Due to the non-availability of a good diagnostic tool, outbreaks however remain undiagnosed in the country.

Transcription

1 1. INTRODUCTION Classical swine fever (CSF), also known as hog cholera, is a highly contagious viral disease of pigs leading to substantial economic losses which can spread in an epizootic form as well as establish enzootic infections in domestic and wild pig populations (Edwards et al., 2000). The presence of CSF virus (CSFV) in pig herds can have a severe economic impact on the meat production industry due to wide-spread animal deaths because of disease as well as trade restrictions on meat exports. This disease is a notifiable to the World organization for animal health or Office International des Epizooties (OIE). At the end of the 20 th century, CSF remains widespread in many parts of the globe. Successful eradication has been achieved in many countries, including North America, Australia and parts of North Europe and many such countries have successfully maintained freedom in the absence of vaccination. The disease continues to be a problem in most of the pig producing states of India and is considered to be the major constraint for the growth of piggery in the North-eastern states of India. Classical swine fever (CSF) or hog cholera, is caused by a Pestivirus known as Classical swine fever virus (CSFV). Pestiviruses represent a group of genetically and antigenically related non-arthropod borne viruses. They are spherical, enveloped, single stranded, positive sense RNA viruses about 40-60nm in diameter with a non-helical and probably an icosahedral nucleocapsid (Horzinek et al., 1973; Bielefeldt-Ohmann, 1990; Heinz et al., 2000). The disease caused by pestiviruses are considered to be one of the most economically important endemic viral diseases of cattle in Europe. The closely related pestivirus of pigs, classical swine fever virus (CSFV), recently has caused losses in Holland that far exceeded the cost of the recent foot and mouth disease virus outbreak in UK (Saatkamp et al., 2000). The term pestivirus was used to group together three antigenically related enveloped RNA viruses: hog cholera virus, now known as classical swine fever virus (CSFV), that infects pigs, bovine viral diahorrea virus (BVDV), that predominantly infects cattle (Horzinek, 1973) and Border disease virus (BDV) of sheep based on the discovery of a close antigenic relationship between CSFV and BVDV (Darbyshire, 1960) and BDV and BVDV (Acland et al., 1972; Gardiner and Barlow, 1972). These viruses were grouped within the family Flaviviridae (Horzinek, 1991). 1

2 Classical swine fever (CSF) is an important disease in pigs caused by a small enveloped RNA virus, the CSF virus (CSFV), which belongs to the genus Pestivirus within the family Flaviviridae. The disease is characterized by a peracute, acute, subacute, chronic, atypical, or in-apparent course (Van Oirschot, 1986). Chronic CSF is a lethal disease with duration of at least 30 days. Chronic form of CSF has three phases of illness: 1) Depression, fever, anorexia; 2) Clinical improvement; 3) Terminal exacerbation of disease. There is often little evidence of CSF in terms of petechial hemorrhages in most organ systems, but a single organ system (lung, gastrointestinal tract, and central nervous system) may be predominantly affected. Acute form of CSF is characterized by short incubation period, severe depression, high fever, anorexia, conjunctivitis, constipation, diarrhea, convulsions, in-coordination and hemorrhages of skin and death within days. The clinical signs may often be uncharacteristic and misleading; therefore, chronic CSF was erroneously called atypical CSF. Chronic CSF has been observed under both natural and experimental conditions. Although the pathogenesis of chronic CSF is poorly understood, it is usually associated with low virulent strain infection or host factors. Low virulent strains generally induce chronic CSF, eventually leading to death or recovery, and may usually be disseminated during a longer period than are high virulent CSFV strains. Outbreak of chronic CSF caused by low virulent strains is more difficult to recognize, and this favors the spread of these strains. These chronically infected pigs are important in the epidemiology of the disease because they act as sources of infection in susceptible pigs. Classical techniques used to diagnose CSFV infections include the detection of viral antigen using fluorescent- or peroxidase-labeled conjugates, either in cryostat sections of tissues or following up to three serial passages in a cell monolayer. But low virulence CSFV is difficult to isolate in PK-15 cell culture and detect in frozen tissues by fluorescent antibody test. Laboratory diagnostic techniques must be used to detect chronic CSFV infection if control or eradication is required. The distribution of CSFV antigen in infected tissues over time is well documented. In pigs acutely infected with highly virulent strains of CSFV, viral antigen was detected in tonsillar cryptic epithelial cells and macrophages, reticular and endothelial cells in spleen and lymph nodes, and smooth muscle cells of blood vessels. By comparison, 2

