Canine Parvovirus-2 - Europe PMC

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of canine parvovirus-2, was a successful means of reproduc- ing gastroenteric signs of canine parvovirus-2 infection. Twenty- one of 24 dogs, which had pre-.
Successful Experimental Challenge of Dogs with Canine Parvovirus-2 S. Carman and C. Povey* 48 heures apres leur infection buccale avec un broyat de la muqueuse intestinale d'un chien infecte experimentalement lui aussi avec le parvovirus canin-2, s'av6ra un moyen efficace de reproduire les signes cliniques de la gastro-enterite imputable a ce virus. Vingt-et-un des 24 chiens auxquels on avait antkrieurement administre divers vaccins contre l'enterite du vison ou qui n'avaient revu aucun vaccin, mais qu'on avait prives de nourriture au temps de leur infection experimentale, eliminbrent des fices pateuses ou liquides, avec ou sans mucus. C'est au ile jour apres l'infection qu'on observa le plus souvent des feces anormales. Sept chiens e'liminerent du sang dans leur feces, a une ou plusieurs occasions, et sept autres vomirent a quelques reprises. On enregistra de la pyrexie chez 71,6% des chiens, le sixieme jour apres leur infection, et une lymphopenie, chez 50% de ceux chez qui on la recherchait, aux cinquieme et sixicme jours apres leur infection. Par contre, quatre chiens non prives de nourriture au temps de leur infection, ne manifesterent pas de signes cliniques de gastro-entkrite, a l'exception d'un seul qui elimina des feces pateuses et un peu de mucus, le sixieme jour apr&s son infection. Quatre autres chiens auxquels on avait administre un vaccin inactive contre le parvovirus canin-2 et qu'on avait prives de nourriture, au temps de leur infection, ne manifesRtSUM: terent pas de signes cliniques. A Le fait de retirer la nourriture part ces derniers, tous les autres a des chiens, 24 heures avant et eliminerent le parvovirus canin-

ABSTRACT Withholding food from dogs for 24 hours prior to, and for 48 hours following oral challenge with a gut mucosal homogenate of canine parvovirus-2, was a successful means of reproducing gastroenteric signs of canine parvovirus-2 infection. Twentyone of 24 dogs, which had previously received various vaccine preparations of mink enteritis virus or were unvaccinated, and which were starved at challenge, developed soft or liquid feces with or without large clots of mucus. Altered feces were most frequent on postexposure day 11. Seven dogs passed frank blood in their stools on one or more occasions and seven dogs vomited sporadically. Pyrexia was noted in 71.6% of the dogs on postexposure day 6 and lymphopenia was detected on postexposure day 5 or 6 in 50% of the dogs monitored. In contrast, four dogs not starved at the time of challenge remained free of gastrointestinal signs apart from one dog which passed a soft stool with scant mucus on one day, postexposure day 6. Also four dogs vaccinated with a killed canine parvovirus-2 vaccine preparation and subsequently starved at the time of challenge, remained clinically healthy. Apart from these last mentioned four dogs, all others shed canine parvovirus-2 in their feces following challenge.

2 dans leur feces, a la suite de leur infection experimentale.

INTRODUCTION The recently described canine parvovirus (CPV) associated with gastro-enteritis in dogs of all ages (15, 22, 26, 35) and myocarditis of pups (18, 19,23,26) has been shown to be distinct (12) from minute virus of canines (MVC), a canine parvovirus isolated by Binn in 1970 (8). For clarity MVC should be designated canine parvovirus-1 (CPV-1) and the newly recognized isolate, canine parvovirus-2 (CPV-2). A distinct, defective canine parvovirus, dependent on adenovirus for its replication and best labelled canine adeno-associated virus (CAAV), has also been described (33). Apart from some preliminary success in reproducing clinical enteritis in two dogs inoculated orally with a fecal suspension containing CPV-2 by Gagnon and Povey, (16) and the intravenous infection of puppies by Robinson et al (32) using 1000 hemagglutinating units of canine parvovirus initially isolated from the myocardium of a dog, there have been failures to establish the clinical signs of the natural disease in dogs infected with 104 TCID5o of CPV-2 given by stomach tube (28); with 104 TCID50 given intranasally or with 105-5 TCID50 given intravenously (6). This last route of inoculation produced lymphopenia and elevated body temperature, but not diarrhea or other clinical signs of severe illness. We wish to report our success

*Clinical Research, Department of Clinical Studies, Ontario Veterinary College, Guelph, Ontario NlG 2W1. Submitted January 26, 1981.

