Isolation and characterization of canine distemper virus from clinical domestic dogs in Addis Ababa pet clinics, Ethiopia

Background: Canine Distemper is infectious, highly contagious viral disease of canine and feline species with worldwide occurrence, having characteristics of systemic signs with frequent nervous dysfunction. However, the previous studies have underreported canine distemper status in Ethiopia together with its strain makes this study mandatory. Therefore, the principal aim of the present study was to find out the occurrence of CD from clinical dogs in association with other cases in Addis Ababa pet clinics. Results: Out of the 150 dogs that are brought to different Addis Ababa pet clinics, 70 cases (46.66%) were identified to have signs suggestive of CDV infection. Of all the 70 suspected cases of CD, ocular and nasal discharge swab samples were collected and screened by FASTest DISTEMPER Strip kit test for the presence of canine distemper virus. Of the screened samples, 16 (22.86%) were positive for canine distemper. Samples that were positive for FASTest DISTEMPER Strip kit test were further confirmed using cell culture in Vero cell as gold standard technique. Out of the 16 screened positive samples inoculated in Vero cells, 12 specimen (75%) showed Cytopathic effects (CPE) in the form of Syncytia formation within 24 and 48 hours of inoculation while the rest 4 negative samples were passaged in Vero cell three times before declaring negative. Uninoculated negative controls included in each run did not show any effects. Conclusion: The present study revealed the existence of CD in the study area, showing 16 positive domestic dogs out of 70 cases do not received periodic immunization according to CD strains. Besides this, CD is chief disease of local domestic dogs relative to exotic breed. Therefore isolation of clinical domestic dogs from healthy ones, good management practice, public awareness creation about the disease, routine immunization of animals according to circulating strains of CD is mandatory. Key word : CDV, Cytopathic Samples collected from dogs with clinical signs suggestive of canine distemper were tested by sensitive, reliable and specific immunochromatographic assay, FASTest DISTEMPER Strip, designed to detect canine distemper virus antigen in nasal and ocular discharge. This test is a rapid immunochromatographic screening test for the detection of canine distemper virus antigen in nasal and ocular discharge. The method employs a unique combination of a specific antibody binding protein, which is conjugated on dye particles, and a second monoclonal antibody which is immobilized to the solid phase membrane. As the test samples flows laterally across the membrane, the specific binding antibody protein –dye conjugate bind to canine distemper antigens in the sample. Then, if the sample contains any antigen to canine distemper, the complex binds to the antibody on the solid phase in the test zone producing a pink/purple band. In the absence of canine distemper antigen, there is no line in the test zone. The liquid continues to migrate along the membrane and produces a pink /purple band in the control zone demonstrating that the reagents are functioning properly and that the next test has been carried out correct. The test components includes specimen tubes, dipsticks distemper tests in foil pouches, sample collection swabs, dropper bottle A containing 5.0 ml buffer diluents