3 CSF viral nucleic acid and antigen were found predominantly within mononuclear cells and lymphocytes of lymph node and spleen from pigs with chronic CSFV infection. The difference in the distribution of CSFV in pigs with chronic CSFV infection when compared with pigs that are acutely infected may be related to different immune responses. Pigs with acute CSF mount neutralizing antibody response to the virus, whereas the neutralizing antibody response can be absent, impaired, delayed, or only transiently detectable in pigs with chronic CSF. Differences in the distribution of CSFV could also be due to differences among strains of virus, in the age of the pigs, in the duration of infection, or in the susceptibility of the breed. Thymic atrophy and lymphoid depletion in lymphoid tissues indicate impaired immunity in pigs with chronic CSF. Infection of macrophages by CSFV could adversely affect the host defense mechanisms. BVDV, which also belongs to the genus Pestivirus, has been reported to modulate functions of immune cells after infection in vitro, with decreased phagocytic activity of infected alveolar macrophages. Secondary bacterial infections are commonly reported in pigs with chronic CSF. Clinical diagnosis of chronic CSFV infection is difficult to make because of the wide variability of clinical signs and gross lesions. The histopathological changes in the lymphoid tissues of pigs with chronic CSFV infection are also nonspecific, and viral inclusions are not seen. The clinical picture of CSF is not generally characterised by febrile disease with typical clinical signs and high mortality (Dahle and Liess, 1992). CSF can occur in four forms namely, percute, acute, subacute and chronic. In percute form, there is high morbidity and death within five days of infection. In acute form, the disease terminates in the animals death in days post infection while sub-acute form, results in death within a month and chronic forms the duration of the disease is more than a month with little or no mortality. The disease was first reported in Ohio, USA in The disease caused severe havoc in many parts of the globe, but currently present in parts of Europe, South America, and Asian sub-continent. The disease has been eradicated in Australia, Canada, New Zealand, USA, and some member states of EU after implementation of strict control measure and regulatory policies. The first report of CSF in India from Aligarh dates back to Subsequently, the disease was reported from several parts of the country. 3

4 Due to the non-availability of a good diagnostic tool, outbreaks however remain undiagnosed in the country. The CSFV structurally and antigenically related to the other two members of the Pestivirus genus, i.e. Bovine Viral Diarrhoea Virus (BVDV) of cattle and Border Disease Virus (BDV) of sheep. Pigs are susceptible to BVDV and BDV infections and natural infection of BVDV in pigs may resemble signs of CSFV. Antibodies induced by infection of animals with one group of virus cross-react with other members of the genus pestivirus. Presence of BVDV infection in apparently normal swine herd is known to interfere in specific sero-diagnosis of CSFV. The traditional agar gel precipitation test (AGPT) or polyclonal antibody based ELISA used for CSF diagnosis, fail to differentiate CSFV from BVDV or other Pestiviruses. In India, a good diagnostic reagent for detection of CSFV specific antibodies is currently not available. The typical course of the disease may not be observed in field cases, due to constant evolutionary change in the viral genome for survival in nature. This may be due to the selection pressure acting on the virus, such as vaccination. Such changes in the pathoepidemiology of CSFV have been described in many parts of the world. Changchun et al. (2001) have described that in China due to mandatory vaccination program, largescale outbreaks are rarely seen. Sporadic epizootics continue to occur every year and infection in piglets under 3 months age has been seen more frequently than in young and adult pigs. One of the most important reasons attributed to this is the genetic variation in the prevalent strains of the virus from the subgroup-1 to subgroup-2. Deng et al. (2005) reported that the group 1 comprises most of the historical strains distributed in different regions of the world but in recent years group 2 viruses are responsible for most of the outbreaks. Suradhat et al. (2007) described that in South East Asia, a change in trend to mild, chronic form of the disease with a long duration, atypical clinical signs and relatively low morbidity has often been observed in endemic areas, even in a certain proportion of vaccinated pigs. Chen et al. (2008) also reported switch in virus population in China. Authors found that subgroup 2.2 viruses have become silent types that rarely cause epizootics, in contrast to those of subgroup 2.1 which have become predominant in south-eastern China. Pig husbandry is a popular practice among the people of NE India. CSF is endemically prevalent in this region and several outbreaks were recorded in the recent past including 4

5 some in vaccinated pig herds (Sarma and Sarma, 1998; Rehman et al., 2001; Barman et al., 2003). North Eastern states of India shares a long international porous boundary with five neighbouring countries, which poses threats to trans-boundary transmission of infection. There is very less information on the genetic characterization of CSF viruses from North Eastern states of India. It is therefore necessary to generate a comprehensive data on the molecular characterization of the isolates prevailing in all the north-eastern states of India The envelop glycoprotein (E2 and E rns )of CSFV, being the most immunogenic outer membrane protein, has been expressed using different plasmid vectors and used in ELISA for specific diagnosis or immunoprophylaxis of CSFV by many workers (Bouma et al., 1999, Van Rijn et al., 1999, Clavijo et al., 2001). Expression of envelop glycoprotein gene of CSFV has been done successfully both in prokaryotic and eukaryotic expression systems for different purposes like ELISA development and as a subunit vaccine. It has been reported that glycosylation of E2 protein is not essential for binding of polyclonal antisera or monoclonal antibodies (Lin et al., 2000). So the present study is aimed at the following objectives 1. To develop a battery of PCR based diagnostic tools for quick and confirmatory detection of CSFV. 2. To carry out molecular epidemiological analysis of field isolates of CSFV. 3. To assess the various genogroups of CSFV circulating in India at diverse geographical location. 4. To express of viral glycoproteins of CSFV in prokaryotic expression system. 5