Can. J. comp. Med. 46: 33-38 (January 1982)

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with a simple experimental chal- SEROLOGY lenge method which does induce Hemagglutination-inhibition clinically apparent enteric disease tests were carried out using a in exposed conventional dogs. This microtitration system.1 work was briefly reported elsePrevaccination, postvaccination where (10). and postchallenge sera samples were initially heat inactivated (56°C, min) and were adsorbed MATERIALS AND METHODS in the 30 first well of the microtitre DOGS plates by adding 100 ,uL of the Thirty-two conventional dogs serum to 100 ,uL of a 50% AGM red were obtained. They were approx- cell suspension (for prevaccination imately three to four months of samples) or 50 ALL of serum to 150 age, of various breeds and mixed ,uL of a 0.5% suspension (for all sex, and with no detectable other samples) and incubated hemagglutinating-inhibition (HI) overnight at 4°C. During this time antibody to CPV-2. Twenty of the red blood cells settled out and a these dogs had received two doses 50 ,uL aliquot of the adsorbed of various inactivated vaccine serum could be transferred to the preparations of mink enteritis next well containinq 50 ,L PBS virus (MEV), as part of a study (pH 7.2) as the dilutent and thus reported separately (11). Eight diluted in a routine manner prodogs were not vaccinated. The four ducing two-fold dilutions. Dupliremaining dogs had been pre- cate rows were used for each viously vaccinated with two doses serum. Four to eight HA units of of a killed adjuvanted CPV-2 vac- CPV-Rae 8 were then added to cine (30) and at the time of chal- each well. Following incubation lenge had CPV-2 HI titres of 1:64 for one hour at 37.5°C 50,uL of a 0.5% suspension of AGM red blood or greater. cells in chilled PBS was added to HOUSING each well, and the plates were All dogs were individually caged further incubated at 4°C for two to in an isolation building of the 18 hours. Appropriate controls were established. A 50% end point Ontario Veterinary College. for inhibition of hemagglutination HEMAGGLUTININ PREPARATION was determined either by direct A local isolate of canine parvovi- observation or extrapolation. rus, CPV-Rae, was used at its eighth passage level. The virus was VIRUS DETECTION AND ISOLATION propagated for two passages in a Fecal samples were stored at canine kidney cell line and for six -20°C until processed by emulsifypassages in a feline kidney cell line ing into a 10% w/v suspension in a (NLFK) using rapidly growing virus transport media which was cell cultures. The hamagglutinin prepared from cell culture media was harvested by three cycles of (Eagles's basal medium with freezing and thawing followed by Hank's salts, supplemented with ultrasonication and low speed cen- 0.5% lactalbumin hydrolysate, 1% trifugation. Harvested virus, at L-glutamine, 20 mL/L of 7.5% the eigth passage had a virus infec- NaHCO3, 125,000 units/L peniciltivity titre of 105.2 CCID50/mL and a lin, 125, 000,ug/L of streptomycin) hemagglutinating (HA) titre of to which 50,000 units/L of myco1:1024 at 4°C using a 0.5% suspen- statin and 2% FCS was added. This sion of African Green Monkey suspension was centrifuged at (AGM) red blood cells with 1% fetal 2000 rpm for 20 minutes and was calf serum (FCS) in phosphate buf- then filtered through a prefilter fered saline (PBS pH 7.2). The (1.2,) and then a 0.45 , filter to virus was stored frozen at -90°C remove bacteria and fecal debris. and thawed and sonicated prior to Fecal filtrates were examined use. for CPV-2 virus specific direct HA 1Dynatech Laboratories Incorporated, Alexandria, Virginia. 34

