llfllfll V T H ES‘. my mpg-van... “W‘k . 332‘ ? b s. ‘1’:- " " M591: H a. E: .. ”‘ 19‘ .—.\i 2 , ' ., g, «‘9‘ ‘9 "a £1.11? 15. fa :3 »& a?» ' ‘3 a: 1‘. .4 Umifiwtwfiy 1'; he. ,l This is to certify that the thesis entitled CAN/Ni: PARVOVllzlLS~ Lineman-rapt) DMGNOMS IND BlOLOGaleAL C§AR9~ C’- T E El ST [C S presented by Arm (3. R wnWNE QOBERTS has been accepted towards fulfillment of the requirements for MS degreein M "P“ Major professor Date f/yI/fv 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records - ‘ "ii/amiifi‘? ¥‘. " “.37.” ' I” ..- ~' 4w ~‘~\\Vr.r'..-» r-Jx 1.43;; 1 7 m} 3.1333 12002 © 1981 ARTHUR WAYNE ROBERTS All Rights Reserved CANINE PARVOVIRUS - LABORATORY DIAGNOSIS AND BIOLOGICAL CHARACTERISTICS BY A. Wayne Roberts A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1980 «is w. 5... ABSTRACT CANINE PARVOVIRUS - LABORATORY DIAGNOSIS AND BIOLOGICAL CHARACTERISTICS BY A. Wayne Roberts Presumptive hemagglutination (HA) tests and virus isolation procedures were used to examine 150 fecal and/or intestinal prepara- tions for the presence of canine parvovirus (CPV). Samples with HA activity 21:32 were almost always positive for CPV isolation, and only a few preparations exhibited nonspecific agglutination of RBC. Virus isolation was more sensitive than HA, as evidenced by the recovery of CPV from 8 specimens with no demonstrable HA activity. Results of fluorescent antibody tests compared favorably with results of virus isolation and HA tests. Canine parvovirus agglutinated erythrocytes of pig, rhesus monkey, cat, and horse, and the virus grew in cell cultures derived from cat, dog, cow, goat, monkey, and human. Replication of CPV was not enhanced by the presence of adenoviruses. DEDICATED TO MY WIFE JOY AND TO MY CHILDREN CURT AND LOURI ii ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. G. R. Carter, who served as my graduate advisor, and to other members of my graduate committee, Dr. Lee Velicer, Dr. Vance Sanger, and Dr. Maria Patterson. I am indebted to Mr. Paul Watkins and Valeria Moojen for their assistance in specimen preparation and FA examinations, and to Ms. Pat Lowrie for electron microscopy. This research used the facilities of the Clinical Microbiology Laboratory, and I am grateful to Dr. Roger Maes, my supervisor, for his patience and understanding during the completion of this.work. iii TABLE OF CONTENTS LITERATURE REVIEW . . . . . . . . . . . Classification and Nomenclature. History of Isolations. . . . . . Virus Replication. . . . . . . . DNA Structure . . . . . . DNA Synthesis and Virus Reproduction. . . Pathogenesis of Parvoviruses . . BIBLIOGRAPHY. O O O O O O O O O O O O 0 ARTICLE: CANINE PARVOVIRUS - LABORATORY DIAGNOSIS AND BIOLOGICAL CHARACTERISTICS. . Summary. . . . . . . . . . . . . Introduction . . . . . . . . . . Materials and Methods. . . . . . Results. . . . . . . . . . . . . Discussion . . . . . . . . . . . References . . . . . . . . . . . iv Page p.- P‘h‘ hJCDGDG>U1PJ 25 26 27 28 34 41 46 LIST OF TABLES Table ‘ Page 1 Physicochemical characteristics of Parvoviridae. . . . . . 2 2 Parvoviridae - member viruses. . . . . . . . . . . . . . . 3 ARTICLE 1 Cell cultures, source, and conditions for growth analysis of CPV. . . . . . . . . . . . . . . . . . . . . . 33 2 Canine parvovirus infection: results of hemagglutina- tion and virus isolation . . . . . . . . . . . . . . . . . 35 3 Canine parvovirus detection: discrepancies noted with FA, EM, HA and isolation results . . . . . . . . . . . . . 37 4 Agglutination of pig, monkey, cat, and horse erythro- cytes by isolates of canine parvovirus . . . . . . . . . . 40 Figure LIST OF FIGURES Page ARTICLE Immunofluorescence of CPV antigen in association with fecal debris O O O C O O O I O O O O I O O C O O O O O O O 38 Immunofluorescence of CRFK cells infected with CPV . . . . 38 Growth curves of CRFK cells and canine parvovirus. . . . . 42 vi LITERATURE REVIEW Classification and Nomenclature Parvoviridae is a family of viruses comprised of two recognized genera, Parvovirus and Densovirus, and an adeno-associated virus (AAV) group to which no genus name has been assigned (76). Virions are nonenveloped, isometric particles with a diameter of 20-30 nm and are unique in that the genome is single stranded (SS) DNA. A summary of physicochemical characteristics of Parvoviridae is provided in Table l. Parvoviruses and AAV are viruses of vertebrates and are placed in separate genera on the basis of the latter's inability to replicate without aid of helper viruses. The Densovirus genus includes those viruses infecting arthropods; their replication, like that of the Parvovirus genus, occurs autonomously. Further distinguishing char- acteristics of the genera are based on type of DNA which is encapsidated. Parvoviruses incorporate only one type of SS DNA (- strand), whereas the DNA encapsidated by individual Virions of AAV and densoviruses are complementary (+ or - strand) and will, upon extraction, anneal spontaneously to form double-stranded products. Numerous viruses isolated from a variety.of sources are now con- sidered to belong to the Parvoviridae family. These are listed in Table 2. Table 1- Physicochemical characteristics of Parvoviridae Characteristic Size (nm) 20-30 Symmetry icosahedral Capsomers 32? Buoyant density 1.38-1.46 g/cm3 Nucleic acid Type 55 DNA Molecular weight 1.5-2.0 x 106 g+c Content 41-53% Infectivity Ether stable yes Heat stable yes Acid stable yes no . whom: one Ho>fla mcflamom A>mov msufl> mfluflummon mmoow ha moomm moo AU>Z I mwcflcmo mo maufl> muscHEV msnfl>o>nmm.ocflcmo« muoofioz mandooum vv mafia Hawo ad um» . >Bm vv mafia Hawo moaaq swan: HHHSA vv mocfla Haoo HOEdu seas: x>9 mHH comamm new um>AH xcee A>mzs maufl> mfluflumucm xcflz mwmnufi> ooumHmu SHHmoflcooHucm hm comamm cummooa Adam I mficmmoxswacmm onwaomv msuH>o>Hmm osaaom a mooom Mano Amsufl> coommi>mmv msufi>o>umm ocw>om vv maaoo mossy mu cues: mmmM momsua> ooumaou >Hamoflcomflucd Hm xooum msufl> onwaoco mo: A>mmv msufi>o>umm ocwouom mm xooum wsufl>ocoom wmsoE Az>zv oofie mo msufi> wanna: MHH mowuano can mmucoomam swan; Ha momsufi> omumaou >Hamoflcmmfiuc4 vHH mossy odomucmammsmuu Hummm amass Hum Hm mHHmo Hoes» ouuflouumo amen: mex mm mums omumouuumoflfimnmmonmoHo>o mo mzu Amumu mo msufl> >£ummofimnmoocm oflmmnuuofiwnv mmm m5 uoEdu UflwadeOHono umu Amsufl> umcflmunomifisqv MIA mm mums owumouuuuz one Smuux vanx Hm Hoes» oaomucmammcmuu mimom amass mum momsufl> omumHou adamoflcwoflucd mo meawxswa maomucoammcmuu one mmeooumm Ho>HH um» A>mv msufl> umm numbed: pwuflcmooom Amsufi> uwuv HIM msufl>o>umm "mofloomm mmxa msufl>o>umm "mscoo .H wocwuwmmm :oflumHOmH mo mousom mmmduw>o>umm momdufl> umoEmE I ompaufi>o>umm .m magma mflcmmoxsmacmm mcflaom 0p UmumHmu ma usn AU>EV mocflcoo mo msufi> muscwe ou mflamofionocdfiafl noumeu uo: ma moon ca .AcoflumoHCSEEoo