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Haemagglutination as a confirmatory test for Peste des petits ruminants diagnosis
Small Ruminant Research 59 (2005) 75–78
Haemagglutination as a confirmatory test for Peste des petits ruminants diagnosis夽 S. Manoharana,∗ , R. Jayakumarb , R. Govindarajanc , A. Koteeswarana a
Vaccine Research Centre-Viral Vaccines, Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai 600 051, Tamil Nadu, India b Department of Animal Biotechnology, Madras Veterinary College, Chennai 7, Tamil Nadu, India c Central University Laboratory, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai 600 051, Tamil Nadu, India Received 4 November 2003; received in revised form 10 May 2004; accepted 8 December 2004
Abstract Seven Peste des petits ruminants (PPR) viruses and a RPV (vaccine strain) and their respective antiserum raised in rabbits were used for reciprocal cross neutralization test and haemagglutination (HA) and haemagglutination inhibition (HI) test employing 0.5% chicken RBC. Although many differential diagnostic methods for PPR and RP (rinderpest) are available viz., immunocapture ELISA, ‘N’ gene radioactive cDNA probes, non radioactively labelled biotinylated cDNA probes, reverse transcription-polymerase chain reaction (RT-PCR) for ‘F’ gene along with reciprocal cross neutralization test, HA and HI tests could be used as a reliable alternative in field conditions particularly in places where RP and PPR are co-existing. The advantages of the tests have been discussed in comparison with reciprocal cross neutralization tests. © 2004 Published by Elsevier B.V. Keywords: Peste des petits ruminants virus; Rinderpest virus; Haemagglutination; Haemagglutination inhibition; Chicken red blood cell; Reciprocal cross neutralization test
1. Introduction Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by a RNA virus of genus Morbillivirus in the family Paramyxoviridae. The etiological agent PPRV is serologically 夽 Part of Ph.D. thesis submitted by the first author to Tamil Nadu Veterinary and Animal Sciences University in the year 1999. ∗ Corresponding author. Fax: +91 044 2555 1573. E-mail address: [email protected] (S. Manoharan).
0921-4488/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2004.12.002
closely related to rinderpest virus (RPV), another member of the Morbillivirus causing clinically indistinguishable disease in large and small ruminants (Scott, 1990). Methods to differentiate these two viruses are reciprocal cross neutralization test (Taylor, 1979; Taylor and Abegunde, 1979; Furley et al., 1987; Chandran et al., 1995), immunocapture ELISA (Libeau et al., 1994), mobility of ‘N’ protein in PAGE (Diallo et al., 1987), radiolabelled cDNA probe from ‘N’ gene (Diallo et al., 1989), non radioactively labelled bi-
1.0 2.2 256 – 2.73 0.7 512 – 2.5 0.5 512 – 2.42 – 512 – 2.43 0.8 256 – 2.2 1.0 512 – 2.3 0.2 512 – 2.33 0.5 1024 – 6.25 6.4 32 Nil Goat Bovine
128 Nil
0.7 0.7 0.8 256 256 128 2.33 2.33 2.66 512 256 512 2.57 2.57 2.63 512 256 1024 2.51 2.51 2.43 512 1024 1024 2.73 2.73 2.51 512 512 256 2.43 2.73 2.33 512 1024 1024 2.73 2.63 2.2 1024 1024 512 2.81 2.51 2.66 1024 512 512 6.25 7.5 5.67 Goat Sheep Goat
PPRV/IND/TN/95/7 (Tirunelveli) PPRV/IND/TN/87/1 (Arasur) PPRV/IND/NAH797/I (Maharashtra) PPRV/IND/ORI/97/ 1 (Orissa) Vaccine strain of rinderpest virus (RBOK)
32 64 oculonasal extract 32 Not done 16 Goat Goat
128 64 64
0.8 1.2 256 256 2.43 2.51 512 256 2.3 2.2 256 512 2.43 2.2 256 128 2.33 2.43 512 256 2.2 2.2 512 1024 2.43 2.43 1024 1024 2.43 2.33 1024 1024 6.5 7.0
6.0 64 32
128 64
PPRV/IND/ ORI/97/1 (Orissa) HI SNT 512 2.7 PPRV/IND/N AH/97/1 (Maharashtra) HI SNT 256 2.42 PPRV/IND/ TN/87/1 (Arasur) HI SNT 512 2.2 PPRV/IND/ TN/95/7 (Tirunelveli) HI SNT 512 2.33 PPRV/IND/ TN/96/2 (Otteri) HI SNT 1024 2.2 PPRV/IND/ TN/96/1 (Dindugul) HI SNT 1024 2.57 PPRV/IND/ TN/97/1 (Coimbatore) HI SNT 1024 2.73
Haemagglutination titre/50% neutralization end point in log10
Antiserum to different viral isolates
Virus titre TCID50 log10 Tissue culture fluid
Goat
Homologous antiserum for all the PPRV isolates and RPV was raised in rabbits as described by Obi et al. (1990). Briefly, Vero adapted PPRVs and RPV (2 ml) were homogenised with Freund’s adjuvant and injected subcutaneously in the flank region. Subsequent three injections were given with incomplete adjuvant intramuscularly (2 ml) at 2 weeks interval. The blood was collected 7 days after last injection and the serum was separated and stored at −20 ◦ C till use.