activity in the microtiter system, using AGM red blood cell suspension as above, and incubating overnight at 4°C. Appropriate controls were established and a 50% endpoint recorded. Also, 1 mL aliquots of the individual filtrate samples were inoculated into young (four to eight hour) monolayers of NLFK cells, maintained with cell culture media supplemented with 6% FCS, in regular culture tubes, initially with 5 x 8 mm coverslips. Cultures were examined for direct HA activity after five days of incubation at 37.5°C, by the addition of 50 ,u L of a chilled 0.5% AGM red blood cell suspension to 150 ,uL of tissue culture supernatant. Initially coverslips were stained by hematoxylin and eosin, following Bouin's fluid fixation, to examine for the presence of typical parvovirus intranuclear inclusions (20). No samples were found to have inclusions present without HA activity. Negative cultures were passaged by monolayer trypsinization and replanting at least once before being considered negative for CPV-2 isolation. Individual fecal filtrate samples from postexposure day (p.e.d.) 2 and 3 of all starved dogs were also examined for the presence of canine coronavirus (CCV). One millilitre aliquots of the individual filtrate samples were inoculated into confluent monolayers of Crandell feline kidney cells in regular culture tubes with 5 x 8 mm coverslips and incubated at 37.5°C. At three and ten days of incubation all cultures were passaged by monolayer trypsinization and replanting into culture tubes with coverslips. Coverslips from postinoculation days 3, 10 and 17 were stained by hematoxylin and eosin following Bouin's fixation. Coverslips from all three occasions were examined under a light microscope for the presence of CCV cytopathic effect (CPE) as described by Binn (9). CHALLENGE MATERIAL

Virus infected material for chal-

lenge consisted of homogenized intestinal mucosa (10% w/v in PBS) of a dog infected orally five days previous with filtered (0.45u ) feces from field cases of CPV-2 infection, confirmed by virus isolation (16). Histological examination of the small intestine (Fig. 1) and other body tissues of this dog demonstrated lesions consistent with enteric and systemic infection with CPV-2 (22). This homogenate was examined by electron microscopy and cultured for the detection of bacterial pathogens (Salmonella and Campylobacter organisms) and other viral contaminants (CCV). No other canine viruses or known bacterial pathogens could be demonstrated. Canine parvovirus-2 was observed in the feces of the infected dog by electron microscopic examination and was reisolated in tissue culture (16). EXPERIMENTAL PLAN

All thirty-two dogs were challenged orally (using a syringe) with 10.0 mLs of challenge material. All vaccinated dogs and four of the nonvaccinated dogs were fasted for 24 h prior to challenge and 48 h following challenge. They

were then returned to ad lib feeding with a commercial dry dog ration. Four remaining dogs were not starved at the time of challenge. Dogs were observed daily for clinical signs. Feces were collected for virus detection and isolation. Rectal temperatures of the dogs were recorded for two days prior to challenge and for ten days thereafter. Uncoagulated blood samples were collected for ten days and total white blood cell counts were performed. Differential leukocyte counts were performed daily on the four unvaccinated starved dogs only. Serum samples were obtained prior to vaccination, prior to exposure and at intervals, postchallenge. They were examined for CPV HI antibodies as previously described. At the end of the fourteen day observation period two CPV-2 vaccinated dogs were euthanized and their body tissues examined histologically for evidence of disease. Tissue sections were stained with hematoxylin and eosin. Selected tissue sections from the colon and small intestine were stained with Warthin-Starry silver stains. Feces were examined for the presence of Salmonella and

Fig. 2. Intact mucosal epithelium with marked hyperplasia of lymphoid tissue of Peyer's patches of the small intestine 14 days postexposure in a CPV-2 vaccinated dog. H & E. X6.

patches, with reactive lymphoid tissue filling the entire submucosa, was present at multiple sites within the small intestine (Fig. 2). Plasma cells were present in the