Hmcomumm .ccwm .2 .AV moufl> mfiuwoumoo>fi one mfiuwumuco mo mesmuusooo wofl3©Hu03 unmomu on» spas omumfloommm A>mov msufl>o$umm wcflcmo one t vm xooum maufl>ocmoo wowsvm >«d wcfisvm mm xooum msuw> mfiuflummo: mowcmo >¢¢ ocflcmu muonswz manmnoum mm xooum msufl> mfiufinoconn Hamsv ><4 cmw>¢ Nb gooum H mnufl>ocmom ocw>on Abxv >¢< ocfi>om hm xooum ma msufl> cmHEHm . v om>u >¢¢ av xooum h m5uw>ocoom amass m mm>u >4¢ mo xooum NH msufl>ocoom amass m max» >¢d h xooum ma msufi> coflsflm a mmxu >m¢ mumosoz ooNflcmoowm H mam» >m¢ "mmwommw umme A><¢vlwsufl> owDMHUOmmmiocooa "mscww .m 4 v0 om>uoa meHOU mwcocsm msufl> MACOCSH vm wm>uma MHNmQONNmE owemwamu wsufi> mfimooHUSCOmcoa Amfiuoaammv m9hw> mflmooHoDCOmcmo "mofiome make msufl>omcma ”mscmw .m mm mmomw DHQQMH msuw>o>umm mcwmmq mHH mDOEDD one mmusmomHm amass. mm mm cmoamm xcda mmommflo xcfiE cmfluswad vm mooom moo A>mov msuw>o>umm ocflcm0« mm :05 mo msufl> mfluflumusoouummw up one no msuw> d mwufiummom oucouomwm cofiumHomH mo mousom momsufi>o>umm inescaueooc N magma 5 History of Isolations Kilham and Oliver in 1959 (66) described the first partial char- acterization of a parvovirus. While attempting to demonstrate oncogenic properties of papovaviruses, they recovered from tumorous tissue a virus which produced cytopathic effects (CPE) in rat embryonic cultures and agglutinated guinea pig erythrocytes. The virus was referred to as rat virus (RV). In 1960, Toolan et al. (114) isolated a virus from a human transplantable tumor (Hep-1) and designated the virus H-l. When inoculated into young hamsters, the virus caused a variety of malforma- tions, including dwarfism, flat face, domed head, protruding eyes, and fragile bones. In subsequent studies, Toolan (113) was able to isolate H—l virus from hamster neonates inoculated with emulsions of various. tissues of cancer patients and human embryos. He also recovered another parvovirus (HT), which was serologically related to H-1, and an additional virus (HB), which was unrelated serologically to H-1 and RV. Additional rodent parvoviruses were isolated (21,31,36,44,73,88) and, owing to their similar effects when inoculated into young hamsters, these viruses were often referred to as hamster osteolytic viruses. Atchison et al. (7) isolated a 20- to 25-nm agent from stock simian adenovirus (SV-lS) and preposed the name adenovirus-associated virus (AAV). Although other investigators (5,23) had previously noted by electron microscopy small particles associated with adenoviruses, Atchison and co-workers clearly demonstrated that these particles were distinct viruses unrelated immunologically and structurally to adeno- viruses. They also demonstrated that the viruses contained DNA and were dependent on adenoviruses for replication, although replication was not limited to a particular adenovirus. Subsequently, it was shown that AAV of human and simian origin represented 4 serotypes (48). In 6 addition, AAV of avian, bovine, canine, and equine were described (33,34,35,72). Although Johnson and Cruickshank (59) were first to present evi- dence that feline panleukopenia virus (FPV) was a parvovirus, the disease which it causes was known as early as 1900. It was thought to be caused by a virus after Verge and Christoferoni (116), in 1928, were able to produce the disease in healthy cats with bacteria-free filtrates prepared from eulsified organs of diseased cats. Later, Lawrence et al. (68) and Lucas and Riser (71) demonstrated that a variety of clinical syndromes, including agranulocytosis and feline distemper, were associated with the same virus. 'It was noted by Schofield (101) in 1949 and Wills (118) in 1952 that serious epizootics of enteritis which occurred in commercially raised mink in Canada exhibited clinical features similar to those observed with FPV. Subse- quently, the virus was isolated and a close immunological relationship between FPV and mink enteritis virus was observed (58). In 1961, Abinanti and Warfield (l) isolated a virus from the intestinal tract of normal calves. The virus grew in cell cultures of bovine embryonic kidney and agglutinated erythrocytes of guinea pigs and humans. Infected cell cultures were also noted to adsorb erythro- cytes of these species, prompting Abinanti and Warfield to refer to the previously undescribed virus as hemadsorbing enteric (HADEN) virus of calves. Data obtained by Storz and Warren (117) and Bachmann (8) permitted the classification of HADEN virus as bovine parvovirus. The electron microsc0pic observation of viral particles similar to Kilham rat virus in hog cholera infected pig kidney cell cultures was noted by Mahnel (74) in 1965. Cartwright and Huck (26) recovered a small DNA-containing virus from aborted and stillborn piglets, and Mayr and co-workers (80) isolated similar viruses from "normal" cell cultures derived from.pig kidneys. These latter investigators observed that these viruses shared many properties with viruses of the parvo- virus group. Later, Cartwright et al. (27) suggested that the viruses described by Mayr et al., as well as those isolated by other investi- gators (18,51),were identical or at least immunologically closely related to the virus which she and Huck had earlier isolated. Binn and co-workers (17) in 1967 reported the isolation of a small virus from feces of asymptomatic dogs. The virus was referred to as "minute virus of canines" (MVC), and in 1970 Binn et a1. (18) pro- vided evidence for classification of the agent as a parvovirus. The virus was difficult to cultivate and only agglutinated erythrocytes of rhesus and African green monkeys. specific antibodies determined by hemagglutination inhibition tests were found in approximately 70% of dogs examined. In 1977, Eugster and Nairn (38) observed, by electron microscopy, parvoviruses in the feces of young puppies with diarrhea. No other clinicalsigns were present, and recovery was uneventful. Beginning in mid-1978, a severe gastroenteritis associated with parvo- virus was described (2). Subsequent reports (3,4,19,28,37,49,4l,55, 60,65,86,109) revealed the disease to be of pandemic proportions. Also, a fatal myocarditis associated with the virus was noted (24,47, 50,55,56,70,86,112). The canine parvoviruses (CPV) associated with enteritis and myocarditis were identical or at least closely related immunologically but were distinct from MCV (T. N. Binn, personal communication). Neither MCV nor CPV were related to canine adeno- associated virus, but CPV was closely related immunologically to feline panleukopenia virus (T. N. Binn, personal communication). 