Introduction
Classical swine fever (CSF) or hog cholera is an important infectious disease of pigs, with considerable economical implications in the swine industry worldwide. It affects domestic pigs, wild boars and feral pigs. The first outbreak of the disease, observed in France in 1822 (Cole et al., Reference Cole, Henley, Dale, Mott, Torrey and Zinober1962), was thought to be caused by a bacterium termed the ‘hog cholera bacillus’. It is now known that the CSF virus (CSFV) is closely related to the bovine viral diarrhea virus (BVDV) and ovine border disease virus (BDV). Infection with CSFV is listed as reportable to the World Organisation for Animal Health (OIE), so every suspected case should be investigated and the OIE should be notified of positive cases (OIE, 2014).
Acute cases of CSF caused by virulent virus can be diagnosed relatively easily, but not the infections caused by less virulent viruses. In some cases, dullness and poor reproductive performance are noticed. Due to a wide range of clinical signs and similarity to other diseases, it has become a challenging task to diagnose the CSF accurately. In spite of a number of efficacious and safe vaccines available to control CSF, the disease is prevalent in Europe, Asia and South America. However, the African continent considered CSF-free except for Madagascar. The disease is endemic to India, where it is prevalent in most of the states. This review is aimed to provide the latest information of all the aspects of CSF including the status of the disease in India, latest diagnostic tools, immunoprophylactic measures and control strategies for the benefit of the swine industry.
Etiological agent
CSFV is classified under the genus Pestivirus within the family Flaviviridae (ICTV, 2012). The capsid of this enveloped virus is hexagonally shaped, 45 nm in diameter and encloses the single-stranded positive sense RNA genome of 12.3 kb. The CSFV genome contains one open reading frame (ORF), which encodes one large polyprotein that is cleaved by proteases to yield mature viral proteins (Moormann et al., Reference Moormann, Schagemann, Dahle, Greiser-Wilke and Leder1990). The ORF is flanked by a 5′ untranslated region (UTR) of almost 400 nt and a 3′-UTR of about 200 nt. The 5′-UTR functions as an internal ribosome entry site (IRES) for translation of the polyprotein of about 3900 amino acids. The order of the gene products is N–C–Erns–E1–E2–P7–NS2.3–NS4a–NS4b–NS5A–NS5B–COOH, with coding regions for the capsid protein and envelopes the glycoprotein Erns–E1–E2 at the N terminus and non-structural proteins at the C terminus. The 5′ and 3′ NTRs of the CSFV are thought to act together in cis to induce apoptosis, and this NTR-mediated apoptosis requires double-stranded RNA and translation shutoff through activation of protein kinase R (Hsu et al., Reference Hsu, Chen, Huang, Wu, Chen, Nadar, Su and Tsai2014). The Erns protein shows RNase activity and is immunosuppressive in nature in vitro. The E2 is the most immunodominant protein and is composed of two independently formed antigenic domains. The NS2.3 is the most conserved among the pestiviruses. Other members under the genus Pestivirus include BVD-1, BVD-2, BDV and a pestivirus isolated from a giraffe. CSFV differs from BVDV and BDV in sequences at the 5′-UTR and the E2 gene (Katz et al., Reference Katz, Ridpath and Bolin1993; Vilcek et al., Reference Vilcek, Herring, Nettleton, Lowings and Paton1994; Van Rijn et al., Reference van Rijn, van Gennip, Leendertse, Bruschke, Paton, Moormann and Van Oirschot1997). CSFV is genetically more stable than BVDV and different strains can be grouped on the basis of differences in the nucleotide sequences of the 5′ NTR, the N terminal of E2 and a region of NS5B. The amino acid similarity between CSFV and BVDV is about 70% with the highest homology between NS3 of the NS2.3 protein and the lowest in the E2 protein (Paton, Reference Paton1995).
There is only one CSFV serotype, but a number of strains of variable virulence and antigenicity can be distinguished with monoclonal antibodies (Wensvoort et al., Reference Wensvoort, Terpstra and De Kluyver1989; Edwards and Sands, Reference Edwards and Sands1990; Edwards et al., Reference Edwards, Moennig and Wensvoor1991). Classification of CSFV strains and isolates currently is mainly based on 190 nt of the E2 envelope glycoprotein gene, but also with 150 nt of the 5′-UTR and 409 nt of the NS5B polymerase gene. On the basis of phylogenetic analysis, CSFV can be divided into three groups with three or four subgroups: 1.1, 1.2, 1.3; 2.1, 2.2 and 2.3; 3.1, 3.2, 3.3 and 3.4. These groups and subgroups show distinct geographical distribution patterns. All of the groups have been found in Asia, but group 3 mainly occurs in Asia, group 2 in the European Union and group 1 in South America, Central America and the Caribbean (Paton et al., Reference Paton, Mcgoldrick, Greiser-Wilke, Parchariyanon, Song, Liou, Stadejek, Lowings, Bjorklund and Belak2000a). CSFV genomes can also be clustered on the basis of their codon pair bias, which correlates with the genotype rather than with the virulence of the isolates (Leifer et al., Reference Leifer, Hoeper, Blome, Beer and Ruggli2011). Most of the highly virulent CSFV strains and the vaccine strains belong to genotype 1. Genotypes 2 and 3 are the moderately virulent strains, and the genetic variability within this strains is comparatively higher than genotype 1 (Uttenthal et al., Reference Uttenthal, Storgaard, Oleksiewicz and de Stricker2003; Rasmussen et al., Reference Rasmussen, Reimann, Uttenthal, Leifer, Depner, Schirrmeier and Beer2010). Both 1.1 and 2.2 subgroups have been reported in India, with the predominance of 1.1 and involvement of both subgroups in outbreaks (Patil et al., Reference Patil, Hemadri, Shankar, Raghavendra, Veeresh, Sindhoora, Chandan, Sreekala, Gajendragad and Prabhudas2010).
Pathology and pathogenesis
Under natural conditions, CSFV enters the host through oral, nasal, conjunctival and genital mucous membranes. The spread of highly virulent CSFV strains in pigs is characterized by lymphatic, viremic and visceral phases. The virus initially infects epithelial cells of the tonsillar crypts, followed by the regional lymph node and the efferent blood capillaries, leading to viremia. Virus proliferation occurs in the spleen, visceral lymph node, lymphoid tissues and bone marrow in acute CSF and persists until the death of the animal. The virus has distinct affinity for vascular endothelium and cells of the immune system. The widespread hemorrhages in acute CSF are due to degeneration and necrosis of endothelial cells, thrombocytopenia and disturbances in fibrinogen synthesis (Pauly et al., Reference Pauly, Konig, Thiel and Saalmuller1998; Gómez-Villamandos et al., Reference Gómez-Villamandos, Ruiz-Villamor, Bautista, Sánchez, Sánchez-Cordón, Salguero and Jover2001).