Background
Canine distemper (CD) is a highly contagious infectious disease of canine and feline species caused by canine distemper virus (CDV) [6]. Canine distemper is a worldwide occurring infectious disease, caused by a morbillivirus of the family paramyxoviridae [10].
Besides canine distemper virus (CDV), the genus morbillivirus comprises measle virus, rinderpest virus, dolphin morbillivirus, pest-despetits ruminants virus, porpoise morbillivirus and phosine distemper virus [33]. Canine distemper virus is an enveloped, single stranded, negative-sense RNA virus [32], which contains six structural proteins: two membrane glycoproteins, the fusion(F) and hemagglutinin(H), the envelopeassociated matrix (M) protein, the phosphoprotein(P), the large polymerase(L) and the nucleocapsid (N) protein [4]. Additional accessory genes, the C and V proteins are found mostly as extra transcriptional units, with in the P gene. The lipid envelope, surrounding the virion contains the two surface glycoproteins F and H, which mediate viral entry and exit from the host cell. Furthermore, the helical neoclocapsid core, containing the N, P, and L protein, initiates intracellular replication and is located within the envelope. The viral M protein connects the surface glycoproteins and nucleocapsid during viral maturation [25].
Host range of canine distemper virus encompasses all species of the families canidae (dog, dingo, fox, jackal, wolf), procyonidae (raccon, coatimundi, panda), mustelidae (weasel, ferret, fishers, mink, badger, marten, otter), the large members of the family felidae (lions, leopards, cheetahs, tigers) and the collared peccary (tayassu tajacu). At least seven distinct lineages of canine distemper are recognized worldwide, based on the sequence analysis of the H genes: Asia-1, Asia-2, American-1, American-2, Arctic-like, European wild life, and Europe. Additional lineage probably will be identified in the future [29]. Genetic analysis of strains causing outbreak shows that CDV does not become more virulent and spread to new host species in a region, but the same strain circulates among susceptible animals of several host species in a given geographic area [18].
CD is highly infectious and frequently lethal disease in dogs and has a high mortality rate after rabies. The disease is transmitted through aerosol and the virus has high affinity for lymphocytes and macrophages. The duration and severity of the disease depends mainly on the ability of the infected animal to quickly mount an immune response to CDV. If the serum antibody titer high level within 8-9 days of infection, the virus disappear from the lymphatic and the other tissues and the infection remains subclinical or mild. However, if the immune response is weak or delayed, the virus disseminates to many tissues causing an acute or chronic disease with high mortality [17]. Immunologically naïve populations may experience high death rates. The mortality rates due to CDV infection vary among susceptible species [3] and could be as high as 100% in ferrets [40]. Domestic dogs mortality rates will largely depends on the immune status of the animal ranging up to 50% [11]. Outbreak in Africa wild dogs have led to mortality rate up to 95% [39].
Canine distemper virus is spread by the respiratory route with initial viral replication in respiratory epithelium and alveolar macrophages [21]. The initial infection is in epithelial cells and lymphoid tissue in the nasopharynx [20]. After multiplication in regional lymph nodes, the virus enters the blood stream, where it circulates with in infected B and T-cells.
Primary viremia is synchronous with the first bout of fever and virus that is spread to lymphoid tissue throughout the body, circulating gut-associated lymphoid tissues, and fixed tissue macrophages such as kuffer cells in the liver. Virions formed in these sites are carried by blood mononuclear cells during second viremia that coincides with the second peak of fever. Epithelium cells do not posses CD150 (SLAM) and the receptor that facilitates virus entry in to epithelial cells is yet to be defined [29].
Canine distemper virus may be shed in virtually all body secretions and excretions depending on the stage of infection. Transmission most commonly occurs through inhalation of air borne virus or direct contact between susceptible and actively infected dogs [3]. Fomite or environmental transmission of canine distemper virus is also possible, but the virus does not remain infectious for more than hours to a few days depending on the ambient temperature and other conditions. Fomites and environmental contamination is therefore of less importance for disease transmission than for a hardier virus such as canine parvovirus. Because the virus does not persist long in the environment, mildly affected and recovering animals plays an important role in maintaining transmission cycles in shelters [30].