PPRV/IND/TN/97/1 (Coimbatore) PPRV/IND/TN/96/1 (Dindugul) PPRV/IND/TN/96/2 (Otteri)
2.2. Antiserum production
Haemagglutination titre
The source of PPR viruses was from different outbreaks in Tamil Nadu and from other states of India (Table 1). Initially, the suspected tissues/infected tissue culture fluid of PPR viruses were confirmed for the presence of antigen and F gene by immunocapture ELISA and RT-PCR, respectively. The viruses were isolated (Libeau et al., 1994; Forsyth and Barrett, 1995) and adapted in Vero cells. The source of RPV was a vaccine strain in primary lamb kidney cells (RBOK strain) obtained from Institute of Veterinary Preventive Medicine, Ranipet, Tamil Nadu, India.
Tissue suspension
2.1. Viruses
Species
2. Material and methods
Viruses
otinylated cDNA probes (Pandey et al., 1992) and reverse transcription-polymerase chain reaction (RTPCR) for ‘F’ gene conserved region (Forsyth and Barrett, 1995). Among this, immunocapture ELISA and reverse transcription-polymerase chain reaction are routinely used for differentiation (OIE, 2000). A simple haemagglutination (HA) and its corresponding haemagglutination inhibition (HI) tests are accepted valid alternatives for differentiating PPRV and RPV (Wosu, 1985, 1991) since the other differentiating methods are cost prohibitive and time consuming for routine screening of large number of samples; a simple screening method to differentiate PPRV and RPV is the need of the hour in countries where rinderpest is reported along with Peste des petits ruminants. Hence, attempts were made to study the usefulness of the HA and HI tests, as a differentiating method in field conditions by comparing with the reciprocal cross neutralization test.
Vaccine strain of rinderpest virus (RBOK) HI SNT 512 1.0
S. Manoharan et al. / Small Ruminant Research 59 (2005) 75–78
Table 1 Haemagglutination, haemagglutination inhibition and 50% serum neutralization titres of different PPRV isolates and RPV with their homologous and heterologous antiserum
76
S. Manoharan et al. / Small Ruminant Research 59 (2005) 75–78
2.3. Reciprocal cross neutralization test This test was performed by following the microtitre techniques of Rossiter and Jessett (1982) and the method of Taylor and Abegunde (1979). Different PPRVs, RPV and their respective antiserum were used in the tests. Hundred TCID50 of each virus was used against two-fold dilution of the antiserum. Serum dilutions were made in triplicate. Observations were made upto 12 days before final readings were recorded. 2.4. Haemagglutination test Haemagglutination test was conducted as per the procedure described by Wosu (1985) with 0.5% chicken RBC in ‘V’ bottom microtitre plate. Briefly two-fold dilution of the virus in tissue suspension or tissue culture fluid was made in 50 l of PBS (pH 7.2) and 50 l of 0.5% chicken RBC was added to all wells and incubated at 37 ◦ C for 45 min. 2.5. Haemagglutination inhibition test Haemagglutination inhibition test was performed as described by Ramachandran et al. (1993). The serum was treated with kaolin to avoid non specific inhibitors. The heat-inactivated serum (56 ◦ C for 30 min) was diluted to 1:4 in PBS (pH 7.2). Twenty-five grams of kaolin was added to 100 ml of 1N HCl and left for 20 min, at room temperature. It was then centrifuged at 2000 rpm for 5–10 min; resuspended in PBS (pH 7.2) again and the washing was repeated until the pH was 7.2. Equal volume of washed 25% kaolin was added to the 1 ml of the diluted serum and left at room temperature for 20 min. After the centrifugation, the supernatant serum in 1:8 dilution was collected. The test was done with positive and negative controls.