8 A virus associated with Aleutian disease of mink was recently isolated (89) and shown to have properties consistent with parvoviruses. The virus (ADV) could only be propagated on initial isolation at a reduced temperature of 31.8 C. After several subcultures at this temperature, optimum temperature for replication was 37 C. Shahrabadi and co-workers (103), using ADV purified from tissues of infected mink, provided evidence that the DNA was single stranded. Matsunaga et al. (75) isolated and characterized a parvovirus from feces of rabbits. The virus grew in rabbit kidney cell cultures and agglutinated human group O erythrocytes at 4 C. No relationship to KRV was observed. The virus was not associated with disease, and a high proportion of commercially obtained rabbits exhibited antibody. Virus Replication DNA Structure The DNA extracted from several autonomous parvoviruses was shown to be single stranded (SS) by formaldehyde reaction (30,99,115). As a result of hydrogen bonding between base pairs, double stranded (DS) DNA lacks free amino groups. On the cther hand, free amino groups exist in SS DNA and react with formaldehyde resulting in an increase of absorption at 260 nm. Nearest neighbor analysis was used by Salzman et al. (100) and McGeoch and co—workers (82) to confirm the SS nature of H-1 and MVM viral DNA, and Mayor and Melnick (78) showed with acri- dine orange staining that the DNA of X14 was single stranded. Unequal ratios of A-T and G-C also indicated that DNA of autonomous parvoviruses was single stranded (98). Originally, a controversy existed over the type of DNA present in' the defective parvoviruses (AAV). Rose et al. (95) extracted DNA from 9 AAV and found evidence of DS DNA, but acridine orange staining performed by other investigators (12,78) indicated that the DNA was SS both inside cells and in intact virions. The controversy was resolved when Crawford et al. (30) proposed and Rose and co—workers (93) confirmed with density shift experiments that AAV contained two types of DNA, either + or - strands, which annealed after extraction to form DS products. The density shift experiment was conducted by growing virus in the presence of bromodeoxyuridine (Budr), which substitutes for thymidine in DNA, thus making the DNA more dense in cesium chloride (CsCl). When the DNA was extracted from such virions and mixed with DNA obtained from virions grown in the absence of Budr, hybrid density molecules were observed in CsCl gradients. Subsequently, Berns and Rose (16) were able to separate + and - strands by similar means. They grew virus in the presence of. Budr and extracted DNA, which was then subjected to conditions to mini- mize annealing; heavy and light bands were noted in CsCl gradients. Analysis of the base composition of the DNA revealed the two strands to be of unequal thymidine content. The - strand contained approximately 27%, whereas the + strand contained about 21% (94). Berns and Adler (13) used a less cumbersome method to isolate + and - strands by sepa- rating intact virions (grown in the presence of Budr) with CsCl gradients and then extracting the DNA. More recently, agarose-acrylamide gels were used to separate + and - strands directly from denatured DNA (46). DNA configuration in mature virions of both autonomous parvoviruses and AAV was determined to be linear. Salzman et al. (100) used exo- nuclease I and electron microsc0pic (EM) examination to verify the linear nature of KRV, and EM examination was used to detenmine linearity of DNA extracted from MVM and Lu III (30,104). Gerry et al. (42) demonstrated the linear nature of AAV DNA. Although the DNAs of 10 parvoviruses and AAV are linear, both termini of viral strands are able to form duplex hairpin structures. The terminal sequences of AAV are repetitious, and it was suggested by Gerry et al. (42) to represent a palindrome of the type 122'1...122'1; evidence to support such a model was provided by Fife et al. (39). Autonomous parvoviruses, unlike AAV, apparently lack terminal repetition but do contain sequences at both termini with properties of palindromes (22). DNA Synthesis and Virus Reprgduction Berns and Hanswirth (14) suggested that these differences in terminal sequences of AAV and autonomous parvoviruses might account for replica- tion and subsequent + or - strand encapsidation by AAV and only - strand by autonomous parvoviruses. It was suggested that DNA synthesis is primed by a 3'terminal hairpin structure and consequently AAV DNA would be expected to have identical termini in order for both strands to be synthesized equally. Since a distinction would need to be made for + and - strand synthesis with autonomous parvoviruses, their DNA would require nonidentical termini. The precise mechanism by which autonomous parvoviruses replicate is unknown. They usually require rapidly dividing cells in order to replicate (105,110,111), and replication appears to be related to cellu- lar DNA synthesis (9). Although Salzman (96) reported that KRV contained a virion associated DNA polymerase, this finding could not be confirmed by other investigators (9,91). Bates et a1. (9) demonstrated a concomi- tant rise in viral infectivity of bovine parvovirus and cellular DNA * polymerase 0 activity, suggesting that cellular DNA polymerase was involved in viral replication. They also noted a slight elevation of * * DNA polymerase 8 , but no increase in polymerase Y. * For nomenclature of eukaryotic DNA polymerases, see ref. 118. ll Adeno-associated viruses are defective and require an adenovirus "helper" for productive infection (7). The nature of this helper function is unknown, but apparently it does not require adenovirus DNA synthesis, as DNA temperature sensitive mutants of adenovirus were also functional (54). Herpesviruses provide a partial helper function, permitting an abortive infection with AAV (6,20). The defect in herpesvirus helper function is believed to be at the level of DNA encapsidation (79). The infectious cycle of autonomous parvoviruses and AAV appears to be essentially identical in the early stages. Adsorption is completed in approximately two hours for parvoviruses (11) as well as for AAV (15) and adSorption of AAV occurs without the aid of helper viruses (15). Also, AAV penetration to the cell nucleus and subsequent uncoating are unaffected by the absence of adenoviruses (15). AAV DNA will, in fact, at least in some instances, form a stable relationship within the cell and can be induced to form mature virions by subsequent infection with adenoviruses (45). This stable relationship was demonstrated to result from integration of the AAV genome into cellular DNA (45). As previously mentioned, the cellular function responsible for autonomous parvovirus replication and the mechanisms by which adeno- viruses aid AAV replication are unknown. Of interest was the observa- tion that replication of some autonomous parvoviruses was stimulated by certain adenoviruses. VLedinko and Toolan (69) observed a pronounced effect with H-l virus. They demonstrated that H-l virus, which normally does not grow in human embryonic lung cells, does so in the presence of human adenovirus type 12. 