Slov Vet Res 2008; 45 (2): 73-8 UDC 636.7.09:578.822:616.34-002:616-091 Case Report RETROSPECTIVE STuDY OF CANINE PARVOVIROSIS IN SLOVENIA Mitja Gombač1*, Tanja Švara1, Marko Tadic2, Milan Pogačnik1 1 Institute of Pathology, Forensic and Administrative Veterinary Medicine, Veterinary Faculty, Gerbiceva 60, 1000 Ljubljana, Slovenija; 2 Veterinary Faculty, Heinzelova 55, 10000 Zagreb, Croatia 'Corresponding author, E-mail: mitja.gombac@vf.uni-lj.si Summary: The aim of this retrospective study, based on the data from the archive of the Institute of Pathology, Forensic and Administrative Veterinary Medicine of Veterinary Faculty in Ljubljana, was to estimate the correlation between epide-miological parameters such as gender, age and breed of dogs that died of canine parvovirosis, and the months of a year in which they died. 84 dogs, aged 1 month to 4 years, of both genders and 27 different breeds, which died of canine parvovirosis from January 1987 to January 2005, were included in the study. Results of this descriptive study suggest that the age of dogs and the time of year when death occurred were correlated with canine parvovirosis while there was no correlation between gender or breed and incidence of death due to canine parvovirosis. Key words: veterinary medicine; pathology; canine parvovirosis; parvoviral enteritis; dogs Introduction Canine parvovirosis is a contagious viral disease of dogs. First cases of canine parvovirosis had been almost simultaneously detected for the first time in the USA, Australia and Europe in 1978 (1, 2, 3). In the following three years the disease had spread to all continents (4, 5, 6). The first case of canine parvovirosis in Slovenia was diagnosed in July 1980 (7). The disease is caused by a virus belonging to the group of parvoviruses, which also includes feline panleukopenia virus (FPV; 6). It is presumed that outbreak of parvovirosis in dogs was a consequence of FPV mutation (6). Canine parvovirosis is caused by two dog specific parvoviruses - canine parvovirus type 1 (CPV1) and canine parvovirus type 2 (CPV2). In contrast to low pathogenicity of CPV1, which rarely causes canine parvovirosis, CPV2 is highly pathogenic (6, 8, 9). Several species of wild canids, e.g. coyotes, raccoons, foxes and wolves, are also susceptible to canine parvovirus infection (6). The most typical form of the canine parvovirosis is exhibited in the form of enteritis, but the disease Received: 5 March 2008 Accepted for publication: 26 May 2008 can also manifest as parvoviral myocarditis or as a mixed form (6). All three forms have often fatal outcome (6). Young dogs from six weeks to one year of age are the most susceptible group for canine par-vovirosis, afterwards the morbidity decreases. Puppies younger than six weeks are protected by maternal immunity (10). Materials in methods The necropsy reports from the archive of the Institute of Pathology, Forensic and Administrative Veterinary Medicine of Veterinary Faculty in Ljubljana were reviewed in order to find out causes of death in dogs dissected in the last 18 years, from January 1987 to January 2005. Eighty four dogs in which parvovirosis was diagnosed by histopatho-logical analysis were included in this study. In 18 dogs, parvovirosis was also confirmed by immuno-cytochemistry. Immunohistochemical staining Polyclonal rabbit antibody against canine par-vovirus was used for immunolabelling. Serum was 74 M. Gombač, T. Švara, M. Tadic, M. Pogačnik raised in rabbits which were immunised with the suspension of canine parvovirus, isolat LJ-80, which was replicated on the cell culture of cell line FER (Feline Embrional Kidney), ECACC, No 90031401. Hemagglutination titer of viral suspension was 1: 1024 (11). Four micrometers thick, deparafinised tissue sections ofjejunum, ileum, liver, kidney, spleen, thymus, lymph nodes and lung were incubated in citrate buffer (pH= 6.0) in the microwave oven for 15 minutes to expose the antigens, cooled to the room temperature and incubated in 3% hydrogen peroxide for 1 hour to inhibite the endogenous peroxidase. Polyclonal rabbit antibodies against canine parvovirus, were diluted 1: 400 in 5% bovine serum albumin in phosphate buffer saline (PBS). Tissue sections were incubated with primary antibodies at room temperature for 30 minutes, followed by washing in PBS for 5 minutes, incubated with biotinylated mixture of goat anti-rabbit and goat anti-mouse secondary antibodies (LSAB®2 HRP visualization system, DAKO) for 20 minutes, rewashed with PBS, incubated in strepta-vidin conjugated with peroxidase (DAKO) and finally incubated in 1% 3-diaminobenzidine tetrahydro-chloride, containing 0.03% hydrogen peroxide, for 7 minutes. Sections were countrastained with haema-toxylin, dehydrated and mounted with resin (11). Statistical analyses Data about gender, age and breed of dogs and month of their death was determined from the records about anamnesis. For determination