Moderate or low-virulence CSFV strains can induce persistent infections designated as chronic or late onset swine fever. Initially, the virus disseminates throughout the body at a slower rate with low concentrations of virus in serum and organs. Secondly, the virus is limited to epithelial cells of the tonsils, ileum, salivary glands and kidney. The antigen–antibody complex is deposited in the kidney leading to glomerulonephritis. Finally, the virus disseminates throughout the body with secondary bacterial infections (Cheville et al., Reference Cheville, Mengeling and Zinober1970).
Late onset CSF occurs when low virulent CSFV infects a fetus during the first 40 days of gestation. Such pigs have a lifelong viremia with spread of the virus in epithelium, lymphoid and reticuloendothelial tissues (van Oirschot, Reference van Oirschot1979). The pathogenesis of CSFV is depicted in Fig. 1.
Fig. 1. Pathogenesis of classical swine fever virus.
The multiplication of low-virulence CSFV following postnatal infection is mainly restricted to tonsils and lymph nodes. In pregnant sows, the outcome of the fetal infection depends on the age of the fetus and the virulence of the virus (Radostitis et al., Reference Radostitis, Gay, Hinchcliff and Constable2007). Transplacental infection with both field and vaccine strains of the virus may induce abnormalities such as hypoplasia of lungs, malformation of the pulmonary artery, micrognathia, arthrogryposis, fissures in the renal cortex, multiple septa in the gall bladder and malformations in the brain. Earlier infection leads to more severe abnormalities and may result in abortions, stillbirths, mummification and malformations, whereas infections at 50–70 days leads to birth of persistently viremic piglets that are clinically normal at birth but die after several months. These animals have retarded growth (runts) and shed a large number of virus particles. Infection occurring at a later stage of gestation may lead to development of immunotolerance (van Oirschot, Reference van Oirschot1979; Meyer et al., Reference Meyer, Liess, Frey, Hermanns and Trautwien1981). Immunity develops rapidly in immunocompetent swine that survive acute infections (Susa et al., Reference Susa, Konig, Saalmuller, Reddehase and Thiel1992; Summerfield, Reference Summerfield, Knotig and McCullough1998a, Reference Summerfield, Hofmann and McCulloughb; van Oirschot, Reference van Oirschot, Coetzer and Tustin2004; van Oirschot et al., Reference van Oirschot, Coetzer and Tustin2004; Radostitis et al., Reference Radostitis, Gay, Hinchcliff and Constable2007).
In peracute cases, no gross changes are found. Hemorrhages occur in various organs throughout the body in case of acute and subacute infection due to capillary endothelium degeneration and thrombocytopenia. Lymph nodes are swollen, edematous and appear as red or black due to diffuse hemorrhages. The heart, larynx, urinary bladder, intestinal mucosa, serosa and skin show petechial to echhymotic hemorrhages. It has also been observed that disseminated intravascular coagulation (DIC) caused by the CSFV may not have an effective role in the pathogenesis of the disease (Blome et al., Reference Blome, Meindl-Böhmer, Nowak and Moennig2013). Kidneys on removal of the capsule show petechiae in the cortex and have an appearance known as ‘turkey egg’. The skin may be cyanotic. The presence of splenic infarcts up to 10 mm in size is considered to be pathognomonic for acute swine fever. Intestines appear hyperemic with diphtheroid inflammation, and large intestines show characteristic ‘button ulcers’ in subacute and chronic CSFV infection. Lesions occur in the brain and spinal cord, and congestion occurs in the liver, bone marrow and lungs. Hemorrhages and infarctions are less pronounced or absent in persistent infection. Thymic atrophy and depletion of lymphocytes in lymph nodes, tonsils and spleen are also observed (Van Oirschot, Reference van Oirschot2000, Reference van Oirschot, Coetzer and Tustin2004).
In a proteomic analysis study of serum from pigs with CSF, lower levels of apolipoprotein A-I (apo A-I), vitamin D-binding protein (VDBP) and haptoglobin (Hp) were observed, and proteins observed at higher levels included retinol-binding protein 4 (RBP4) and serotransferrin. Up and downregulation of these proteins has different implications on the pathogenesis of the disease due to various functions of these proteins, as Apo A-I may affect vascular endothelium repair. Hp, which is a positive acute phase protein, affects angiogenesis and vascular repair. RBP4 in CSFV-infected pigs may be involved in kidney injury caused by CSFV infection. Serotransferrin in CSFV-infected serum suggests that iron is needed for efficient CSFV replication (Sun Jin-fu et al., Reference Sun, Shi, Guo, Li and Tu2011).
Epidemiology
Both domestic pigs (Sus scrofa domesticus) and wild boar (Sus scrofa scrofa) are equally susceptible to CSFV infection. All the secretions and excretions, notably saliva, nasal and ocular discharges, urine, semen and feces, are rich sources of the virus, and pigs are susceptible to infection through ingestion and inhalation of contaminated products. Movement of the pigs which are incubating the disease or persistently infected is the most common cause of spreading the infection (de Smit et al., Reference de Smit, Bouma, Terpstra and van Oirschot1999). The virus can survive for long periods as it is quite resistant to many chemical and physical conditions. Pigs infected with the virulent virus may shed the virus before the onset of clinical signs causing high morbidity and mortality in a herd until they die or recover. Pigs are also infected during transport in contaminated trucks, through inanimate objects and mechanical vectors like flies and mosquitoes (de Smit et al., Reference de Smit, Bouma, Terpstra and van Oirschot1999). Experimental transmission of CSFV to goats, sheep, cattle, peccaries (Tayassu tajacu) and rabbits was successful whereas other vertebrates such as racoons, mice and pigeons did not support the propagation of the virus. Pork and pork products may also harbor the virus for several months under frozen conditions (Dunne, Reference Dunne, Dunne and Leman1975).
Immune mechanisms
CSF affects the immune system, mainly by causing generalized leukopenia. Neutralizing antibodies are observable after 9 days in recovering animals and after 15 days in fatally infected animals. Neutralizing antibodies are important in terms of protection and are detectable during the partial recovery phase, 3–6 weeks after infection in chronic cases. Maximum antibody titers occur 3–4 weeks after infection and may persist for 6 months.