12 Transcription and subsequent protein synthesis were reviewed by Carter (25) and Tattersall (108), respectively, and appear to be similar for both non-defective and defective parvoviruses. The RNA of both virus types undergoes several post-transcriptional modifications, including polyadenylation and methylation. The mRNA is small and can only specify one protein of approximately 100K (15). The three poly- peptides that are evident in electrophoretic analysis of disrupted virions were shown to be related and presumably derived from a single translation product (61). Pathogenesis of Parvoviruses The in vitro Observation that parvoviruses generally require rapidly dividing cells in order to replicate is consonant with their in vivo predilection for mitotically active tissues. As a consequence, these viruses have usually been recovered from the intestinal tract in adult animals. Most parvovirus infections in adult animals produce no overt clinical signs; exceptions are feline panleukopenia virus and canine parvovirus, both of which may produce a severe gastroenteritis in animals of all ages (2,63). Similarly, the "Norwalk" agent, which is probably a parvovirus, is associated with gastroenteritis in humans (32). Infection of pregnant animals may result in subsequent infection of the fetus, causing abortion and mummification, as evidenced strikingly by porcine parvovirus infection (83). The mongoloid deformities caused by hamster osteolytic viruses (114) inoculated into young hamsters result from virus replication in developing dental and skeletal tissues as well as in rapidly dividing cells of the cerebellar cortex. Likewise, spontaneous ataxia (cerebellar hypoplasia) associated with feline pan- leukopenia virus infection in young kittens is; a result of viral 13 damage to the cerebellar cortex. The pathogenesis of the important parvoviruses is discussed more fully below. Feline panleukopenia virus was initially recognized as a cause of feline infectious enteritis and was presumed to be of viral origin as early as 1928 (116). Later, the virus was associated with several other disease syndromes, including leukopenia, fetal death, and cere- bellar ataxia. In acutely infected cats, the virus is present in saliva, feces, vomitus, and urine, and spread of the virus occurs by fomites and aerosols (43). Following infection, the virus is initially thought to replicate in pharyngeal lymphoid tissue (43), followed by a viremia and spread to secondary target organs. The organs invariably affected are those containing highly mitotically active cells such as lymph nodes, spleen, thymus, bone marrow, and Peyer's patches, as well as crypt cells of the intestinal mucosa (63). Viral damage to lymphoid tissues results in lymphopenia and neutropenia and may lead to mild immunodeficiency (102). Although absorptive cells of the resulting villi are not directly affected, extensive damage to crypt cells occurs. The inability of these damaged crypt cells to migrate and replace sloughed villous absorptive cells leads to malabsorptive disease (63). The enteritic form of the disease is less severe in germ-free cats and this is thought to be a result of depressed mitotic activity of intestinal crypt cells in the absence of bacterial flora (63). The pathogenesis of feline panleukopenia was recently reviewed by Kahn (63). The clinical disease caused by canine parvovirus (CPV) is strikingly similar to the disease in cats caused by FPL (2). Enteritis with vomiting, diarrhea, and a marked leukopenia.with resulting pathological changes similar to those observed with FPL suggest that the pathogenesis 14 of CPV is analogous to the pathogenesis of FPL. However, fetal infec- tion and cerebellar hypoplasia have not been described for CPV. The myocarditis associated with CPV is difficult to explain, since myocardial cells have little or no mitotic activity. It has been suggested that changes in the myocardium may result from an autoimmune reaction and that infection with CPV may occur with the aid of a helper virus (90); however, the latter possibility seems unlikely, in that CPV was not influenced by canine adenoviruses in vitro (Personal observation). Porcine parvovirus causes no apparent clinical disease in young or adult animals but may.result in reproductive failure in the sow or gilt (62). Transplacental infection occurs, and fetal mummification often results if the dam is infected before the 56th day of gestation (62,83), the time of fetal immune competence. Owing to the nature of the swine uterus, i.e., separate blood supply for each fetus, both infected and uninfected fetuses may be present in the same litter (62). Although bovine parvovirus (BPV) has been isolated from the intes- tinal tract of calves on numerous occasions (1,10,52), its role in clinical disease has not been clearly defined. Storz and co-workers (106) administered a cell culture propagated strain of BPV orally and intravenously (IV) to young calves. Enteritis developed 24 to 48 hours after infection and was more severe in IV inoculated calves. Attempts to relate bovine parvoviruses to reproductive prOblems have generally been unsuccessful, although Moojen et al. (85) reported the electron microscopic observation of parvovirus-like agents in tissues of two aborted bovine fetuses. Aleutian disease of mink is presently thought to be caused by a parvovirus (89). Infection is persistent, and the disease is charac- terized by plasmacytosis, highly elevated levels of immunoglobulins, 15 and the presence of circulating immune complexes. These circulating immune complexes result in glomerulonephritis and necrotizing arteritis. Severity of the disease can be alleviated by administering immuno- suppressive drugs. Aleutian disease was reviewed by Ingram and Cho (53). BIBLIOGRAPHY 10. ll. BIBLIOGRAPHY Abinanti, F. R., and M. S. Warfield. 1961. Recovery of a hemad- sorbing virus (HADEN) from gastrointestinal tract of calves. Virology 14, 288-289. Appel, M. J., B. J. Cooper, H. Greisen, and L. E. Carmichael. 1978. Status report: canine viral enteritis. J. Am. Vet. Med. Assoc. 173, 1516-1518. Appel, M. J. G., B. J. Cooper, Greisen, H., et al. 1979. Canine viral enteritis. I. Status report on corona- and parvo- 1ike viral enteritides. Cornell vet. 69, 123-133. Appel, M. J. G., F. W. Scott, and L. E. Carmichael. 1979. Isola- tion and immunization studies of a canine parvo-like virus from dogs with haemorrhagic enteritis. Vet. Rec. 105, 156-159. Archetti, I., and D. S. Bocciarelli. 1964. On the structure of a small simian virus. Pr0c. 3rd Europ. Reg. Conf. Electron Microscopy, Prague, p. 343. Atchison, R. W. 1970. The role of herpesvirus in adeno- associated virus replication in vitro. Virology 42, 155-162. Atchison, R. W., B. C. Castro, and W. McD. Hammon. 