of effects of chosen epidemio-logical parameters on canine parvovirosis, dogs were divided into two groups by gender: 70 males and 14 females; in three groups by age: 50 puppies up to 6 months of age, 19 dogs from 6 months to 1 year and 5 dogs older than 1 year; into four groups by breed: 14 German Shepherds, 9 Rottweilers, 6 Labrador Retrievers and 20 mixed breeds. Regarding the month of the death, dogs were divided into 12 groups, and by the year of death into 18 groups. Relative ratio of deaths caused by parvovirosis from the total number of dissected dogs (2486) was calculated individually for each of the above groups. The differences between groups for each epidemio-logical parameter were determined using standard statistical methods: correlation and regression analysis, t-test and chi test, with p < 0.05, which were considered as statistically significant. Results Anamnesis and diagnosis of canine parvovirosis In the last 18 years, 2486 dog carcasses underwent pathological examination at the Institute of Pathology, Forensic and Administrative Veterinary Medicine of Veterinary Faculty in Ljubljana in the last 18 years. Typical macroscopic lesions in the small intestine (mostly hemorrhagic enteritis), which were diagnosed as parvoviral enteritis using histopathological examination, were found in 84 dogs (3.4 % of all examined dogs). In 55 dogs that died due to parvovirus infection, sudden onset with vomiting, bloody diarrhea, depression and loss of appetite were noticed by their owners or veterinarians. All these dogs died in one or at most two days after the onset, or were euthanized due to severe illness. Five dogs died suddenly, without previous clinical signs. For 24 dogs there was no anamnesis concerning the clinical status prior to death. Ten dogs were vaccinated against canine parvovirus, three dogs were not vaccinated while for 71 dogs there were no data concerning the vaccination. Epidemiological analysis Sex of dogs Seventy dogs (83.3 %) that died due to canine parvovirosis were males and 14 (16.7 %) were females (Diagram 1). Statistical analysis revealed significant difference between sexes in dogs that died due to parvovirosis (P < 0.01). 80 « 70 « 60 ^ 50 2 40 = 20--■ C 10--1 0 -I-^-1- male female sex Diagram 1: Sex ratio of dogs that died due to canine parvovirosis The age of dogs Fifty dogs (67.6 %) that died of canine parvovirosis were younger than six months, 19 dogs (25.7 %) Retrospective study of canine parvovirosis in Slovenia 75 were aged from six months to one year, and only five dogs (6.8 %) were older than one year. For 10 dogs included in our study there were no data about their age (Diagram 2). Comparison between age groups and the death due to canine parvovirosis revealed statistical significance between groups (P < 0.01). 60 50 D1 -§ 40 30 20 0.05). The year of the death Twenty dogs died due to canine parvovirosis in 1991 (10.3 % of all dogs). In 1987 and from 2001 to 2003 there were no deaths due to canine parvoviro-sis. The percentage of deaths in other years ranged from 1.29 to 8.4 %. Diagram 3: Number of dogs that died due to canine parvo-virosis in the last 18 years Diagram 4: Number of dogs that died due to canine parvo-virosis by the month in which they died. Discussion In the last 18 years, 2486 dogs were dissected at the Institute of Pathology, Forensic and Administrative Veterinary Medicine of Veterinary Faculty in Ljubljana and among them, 84 died or were euthanized due to canine parvovirosis. In all cases, canine parvovirosis was suspected due to clinical signs and/or characteristic intestinal lesions, and was confirmed by histopathological examination of the intestine and in 18 dogs also with immuno-histochemical reaction using polyclonal antibodies against canine parvovirus (11). Several authors described microscopic changes in small intestine, i.e. necrosis of crypt's epithelia with crypt's dilatation, atrophy of intestinal villi, necrotic enterocytes with enlarged nucleus and focal mucosal collapse, as typical for parvovirosis (6, 7, 18). Similar lesions were found also in the small intestines of dogs included in our retrospective study. Ruiz Romero et al. (19) also found necrosis of crypts and atrophy of villi in small intestine of dogs and confirmed parvoviro-sis by immunohistochemical staining (19). Since 1980, when the first case of canine parvo-virosis has been diagnosed and described in Slov- 76 M. Gombač, T. Švara, M. Tadic, M. Pogačnik enia (7), deaths due to canine parvovirosis were diagnosed almost every year. The number of deaths increased in 1990, 1991 and in 1992, while a slow fall was noticed afterwards until 2001, when no case of death due to canine parvovirosis was diagnosed. The first new outbreak occurred in November 2004. There is no data in the literature concerning the disease frequency in the world during the last decades. The most likely reason for decreased number of deaths due