3. Results and discussion Haemagglutination, haemagglutination inhibition and reciprocal cross neutralization test results are presented in Table 1. For the original tissue suspension the HA titre was found to range from 16 to 64 and for the culture adapted viruses the titre ranged from 64 to 128, whereas the HI titre of the homologous and heterolo-
77
gous antisera of different PPRV isolates ranged from 128 to 1024 and the titre of RPV antiserum with PPRV isolates was from 128 to 512. The results of the reciprocal cross neutralization confirmed the identity of the PPR viruses. The 50% log10 neutralization end point for the homologous and heterologous antisera of different PPRV viruses varied from 2.2 to 2.81, whereas the SN50 titre of RPV antiserum with different PPRV isolates and various PPRV antiserum with RPV virus ranged from 0.2 to 1.2, but with RPV antiserum against RPV the titre was 2.2. The HA activity of PPR suspected samples and tissue culture adapted PPR viruses was demonstrated previously by Chandran et al. (1992) and Ramachandran et al. (1993). In this study, seven PPRV isolates and a RPV vaccine strain (RBOK strain) were subjected to HA test. The PPRV isolates confirmed by immunocapture ELISA/RT-PCR were further confirmed by the results of reciprocal cross neutralization test. Haemagglutination activity was found only with the PPRV isolates and not with RPV as anticipated (Wosu, 1991; Ramachandran et al., 1993). HA test was performed with 0.5% chicken erythrocytes (Ramachandran et al., 1993) instead of 0.6% piglet erythrocytes (Wosu, 1985) or 0.5% monkey erythrocytes (Chandran et al., 1992), due to ease of its availability and adaptability under field conditions (Ramachandran et al., 1994). Haemagglutination activity for PPR suspected tissue specimens was found to be ranging between 32 and 64 and for the tissue culture adapted PPR viruses was found to range between 64 and 128. The HA titre range for tissue specimens and tissue culture fluid were comparable with the results of Wosu (1985, 1991). This test was employed to differentiate the PPRV and RPV isolates by showing HA activity in all the PPR viruses and was absent in RPV. Specificity of the HA activity of the PPR viruses was confirmed by haemagglutination inhibition test using polyclonal antiserum against PPRV isolates and RPV. The HI titre of the PPR antisera to PPR isolates was from 128 to 1024 and the titre with RPV antiserum to PPR isolates was from 128 to 512 (Table 1), which revealed the serological relatedness of PPRV isolates and RPV. The results of cross neutralization test were in accordance with that of Taylor and Abegunde (1979) who showed that this test could be accurately used to differentiate PPRVs and RPV based on serum neutralization end point titre (SN50 ) against homologous and heterol-
78
S. Manoharan et al. / Small Ruminant Research 59 (2005) 75–78
ogous viruses. This test is cost prohibitive and time consuming. The above results indicate the usefulness of HA and HI test in differentiating RPV and PPRV. The test results were available with in a day and did not require sophisticated facilities or equipments as required for immunocapture ELISA, SNT or RT-PCR. The ease and economy of this test make it as a candidate test for routine screening at field level as observed earlier (Ramachandran et al., 1994). Hence, an agar gel precipitation test/counter immunoelectrophoresis (Obi and Patrick, 1984) using either RP and PPR hyperimmune serum followed by HA and HI test can be employed for routine screening purposes in control programmes, whereas RT-PCR could be used for further confirmation and molecular studies. 