Treatment of Canine Parovirus Infection with recombinant Feline Interferon - ω

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1 Treatment of Canine Parovirus Infection with recombinant Feline Interferon - ω Tomiya Uchino, DVM MS PhD; Hirotaka Matsumoto, DVM MS PhD; Toshiko Sakamoto, BA; Toru Sakurai, MS A clinical study was conducted to evaluate the efficacy of recombinant feline interferon-ω (rfeifn-ω) against canine parvovirus (CPV) infection. The efficacy rates assessed by a veterinarian on day 7 were 70.1% (1.0 MU/kg group), 66.7% (2.5 MU/kg group) and 38.5% (control group) and the survival rates were 83.5%, 76.2% and 46.2%, respectively. These data suggested that rfeifn-ω treatment significantly and safely improved disease prognosis and could be used in the treatment of canine CPV infections regardless of other concurrent therapies. Key Words: Canine Parvovirus, Dog, Feline Interferon-ω Introduction anine parvovirus (CPV) infection is C an acute systemic disease that is characterized primarily by cataplectic emesis, diarrhea, dehydration and leucopenia. Even though various treatments are available e.g., electrolyte infusion, blood transfusion, administration of antiemetics, hyperimmune serum, recombinant granulocyte colony-stimulating factor, general antibiotics for inapparent infection such as Campylobacter sp., Salmonella sp. and Escherichia sp, and anti-bacterial endotoxicosis immunotherapies, including antiendotoxin,the infection can be fatal in serious cases. 4,7 Since vaccination against CPV only confers a 50% survival in young dogs a more effective treatment strategy for this highly-susceptible population is required. 2 Recombinant feline interferon-ω (rfeifn-ω) a was purified from a baculovirus vector-transfected silkworm larva. 11,15 rfeifn-ω demonstrated antiviral activity in vitro and was demonstrated to be effective against feline calicivirus (FCV) infections. 10,12,18 After rfeifn-ω was licensed for use in 1993 for the treatment of FCV it was tested in the treatment of other viral infections including feline leukemia virus (FeLV) and the feline immunodeficiency virus (FIV). 3 In 2004 it was approved by the European Union (EU) for the treatment of FeLV and FIV infections. More recently the effects of rfeifn-ω on canine cells has been demonstrated in vitro and its therapeutic effects in the treatment of CPV infections have been reported in clinical settings. 2,6,8,9,13,16,17 In 1997 and 2001 Japan and the EU, respectively approved its use for the treatment of CPV infections. In this report we describe the results of a clinical study that was designed to assess the efficacy of a rfeifn-ω preparation against CPV infection in dogs. Materials and Methods Animals The inclusion criteria for this study were as follows: a clinical diagnosis of a CPV-associated symptoms (e.g., dehydration, decreased appetite, thirst, decreased vitality, diarrhea, salivation, dyspnea and emesis) and a positive CPV confirmation by testing for hemagglutination assay method 1 using fecal samples collected on day 0 and to the virus test with a commercially available CPV detection kit. b Dogs in the rfeifn-ω treatment group treated with an immunomodulator (including a corticosteroids, dogs that underwent blood transfu- From the Veterinary ME Research Center, 482-3, Ina, Akiruno-shi, Tokyo , Japan (Uchino); Laboratory of Veterinary Internal Medicine, Nippon Veterinary and Life Science University, 7-1 kyonan-cho 1-chome, Musashino, Tokyo , Japan (Matsumoto); Toray Industries, Inc. Head Office, 2-1, Nihonbashi-Muromachi 2-chome, Chuo-ku, Tokyo , Japan (Sakamoto, Sakurai) Correspondence: Dr. Tomiya Uchino XLN07445@nifty.ne.jp 130 JVCS, Vol. 1, No. 4, October 2008