1965. 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ARTICLE CANINE PARVOVIRUS Laboratory Diagnosis and Biological Characteristics to be submitted to American Journal of Veterinary Research 25 SUMMARY Presumptive hemagglutination (HA) tests and virus isolation procedures were used to examine 150 fecal and/or intestinal prepara- tions for the presence of canine parvovirus (CPV). Samples with HA activity 21:32 were almost always positive for CPV isolation, and only a few preparations exhibited nonspecific agglutination of RBC. Virus isolation was more sensitive than HA, as evidenced by the recovery of CPV from 8 specimens with no demonstrable HA activity. Results of fluorescent antibody tests compared favorably with results of virus isolation and HA tests. Canine parvovirus agglutinated erythrocytes of pig, rhesus monkey, cat, and horse, and the virus grew in cell cultures derived from cat, dog, cow, goat, monkey, and human. Replication of CPV was not enhanced by the presence of adenoviruses. 26 27 INTRODUCTION Binn and co-workers (4) in 1967 reported the isolation of a small virus from feces of asymptomatic dogs. The virus was referred to as "minute virus of canines" (MVC), and in 1970 Binn et al. (5).provided evidence for classification of the agent as a parvovirus. The virus was difficult to cultivate and only agglutinated erythrocytes of rhesus and African green monkeys. Specific antibodies determined by hemag- glutination inhibition tests were found in approximately 70% of dogs examined. In 1977, Eugster and Nairn (11) observed, by electron microscopy, parvoviruses in the feces of young puppies with diarrhea. No other clinical signs were present, and recovery was uneventful. Beginning in mid-1978, a severe gastroenteritis associated with parvo- virus was described (1). Subsequent reports (2,3,6,10,11,13,14,l7, 19,21,24,25) revealed the disease to be of pandemic proportions. Also, a fatal myocarditis associated with the virus was noted (9,15,16,17,18,22,24,26). The canine parvoviruses (CPV) associated with enteritis and myocarditis were identical or at least closely related.inmunologically but were distinct from MCV (T. N. Binn, personal communication). Neither MCV nor CPV were related to canine adeno-associated virus, but CPV was closely related immunologically to feline panleuk0penia virus (T. N. Binn, personal communication). At Midhigan State University, we initially used a hemagglutination test (HA) directly on extracts of fecal material and intestinal contents to presumptively diagnose CPV infections. We later propagated the virus and prepared a fluorescent antibody (FA) conjugate which replaced our presumptive HA test. Electron microscopic (EM) examinations were performed on some cases. Occasional discrepancies between FA and EM examinations prompted the present report, in which we describe the 28 reexamination of 150 cases using isolation and HA procedures. Bio- logical characteristics, including susceptibility of erythrocytes of different animal species to agglutination by CPV and the susceptibility of various cell cultures to infection with CPV, are also presented. MATERIALS AND METHODS Preparation of Specimens An approximate 10% emulsion of feces or intestine was prepared with a mortar and pestle using balanced salt solution as diluent. As these preparations were used for both HA tests and viral isolation procedures, high concentrations of antibiotics (potassium penicillin-G, 1000 units/ml; polymyxin B sulfate, 500 units/ml; and streptomycin sulfate, 1000 ug/ml) were included in the diluent. Preparations were stored at -20 C and subsequently thawed and clarified by centrifugation at approximately 2000 x g for 5 min immediately before use. Hemagglutination (HA) Tests Porcine and rhesus monkey erythrocytes (RBC) were used routinely. Blood from pigs was collected in Alsever's solution and stored at 4 C; monkey RBCs were purchased commerciallya as a 25% suspension in Alsever's solution. Two-fold dilutions (50 ul) of supernatant fluids from prepared specimens were made using standard microtiter procedures, and 50 ul of a cold 1% suspension of RBC was added to each well. Plates were shaken lightly to mix the contents and incubated at 4 C until controls settled, usually within 1 to 2 hours. The diluent used for making dilutions and preparing 1% RBC suspensions was 0.1% bovine serum * Microbiological Associates, Bethesda, MD. 29 albumdn in 0.85% NaCl. Microtiter plates were 96 well flexible U plates,b and HA tests on each specimen were performed simultaneously with both pig and monkey RBC. In subsequent comparative studies with selected isolates of CPV, RBCs from other animal species were used. The source of RBC and variations from the above procedure are reported in the results. Virus Isolation The cells used routinely for virus isolation were Crandell-Rees feline kidney cells (CRFK). Eagle's minimum essential medium with 0.5% lactalbumin hydrolysate, 1 mM sodium pyruvate, 2X concentration of non-essential amino acids, and 10% bovine fetal serum (BFS) was used for propagating the cells. Cells were seeded (1.5 m1) into 16 x 150 mm culture tubes containing coverglasses at a cell concentra- tion of approximately 1.3-1.4 x 105 per ml or a split-ratio of 1 to 4. This concentration of cells resulted in about 80% confluency within 40 hours, at which time medium was replaced with fresh medium containing only 2% BFS. Freshly refed cells were inoculated with 0.2 ml of supernatant fluid from specimens prepared as described earlier. Two tubes were used for each specimen, and inoculated cells were incubated in a stationary position at 36 C and examined each day for cytopathic effects (CPE). Coverglasses were removed at day 2 and day 5 and stained with a fluorescent antibody conjugate prepared as described below. Positive cultures were frozen at day 7 or earlier if the CPE was severe. * Cooke Microtiter Plates, Microbiological Associates, Bethesda, MD. 30 Preparation of Hyperimmune Serum Hyperimmune serumwwas prepared by inoculating a young adult New Zealand White rabbit with a cell culture isolate of CPV obtained from the intestine of a dog that died from gastroenteritis. The isolate (VC-13-79) had been subcultured 3 times in CRFK cells and had a HA titer of 1:4,096 per m1 of cell culture fluid. One milliliter of a mixture of viral fluid and Freund's complete adjuvantC was administered at 2 subcutaneous sites and l intramuscular site. Two weeks later 0.5 m1 of virus without adjuvant was administered intravenously. Serum collected at this time had a HA titer of 1:64 against 20 HA units of CPV. The HI titer was 1:2,048 5 days after the IV injection, and 50 m1 of blood was collected via cardiac puncture. The rabbit was then humanely killed