4. Conclusion From the study it was found that the HA and HI test are specific and early in the differential diagnosis of PPR and RP in the field condition. Hence, it is suggested that for routine screening of PPR suspected samples an agar gel precipitation/counter immunoelectrophoresis tests for confirming Morbillivirus identity followed by HA test with 0.5% chicken RBC and specific inhibition by PPR antiserum could be employed for specific diagnosis. Acknowledgement The first author sincerely thanks the Council of Scientific and Industrial Research, New Delhi, India for granting Senior Research Fellowship for his Ph.D. programme. References Chandran, N.D.J., Kumanan, K., Albert, A., Venkatesan, R.A., 1992. Haemagglutinating attributes of Peste des petits ruminants virus. Indian J. Virol. 8, 53. Chandran, N.D.J., Kumanan, K., Venkatesan, R.A., 1995. Differentiation of Peste des petits ruminants and rinderpest viruses by
neutralization indices using hyperimmune rinderpest antiserum. Trop. Anim. Health Prod. 27, 89–92. Diallo, A., Barrett, T., Barbron, M., Shaila, M.S., Taylor, W.P., 1989. Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA probes. J. Virol. Methods 23, 127–136. Diallo, A., Barrett, T., Lefevre, P.C., Taylor, W.P., 1987. Comparison of proteins induced in cells infected with rinderpest and peste des petits ruminants viruses. J. Gen. Virol. 68, 2033–2038. Forsyth, M.A., Barrett, T., 1995. Evaluation of polymerase chain reaction for the detection and characterization of rinderpest and peste des petits ruminants viruses for epidemiological studies. Virus Res. 39, 151–163. Furley, C.W., Taylor, W.P., Obi, T.U., 1987. An outbreak of peste des petits ruminants in a zoological collection. Vet. Rec. 121, 443–447. Libeau, G., Diallo, A., Colas, F., Guerre, L., 1994. Rapid differential diagnosis of rinderpest and peste des petits ruminants using an immunocapture ELISA. Vet. Rec. 134, 300–304. Obi, T.U., McCullough, K.C., Taylor, W.P., 1990. The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis. J. Vet. Med. 37, 345–352. Obi, T.U., Patrick, D., 1984. The detection of peste des petits ruminants (PPR) virus antigen by agar gel precipitation test and counter-immunoelectrophoresis. J. Hyg. 93, 579–586. OIE, 2000. Manual of Standards Diagnostic Test and Vaccines. Chapter 2.1.5. Pandey, K.D., Baron, M.D., Barrett, T., 1992. Differential diagnosis of rinderpest and PPR using biotinylated cDNA probes. Vet. Rec. 131, 199–200. Ramachandran, S., Hegde, N.R., Shaila, M.S., 1993. Haemagglutination by Peste des petits ruminants virus (PPRV). In: Proceedings of the Third International Sheep Veterinary Society Conference, Edinburgh, Scotland, UTK, 27th June–1st July. Ramachandran, S., Shaila, M.S., Shyam, G., 1994. Haemagglutination and haemadsorption by Pesti des petits ruminants virus (PPRV). In: Proceedings of the Third International Conf. Eur. Soc. Vet. Virol., Interlaken, September 1994, p. 513. Rossiter, P.B., Jessett, D.M., 1982. Detection of rinderpest virus antigens in vitro and in vivo by direct immunofluorescence. Res. Vet. Sci. 33, 198–204. Scott, G.R., 1990. Peste des petits ruminants (goat plaque) virus. Virus Infect. Vertebr. 3, 355–361. Taylor, W.P., 1979. Serological studies with the virus of peste des petits ruminants in Nigeria. Res. Vet. Sci. 26, 236–242. Taylor, W.P., Abegunde, A., 1979. The isolation of peste des petits ruminants virus from Nigerian sheep and goats. Res. Vet. Sci. 26, 94–96. Wosu, L.O., 1985. Agglutination of red blood cells by peste des petits ruminants (PPR) virus. Nig. Vet. J. 14, 56–58. Wosu, L.O., 1991. Haemagglutination test for diagnosis of peste des petits ruminants disease in goats with samples from live animals. Small Rum. Res. 5, 169–172.