Campylobacter by bacteriological lamina propria. The four unvaccinated dogs, techniques, and the presence of other canine enteric viral patho- which were not starved at the time of challenge, also remained free of gens by electronmicroscopy. clinical disease except for one dog which passed an unformed stool with mucus, during the early part RESULTS of the observation period, on p.e.d. The four dogs vaccinated with 6. All four dogs showed a decrease killed adjuvanted CPV-2 vaccine, in leukocytes on one or more of which were starved at the time of p.e.d. 5, 6 and 7. Canine parvochallenge, remained clinically virus-2 was recovered from the healthy following challenge, did feces of these dogs from p.e.d. 3 to not shed virus in their feces and did p.e.d. 7. All four dogs developed not show a rise in serum HI anti- serum HI antibody titres of 1:1024 bodies subsequent to challenge. All or greater following challenge. Of the remaining twenty-four dogs had a marked increase in circulating lymphocytes on p.e.d. 5. dogs (twenty MEV vaccinated, Two of these four dogs passed feces four nonvaccinated) that were with mucus on one day each near starved at the time of challenge, all the end of the fourteen day obser- but two dogs (one MEV vaccinated vation period (one on p.e.d. 12 and and one control dog) showed some the other on p.e.d. 14). No patho- or all signs of clinical disease genic organisms were identified at including pyrexia (> 39.4°C), euthanasia on p.e.d. 14 and no his- vomition, leukopenia ( 39.40C) on each postPOST EXPOSURE DAY recorded in detail in a separate exposure day. Fig. 4. Proportion of the 24 starved dogs paper (11). Following challenge, showing changes in the character of all but three dogs developed detec14 day observation period. Four table antibody to CPV-2 by p.e.d. dogs showed pyrexia as early as their feces on each postexposure day. 21. p.e.d. 1. Fifteen dogs showed pyrexia on one or more of days 4, 5, 6 with the greatest number of dogs ST EXPOSURE DAY showing pyrexia on p.e.d. 6. Pyrexia recurred in nine dogs on VACCINE I DOGI 0 11 21314 5 Ir61 71 819 110111112113114 I p.e.d. 9 and 10 (Fig. 3). Two dogs, -II -I I I I_ +1+~~~ + r7 Il Q I I both vaccinated, developed an T v absolute leukopenia, one on p.e.d. 7 + I and one on p.e.d. 9. Two of the four 1+++ control animals monitored showed ++ AW_ ++I +1 an absolute lymphopenia on either + + + +..7 + F + 0 p.e.d. 5 or p.e.d. 6, without demon1 4 i i strating an absolute leukopenia. R I + + + +I+ . Seven dogs (five vaccinated, two KI I--II I Icontrols) vomited sporadically 11 over the 14 day observation period. ~1 +~~ w One control dog vomited on two separate days postexposure. All T I i I7 0 + + + 0 K I I but three dogs (two MEV vaccinated, one control) showed changes in the character of their feces. The main changes were to soft or liquid feces with or without mucus clots. Seven different dogs (six vaccinated, one control) passed frank t 0 p+1+;+l+l+l+ + +1 t D KV blood in their feces on one or more days. On p.e.d. 11 and 12, seven dogs did not pass any feces. The greatest number of dogs showed changes in the character of their + +. fecal excretion on p.e.d. 11 (Fig. 4). All fecal samples were examined +|+|+||r +| + | + +| Nonvac DU | | + Non-vacc.++++ for presence of CPV-2 by direct HA, and titers > 1:16 were detected in the feces of dogs only on T E++ +++ + + p.e.d. 6 (four of 24) and p.e.d. 7 (six Controls viral maximal of 24), suggesting +0 ++ + + J I shedding occurred on these days. This is similar to the data pre0 no sample viously reported (12). Isolation in Fig. 5. Isolation of CPV-2 in cell culture from the feces of individual starved dogs. tissue culture proved to be a much (Vaccine Groups: I formalin inactivated and adjuvanted; II Formalin inactivated, more sensitive technique for the not adjuvanted; III Beta-propiolactone inactivated and adjuvanted; IV Betadetection of CPV-2 in feces. Thus propiolactone, not adjuvanted; Nonvaccinated controls). 50

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DISCUSSION In this challenqe experiment dogs orally exposed to CPV-2 contaminated gut mucosal scrapings developed clinical disease with pyrexia, vomition and diarrhea, only when exposed following a period of starvation immediately before and after exposure. Dogs which were not starved at the time of exposure remained clinically healthy despite CPV-2 seroconversion and virus shedding, except for one dog which passed an unformed stool with mucus on one day, but without other signs of clinical disease.