by injecting pentobarbital directly into the heart. Preparation of FA Conjugate Fluorescent antibody conjugate was prepared essentially as described elsewhere (7). Briefly, immunoglobulins were precipitated from cold serum by the addition of 1/2 volume of saturated ammonium sulfate. The globulin fraction was collected by centrifugation at approximately 2000 x g, resuspended in distilled water adjusted to pH 7.5 with 0.1 M NA HPO 2 4, and reprecipitated with ammonium sulfate. After 3 precipitations, the globulin was dialyzed overnight against several changes of 0.85% NaCl. Protein concentration was determined by the Kalb procedure (20), and 0.05 mg of FITCd per mg protein was added. Conjugation was allowed to proceed overnight at 4 C with constant stirring, after which free fluorescein was removed by passing c . . . Difco Laboratories, Detr01t, MI. dFluorescein isothiocyanate, Grand Island Biological Company, Grand Island, NY. 31 the conjugate through a column of coarse G-25 Sephadex. The conjugate was then absorbed with rabbit liver powder. Performance of FA Test Fluorescent antibody tests were performed on 8 um thick frozen sections of intestine, fecal preparations made by rolling a swab with collected fecal material onto a microscope slide, and cell cultures growing on coverglasses. Preparations were fixed for 10 min at room temperature (~23 C) with acetone, overlaid with conjugate, incubated for 30 min in a 35 C moist chamber, and washed with 0.85% NaCl for 10 min. Examinations were made using a Zeiss microscope equipped with a transmitted 100 watt halogen lamp source and FITC interference filter. Barrier filters were Schott 0G 4 and 0G 5 used in combination. Growth Rate of CRFK Cells and Growth Rate of CPV The growth rate of CRFK cells under conditions used for isolation of CPV was determined. Plastic flasks (25 cm2) were seeded with 5 m1 of a cell suspension containing 1.37 x 105 cells per m1 and incubated at 36 C. Duplicate counts of cells collected from 2 flasks were made in a hemacytometer at 12 hour intervals for the first 48 hours and thereafter at 24-hour intervals. At 36 hours after seeding, when the cell monolayer was essentially 80% confluent, medium was replaced in a corresponding set of flasks with 0.5 m1 of CPV (VC-l3-79). The inoculum contained approximately 2,048 HA units per m1 and was allowed to adsorb for 90 min at room temperature. Following adsorption, the monolayers were washed twice and refed with medium containing 2% BPS. Uninoculated control flasks used for growth rate study were treated in a similar manner. The amount of CPV HA activity was determined at 12 hour intervals for the first 24 hours and thereafter at 24-hour intervals. 32 Cell Culture Host Range of CPV Susceptibility of cell cultures derived from a variety of animal species to CPV infection was determined. Growth conditions for the various cells were analogous to those described earlier for CRFK cells. Cell types used and their source are listed in Table 1. Plastic flasks (25 cm2) and 16 x 150 mm culture tubes containing rapidly dividing cells were inoculated with 0.5 ml and 0.2 ml, respectively, of CPV (VC-l3-79) containing approximately 2,048 HA units per m1. Inocula in plastic flasks were allowed to adsorb for 90 min, as described directly above; inocula were not removed from tubes. The ability of cell cultures to support the growth of CPV was determined by FA and HA examinations. Effect of Adenoviruses on CPV Replication The effect of canine adenoviruses types 1 and 2 on CPV replication was determined by the simultaneous inoculation of CPV (VC-l3-79) and adenovirus onto rapidly dividing canine kidney cells (MDCK). The inoculum contained 2,048 HA units per ml of CPV and 105 TCIDSO per m1 of the respective adenoviruses. Inoculation of plastic flasks and tubes was done as described under cell culture host range. Fluorescent antibody examinations were performed at 40 hours after infection, and cultures were frozen at 72 hours due to extensive adenovirus CPE. Hemagglutination activity and viral infectivity were determined. Infectivity analysis was performed in CRFK cells to avoid cellular destruction by adenovirus and was conducted using microtiter slide chamberse to achieve greater accuracy of the FA test used to ascertain the presence of infected cells. Controls consisted of cells infected eBellco Glass, Inc., Vineland, NJ. 33 Table 1. Cell cultures, source, and conditions for growth analysis of CPV Cell Culture Source "Helper" Virus Feline kidney (CRFK) W. A. Nelson-Rees1 none Canine kidney (MDCK) ATCCZ none Canine kidney (MDCK) ATCC canine adenovirus l Canine kidney (MDCK) ATCC canine adenovirus 2 Monkey kidney (VERO) . ATCC ' none Monkey kidney (VERO) ATCC simian adenovirus (SV-37) Human (HeLa) ' ATCC none Human (HeLa) ATCC human adenovirus 8 Human (HeLa) ATCC human adenovirus 31 Mouse (L cells) ATCC none Bovine kidney MSU3 none Bovine kidney MSU bovine adenovirus 3 Caprine kidney MSU none Caprine kidney MSU caprine adenovirus (CH-8)5 Caprine kidney MSU caprine adenovirus (CH-38)S Ovine kidney MSU none Equine dermal Kanitz4 none Equine dermal Kanitz equine adenovirus (E-268)5 1Naval Biomedical Research Laboratory, Oakland, CA. 2American Type Culture Collection, Rockville, MD. 3Michigan State University, Clinical Microbiology Laboratory. 4College of Veterinary Medicine, Purdue University. 5MSU isolates: CH-8 and CH-38 are previously undescribed adenoviruses of goats. Other viruses were obtained from ATCC. 34 with CPV alone and adenovirus alone. The effect of additional adeno- viruses on the replication of CPV in other cell types was also investigated. The particular virus and cell type are presented in Table 1. RES ULTS Clinical Cases - Diagnosis of CPV Infection by HA and Virus Isolation Of 150 specimens (intestine/feces) examined, 30 exhibited HA activity to both pig and rhesus monkey RBC with HA titers ranging from 1:8 to greater than 1:65,536. Thirteen additional samples at dilu- tions 51:4 reacted with pig RBC and 8 of these reacted similarly with monkey RBC. However, retesting of these samples yielded negative HA results in most instances. Of interest was the finding of one sample with HA activity of 1:4,096 for pig RBC but no activity for monkey RBC. Similar but less dramatic differences were noted in another sample in which pig RBC were agglutinated at 1:256 and monkey RBC were agglutinated at 1:16. Only 1 sample agglutinated rhesus monkey RBC but failed to agglutinate pig RBC. The HA activity was low (1:16) and inconplete. Results are summarized in Table 2. Canine parvoviruses were isolated from 34 of the 150 specimens examined. Twenty-four of these positive samples corresponded to 24 of the 30 samples in which HA activity to both pig and monkey RBC was demonstrated. Five of the remaining 6 samples which