Haemagglutination as a confirmatory test for Peste des petits ruminants diagnosis夽 S. Manoharana,∗ , R. Jayakumarb , R. Govindarajanc , A. Koteeswarana a
Vaccine Research Centre-Viral Vaccines, Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai 600 051, Tamil Nadu, India b Department of Animal Biotechnology, Madras Veterinary College, Chennai 7, Tamil Nadu, India c Central University Laboratory, Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai 600 051, Tamil Nadu, India Received 4 November 2003; received in revised form 10 May 2004; accepted 8 December 2004
Abstract Seven Peste des petits ruminants (PPR) viruses and a RPV (vaccine strain) and their respective antiserum raised in rabbits were used for reciprocal cross neutralization test and haemagglutination (HA) and haemagglutination inhibition (HI) test employing 0.5% chicken RBC. Although many differential diagnostic methods for PPR and RP (rinderpest) are available viz., immunocapture ELISA, ‘N’ gene radioactive cDNA probes, non radioactively labelled biotinylated cDNA probes, reverse transcription-polymerase chain reaction (RT-PCR) for ‘F’ gene along with reciprocal cross neutralization test, HA and HI tests could be used as a reliable alternative in field conditions particularly in places where RP and PPR are co-existing. The advantages of the tests have been discussed in comparison with reciprocal cross neutralization tests. © 2004 Published by Elsevier B.V. Keywords: Peste des petits ruminants virus; Rinderpest virus; Haemagglutination; Haemagglutination inhibition; Chicken red blood cell; Reciprocal cross neutralization test
1. Introduction Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by a RNA virus of genus Morbillivirus in the family Paramyxoviridae. The etiological agent PPRV is serologically 夽 Part of Ph.D. thesis submitted by the first author to Tamil Nadu Veterinary and Animal Sciences University in the year 1999. ∗ Corresponding author. Fax: +91 044 2555 1573. E-mail address: [email protected] (S. Manoharan).
0921-4488/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2004.12.002
closely related to rinderpest virus (RPV), another member of the Morbillivirus causing clinically indistinguishable disease in large and small ruminants (Scott, 1990). Methods to differentiate these two viruses are reciprocal cross neutralization test (Taylor, 1979; Taylor and Abegunde, 1979; Furley et al., 1987; Chandran et al., 1995), immunocapture ELISA (Libeau et al., 1994), mobility of ‘N’ protein in PAGE (Diallo et al., 1987), radiolabelled cDNA probe from ‘N’ gene (Diallo et al., 1989), non radioactively labelled bi-
1.0 2.2 256 – 2.73 0.7 512 – 2.5 0.5 512 – 2.42 – 512 – 2.43 0.8 256 – 2.2 1.0 512 – 2.3 0.2 512 – 2.33 0.5 1024 – 6.25 6.4 32 Nil Goat Bovine
128 Nil
0.7 0.7 0.8 256 256 128 2.33 2.33 2.66 512 256 512 2.57 2.57 2.63 512 256 1024 2.51 2.51 2.43 512 1024 1024 2.73 2.73 2.51 512 512 256 2.43 2.73 2.33 512 1024 1024 2.73 2.63 2.2 1024 1024 512 2.81 2.51 2.66 1024 512 512 6.25 7.5 5.67 Goat Sheep Goat
PPRV/IND/TN/95/7 (Tirunelveli) PPRV/IND/TN/87/1 (Arasur) PPRV/IND/NAH797/I (Maharashtra) PPRV/IND/ORI/97/ 1 (Orissa) Vaccine strain of rinderpest virus (RBOK)
32 64 oculonasal extract 32 Not done 16 Goat Goat
128 64 64
0.8 1.2 256 256 2.43 2.51 512 256 2.3 2.2 256 512 2.43 2.2 256 128 2.33 2.43 512 256 2.2 2.2 512 1024 2.43 2.43 1024 1024 2.43 2.33 1024 1024 6.5 7.0
6.0 64 32
128 64
PPRV/IND/ ORI/97/1 (Orissa) HI SNT 512 2.7 PPRV/IND/N AH/97/1 (Maharashtra) HI SNT 256 2.42 PPRV/IND/ TN/87/1 (Arasur) HI SNT 512 2.2 PPRV/IND/ TN/95/7 (Tirunelveli) HI SNT 512 2.33 PPRV/IND/ TN/96/2 (Otteri) HI SNT 1024 2.2 PPRV/IND/ TN/96/1 (Dindugul) HI SNT 1024 2.57 PPRV/IND/ TN/97/1 (Coimbatore) HI SNT 1024 2.73
Haemagglutination titre/50% neutralization end point in log10
Antiserum to different viral isolates
Virus titre TCID50 log10 Tissue culture fluid
Goat
Homologous antiserum for all the PPRV isolates and RPV was raised in rabbits as described by Obi et al. (1990). Briefly, Vero adapted PPRVs and RPV (2 ml) were homogenised with Freund’s adjuvant and injected subcutaneously in the flank region. Subsequent three injections were given with incomplete adjuvant intramuscularly (2 ml) at 2 weeks interval. The blood was collected 7 days after last injection and the serum was separated and stored at −20 ◦ C till use.