Lymphoid organs are also affected by CSFV infections, leading to a deficiency of B lymphocytes. The cell-mediated immune response plays a key role in CSF pathobiology and prevention through quantitative changes in the T-lymphocyte population and qualitative changes in cytokine expression by these cells in the serum, thymus and spleen (Gómez-Villamandos et al., Reference Gómez-Villamandos, Ruiz-Villamor, Bautista, Sánchez, Sánchez-Cordón, Salguero and Jover2001; Sánchez-Cordon et al., Reference Sánchez-Cordón, Romanini, Salguero, Nún~ez, Bautista, Jover and Gómez-Villamandos2002, Reference Sánchez-Cordón, Romanini, Salguero, RuizVillamor, Carrasco and Gómez-Villamandos2003). The lymphoid depletion is caused by lymphocyte apoptosis induced by chemical mediators from monocyte–macrophage cells (Sánchez-Cordón et al., Reference Sánchez-Cordón, Romanini, Salguero, Nún~ez, Bautista, Jover and Gómez-Villamandos2002). In CSF, differentiation and maturation of T lymphocytes in the thymic cortex are potentiated, although T-lymphocyte apoptosis impairs the effectiveness of the non-specific immune response (Sánchez-Cordón et al., Reference Sánchez-Cordón, Salguero, Carrasco and Gómez-Villamandos2005). The non-arrival of T lymphocytes from the thymic cortex might prevent any recovery of T-lymphocyte populations in the spleen. From the onset of the disease, an increase is noted in the number of CD4+ and CD8+ lymphocytes (Narita et al., Reference Narita, Kawashima and Shimizu1996, Reference Narita, Kawashima, Kimura, Mikami, Shibahara, Yamada and Sakoda2000), and the CD8+ population was found to be relatively high compared to CD4+ T cells at advanced stages (Narita et al., Reference Narita, Kawashima and Shimizu1996, Reference Narita, Kawashima, Kimura, Mikami, Shibahara, Yamada and Sakoda2000; Pauly et al., Reference Pauly, Konig, Thiel and Saalmuller1998; Lee et al., Reference Lee, Wang and Chien1999; Sánchez-Cordón et al., Reference Sánchez-Cordón, Salguero, Carrasco and Gómez-Villamandos2005). This increase, despite intense lymphoid depletion, may be due to activation and differentiation of lymphocytes still remaining in the organs examined, which may have played a role in inducing a cell-mediated response to the virus (Hernández et al., Reference Hernández, Garfias, Nieto, Mercado, Montan~o and Centeno2001). The increase in the number of CD8+ T cells is more closely linked to expression of IL-6 (Van Snick, Reference Van Snick1990; Sánchez-Cordón et al., Reference Sánchez-Cordón, Romanini, Salguero, Nún~ez, Bautista, Jover and Gómez-Villamandos2002) and may also be related to the clonal expansion (Tizard, Reference Tizard and Tizard1998). CD8+ T cells are cytotoxic for CSFV-infected cells, and their increase during the disease may therefore be a part of the defense mechanism (Doherty et al., Reference Doherty, Allan and Eichelberger1992; Pauly et al., Reference Pauly, Elbers, König, Lengsfeld, Saalmüller and Thiel1995; Summerfield et al., Reference Summerfield, Rziha and Saalmüller1996). This has been demonstrated when vaccinated swine remained protected from the virus despite the absence of neutralizing antibodies (Rümenapf et al., Reference Rümenapf, Stark, Meyers and Thiel1991; Suradhat et al., Reference Suradhat, Intrakamhaeng and Damrongwatanapokin2001). IL-2 synthesis may be enhanced by secretion of IL-1 and IL-6 from macrophages, with IFN-γ inducing an autocrine effect on the production of this cytokine (Biron and Sen, Reference Biron, Sen, Knipe, Howley, Griffin, Marti, Roizman and Straus2001). Early apoptosis of infected monocyte–macrophage cells and phagocytosis of apoptotic bodies by other monocyte–macrophage cells may play a decisive role in the spread of the virus and in its initial evasion of the immune response (Gómez-Villamandos et al., Reference Gómez-Villamandos, Ruiz-Villamor, Bautista, Sánchez, Sánchez-Cordón, Salguero and Jover2001; Sánchez-Cordón et al., Reference Sánchez-Cordón, Romanini, Salguero, RuizVillamor, Carrasco and Gómez-Villamandos2003). A general dysfunction of the T-lymphocyte activity has also been observed. Follicular depletion of lymphocytes and/or necrosis in pigs infected with a virulent CSFV strain are observed in histopathological examinations.
Antibodies are produced against NS3, E2 and Erns viral proteins. Antibodies against E2 and Erns are protective. Although CSFV has little tendency to accumulate mutations in contrast to other RNA viruses, antigenic variation is observed among CSFV isolates due to the highly variable antigenic region of the E2 gene but not to the extent observed with ruminant pestiviruses. Convalescent animals therefore have a stable and long-lasting immunity against all variants of CSFV (Moennig and Greiser-Wilke, Reference Moennig and Greiser-Wilke2008).
The number of CD4+ and CD8+ T lymphocytes is significantly higher in pigs infected with virulent strains of CSFV. Mature granulocytes (SWC3+; SWC8+) are not susceptible to CSFV infection, whereas the less differentiated myeloid progenitor cells (SWC3low; SWC8−) are infected, thus explaining the presence of CSFV in peripheral blood mature SWC8+ cells (Summerfield et al., Reference Summerfield, Knotig and McCullough1998a, Reference Summerfield, Hofmann and McCulloughb, Reference Summerfield, Zingle, Inumaru and McCullough2001). As much as 90% of total T cells are depleted in the final stages of the disease, depending on the virulence of the viral strain (Pauly et al., Reference Pauly, Konig, Thiel and Saalmuller1998). Increased numbers of necrotic and apoptotic uninfected cells have been identified in the bone marrow of CSFV-infected pigs (Summerfield et al., Reference Summerfield, Knotig, Tschudin and McCullough2000). Immunosuppression can be detected much earlier than seroconversion and clinical signs of the disease, which is relevant both for early diagnosis and for the study of viral pathogenesis (Pauly et al., Reference Pauly, Konig, Thiel and Saalmuller1998; Summerfield et al., Reference Summerfield, Knotig and McCullough1998a, Reference Summerfield, Zingle, Inumaru and McCullough2001; Ganges et al., Reference Ganges, Barrera, Nunez, Blanco, Frias, Rodriguez and Sobrino2005). In one study, it was observed that during CSFV infection there may be inhibition of expression of MHC class II molecule SLA-DR, which is primarily involved in antigen presentation (Feng et al., Reference Feng, Li, Li, Li, Meng, Zhang, Liang, Li, Sun, Cai, Su, Yin, Li and Luo2012).
When the immune responses are insufficient to clear the virus from the body, persistence of CSFV in the host may occur. Persistent infections can be established even in the presence of neutralizing antibodies. Congenital infection with CSFV can lead to persistently infected animals that do not develop specific antibodies against the virus (van Oirschot, Reference van Oirschot1979; de Smit et al., Reference de Smit, van Gennip, Miedem, van Rijn, Terpstra and Moormann2000), probably due to the immunotolerance developed during fetal lymphocyte differentiation. Specific immune unresponsiveness may occur during intrauterine infection of the piglets which are persistently viremic and may live for several weeks, but usually die at 3 weeks of age (Radostitis et al., Reference Radostitis, Gay, Hinchcliff and Constable2007). The animals with persistent infection continuously shed the virus and are a potential source of new CSF outbreaks (Vannier et al., Reference Vannier, Plateau and Tillon1981; Carbrey, Reference Carbrey and Liess1989), as well as creating problems in diagnosis. Viral destruction of the germinal centers in lymphoid tissues leads to the B-lymphocyte deficiency and is the most significant immunopathologic consequence of acute infection with the virulent strain of CSFV. Thus, CSFV appears to alter the immunity of the pigs by causing the B-cell reaction and increased T lymphocytes in chronic stages (Narita et al., Reference Narita, Kawashima, Kimura, Mikami, Shibahara, Yamada and Sakoda2000).