were positive by HA but negative\by isolation had HA titers of 1:16 or less. The other 10 specimens positive by isolation included the 1 sample which exhibited HA activity to only porcine RBC and 9 which were HA negative for both porcine and monkey RBC. Cytopathic effects (CPE) were directly related Table 2. Canine parvovirus infection: virus isolation 35 results of hemagglutination and HA Case No. pig RBC monkey RBC Isolation C-1549-78 1:64 1:128 + C-l728-78 --- --- + C-1992-78 1:64 1:32 + VC-4-79 1:65,536 1:65,536 + VC-13-79 1:256 1:256 + VC-16-79 -- --- + VC-17-79 --- --- + VC-26-79 1:32 1:32 + VC-27-79 1:65,536 1:65,536 + VC-31-79 l:32,768 l:l6,384 + VC-42-79 1:4096 1:4096 + VC-45-79 1:8192 1:8192 + VC-52-79 1:1024 1:1024 + *VC-56-79 1:16 1:16 - VC-82-79 1:128 1:64 - VC-90-79 1:1024 1:1024 + VC-93-79 --- -—- + VC-lOl-79 --- --- + VC-104-79 1:16.384 1:16.384 + VC-109-79 1:512 1:512 + VC-ll4-79 1:2048 1:2048 + VC-116-79 1:1024 1:2048 + VC-13l-79 --— --- + *VC-140-79 1:8 1:8 - *VC-l46-79 1:8 1:8 - VC-150-79 1:256 1°16 VC-160-79 --- --- . + VC-l62-79 l:32,768 l:32,768 + VC-l69-79 --- --- + *VC-l77-79 1:8 1:8 - VC-187-79 1:4096 1:8192 + VC-l9l-79 --— --- + VC-193-79 1:8192 1:8192 + *VC-194-79 1:16 1:16 - VC-200-79 1:4096 --- + VC-202-79 1:1024 1:2048 + VC-254-79 1:512 1:1024 + VC-262-79 1:16,384 l:32,768 + VC-272-79 1:4096 1:2048 + *VC-277-79 --- l:l6(inc.) - VC-126-80 1:64 1:64 * HA activity was not removed in the presence of specific CPV antiserum. 36 to degree of HA activity and were minimal or nil in low titer and negative HA samples. Results are summarized in Table 2. Electron microscopic (EM) results were available for comparative purposes in 30 of the 150 cases, and FA results were available in 86. Electron microscopic results were in agreement with isolation and HA findings in 25 of the 30 cases, and FA examination of fecal smears or frozen sections of intestine correlated with HA and isolation results in 76 of the 86 specimens examined. Differences in FA, EM, HA, and isolation results are summarized in Table 3. Four samples were posi- tive by EM which were negative by HA and isolation; the converse was true for the other sample.v Six samples positive by FA were negative for HA activity and in 4 of these virus was not isolated. Of 4 samples negative by FA, 3 were positive for HA and all 4 were positive by isolation. Fecal preparations were occasionally difficult to interpret due to lack of intact cells, although a generalized fragmented fluorescence in association with fecal debris was often noted in samples with high HA activity (Figure 1). Biological Characteristics of Canine Parvovirus Erythrocyte Agglutination The VC-13-79 isolate of CPV which had been subcultured 3 to 5 times in CRFK cells was used in preliminary HA studies. This particular isolate agglutinated RBC from pig, rhesus monkey, cat, and horse but not those of sheep, dog, goat, cow, rabbit, guinea pig, chicken, and human. Agglutination occurred at 4 C and at room temperature with RBC of pig and monkey, but only at 4 C with those derived from cat and horse. Erythrocytes from pig and monkey were agglutinated essentially to the same degree by CPV and approximately 2.0 to 4.0 times more 37 Table 3. Canine parvovirus detection: discrepancies noted with FA, HA and isolation results HA Results Isolation Results + - + - +(4) 0 4 0 4 EM Results -(1) 1 0 l 0 +(6) 0 6 2 4 FA Results -(4) 3 1 4 0 38 Figure l. Immunofluorescence of CPV antigen in association with fecal debris. Figure 2. Immunofluorescence of CRFK cells infected with CPV. 39 efficiently than those from cat and horse. Erythrocytes of cat and horse eluted when test plates were removed from 4 C and placed at room temperature. No e1ution was noted with RBC of pig and monkey, except at the higher dilutions of antigen. The HA activity of CPV was not appreciably altered over a.wide pH range (pH 5.5-7.5). Sig- nificant differences between RBC of different swine breeds were not noted, although RBC that failed to settle properly were occasionally encountered. Seven other CPV isolates subcultured 1 to 3 times in CRFK cells were compared with VC—13-79. Results are presented in Table 4. Cell Culture Susceptibility The ability of CPV (VC-13-79) to replicate in various cell cultures was examined. Successful replication, as determined by transfer ability of CPV through 3 subcultures, was noted in cell cultures derived from dog, cat, cow, goat, human, and monkey. Virus did not replicate in cell cultures derived from sheep, pig, horse, and mouse. The virus grew best in cells of cat (Figure 2) and dog, to a lesser extent in those derived from goat and cow, and poorly in human and monkey cells. Obvious CPE was not observed in cells other than those derived from cat. Adenovirus "Helper" Function Effect of canine adenoviruses, types 1 and 2, on CPV replication was nonexistent. No differences were noted in HA activity or infecti- vity. Similarly, CPV caused no positive or negative effect on canine adenovirus replication. Canine parvovirus replication in cell cultures derived from animal species other than dog (Table l) was not benefited by the presence of other adenoviruses. 40 Table 4. Agglutination of pig, monkey, cat, and horse erythrocytes by isolates of canine parvovirus Erythrocytes From Case No. Pig Monkey Cat* Horse* VC-13-79 1:2,048 1:4,096 1:64 1:16 VC-27-79 1:16,384 1:16,384 1:256 1:256 VC-3l-79 1:2,048 1:2,048 1:128 1:32 VC-42-79 1:4,096 1:4,096 1:128 1:16 VC-45-79 1:4,096 1:4,096 1:128 1:16 VC-114-79 1:1024 1:512 1:32 1:8 VC-102-79 1:16,384 1:16,384 1:256 1:32 VC-128-80 1:512 1:512 1:32 1:16 * Erythrocytes from cat and horse did not agglutinate completely and appeared as a rough fringed button. 41 Growth Rate of CRFK Cells and CPV The growth rate of CRFK cells is shown graphically in Figure 3. At a seeding rate analogous to that used in isolation studies, the lag period lasted for about 24 hours and maximum growth was obtained between 36 and 96 hours. Confluency was reached in approximately 60 hours and cells attained their saturation density at 96 hours. Figure 3 also depicts the viral growth curve of CPV in a cor- responding set of CRFK cells infected at 36 hours post-seeding. Approximately 50 to 70% of the virus was absorbed in 90 min at room temperature, as indicated by remaining HA activity in the inoculum. No intracellular HA activity was detected prior to 12 hours after infection, and HA activity in supernatant fluid did not appear until 24 hours had elapsed. Most CPV replication occurred within 72 hours, and maximum HA titers of intracellular and extracellular virus were Obtained 120 hours after infection. DISCUSSION One hundred fifty fecal and/or intestinal preparations were examined for the presence of CPV using a presumptive HA test and virus isolation. In agreement with recently published results of Carmichael et a1. (8), HA titers 31:32 correlated well with positive isolation results. Samples which exhibited HA activity of 1:16 or less and were negative by isolation were presumed to be nonspecific and not related to CPV, as specific antiserum failed to remove this activity in parallel hemagglutination inhibition (HI) tests. Similarly, the 2 samples (VC-82-79 and VC-150-79) with moderate HA activity and the 1 sample (VC-200-79) whiCh agglutinated only porcine RBC were unaffected by the presence of specific antiserum. The cause of these nonspecific 5) Cells per ml (x 10 42 N o ‘_ ..