to canine parvovirosis is greater owner's awareness and regular preventive vaccination against canine parvovirus. The new outbreak of canine parvovirosis could be related to the new canine parvovirus mutant that has been just found in Italy (12). This hypothesis is supported by the facts that this was the first outbreak of disease after three years and, that all three puppies were vaccinated with standard vaccine against canine parvo-virus. The second possible explanation for the new outbreak is inappropriate time of vaccination. It is known that there is a critical period from two to four weeks, when vaccination is fairly unsuccessful. In this period, the maternal antibody titer is so high, that a puppy is unsusceptible to preventive vaccination, but at the same time too low to protect the animal against infection. For that reason the vaccination in the above cited period is ineffective. Therefore it is suggested that the assessment of the titer of antibodies against canine parvovirosis should be determined before preventive vaccination (13). 83.3 % of dogs, which died due to canine parvovirosis, were males, and 16.7 % were females. The difference between genders in the death ratio due to canine parvovirosis is statistically significant. In spite of this result we cannot conclude that males are more susceptible for canine parvovirus infection. The major susceptibility of male dogs will be confirmed, if there will be an equal number of male and female dogs in the Slovenian dogs' population. Unfortunately, we don't have the data about the exact number of male and female dogs in Slovenia. Other authors (4) reported that the canine parvovi-rus infection was more frequent in male dogs, but statistical analyses could not confirm significant differences between sexes and morbidity (4, 5). The highest percentage of deaths, 67.7 %, was noticed in dogs younger than six months, and the lowest, 6.7 %, in dogs older that one year. From six months to one year 25.7 % of dogs died due to canine parvovirosis. Comparison between the age groups and death due to canine parvovirosis showed statis- tical significance. We have demonstrated that age affects the death due to canine parvovirosis. Very high percentage of deaths, 80 %, was found in puppies younger than six months by Pospischill and Yama-ho (14) and 74.5 % morbidity was reported by Ernst et al. (4) at the same age. The most likely reason that dogs younger than six months are at the greatest risk is very high affinity of canine parvovirus for tissues with high mitotic activity (10). Puppy's immune system, which depends on the maternal antibodies, also has an important role in the fight against canine parvovirus (15). Maternal immunity which lasts two to three months makes morbidity of puppies at this age very low. Singular cases of canine parvovirosis in puppies are probably a consequence of subclini-cal form of canine parvovirosis during the mother's pregnancy (4). The mitotic activity in the cells decreases with age, therefore the percentage of older dogs that die due to canine parvovirosis is very low. Deaths in older dogs are mostly related to the high invasions with intestinal parasites, bacterial and viral infections that all cause immunosuppresion, damage of intestinal mucosa and consequently provoke increased mitotic activity of intestinal cells (4). The statistically significant difference between the age of dogs and morbidity due to canine parvoviro-sis was reported in the study of Ernst et al. (4). 23.8 % of Slovenian dogs, which died due to canine parvovirosis, were mixed breeds, followed by German Shepherds, Rottweilers, Labrador Retrievers and Poodles. Using statistical analysis we found that the deference between groups of different breeds and the number of deaths due canine par-vovirosis was not statistically significant. We have demonstrated that breed does not have influence on death due to canine parvovirosis. Several authors wrote about greater susceptibility of some pure breeds, i. e. Setters (4), Pointers (4), Doberman Pinschers (4, 16, 17) and Rottweilers (4, 16). Pospischill and Yamaho (14) and Houston et al. (17) established that German Shepherds are the most susceptible breed for canine parvovirosis. According to statistical data from the year 1996, we noticed that German Shepherds and Rottweilers, followed by Labrador Retrievers and Poodles were the most numerous amongst pure breed's offspring in Slovenian dog's population. In our study we found out that these are breeds with the highest percentage of death due to canine parvovirosis. Most of Slovenian dogs died due to canine parvovirosis from August to January. The highest percentage of deaths was reported in August, which is one Retrospective study of canine parvovirosis in Slovenia 77 of the warmest months in Slovenia, followed by September. High daily temperatures induce loss of appetite, decrease function of the immune system and