Diagnosis
Recognition of clinical signs by veterinary practitioners in the field, and of gross pathological lesions after post mortem examination, is important in diagnosis of CSF. A laboratory-based diagnostic test validates the presence of the CSFV and helps to differentiate the disease in the presence of similar signs attributable to others diseases.
Clinical diagnosis
The diversity of clinical signs in CSF under field and experimental conditions makes it difficult to diagnose the disease. Furthermore, BVDV and BDV infections can seriously interfere with the clinical and laboratory diagnosis of the disease.
Laboratory diagnosis
Antigen detection
CSFV antigens can be detected by the direct fluorescent antibody test (DFAT) or immunohistochemistry by demonstration of the virus in the tonsillar crypt and germinal center besides the spleen, lymph node, kidney, thymus, tonsil, brain and lower ileum. To rule out BVDV and BDV, a panel of monoclonal antibodies (MAbs) is required. Antigen can also be tested with the agar gel precipitation test (AGPT) (Nandi et al., Reference Nandi, Kumar and Manohar2011b), MAbs detected with an avidin–biotin complex (ABC) immunoperoxidase test (Porntrakulpipat et al., Reference Porntrakulpipat, Depner and Moennig1998) and sandwich enzyme-linked immunosorbent assays (ELISAs) with polyclonal or monoclonal antibody to capture and detect CSFV antigen in serum, blood or WBCs for rapid herd screening (Leforban et al., Reference Leforban, Edwards, Ibata and Vannier1990; Moser et al., Reference Moser, Ruggli, Tratschin and Hofmann1996; Clavijo et al., Reference Clavijo, Zhou, Vydelingum and Heckert1998). The sensitivity of ELISA is comparatively higher and less cumbersome than that of virus isolation (Clavijo et al., Reference Clavijo, Zhou, Vydelingum and Heckert1998).
Detection of infectious virus
Virus can be isolated from tissue or blood and is a sensitive in vitro method for detection of CSFV in the early phase of infection. The porcine kidney cell lines (PK-15, SK-6) or swine testicular cells are commonly used for isolation of virus. CSFV is almost always non-cytopathic, so fluorescent antibody tests (FAT) or immunoperoxidase staining after incubation for 1–3 days is required to detect the CSFV antigen (OIE, Reference Drew2008). The virus isolation is more sensitive than DFAT on frozen tissue sections (de Smit, Reference de Smit2000).
Detection of antibodies
CSFV infection mainly induces antibodies against viral proteins E2, Erns and NS3. Detectable levels of antibodies appear 2–3 weeks post-infection and persist lifelong. Paired sera samples are to be collected from convalescent pigs for testing. Among diagnostic techniques, the virus neutralization test (VNT), namely the neutralization peroxidase linked assay (NPLA), is most commonly used in Western Europe. In Japan, the END method (exaltation of the Newcastle disease virus) to assay most field viruses has been used, including the END neutralization test (Shimizu et al., Reference Shimizu, Kumagai, Ikeda and Matumoto1964). The immunoperoxidase monolayer assay (IPMA) is often used in North America and Latin America (Afshar et al., Reference Afshar, Dulac and Bouffard1989). To avoid false-positive results, parallel assays with BVDV/BDV and analysis by differences in the antibody titer are usually taken into consideration. OIE–recommended tests for international trade use blocking or indirect ELISAs to detect antibodies to E2, Erns or NS3 protein (Muller et al., Reference Muller, Depner and Liess1996; Langedijk et al., Reference Langedijk, Middel, Meloen, Kramps and de Smit2001). The sensitivity and specificity of E2-based ELISA are 90–99 and 99%, respectively, compared to VNT. In general, ELISAs are cross-reactive leading to false positive results but are well suited for mass screening of animals, and testing of individual animals should be avoided. Tissue, blood and serum samples can be diagnosed by an antigen capture ELISA based on the CSFV Erns glycoprotein.
Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR is a more rapid, sensitive and specific test than FAT, ELISA or virus isolation and is useful in preclinical and clinical diagnosis of CSF (McGoldrick et al., Reference Mcgoldrick, Lowings, Ibata, Sands, Belak and Paton1998; Paton et al., Reference Paton, Mcgoldrick, Bensaude, Belak, Mittelholzer, Koenen, Vanderhallen, Greiser-Wilke, Scheibner, Stadejek, Hofmann and Thuer2000b; Risatti et al., Reference Risatti, Callahan, Nelson and Borca2003, Reference Risatti, Holink, Lu, Kutish, Callahan, Nelson, Brea Tio and Borca2005; Hoffmann et al., Reference Hoffmann, Beer, Schelp, Schirrmeier and Depner2005). Care should be taken to avoid false-positive results due to contamination during processing and false-negative results due to nucleic acid degradation and inhibitors in the sample (OIE, Reference Drew2008). RT-PCR followed by nucleotide sequencing is helpful in differentiating other pestivirus infections beside grouping and tracing the outbreak strain during epidemics for molecular epidemiology (Lowings et al., Reference Lowings, Ibata, Needham and Paton1996; Vilcek et al., Reference Vilcek, Stadejek, Ballagi-Pordany, Lowings, Paton and Belak1996; Greiser-Wilke et al., Reference Greiser-Wilke, Dreier, Haas and Zimmermann2006; Sarma et al., Reference Sarma, Mishra, Vilcek, Rajukumar, Behera, Nema, Dubey and Dubey2011; Patil et al., Reference Patil, Hemadri, Veeresh, Sreekala, Gajendragad and Prabhudas2012). Nested RT-PCR is generally considered the most sensitive method for the detection of CSFV. A multiplex RT-PCR assay is useful for rapid and differential diagnosis of CSFV among other Pestivirus infections based on the NS5B gene and 5′-UTR (de Arce et al., Reference de Arce, Perez, Frias, Rosell, Tarradas, Nunez and Ganges2009). A pan-Pestivirus-specific single-tube nested PCR and a CSFV-specific fluorescent probe allow detection of pestiviruses as well as CSFV in clinical samples (McGoldrick et al., Reference Mcgoldrick, Lowings, Ibata, Sands, Belak and Paton1998). A real-time RT-PCR has also been developed for detection and genotyping of CSFV without inter-genotypic cross-reactivity among different CSFV strains or with other swine pathogens (Huang et al., Reference Huang, Pang, Pan, Chen, Jong, Huang and Jeng2009). TaqMan-based real-time PCR using lyophylized RT-PCR reagents and portable instruments has been developed as a pen-side assay for rapid on-site detection of CSFV (Risatti et al., Reference Risatti, Callahan, Nelson and Borca2003). RT-PCR is less vulnerable to sample degradation with on-site detection, because the average half-life of viral RNA is 1–3 days. The tonsil and spleen are appropriate samples for the detection of infectious virus and viral RNA both in fresh and degraded samples (Weesendorp et al., Reference Weesendorp, Willems and Loeffen2010), but blood may be considered as the most appropriate sample for early detection of CSFV by RT-PCR (Shivaraj et al., Reference Shivaraj, Patil, Rathnammal, Hemadri, Geetha, Narayanaswamy, Manjunatha Reddy, Sharada, Aralikatti and Rahman2013). Whole blood and tonsil scrapings may be considered as the samples of choice for quick and early CSFV detection in live pigs irrespective of the virulence of the CSFV strain (Donahue et al., Reference Donahue, Petrowski, Melkonian, Ward, Mayr and Metwally2012).