PPRV/IND/TN/97/1 (Coimbatore) PPRV/IND/TN/96/1 (Dindugul) PPRV/IND/TN/96/2 (Otteri)
2.2. Antiserum production
Haemagglutination titre
The source of PPR viruses was from different outbreaks in Tamil Nadu and from other states of India (Table 1). Initially, the suspected tissues/infected tissue culture fluid of PPR viruses were confirmed for the presence of antigen and F gene by immunocapture ELISA and RT-PCR, respectively. The viruses were isolated (Libeau et al., 1994; Forsyth and Barrett, 1995) and adapted in Vero cells. The source of RPV was a vaccine strain in primary lamb kidney cells (RBOK strain) obtained from Institute of Veterinary Preventive Medicine, Ranipet, Tamil Nadu, India.
Tissue suspension
2.1. Viruses
Species
2. Material and methods
Viruses
otinylated cDNA probes (Pandey et al., 1992) and reverse transcription-polymerase chain reaction (RTPCR) for ‘F’ gene conserved region (Forsyth and Barrett, 1995). Among this, immunocapture ELISA and reverse transcription-polymerase chain reaction are routinely used for differentiation (OIE, 2000). A simple haemagglutination (HA) and its corresponding haemagglutination inhibition (HI) tests are accepted valid alternatives for differentiating PPRV and RPV (Wosu, 1985, 1991) since the other differentiating methods are cost prohibitive and time consuming for routine screening of large number of samples; a simple screening method to differentiate PPRV and RPV is the need of the hour in countries where rinderpest is reported along with Peste des petits ruminants. Hence, attempts were made to study the usefulness of the HA and HI tests, as a differentiating method in field conditions by comparing with the reciprocal cross neutralization test.
Vaccine strain of rinderpest virus (RBOK) HI SNT 512 1.0
S. Manoharan et al. / Small Ruminant Research 59 (2005) 75–78
Table 1 Haemagglutination, haemagglutination inhibition and 50% serum neutralization titres of different PPRV isolates and RPV with their homologous and heterologous antiserum
76
S. Manoharan et al. / Small Ruminant Research 59 (2005) 75–78
2.3. Reciprocal cross neutralization test This test was performed by following the microtitre techniques of Rossiter and Jessett (1982) and the method of Taylor and Abegunde (1979). Different PPRVs, RPV and their respective antiserum were used in the tests. Hundred TCID50 of each virus was used against two-fold dilution of the antiserum. Serum dilutions were made in triplicate. Observations were made upto 12 days before final readings were recorded. 2.4. Haemagglutination test Haemagglutination test was conducted as per the procedure described by Wosu (1985) with 0.5% chicken RBC in ‘V’ bottom microtitre plate. Briefly two-fold dilution of the virus in tissue suspension or tissue culture fluid was made in 50 l of PBS (pH 7.2) and 50 l of 0.5% chicken RBC was added to all wells and incubated at 37 ◦ C for 45 min. 2.5. Haemagglutination inhibition test Haemagglutination inhibition test was performed as described by Ramachandran et al. (1993). The serum was treated with kaolin to avoid non specific inhibitors. The heat-inactivated serum (56 ◦ C for 30 min) was diluted to 1:4 in PBS (pH 7.2). Twenty-five grams of kaolin was added to 100 ml of 1N HCl and left for 20 min, at room temperature. It was then centrifuged at 2000 rpm for 5–10 min; resuspended in PBS (pH 7.2) again and the washing was repeated until the pH was 7.2. Equal volume of washed 25% kaolin was added to the 1 ml of the diluted serum and left at room temperature for 20 min. After the centrifugation, the supernatant serum in 1:8 dilution was collected. The test was done with positive and negative controls.