In dogs, canine distemper virus infection may result in subclinical infection or clinical disease. It is estimated that 75% of the infections occur as subclinical infections. The clinical disease has been characterized by systemic signs (dermatological, respiratory and gastrointestinal) with frequent nervous disfunction [18]. A transient fever usually occurs 3-6 days after infection and there may be a leucopenia (especially lymphopenia) at this time but these signs may go unnoticed. The fever subsides for several days before a second fever occurs, which lasts < 1 weeks. This may be accompanied by serous nasal discharge, mucoprulent ocular discharge, and anorexia. Hyperkeratosis of foot pads (hard pad disease) and epithelium of the nasal plane may be seen [1]. Canine distemper virus affects both white and gray matter in the central nervous system. Thus, various neurological signs may be observed including behavioral changes, seizures, cerebellar and vestibular signs, visual deficits, paresis, paralysis, limb weakness, tremors, and myoclonus ( is a gray matter sign characterized as a rhythmic jerking of single muscle or muscle groups) [2]. Seizure and myoclonus are typically gray matter signs, while visual deficits and different motor impairment are mainly signs of white matter dysfunction [18]. Signs of leptomeningitis, such as cervical rigidity and generalized hyperesthesia, may also occur [24].
In suspected cases of distemper, a complete blood count and thoracic radiographs used to access leukocyte responses and pneumonia respectively. In dogs presenting with neurologic disease suspected to be due to CDV, routine cerebrospinal fluid (CSF) analysis carried out to distinguish CDV infection from other diseases. The presence of CDV-specific antibody in CSF can confirm the diagnosis but requires special laboratory [36]. Routine diagnosis of canine distemper virus by immunoflourescence (IF) is applied to various specimens, including conjuctival, nasal, and vaginal smears, using polyclonal or monoclonal antibodies. This tests is not sensitive and can detect canine distemper virus antigens only within 3 weeks after infection, when the virus is still present in the epithelial cells [4]. Serological methods such as ELISA and seroneutralization (SN) assays, have a little diagnostic value because high titers of antibodies to canine distemper virus may be the result of previous vaccination or subclinical or clinical infection [23]. Definitive diagnosis of canine distemper by virus isolation on canine cells is fastidious and time consuming, taking several days to weeks, notably when applied to clinical specimen [14,37]. During life, clinical diagnosis can be confirmed by finding typical cytoplasmic and intranuclear inclusion bodies in the smears of cells of the respiratory epithelium and peripheral blood. Unfortunately, these inclusion are not present in all cases, hence their absence does not preclude the diagnosis of distemper [21]. Distemper inclusions in canine erythrocytes are irregular to round to ring shaped and stain magenta with Romanowsky and Diff-Quik stains (the inclusions may stain with other rapid blood stains). Distemper inclusions are transient [5]. These inclusions appear as homogeneous, red to purplish, red or pale blue, pleomorphic but homogeneous cytoplasmic inclusions; they are found in neutrophils, monocytes, lymphocytes and erythrocytes. They probably occur in the early viremic stage and before clinical illness [38]. RT-PCR has been applied successfully to diagnosis of canine distemper [34,37].
Control of canine distemper virus infection is based on adequate diagnosis, quarantine, sanitation, and vaccination with attenuated canine distemper virus vaccines. The virus is very fragile, and susceptible to standard disinfectants. Thorough disinfection of premises, however, can be very challenging. For treatment, hyper immune serum or immune globulin can be used prophylactically immediately after exposure. Antibiotic therapy generally has a beneficial effect by lessening the effect of secondary opportunistic bacterial infections [29]. Canine distemper virus is monotypic virus as defined by polyclonal antisera and a single exposure to the virus normally confers long-lasting immunity in dogs. In general, the introduction of live attenuated canine distemper virus vaccines in the 1950s and their extensive use drastically reduced the incidence of the canine distemper in dogs [15].
Therefore, previous underreporting of canine distemper in Ethiopia together with its strain makes this study mandatory. The principal objectives of the present study were to assess the occurrence of canine distemper relative to other contagious infectious and non infectious disease of dogs in Addis Ababa, and to isolate and characterize CDV from clinical cases of canine distemper.