A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the CSFV 5′-UTR was more recently developed (Wongsawat et al., Reference Wongsawat, Dharakul, Narat and Rabablert2011). The benefits of LAMP compared with other nucleic acid amplification techniques are ease of operation, no need for specialized equipment like a thermocycler or electrophoresis apparatus, superior sensitivity, lower risk of contamination, suitability for high-throughput DNA detection and visualization of results by naked eye (Mori et al., Reference Mori, Nagamine, Tomita and Notomi2001).
The OIE-prescribed tests for serological diagnosis or surveillance and international trade include NPLA, fluorescent antibody virus neutralization (FAVN) and ELISA. The virus neutralization test (VNT), although slow and laborious, is considered to be the best method for antibody detection in terms of sensitivity and the ability to distinguish antibodies directed against CSFV from antibodies against other pestiviruses. VNT does not discriminate infected from vaccinated animals, although it may be used as a confirmatory test in ELISA-positive cases from areas free of CSF. Thus, to screen large amounts of sera for antibody, ELISA is the best method and the doubtful cases may be confirmed by VNT (Blome et al., Reference Blome, Meindl-Böhmer, Loeffen, Thuer and Moennig2006).
Differential diagnosis
CSF needs to be differentiated from several viral and bacterial diseases that affect swine. Splenomegaly, edema of the gall bladder and bile ducts, subpleural and interlobular lung edema and hematoma-like lymph nodes are indicative of African swine fever (ASF), a disease that is similar to CSF. Other diseases affecting swine such as pasteurellosis, erysipelas, streptococcosis, porcine reproductive and respiratory syndrome (PRRS), cumarin poisoning, porcine dermatitis and nephropathy syndrome (PDNS), salmonellosis and Glasser's disease (due to Haemophilus parasuis infection) have CSF-like signs. In the case of runt pigs, diarrhea, occurring usually in the chronic phase of CSF, must be differentiated from other etiologies such as Escherichia coli and Salmonella infections. CSF intestinal inflammatory lesions must be differentiated from those due to campylobacteriosis, clostridial infections and swine dysentery. Reproductive inefficiencies such as abortions, mummified fetuses and stillbirths can also be found in the cases of pseudorabies infection, parvovirus and PRRS. CSF and pasteurellosis often are concurrently associated with swine mortality (Kumar et al., Reference Kumar, Mahajan, Sharma, Alka Singh, Arora, Banga, Verma, Kaur, Kaur, Meenakshi and Sandhu2007). Based on the epidemiology of CSF in a particular area, differentiating tests can be used reliably to effectively diagnose the disease in the presence of other diseases.
Vaccines and vaccination
In endemic countries, the introduction of CSFV can be prevented by purchasing pigs from CSF-free herds and implementing a quarantine period of 4 weeks followed by testing for CSFV. Strict hygiene and thorough cleaning and disinfection of pens also help in preventing virus entry into a herd. Furthermore, live attenuated vaccines (LAV), namely the lapinized Chinese (LC) strain, Japanese GPE-strain, French PK-15 cell-adapted Thiverval strain or PAV-250 strain, are used to control classical swine fever. The LC strain has an insertion of 13 nucleotides in the 3′-UTR compared to virulent virus whereas the GPE strain differs in 225 nucleotides from its parental strain. The LC strain, attenuated by 100 passages in rabbits, is very efficacious and widely used as it provides solid immunity against clinical signs, virus replication and excretion within a week of vaccination (Biront et al., Reference Biront, Leunen and Vandeputte1987); this is a vaccine of choice for an emergency vaccination (Van Oirschot, Reference van Oirschot2000, Reference van Oirschot, Coetzer and Tustin2004). This vaccine can block transmission of virulent CSFV to in-contact pigs 7 days after vaccination, it does not persist in pigs beyond 2–3 weeks (Lorena et al., Reference Lorena, Barlic-Maganja, Lojkić, Madić, Grom, Cac, Roić, Terzić, Lojkić, Polancec and Cajavec2001) and it confers protection for 3 years to life time (van Oirschot, Reference van Oirschot, Coetzer and Tustin2004; van Oirschot et al., Reference van Oirschot, Coetzer and Tustin2004). The vaccine types available in India, and that are mostly used, include the tissue culture LC strain vaccine, although it is still in the developmental phase (Bett et al., Reference Bett, Deka, Padmakumar and Rajasekhar2012). Piglets vaccinated in the presence of maternal antibodies need to be revaccinated at 8 weeks of age. Multiplication of the vaccine strains is mainly restricted to lymphoid organs, notably tonsils, and can cross the placenta without any abnormalities in fetuses (van Oirschot, Reference van Oirschot, Coetzer and Tustin2004; van Oirschot et al., 2004). Vaccination does not always prevent replication of virulent CSFV upon challenge.
In countries free of CSF, preventive vaccination is very rarely practiced. However, if an outbreak occurs, regional vaccination may be necessary for effective control of CSF. Oral CSF-MLV vaccination was introduced by the European countries for the purpose of controlling CSF in wild boars (Van Oirschot, Reference van Oirschot2003). To obtain complete protection, a higher dose of the oral CSF vaccine along with a stabilizer is necessary (Kaden et al., Reference Kaden, Lange, Fischer and Strebelow2000). Subunit vaccines based on the E2 protein in a double water–oil emulsion also reduced clinical signs and mortality in pigs challenged with virulent CSFV. A recombinant truncated E2 protein conferred protection with reduced clinical signs and mortality, but was unable to prevent transplacental transmission (Reimann et al., Reference Reimann, Depner, Trapp and Beer2004).
Marker vaccines
The concept of marker vaccines has come into existence to differentiate infected from vaccinated animals with the help of companion serological tests. One such vaccine is based on CSFV E2, which elicits a neutralizing antibody response in pigs. The N-terminal half of E2 forms two structural subunits, one consisting of domains B and C and the other consisting of domains A and D (van Rijn et al., Reference van Rijn, Miedema, Wensvoort, van Gennip and Moormann1994). Antigenic domains B and C are non-conserved while antigenic domain A is highly conserved (van Rijn et al., Reference van Rijn, Bossers, Wensvoort and Moormann1996). Neutralizing MAbs have shown that synergistic neutralization effects have been observed in domains B and A, and in domains C and A, but not in domains B and C. The baculovirus-expressed recombinant E2 protein, in which the antigenic unit B/C or A has been deleted, is capable of protecting pigs from lethal CSFV challenge (van Rijn et al., Reference van Rijn, Bossers, Wensvoort and Moormann1996). Bouma et al., (Reference Bouma, de Smit, de Kluijver, Terpstra and Moormann1999) reported that 3 weeks after a single vaccination with 32 μg of baculovirus-expressed E2 in a water–oil–water emulsion, clinical signs and mortality can be prevented after challenge with virulent CSFV. This E2 subunit marker vaccine could significantly reduce transplacental transmission of a moderately virulent CSFV challenge for up to 13 months (Ahrens et al., Reference Ahrens, Kaden, Drexler and Visser2000; de Smit et al., Reference de Smit, van Gennip, Miedem, van Rijn, Terpstra and Moormann2000, Reference de Smit, Bouma, de Kluijver, Terpstra and Moormann2001). Two commercially produced ELISAs which detect antibodies to Erns could also be successfully used as a companion test (Floegel-Niesmann, Reference Floegel-Niesmann2001).