2 sions or were treated with an immunoglobulin preparation) were excluded from the study. rfeifn-ω was purified from silkworms transfected with the gene encoding rfeifn-ω 6,7 and was freeze-dried until use at a concentration of 10 million units (MU) per vial and stabilized with purified gelatin and D-sorbitol. The freeze-dried preparation was dissolved in 1.0 ml of sterile physiological saline solution immediately before injection. Treatment protocol The dogs were recruited from 24 animal hospitals in Japan. Thirty-six different breeds were enrolled in the study, including mongrels (30), Shiba inu (17), Miniature Dachshund (16), Shih-tzu (13), Dachshund (14), Papillon (8), Welsh Corgi (6), Golden Retriever (6), Labrador Retriever (6), Maltese (6), Tosa inu (5), Chihuahua (6), Pug (5), Beagle (5), Yorkshire Terrier (5), Shetland Sheepdog (3), Great Pyrenees (3), West Highland White Terrier (2), Cavalier King Charles spaniel (2), Siberian Husky (2), Dalmatian (2), Toy Poodle (2), Bernese Mountain dog (2), Poodle (2), Pomeranian (2), Kishu-inu (2), Old English Sheepdog (1), Samoyed (1), Spitz (1), Flat-coated Retriever (1), Miniature Pinscher (1), Schnauzer (1), Pekingese Efficacy of rfeifn-ω on CPV infection (1), Boxer (1), Boston Terrier (1) and Lhasa apso (1). At day 0 rfeifn-ω was administered at either 1.0 or 2.5 MU/kg intravenously once daily for three consecutive days. For these two treatment groups, rehydration therapy and the use of spasmolytics, antiemetics, antibiotics, intestinal demulcents, gastromucosal protectants, antidiarrheals, hemostatics or vitamins were allowed as concurrent therapies. Untreated control animals (one dog was recruited from each hospital) were treated in the same fashion for CPV infections with the exception of rfeifn-ω or corticosteriod treatments. The dogs were assigned to their respective groups at the discretion of the veterinarian and informed consents (including treatment costs) were obtained from dog owners prior to commencing the study. Evaluation The following clinical signs were evaluated based on the clinical sign evaluation criteria shown in Table 1: dehydration, decreased appetite, thirst, decreased vitality, diarrhea, salivation, dyspnea or emesis. In addition, overall therapeutic efficacy was classified into one of five levels according to the evaluation criteria for therapeutic efficacy described in Table 2 on the final day of the study (day 7). Table 1. Evaluation of clinical signs Clinical Sign Clinical Score Dehydration(%) Normal Appetite Normal 1/4 anorexia 1/2 anorexia Refusal to eat NA 1 Thirst Normal NA No thirst Abnormal Water Consumption NA Vitality Normal Decreased vitality loss Loss of vitality NA NA Diarrhea Normal Soft stool Diarrhea Diarrhea with teneamus Bloody mucus Salivation Normal Reduced amount Large amount NA NA Dyspnea None Nostril breathing Dyspnea Two-stage breathing NA Emesis None Sporadic Occasional Frequent Continuous JVCS, Vol. 1, No. 4, October

3 Uchino et al Table 2. Evaluation criteria for overall therapeutic efficacy Evaluation Excellent Good Fair No effect Definition Improvement of diarrhea by day 5, improvement of appetite, emesis frequency and dehydration by day 3, and normalization of all symptoms by day 7. Improvement of diarrhea by day 7 and improvement of appetite, emesis frequency and dehydration by day 5. Improvement of diarrhea, appetite, emesis frequency and dehydration, but below the levels observed or the good evaluation. No improvement of symptoms and death by day 3 or after among animals that died. Worse Exacerbation of symptoms compared with those at the start of treatment and death by day 2. The same veterinarian performed the scoring of the clinical signs throughout the course of the study. The efficacy rate was expressed as follows: (number of excellent + number of good cases)/total number of cases. Body temperature measurements, body weight, a complete blood count analysis and a serum chemistry panel were performed on days 0, 1, 2, 3, 5 and 7. White blood cell (WBC) count was also measured on days 0, 1, 2, 3, 5 and 7. Statistical analysis Statistical analyses were conducted using a commercial package. c Survival analysis between groups was analyzed using the chi-square test. Therapeutic efficacies between groups were compared using the chi-square, the Mann-Whitney U and the Student s t-tests. The Mann-Whitney U test was used for 5-grade therapeutic efficacy analysis (excellent, good, fair, no effect or worse) and the chi-square test was used for 2-grade evaluations. The Student s t-test was used to assess improvement of clinical signs over time between two groups. The outcome