Laboratory research into canine parvovirus disease and its control has been hampered by the inability of investigators to reproduce clinical signs, consistent with the field disease, in dogs infected with various preparations of CPV-2, at different dosages and by different routes. Discussion of the reasons for these failures has included suggestions that, in the field, intercurrent infection of dogs with other viruses, bacteria such as Campylobacter, or endoparasites such as coccidia and hookworms may be important. It has also been observed that many of the severe clinical cases occur in dogs which have been apparently stressed by factors such as being boarded, attending shows or having strayed. The requirement of parvoviruses for actively dividing cells and a high level of host cell DNA syn-

thesis for viral replication has been well documented (21, 31, 34). The late S-phase of cell mitosis is most important (31) and this explains the tropism of parvoviruses for tissues where large numbers of actively dividing cells are present, such as gut crypt epithelium, and lymphoid tissue. Epithelial cell replication in the gut mucosa is carried on at a variable level, influenced positively by factors such as bacterial flora (1, 25) and food intake (13, 14, 17). This latter effect is mediated via endogenous factors in bile, gastric, duodenal and pancreatic juices (3, 4). Starvation followed by refeeding results in an enhanced rate of entry of cryptal epithelial cells of the gut into mitosis (2, 5). We postulated, therefore, that by starving dogs for a period, around the time of virus challenge, and then refeeding ad lib, the virus would be infecting a gut in which an enhanced epithelial cell turnover rate would increase the potential for production of clinical enteritis. The success of this challenge in producing clinical signs of parvovirus disease may be attributable to the above hypothesis. In the field, the apparent association between stress situations and clinical parvovirus disease may in part be due to reduced food intake at the onset of stress, followed later by resumption of appetite. In subsequent challenges, using similar inocula, where unvaccinated dogs were starved at the time of exposure, clinical signs similar to those described here have occurred (30). Parenteral vaccination of dogs with MEV did not appear to provide protection against oral challenge but may have altered the pattern of clinical disease seen in this group of dogs. The challenge inocula used in this experiment was demonstrated to be specific for CPV-2 in the following ways. No other canine enteric pathogens were demonstrable by standard virological,

lenged dogs were examined. No histological lesions were present in the gut mucosal epithelium of the two CPV-2 vaccinated dogs. However, marked hyperplasia of the gut associated lymphoid tissue occurred. In these two dogs the marked hyperplasia of Peyer's patches of the gut mucosal lymphoid system and presence of plasma cells in the lamina propria, in association with the increase in circulating blood lymphocytes, may be expected following prolonged exposure to specific viral antigen. Following initial oral challenge these dogs were then continuously exposed to virus excreted by littermates housed in the same room. In dogs, parenteral immunization closely followed by repeated oral challenge, has been shown to induce an effective local gut mucosal immune response (29). The appearance of mucus in the feces of these two dogs late in the observation period (p.e.d. 12 or 14), in the absence of histological mucosal epithelial lesions and enteric pathogens and subsequent to repeated exposure to antigen, may have been due to an immunemediated release of mucus from goblet cells in the small intestine. Mucus release by goblet cells can be stimulated by both immune complexes within the gut lumen (36) and by antigen administered orally, in orally sensitized animals (24). It has been suggested that T cells may also be involved in the regulation of goblet cell secretory activity (7). T cells appear to also regulate the number of goblet cells in the gut mucosa (27). ACKNOWLEDGMENTS

This study was supported in part by the Canadian Veterinary Research Trust Fund, in part by Connaught Laboratories and by private donations from many kennel clubs across Canada. One bacteriological, histopathological author (Carman) is a research felor electronmicroscopic methods low of the Medical Research Counwhen the original donor dog was cil of Canada. The authors thank examined nor subsequently when Elmer Ewert, Lloy Osburn and the two CPV-2 vaccinated chal- Susan Campolongo for their tech37

nical assistance and Diane Bending for secretarial assistance. 12.

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