The next generation of marker vaccine candidates for the control of CSF includes recombinant porcine adenovirus expressing the CSFV E2 gene (Hammond et al., Reference Hammond, McCoy, Jansen, Morrissy, Hogson and Johnson2000, Reference Hammond, Jansen, Morrissy, Goff, Muhan, Williamson, Lenghaus, Sproat, Andrew, Coupur and Johnson2001a, Reference Hammond, Jansen, Morrissy, Williamson, Hodgson and Johnsonb), live attenuated chimeric C strain viruses containing marker antigens and replicon vaccines that are non-replicating virus particles produced by infecting the appropriate trans-complementing cell lines with CSFV-E2 and CSFV-Erns deletion mutants (van Gennip et al., Reference van Gennip, Bouma, van Rijn, Widjojoatmodjo and Moormann2002; Frey et al., Reference Frey, Bauhofar, Ruggli, Summerfield, Hoffman and Tratschin2006). Experimental DNA vaccines encoding the full-length CSFV E2 glycoprotein have also been used (Andrew et al., Reference Andrew, Morrissy, Lenghaus, Oke, Sproat, Hodgson, Johnson and Coupar2000; Yu et al., Reference Yu, Tu, Li, Hu, Chen, Li, Zhang and Yin2001; Ganges et al., Reference Ganges, Barrera, Nunez, Blanco, Frias, Rodriguez and Sobrino2005; Wienhold et al., Reference Wienhold, Armengol, Marquardt, Marquardt, Voigt, Buttner, Saalmuller and Pfaff2005; Andrew et al., Reference Andrew, Morris, Coupar, Sproat, Oke, Bruce, Broadway, Morrissy and Storm2006). To enhance the immunogenicity, co-administration of cytokine genes (IL-3, IL-12 and IL-18) or regulatory cell surface molecule genes (CD154 or CD40) was performed (Wienhold et al., Reference Wienhold, Armengol, Marquardt, Marquardt, Voigt, Buttner, Saalmuller and Pfaff2005; Andrew et al., Reference Andrew, Morris, Coupar, Sproat, Oke, Bruce, Broadway, Morrissy and Storm2006). However, to protect pigs against lethal CSFV challenge infection, high dosages and several applications were needed (Wienhold et al., Reference Wienhold, Armengol, Marquardt, Marquardt, Voigt, Buttner, Saalmuller and Pfaff2005). Commercially available Erns or NS3-based antibody ELISAs are also being used as a DIVA (Differentiating Infected from Vaccinated Animals) strategy (Beer et al., Reference Beer, Reimann, Hoffmann and Depner2007). Enhanced IFN-γ and persistent, high IgG2 levels are induced using the CP7_E2alf marker vaccine which was based on the cytopathogenic BVDV strain, ‘CP7’, that carries the structural protein E2 of the CSFV strain, ‘Alfort/187’ (Reimann et al., Reference Reimann, Depner, Trapp and Beer2004), suggesting an important role of cell-mediated immunity in long-term protection against CSFV (Renson et al., Reference Renson, Le Dimna, Gabriel, Levai, Blome, Kulcsar, Koenen and Le Potier2014). Also, in another study, this DIVA vaccine was proposed as an alternative to C-strain-based bait vaccines, after its successful assessment (Feliziani et al., Reference Feliziani, Blome, Petrini, Giammarioli, Iscaro, Severi, Convito, Pietschmann, Beer and Mia2014). CP7_E2gif is another efficient DIVA vaccine which can be used in combination with detection of anti-CSFV E2-specific antibodies (Rosen et al., Reference Rosen, Rangelova, Nielsen, Rasmussen and Uttenthal2014).
Viral vectors based on the pseudorabies virus (PRV) (van Zijl et al., Reference van Zijl, Wensvoort, de Kluyver, Hulst, van der Gulden, Gielkens and Moormann1991; Mulder et al., Reference Mulder, Priem, Glazenburg, Wagenaar, Gruys, Gielkens, Pol and Kimman1994; van Iddekinge et al., Reference van Iddekinge, de Wind, Wensvoort, Kimman, Giel kens and Moormann1996; Peeters et al., Reference Peeters, Bienkowska-Szewczyk, Hulst, Gielkens and Kimman1997), porcine adenovirus (PAV) (Hammond et al., Reference Hammond, McCoy, Jansen, Morrissy, Hogson and Johnson2000, Reference Hammond, Jansen, Morrissy, Goff, Muhan, Williamson, Lenghaus, Sproat, Andrew, Coupur and Johnson2001a, Reference Hammond, Jansen, Morrissy, Williamson, Hodgson and Johnsonb, Reference Hammond, Jansen, Morrissy, Hodgson and Johnson2003, Reference Hammond and Johnson2005), swinepox virus (Hahn et al., Reference Hahn, Park, Song, An and Ahn2001), vaccinia virus (Konig et al., Reference Konig, Lengsfeld, Pauly, Stark and Thiel1995), parapoxvirus (Voigt, Reference Voigt2005) and alphavirus (Zhao et al., Reference Zhao, Sun, Sun, Li and Qiu2009; Sun et al., Reference Sun, Li, Li, Li and Qiu2010, Reference Sun, Li, Tian, Han, Zhang, Li and Qiu2011), expressing the E2 and/or Erns glycoproteins (rPRV-E2, rPAV-E2 or rPPV-E2) have been tested. Chimeric pestiviruses based on the infectious DNA copy of the CSFV vaccine strain C or the BVDV strain CP7 were constructed and characterized in vitro and in vivo (van Gennip et al., Reference van Gennip, van Rijn, Widjojoatmodjo, de Smit and Moormann2000; Reimann et al., Reference Reimann, Depner, Trapp and Beer2004). Pigs immunized with these chimeras were completely protected against lethal CSFV infection, with no virus transmission to contact animals (van Gennip et al., Reference van Gennip, van Rijn, Widjojoatmodjo, de Smit and Moormann2000; de Smit et al., Reference de Smit, Bouma, de Kluijver, Terpstra and Moormann2001).