-' " ‘EE .4- ’,ar a’ 1", V‘ I . o O . . . ..S / . ' a I O Q 0 U1 m 4H t . N O N 5' 13“ P. ..8 ‘05 IN ““8 N u: l «a e. .Ki 0"), ‘2; NP 1 Q A Q I R A L 24 48 72 96 ”120 144 168 viral growth--time(hours)--post inoculation HL 24 53 73 96‘ lib 134 cell growth--time(hours)--post seeding ——-cell number ---intracellular virus ...extracellular virus Figure 3. Growth curves of CRFK cells and canine parvovirus. 43 reactions was not determined, but it seems unlikely that these levels of HA activity, at least for VC-200-79, were due to nonspecific agglutinins. However, attempts to isolate viruses other than CPV using MDCK cells were unsuccessful (data not shown). The CPV isolated from.VC-200-79 was apparently incidental to the HA activity. The reason for lack of CPV specific HA was attributed to the small amount of virus present in the specimen, as evidenced by the presence of only a few FA positive cells in inoculated cultures. The selective partial agglutination of 1 sample (VC-277-79) for rhesus monkey RBC may have been due to the presence of minute virus of canines (MVC), which agglutinates monkey RBC but not those of pig (5). Viral isolation procedures were shown to be a more sensitive technique than direct HA for the diagnosis of CPV. Eight specimens with no demonstrable HA activity were positive by isolation. In each of these cases, only a few FA positive cells were noted in inoculated cultures, and HA activity of supernatant fluids was negligible. It is doubtful that these isolates represent CPV with different HA properties, although this possibility was not eliminated. In general, HA activity of fluid from infected cell cultures correlated well with the HA activity in the original inoculum, indicating that virus "output" was directly related to virus "input." Thus, lack of HA activity was thought to be a result of low amounts of virus in these preparations. Positive EM results and negative HA/isolation results were likely due to the presence of CPV antibody in the intestinal tract; conse- quently, there was no free virus, or the presence of MVC, which is microscopically indistinguishable from CPV, may have interfered. Nega- tive EM results and positive HA/isolation results might occur with samples containing small amounts of virus. However, the 1 sample in 44 this category had a HA titer of 1:8,192, which was considerably higher than some others that were positive by EM. Negative FA results on the 4 specimens from which CPV was iso- lated were thought to be due to the type of specimen utilized. Fecal material was submitted in each of these cases, and difficulty was occasionally encountered in the interpretation due to the absence of intact cells. The presence of intact cells was not necessarily a prerequisite, as a generalized fragmented fluorescence was often observed in association with debris if virus concentration was heavy, i.e., high levels of HA activity. The 4 samples that were positive by FA but fronlwhich no virus was isolated may have been due to the presence of CPV antibody. The presence of antibody would not affect the FA test results, as viable cells would be impermeable, but emulsifying the preparation for isolation studies would disrupt the cells and effective neutralization of CPV could occur. The ability of CPV to agglutinate RBC of pig, rhesus monkey, horse and cat at 4 C is in agreement with results reported by Carmichael and co-workers (8), but e1ution at 23 C did not occur with RBC of pig and monkey except at high dilutions of antigen. In fact, only minor dif- ferences were noted in HA activity with pig and monkey RBC when parallel tests were conducted at 4 C and 23 C. These findings are in agreement with Gagnon and Povey (14), who also reported HA activity of CPV at 23 C. There was no spontaneous agglutination of pig RBC at pH values below 6, as reported by Carmichael et al. (8), although occasional batches of RBC failed to settle properly. No major differences were noted in HA activity of 8 cell culture isolates of CPV. Growth of CPV in cell cultures derived from cat, dog, mink, raccoon, and bovine has been reported (3), and we observed that 45 replication occurred in cells derived from goat, human, and monkey as well. Replication of CPV in monkey cells (vero) was in contrast to a previous negative report (3). Linser et a1. (23) reported that the ability of minute virus of mice to infect cells was dependent upon specific receptor sites, and it is surprising that CPV does not exhibit the restricted host range observed.with most autonomous parvoviruses. Canine parvovirus is associated with myocarditis in young dogs (9,15,16,17,18,22,24), and it has been suggested that the ability of CPV to replicate in heart muscle may be due to a "helper" virus (6). However, we failed to demonstrate any enhancement of CPV replication in MDCK cells when CPV was co-cultivated with either canine adenovirus types 1 or 2. This finding does not necessarily rule out the possi- bility that other viruses could provide helper function, although it seems unlikely. We used an enteric isolate of CPV in our study, and the possibility remains that a myocardial isolate of CPV might behave differently. The growth rate of CPV was maximum during the logarithmic growth of CRFK cells, as was expected. Under conditions which we used for general isolation purposes, maximum virus concentration was attained within 4 to 5 days after inoculation. Therefore, there was no reason for holding cultures for extended periods of time. In conclusion, the results presented herein indicate that an accurate diagnosis of CPV infection is best achieved through virus isolation procedures. Although HA titers of 1:32 or greater are usually indicative of CPV infection, simultaneous HI tests should be conducted to rule out nonspecific reactions. Care should be taken in interpreting FA results of fecal smears, as the fragmented fluorescence 46 associated with fecal debris may be confusing. In such cases, a blocking test would be advisable. The primary advantage of EM examination _is the detection of viruses other than CPV. No dis- tinguishing differences were noted between different isolates of CPV. 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