Study area
The study was conducted from November 2014 to April 2015 in Addis Ababa pet clinics (Akaki kality, Sholla, Kera) in most clinical dogs which were suggestive to canine distemper. Addis Ababa is located at the elevation of 2000-3000 m a.s.i. The mean annual rain fall is 1800 mm with bimodal pattern. There are alternating dry and rainy seasons in the area. The long rainy season extending from June to September contributes about 84% of the annual rainfall while dry season last from October to February. The short rainy season lasts from March to May. The mean annual minimum and maximum temperature are 14 0 C and 21 0 C respectively with an overall average of 17 0 C. The mean relative humidity is 61.37 [9].

Study animal
The study was carried out on local, and exotic breeds of dogs that brought to Addis Ababa small animal clinics (Akaki-Kality, Sholla, and Kera).

FASTest DISTEMPER Strip Kit test
Samples collected from dogs with clinical signs suggestive of canine distemper were tested by sensitive, reliable and specific immunochromatographic assay, FASTest DISTEMPER Strip, designed to detect canine distemper virus antigen in nasal and ocular discharge. This test is a rapid immunochromatographic screening test for the detection of canine distemper virus antigen in nasal and ocular discharge. The method employs a unique combination of a specific antibody binding protein, which is conjugated on dye particles, and a second monoclonal antibody which is immobilized to the solid phase membrane. As the test samples flows laterally across the membrane, the specific binding antibody protein -dye conjugate bind to canine distemper antigens in the sample. Then, if the sample contains any antigen to canine distemper, the complex binds to the antibody on the solid phase in the test zone producing a pink/purple band. In the absence of canine distemper antigen, there is no line in the test zone. The liquid continues to migrate along the membrane and produces a pink /purple band in the control zone demonstrating that the reagents are functioning properly and that the next test has been carried out correct.
The test components includes specimen tubes, dipsticks distemper tests in foil pouches, sample collection swabs, dropper bottle A containing 5.0 ml buffer diluents [13].

Virus isolation
Each swab specimen positive with screening immunochromatographic assay in the field was individually inoculated on sub cultured monolayer Vero cells after centrifugation of specimen at 4000 rpm for 20 minutes in tissue culture flask. The flask incubated at 37 0 C for 60 minutes to allow the virus to absorb on to the cell culture in humidified incubator and examined daily for Cytopathic Effect (CPE) which is observed as gaint multinucleated syncytium formation and detachment of cells. Each sample was inoculated in Vero cells and uninoculated flasks used as negative controls were included in each run. Each sample in the virus isolation passaged in cell culture three times before declaring specimen negative.

Data management and analysis
The raw data was collected and entered in a computer Microsoft-excel spread sheet then was analyzed by using STATA, version 11 software. Chi-square (x 2 ) test was used to determine the association of different risk factors and p-value of less than 0.05 was taken as statistically significant difference. These identified cases other than CD show the relative importance of canine distemper while comparing it with other types of disease during study time (Table-1

Discussion
Canine distemper is endemic in Africa as in other parts of the world. Under reporting is a characteristic of the almost every infectious disease in most developing countries [8].
Several approaches have been used for the diagnosis of canine distemper [35]. In this study, there was no statistical significant difference among age groups of domestic dogs except numerical difference. Thus, puppies (less than 1year) as well as adult (greater than 1year) were equally susceptible to CDV infection. This finding is different from the other reports where the dogs of 3-6 months of age more susceptible [22].
According to Latha and Srinikasan [26], dogs of 1-5 years of age, which means in our study Adult, were susceptible to CDV infection because of the interference of maternal antibodies during primary immunization or poor storage and handling of the vaccines or immune status of the animal, which would result in quick depletion of vaccine antibodies and the lack of the routine vaccination of dogs. The equal susceptibility of the puppy and Adult age groups to CD infection in this study could be because of lack of routine vaccination of dogs. For thus, there were no any maternal derived distemper antibodies that passed to puppies to develop immunity makes equal susceptibility of both age groups to CD. Improved vaccination has reduced the frequency and magnitude of canine distemper outbreaks [7] and Patronek et al. [31], demonstrate that the lack of vaccination against distemper was associated with several hundred-fold in the risk of the disease.
However, 16 (22.85) CDV positive dogs in our study had been unvaccinated against CDV rather than rabies.
Both sexes were equally affected (22:26%) by CDV infection in this study. This is the synonymous result to Headley and Graca [19] in an epidemiological study of 250 cases in Brazil and Gou et al. [16] in Texas, USA. The percentage of the CDV infected cases was high in local breeds of domestic dogs according to this study while according to [35], Brachycephalic dogs have been reported to have a lower prevalence of disease, sequelae and mortality compared with Dolichocephalic breeds. This may be due to the variation of the immune status of both breeds (local and exotic) of dogs and also variability of handling local and exotic breeds of dogs in Ethiopia, management difference among two breeds.
The gold standard for the diagnosis of virus infection has for a long time been virus isolation in cell culture [18]. Virus isolation is important not only to confirm a diagnosis and provide material for direct sequence analysis but also for investigation of the pathogenesis in animal experiments and vaccine improvements [27].  Ethical approval and consent to participate The study protocol was approved by viral serology department of National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta. The purpose of study was explained to clients and verbal agreement was obtained before commencing to the study.