Epitope-based vaccines are one of the current focuses in the development of new vaccines against CSF. Recombinant rE2-ba-based E2 glycoprotein immunized pigs have shown a good response against CSF (Zhou et al., Reference Zhou, Liu, Jiang, Jian-Chao Wei and Chen2011). A yeast-expressed CSFV glycoprotein E2 has been shown to induce a protective immune response against the virus (Cheng et al., Reference Cheng, Wu, Lin, Lee, Chien and Huang2014). The heterologous DNA prime and recombinant adenovirus pSFV1CS-E2/rAdV-E2 boost strategy can induce solid protective immunity with high titers of CSFV-specific neutralizing antibodies and comparable increases in numbers of CD4+ and CD8+ T cells, compared to the pigs receiving immunizations with rAdV-E2 twice (Sun et al., Reference Sun, Li, Li, Li and Qiu2010). A double antigenic marker, live attenuated CSFV strain FlagT4v obtained by combining two genetic determinants of attenuation FlagT4v (a synthetic Flag® epitope positive antigenic marker introduced through a 19mer insertion in the E1 glycoprotein) and a negative marker resulting from mutations of the binding site of the MAb WH303 (mAbWH303) epitope in the E2 glycoprotein have been utilized. Intranasal or intramuscular administration of FlagT4v protected swine against the virulent CSFV Brescia strain at early exposure (2 or 3 days) (Holinka et al., Reference Holinka, Fernandez-Sainz, O'Donnell, Prarat, Gladue, Lu, Risatti and Borca2009).
Recombinant proteins can be used as vaccines. Highly immunogenic mixtures were found to induce protective immunity against CSFV challenge infection. However, in most cases the peptide vaccines failed to provide complete protection from clinical signs, viremia and virus shedding (Dong et al., Reference Dong, Qi, Ying, Chen and Chen2006; Dong and Chen, Reference Dong and Chen2006).
Mutations were introduced to dampen the immunogenicity of the A-domain to render the C-strain suitable as a DIVA vaccine. Antibody response analysis in rabbits elicited shielding of the A-domain by an N-linked glycan had a minor effect on the immune response against the A-domain, whereas a single amino acid targeted deletion severely dampened this response. LC-strain mutants with larger deletions were highly debilitated and incapable of sustained growth in vitro. Genetically stable and replicating LC-strain mutant was produced by virtue of the compensatory evolution that can be serologically differentiated from wild-type CSFV (Kortekaas et al., Reference Kortekaas, Vloet, Weerdmeester, Ketelaar, van Eijk and Loeffen2010). By integration of B and T antigenic sites of CSFV and displaying the B epitopes, three peptide-based systems were designed and produced, and shown to bring about significant enhancements in immunogenicity over the peptides in monomeric form (Zhao et al., Reference Zhao, Sun, Sun, Li and Qiu2009; Monso et al., Reference Monso, Tarradas, de la Torre, Sobrino, Ganges and Andreu2010).
Conventional LAV are still used successfully to control CSF, and are regarded as a ‘gold standard’ because they have proven to be very efficacious and safe for inducing a high level of protection a few days after application. However, LAV does not comply with the DIVA principle, and there is a need to develop vaccines that comply with the DIVA strategy. As the first step, subunit marker vaccines based on the baculovirus-expressed E2 glycoprotein of CSFV have been developed and are available on the market. However, the immune response is delayed and less protective compared with conventional LAV. The same disadvantages can also be seen with the immunogenic peptides, DNA vaccines and trans-complemented replicons. The most promising candidates at the moment are vaccines based on viral vectors or chimeric pestiviruses. They have the potential of inducing a similarly strong immunity as conventional LAV with the DIVA strategy. Nevertheless, all of these new vaccines are genetically modified prototypes and there are many problems with regard to their acceptance and registration, which means the focus of disease control will have to be shifted during an outbreak situation from an indirect (serological testing) to a direct approach (antigen detection) (Beer et al., Reference Beer, Reimann, Hoffmann and Depner2007). Under such conditions, conventional modified live and novel marker vaccines might regain importance.
Prevention and control
There is no specific treatment for CSF. With consistent implementation of zoosanitary control measures, many countries such as United States, Canada, Australia, New Zealand and several European countries have eradicated this disease by adopting a test-and-slaughter policy. This also includes banning the importation of live pigs and pig products from CSF-endemic countries. Furthermore, kitchen leftovers and swill must be treated to destroy CSFV before feeding to pigs. When CSF is diagnosed in a country previously free of it, stamping out policy along with cleaning and disinfection of the premises and bans on movement of pigs are generally followed. Both Japan and Brazil are in the final stages of eradication after carrying out similar control programs in the past decade. In CSF-endemic countries, it is common practice to vaccinate pigs. As per EU guidelines, pig and pig products can only be imported from countries where no CSF has occurred and vaccination is carried out for 12 months. Despite continued efforts to control CSF, outbreaks have occurred intermittently in several European countries and large numbers of pigs were culled. Notably, emergency vaccination has not been used in Western Europe. Oral MLV-based vaccination of wild hogs can prevent the spread of disease to domestic animals under experimental conditions (Gers et al., Reference Gers, Vosloo, Drew, Lubisi, Pardini and Williams2011; Everett et al., Reference Everett, Crooke, Gurrala, Dwarka, Kim, Botha, Lubisi, Pardini, Gers, Vosloo and Drew2011).
Status in India
CSF is enzootic to most of the pig producing states particularly in the North Eastern (NE) region of India. Pigs are reared in the majority of households in NE India (80%), where pork is a key part of their diet. In an epidemiological study conducted in Assam, Mizoram and Nagaland by the International Livestock Research Institute (ILRI), it was shown that Indian pig farmers incur huge economic losses from mortality, treatment, replacement costs, etc., amounting to more than 2 billion Indian rupees (INR) each year (Bett et al., Reference Bett, Deka, Padmakumar and Rajasekhar2012).
In India, the first suspected case of CSF occurred in Aligarh in 1944 (Krishnamurty, Reference Krishnamurty1964). The first documented report on CSF in India dates back to 1962, where an outbreak in a piggery unit in Morol, a suburb of Mumbai, led to widespread outbreaks in other parts of the city (Sapre et al., Reference Sapre, Moghe, Bhagwat, Chaudhry and Purohit1962). The disease has been reported thereafter in a number of states of India (Sarma et al., Reference Sarma, Krishna and Mishri2008). The CSFV diagnosis has been confirmed by RT-PCR and nucleotide sequence data showed the presence of 1.1 and 2.2 subgroups (Chakraborty et al., Reference Chakraborty, Veeregoda, Chandra Naik, Rathnamma, Isloor, Venkatesha, Leena, Veeresh and Patil2011). According to the OIE website data compilation, it could be concluded that there were 1308 outbreaks of CSF in India from 1996 to 2008, which is indicative of the disease burden in this country (Patil et al., Reference Patil, Hemadri, Shankar, Raghavendra, Veeresh, Sindhoora, Chandan, Sreekala, Gajendragad and Prabhudas2010). Because of the sporadic nature of the disease and the lower preference for the pig husbandry (barring NE states) in India, CSF has not been studied systematically and the epidemiology of the disease is largely unknown. In one study, phylogenetic analysis revealed the presence of subgroup 1.1 (Patil et al., Reference Patil, Hemadri, Shankar, Raghavendra, Veeresh, Sindhoora, Chandan, Sreekala, Gajendragad and Prabhudas2010). This was different from the situation prevailing in other Asian countries and E2 and NS5B region analysis placed the Indian isolates in a clearly separated clade within subgroup 1.1 (Sarma et al., Reference Sarma, Mishra, Vilcek, Rajukumar, Behera, Nema, Dubey and Dubey2011). In another study, a total of 594 sera samples from 12 states and 287 tissue samples from 13 states of India were tested using commercial ELISA kits. The mean prevalence of antibodies against CSFV in suspected sera was found to be 63.3% (376/594) and 76.7% (220/287) for the antigen in the CSFV-suspect samples. This suggests that CSF is endemic to India (Nandi et al., Reference Nandi, Muthuchelvan, Ahuja, Bisht, Chander, Pandey and Singh2011a).