measures for efficacy analysis were assessed per protocol. The clinical scores of all the dogs were included in the statistical analyses whether or not they had completed the study. All missing clinical scores were replaced using the last value carry forward rule. 5 The Mann-Whitney U test was used for comparing WBC level. Results One hundred and twenty-seven dogs were assigned to the 1.0 MU/kg dose group, 42 to the 2.5 MU/kg dose group, and 13 to the control group. Case distributions between the hospitals were as follows: 11 hospitals had between 1-5 cases, seven hospitals had between 5-10 cases, three hospitals had between cases and three hospitals had between cases (Table 3). No differences in the clinical scores were observed in the treatment groups prior to therapy. The clinical indices at day 0 were 96.7% for decreased appetite, 95.6% for diarrhea, 90.7% for decreased vitality, 90.1% for emesis, 87.4% for dehydration and thirst, 31.3% for salivation and 23.6% for dyspnea. The dogs received various concurrent therapies at the discretion of the attending veterinarian i.e., antibiotic treatment (117 in the 1.0 MU/kg group, 42 in the 2.5 MU/kg group and 13 dogs in the control group); rehydration therapy (95 in the 1.0 MU/kg group, 30 the in 2.5 MU/kg group and 9 dogs in the control group); intestinal demulcents (40 in the 1.0 MU/kg group, 16 in the 2.5 MU/kg group and 3 dogs in the control group); anti-diarrheals (30 in the 1.0 MU/kg group, 8 in the 2.5 MU/kg group and 4 dogs in the control group); spasmolytics (20 in the 1.0 MU/kg group, 7 in the 2.5 MU/kg group and 3 dogs in the control group); hemostatics (22 in the 1.0 MU/kg group and 2 dogs in the control group); and vitamin supplementation (46 in the 1.0 MU/kg group, 22 in the JVCS, Vol. 1, No. 4, October 2008

4 MU/kg group and 6 dogs in the control group). There were only two dogs that were administered interferon only (without concomitant medication) in the 1.0 MU/kg group. Survival rate analysis demonstrated that the two treatment groups responded significantly (P = ) to the treatments i.e., 83.5% (106/127, 1.0 MU/kg group) and 76.2% (32/42, 2.5 MU/kg group) survival rates compared to 46.2% (6/13) in the control group (Figure 1). To estimate the effect on the survival rates due to prior vaccination against CPV the data were analyzed in the context of the vaccination status. Among the 182 dogs, 47 had been vaccinated (36 in the 1.0 MU/kg group, 9 in the 2.5 MU/kg group and 2 in the control group) and 133 had not been vaccinated (89 in the 1.0 MU/kg group, 33 in the 2.5 MU/kg group and 11 in the control group) and two had an undefined vaccination status. Over the course of the study there were ten deaths in the vaccination group (1 in the 1.0 MU/kg group, 7 in the 2.5 MU/kg group and 2 in the control group). There was a significant difference in the mortality rates between the treatment and control groups (P < ). Twenty-eight of the unvaccinated dogs (14 in the 1.0 MU/kg group, 9 in the 2.5 MU/kg group and 5 in the control group) died and there was a significant difference in mortality rates between vaccinated and treated groups when compared to the vaccinated but untreated controls (chi-square test, P = ). We next examined the correlation between each dog s clinical score prior to therapy to survival. The average clinical score prior to therapy was 8 in 30 dogs (22 in the 1.0 MU/kg dose group, 6 in the 2.5 MU/kg group and 2 in the control group) and > 8 in 152 dogs (105 in the 1.0 MU/kg group, 36 in the 2.5 MU/kg group and 11 in the control group). During the course of the study there were ten deaths in the 8 group (8 in the 1.0 MU/kg group, 1 in the 2.5 MU/kg group and 1 in the control group). There were no significant differences in mortality between the treatment groups and the control Efficacy of rfeifn-ω on CPV infection group. In contrast, there were 28 deaths (13 in the 1.0 MU/kg group, 9 in the 2.5 MU/kg group and 6 in the control group) in the >8 group which was statistically significant (chi-square test, P = ). There was a significant difference in therapeutic efficacy based on evaluation criteria described in table 2 of this study (Table 4). The