increase susceptibility to bacterial, parasitic and viral diseases. In the literature we found out that the highest titer of the antibodies against canine parvovirosis was determined during summer months (4). Pospischill and Yamaho (14) reported the highest incidence of canine parvovirosis in November, December and January and the lowest number in June, July and September. Increased number of deaths during winter could be related to low temperatures which also suppress immune system. Using statistical analysis we demonstrated that the difference between months and the number of deaths due to canine parvovirosis was statistically significant. We can conclude that very warm and very cold months have influence on the death due to canine parvovi-rosis Ernst et al. (4, 5) also demonstrated that the difference between months and morbidity due to canine parvovirosis is statistically significant. References 1. Kelly WR. An enteric disease of dogs resembling feline panleukopaenia. Aust Vet J 1978; 54: 593. 2. Kelly WR, Atwell RB. Diffuse subacute myocarditis of possible viral aetiology: a cause of sudden death in pups. Aust Vet J 1979; 55: 36-7. 3. Osterhaus AD, van Steenis G, de Kreek P. Isolation of a virus closely related to feline panleukopenia virus from dogs with diarrhea. Zentralbl Veterinarmed B 1980; 271: 11-21. 4. Ernst S, Montes S, Martin R. Estudio epidemiologico retrospectivo de los factores de riesgo asociados a la ocurrencia de parvovirosis en una poblacion canina hospitalaria. Arch Med Vet 1988; 20: 38-43. 5. Ernst S, Cid L, Martin R, Thinbaut J. Distribution temporal de la parvovirosis clinica en una poblacion canina hospitalaria de Valdivia, Chile (1981 - 1990). Arch Med Vet 1992; 24: 157-61. 6. Barker IK, Van Dreumel AA, Palmer N. The alimentary system. In: Jubb KV, Keneddy PC, Palmer N, eds. Pa- thology of domestic animals. Vol.2. San Diego: Academic Press, 1993: 475-84. 7. Šenk L, Pogačnik M. Virusni enteritis psov. Zb Biotehn Fak Univ E Kardelja Vet 1982; 19: 17-26. 8. Carman PS, Povey RC . Pathogenesis of canine par-vovirus-2 in dogs: haemathology, serology and virus recovery. Res Vet Sci 1985; 38: 134-40. 9. Carman PS, Povey RC . Pathogenesis of canine par-vovirus-2 in dogs: histopathology and antigen identification in tissues. Res Vet Sci 1985; 38: 141-50. 10. Sherding RH. Diseases of the small bowel. In: Ettinger SJ, ed. Textbook of veterinary internal medicine. 3rd ed. Philadelphia: WB Saunders Company, 1989: 1894-915. 11. Švara T, Juntes P, Pogačnik M, Gombač M, Hostnik P. Imunohistokemično dokazovanje virusnega antigena v organih psov pri parvovirozi. Slov Vet Res 2003; 40 (2): 83-92. 12. Martella V, Cavalli A, Pratelli A, et al. A canine parvovirus mutant is spreading in Italy. J Clin Microbiol 2004; 42: 1333-6. 13. Twedt DC, Magne ML. Diseases of the stomach. In: Ettinger SJ, ed. Textbook of veterinary internal medicine. 3rd ed. Philadelphia: WB Saunders Company, 1989: 1351-3. 14. Pospischil A, Yamaho H. Parvovirus enteritis in dogs based on autopsy statistics 1978 - 1985. Tierarztl Prax 1987; 15: 67-71. 15. Meunier PC, Cooper BJ, Appel MJG, Slauson DO. Pathogenesis of canine parvovirus enteritis: importance of viremia. Vet Pathol 1985; 22: 60-71. 16. Glickman LT, Domanski LM, Patronek GJ, Visin-tainer F. Breed-related risk factors for canine parvovirus enteritis. J Am Vet Med Assoc 1985; 187: 589-94. 17. Houston DM, Ribble CS, Head LL. Risk factors associated with parvovirus enteritis in dogs: 283 cases (1982-1991). 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Anamnestične podatke o raztelešenih psih, poginulih od januarja 1987 do januarja 2005, ter podatke o vzrokih njihovega poginov ali usmrtitev smo vzeli iz arhiva Inštituta za patologijo, sodno in upravno veterinarstvo Veterinarske fakultete v Ljubljani. Parvoviroza je bila diagnosticirana s patohistološko preiskavo črevesja, v katerem parvovirusi povzročajo za to bolezen patognomonične spremembe, pri 18 psih pa smo bolezen potrdili tudi z imunohistokemično preiskavo črevesja in nekaterih parenhimskih organov. Od januarja 1987 do januarja 2005 je bila na Inštitutu opravljena raztelesba 2486 psov, od katerih jih je 84 (3,4 %) poginilo zaradi parvoviroze. Največ poginov zaradi parvoviroze je bilo zabeleženih v avgustu (16,7 %) in septembru (11,9 %), v oktobru, decembru in januarju pa je bilo poginov nekoliko manj (10,7 %). Najmanj psov je zaradi parvoviroze poginilo v februarju. 67,6 % psov je bilo ob poginu starih do 6 mesecev, le 6,7 % psov pa je bilo starejših od enega leta. 83,3 % psov je bilo moškega spola, med čistokrvnimi psi pa je bila parvoviroza najpogosteje diagnosticirana pri nemških ovčarjih, rotvajlerjih in labradorcih. Na osnovi statističnih analiz podatkov smo ugotovili, da so starost psov in meseci v letu v povezavi s poginom zaradi parvoviroze, spol in pasma pa ne. Ključne besede: veterinarska medicina, patologija; parvoviroza; parvovirusni enteritis, psi