CSFV strains have also been isolated from pigs in India. Nine CSFV field isolates were collected from the union territory of Andaman and Nicobar Islands and from the states of Assam, West Bengal and Uttarakhand; and phylogenetic analysis indicated that three isolates belonged to genotype 2.1 and were in close relation to European CSFV strains, and six isolates belonged to genotype 1 based on 5′-UTR sequence analysis and subsequent genetic classification. Based on this study, circulation of both genotypes 1 and 2.1 in NE India was observed (Desai et al., Reference Desai, Sharma, Kataria, Barman and Tiwari2010). An isolate was obtained from an outbreak in Mizoram, a NE state of India; the analysis of the isolate was determined to be Chinese strain Shimen–HVRI, which may be due to the proximity of this state to China and Myanmar (Barman et al., Reference Barman, Gupt, Bora, Kataria, Tiwari and Roychoudhury2010). CSF is also known to cause disease in the pygmy hog which is a rare, small and highly endangered mammal belonging to the Suidae family, found only in the Assam state of India. During investigation of death of pygmy hogs, it was confirmed that they were susceptible to and died as a result of contracting CSF. The phylogenetic analysis revealed that CSFV 5′-UTR sequences were grouped in the Indian CSFV genotype 1.1 cluster, and thus the strains causing infection were closely related to CSFV isolates circulating in domestic pigs, which shows the threat of ‘spillover transmission’ of infectious agents from reservoir domestic populations to sympatric wildlife and vice versa (Barman et al., Reference Barman, Bora, Tiwari, Kataria, Desai and Deka2012). In a study on 16 CSFV isolates from Assam, India, the genetic typing based on 5′-UTR, E2 and NS5B gene sequences showed that all isolates only belonged to subgroup 1.1, indicating their common origin which could have occurred due to slaughter of CSFV infected pigs sold at local markets. These outbreaks occurred in small sized backyard pig farms in Assam, where garbage containing infected pork scraps, feeding swill, is common and vaccination is not practiced (Sarma et al., Reference Sarma, Mishra, Vilcek, Rajukumar, Behera, Nema, Dubey and Dubey2011). Likewise various CSFV isolates recovered from field outbreaks in various parts of India were used for genetic analysis in the NS5B gene region (409 nt), which indicated the continued dominance of subgroup 1.1 strains, and subgroup 2.2 virus detailed analysis indicated the probable Chinese origin of this subgroup. This also provides indirect evidence of routes of CSFV movement within the South East Asia region (Patil et al., Reference Patil, Hemadri, Veeresh, Sreekala, Gajendragad and Prabhudas2012). Recently, in an outbreak among zovawk pigs indigenous to Mizoram, CSFV was confirmed by RT-PCR on representative tissue samples targeting the genomic region of CSFV that encodes the 5′-UTR, NS5B, Erns and E2 genes (Rajkhowa et al., Reference Rajkhowa, Hauhnar and Jamlianthang2013). Out of 12 outbreaks attended in nine different districts of Karnataka suspected for CSF, 9 were confirmed CSF, by antigen-capture ELISA and RT-PCR CSFV NS5B gene-specific primers (Shivaraj et al., Reference Shivaraj, Patil, Rathnammal, Hemadri, Geetha, Narayanaswamy, Manjunatha Reddy, Sharada, Aralikatti and Rahman2013). CSFV-affected states and union territories of India, based on reported disease outbreaks and serological evidence up to 2013, are illustrated in Fig. 2.
Fig. 2. Map of India showing classical swine fever virus-affected states and union territories based on reported disease outbreaks and serological evidence.
A LC strain vaccine is currently being used in India, but the quantity is not sufficient to immunize even 1% of the total pig population (Sarma et al., Reference Sarma, Krishna and Mishri2008). The northeast part of India requires 7.64 million doses of the CSF vaccine, while the total requirement per year in India amounts to 22.26 million doses, and only 1.2 million doses (<1%) are currently available (Bett et al., Reference Bett, Deka, Padmakumar and Rajasekhar2012). CSF outbreaks due to inadequate attenuation of the virus have also been reported in various NE states (Sarma et al., Reference Sarma, Krishna and Mishri2008). The presence of CSFV in the tissues of pigs slaughtered for human consumption has also been reported.
Conclusion
Worldwide, CSF is a highly contagious viral disease of swine with high morbidity and mortality, particularly in young animals. The diversity of clinical symptoms, under both natural and experimental conditions, and similarities to other hemorrhagic diseases make it difficult to diagnose CSF. Secondary infections are common, and may overshadow the presence of CSFV. Furthermore, other pestiviruses are antigenically closely related, and their serology with polyclonal antibody-based diagnostic tests can be confusing. MAb-based serological tests and molecular techniques are useful in diagnosing and differentiating exposure to CSFV from other pestiviruses and pathogens. It is important to check the disease at small scale or village level, especially in parts of India where backyard pig farming is common. Furthermore, surveillance programs should be adopted for laboratory testing of ailing pigs and materials from abattoirs. The application of excellent biosecurity measures in pig farms needs to be emphasized. In all the pig-farming sectors, a proper level of data collection, processing and recording along with the combined effort of the owner, private practitioners, veterinary authorities and welfare organizations should be adopted. In CSF-free counties with intensive pig industries, the control of CSF outbreaks is expected to change from mass culling of pigs to control based on real-time RT-PCR-based diagnosis followed by vaccination with marker vaccines. In endemic areas, mass vaccination with modified live virus vaccines, control of pig movement and epidemiological surveillance might help to control the disease to a great extent. Endemicity of CSFV infections of wild boar populations may also be an important threat to domestic pigs and can pose a threat in reintroducing the disease. Due to the inadequate availability of vaccine doses, other factors that increase the incidence of CSF in India include the lack of timely diagnosis, unrestricted movement of pigs within the country and across the border surrounding the NE states, and poor public awareness. Thus, detailed epidemiological investigations with proper preventive and control measures are needed to fully contain the disease throughout the country. Finally, increased understanding and awareness of CSF among concerned parties would help to control this disease in the near future, which in turn will help to avoid enormous economic losses.