efficacy rates for the 1.0 and 2.5 MU/kg treatment groups (70.1% and 66.7%, respectively) were significantly higher than that for the control group (38.5%) (chi-square test, P = ). In addition, significant differences were observed between the treatment and the control group in the 5-grade efficacy analysis (Mann-Whitney U-test, P = ). There were no significant differences in the efficacy rates and the 5-grade efficacy analysis between the treatment dose groups. Clinical symptom improvement over time is shown in Figure 2 and significant differences in the parameters examined between the treatment and the control group from day 3 to day 7 were observed. No abnormalities in body temperature, body weight at the completion of the study were observed for any group. The level of WBC in the day 0 control group was ± /μl. Furthermore, 1.0 MU/kg group and 2.5 MU/kg group were ± /μl and ± 5,9549 /μl, respectively. The significant difference of the WBC level was not conformed between the control group and the rfeifn-ω administrated groups. On the other hand, the WBC level of the control group after treatment (day 7) was ± /μl. That of 1.0 MU/kg group was ± /μl and 2.5 MU/kg group was ± /μl. The WBC level of the control group did not have a significant difference between before and after treatment. However, the WBC level of 1.0 MU/kg group and 2.5 MU/kg group were significantly elevated than those of before treatment (Mann- Whitney U-test, P = 0.001). JVCS, Vol. 1, No. 4, October

5 Uchino et al Table 3. Admission profile s of the study population * Sex Age Breed Disease onset (days before day 0) Criterion Study group 1.0MU/kg (%) 2.5MU/kg (%) Control (%) Male Female <1 month >1 month-<2 months >2 months-<3 months >3 months-<6 months >6 months-<1 year >1 year-<3 years >3 years-<6 years >6 years-<11 years Unknown Mean±S.D Mongrel Shiba Inu Miniature ddachshund Shih tzu Dachshund Other < >1-< >3-< Unknown Mean±S.D Significance Vaccinated Vaccination Status Unvaccinated N.S. Unknown White blood cell counts (mean ± S.D.) N.S., Clinical score prior to therapy (mean±s.d.) Dehydration Appetite Thirst Vitality Diarrhea Salivation Dyspnea Emesis * Average clinical criteria by breed expressed as the mean ± Standard Deviation (SD), N.S.: Not significant, Chi-square test, Tukeykramer test. N.S. N.S. N.S. N.S. N.S. Table 4. Number of dogs show improvement of clinical signs at day 7 post treatment. Treatment Group Excellent Good Fair No effect Poor Total Mann-Whitney U-test Efficacy rate 1.0 MU/kg % N.S. 2.5 MU/kg % Total dogs treated % p= Controls % N.S.: Not significant Chi-square test N.S. p= JVCS, Vol. 1, No. 4, October 2008

6 Efficacy of rfeifn-ω on CPV infection Figure 1 Survival rates of dogs were 46.2% in the control group, 83.5% in the 1.0 MU/kg group and 76.2% in the 2.5 MU/kg group. Significant differences in the survival rates were observed between the dose groups and the control group. *:p<0.005 Discussion Generally, electrolyte infusion and the administration of antiemetics, antibacterials, endotoxin antagonists, recombinant granulocyte-colony stimulating factor and specific hyperimmune plasma can be used for the treatment of CPV depending on the dog s symptoms and disease severity. 5,14 In the current study, even though most dogs in the treatment and control groups were receiving one of the above-mentioned therapies concurrently during the course of the study, the survival rate of the control group (46.2%) was significantly lower than that observed for the treatment groups (83.5 and 76.2% for the 1 and 2.5 MU/kg treatment groups, respectively ). For dogs whose total clinical score prior to therapy was <8, no significant differences in mortality were observed between the treatment and the control groups. By contrast, a significant difference in mortality was observed between treatment and control groups for dogs whose total clinical score prior to therapy was >8, indicating that for serious cases conventional therapies may be insufficient. Another reason for the differences in mortality rates may have been the lack of 24 h intensive care units available in general animal hospitals in Japan in comparison to hospitals with intensive care units in Europe and the United States. The fact that significant differences survival rates resulting from Figure 2 Time course of improvement rates of clinical signs of dogs in the control group, the 1.0 MU/kg group and the 2.5 MU/kg group. The means and standard deviation for the two dose groups and the control group showed significant differences in all clinical signs. rfeifn-ω treatment used concomitantly with additional anti-cpv therapies suggests that rfeifn-ω should be used for the treatment of CPV infections. In addition, significant differences in the efficacy rates (assessed by veterinarians) on day 7 between treatment groups (70.1 and 66.7% for the 1 and 2.5 MU/kg groups, respectively, compared to 38.5% in the control JVCS, Vol. 1, No. 4, October

7 group) were significant. Significant differences were also observed between the treatment and the control groups in the evaluation of clinical symptom improvements over time (days 3-7). These data suggested that despite the use of additional aggressive anti-cpv treatment regimens the use of rfeifn-ω significantly improved the clinical outcome. De Mari et al. demonstrated that the administration of rfeifn-ω at 2.5 MU/kg per day for three days effectively improved the clinical outcome for dogs infected with CPV and indicated that the survival ratio of rfeifn-ω administrated group (93.0%) was significantly higher than that of placebo ones (71.4%). 2 In our study, rfeifn-ω at 1.0 MU/kg or 2.5 MU/kg per day for three days was used. Because we found no significant differences in the survival and efficacy rates between the two treatment groups we confirmed that the lower dose i.e., 1.0 MU/kg was as effective as the 2.5 MU/kg dose. These results suggested rfeifn-ω therapy for the treatment of CPV infections could be made more cost effective if used at lower doses. Mochizuki et al. previously reported that rfeifn-ω did not inhibit the growth of the feline panleukopenia virus in vitro. 10 Since CPV growth inhibition by rfeifn-ω has not been established as a mechanism of action it has been speculated that rfeifn-ω acted on CPV via a mechanism other than the inhibition of viral growth. Although the inhibitory mechanism(s) are not clear at the moment, we speculate that rfeifn-ω exerted its effects by inhibiting cell growth or increasing immunity. The pleiotropic effects of rfeifn-ω i.e., enhanced phagocytic activity, antiviral properties, endotoxin inhibition and the induction of tissue regeneration resulting from the turnover of gastric mucosal cells are believed to play an important role in the recovery from CPV infections. Uchino et al Furthermore, the WBC level before treatment in the control group and rfeifnω groups were presented within the normal range both. Though control group did not show significant elevation, the level of after treatment WBC of rfeifn-ω groups significantly elevated in comparison with before treatment. From this fact, it was suggested that rfeifn-ω was effective possibility for CPV infected dogs with an extremely low WBC count. Conclusion In this study we demonstrated that treatment of CPV infections with rfeifn-ω for three days at a dose of at least 1.0 MU/kg per day (regardless of additional anti-viral therapies) significantly improved CPV infection prognosis, suggesting that rfeifnω can safely and effectively be used in the treatment of CPV infections. Acknowledgements The authors would like to thank the following veterinarians who collaborated in the clinical study: Drs. Teruhisa Okamoto, Takuya Kubo, Toru Takahashi, Toru Tachibana, Youji Nakagawa, Kiyoshi Ookusa, Shigekatsu Motoyoshi, Kazuhiro Kariya, Harutaka Kumai, Takeshi Sato, Hitoshi Ogasawara, Isamu Kanemoto, Hisahiro Sugiura, Tamaki Suzuki, Koichiro Miyano, Haruo Shibata, Hanao Kumasaka, Susumu Nakatsu, Kouji Nishida, Tetsuya Hasegawa, Tetsuya Shimoda, Terumi Miura, Yoshihisa Yamane, Takashi Inoue. Footnotes a Intercat, Toray Industries, Inc., Tokyo, Japan b TEST-LA-[NIKKEN], Nikken Bio-medical laboratory Inc., Kyoto, Japan c StatView 5, Abacus Concepts, Inc., Berkeley, CA, USA 136 JVCS, Vol. 1, No. 4, October 2008

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