3. Results and discussion Haemagglutination, haemagglutination inhibition and reciprocal cross neutralization test results are presented in Table 1. For the original tissue suspension the HA titre was found to range from 16 to 64 and for the culture adapted viruses the titre ranged from 64 to 128, whereas the HI titre of the homologous and heterolo-
77
gous antisera of different PPRV isolates ranged from 128 to 1024 and the titre of RPV antiserum with PPRV isolates was from 128 to 512. The results of the reciprocal cross neutralization confirmed the identity of the PPR viruses. The 50% log10 neutralization end point for the homologous and heterologous antisera of different PPRV viruses varied from 2.2 to 2.81, whereas the SN50 titre of RPV antiserum with different PPRV isolates and various PPRV antiserum with RPV virus ranged from 0.2 to 1.2, but with RPV antiserum against RPV the titre was 2.2. The HA activity of PPR suspected samples and tissue culture adapted PPR viruses was demonstrated previously by Chandran et al. (1992) and Ramachandran et al. (1993). In this study, seven PPRV isolates and a RPV vaccine strain (RBOK strain) were subjected to HA test. The PPRV isolates confirmed by immunocapture ELISA/RT-PCR were further confirmed by the results of reciprocal cross neutralization test. Haemagglutination activity was found only with the PPRV isolates and not with RPV as anticipated (Wosu, 1991; Ramachandran et al., 1993). HA test was performed with 0.5% chicken erythrocytes (Ramachandran et al., 1993) instead of 0.6% piglet erythrocytes (Wosu, 1985) or 0.5% monkey erythrocytes (Chandran et al., 1992), due to ease of its availability and adaptability under field conditions (Ramachandran et al., 1994). Haemagglutination activity for PPR suspected tissue specimens was found to be ranging between 32 and 64 and for the tissue culture adapted PPR viruses was found to range between 64 and 128. The HA titre range for tissue specimens and tissue culture fluid were comparable with the results of Wosu (1985, 1991). This test was employed to differentiate the PPRV and RPV isolates by showing HA activity in all the PPR viruses and was absent in RPV. Specificity of the HA activity of the PPR viruses was confirmed by haemagglutination inhibition test using polyclonal antiserum against PPRV isolates and RPV. The HI titre of the PPR antisera to PPR isolates was from 128 to 1024 and the titre with RPV antiserum to PPR isolates was from 128 to 512 (Table 1), which revealed the serological relatedness of PPRV isolates and RPV. The results of cross neutralization test were in accordance with that of Taylor and Abegunde (1979) who showed that this test could be accurately used to differentiate PPRVs and RPV based on serum neutralization end point titre (SN50 ) against homologous and heterol-
78
S. Manoharan et al. / Small Ruminant Research 59 (2005) 75–78
ogous viruses. This test is cost prohibitive and time consuming. The above results indicate the usefulness of HA and HI test in differentiating RPV and PPRV. The test results were available with in a day and did not require sophisticated facilities or equipments as required for immunocapture ELISA, SNT or RT-PCR. The ease and economy of this test make it as a candidate test for routine screening at field level as observed earlier (Ramachandran et al., 1994). Hence, an agar gel precipitation test/counter immunoelectrophoresis (Obi and Patrick, 1984) using either RP and PPR hyperimmune serum followed by HA and HI test can be employed for routine screening purposes in control programmes, whereas RT-PCR could be used for further confirmation and molecular studies. 4. Conclusion From the study it was found that the HA and HI test are specific and early in the differential diagnosis of PPR and RP in the field condition. Hence, it is suggested that for routine screening of PPR suspected samples an agar gel precipitation/counter immunoelectrophoresis tests for confirming Morbillivirus identity followed by HA test with 0.5% chicken RBC and specific inhibition by PPR antiserum could be employed for specific diagnosis. Acknowledgement The first author sincerely thanks the Council of Scientific and Industrial Research, New Delhi, India for granting Senior Research Fellowship for his Ph.D. programme. References Chandran, N.D.J., Kumanan, K., Albert, A., Venkatesan, R.A., 1992. Haemagglutinating attributes of Peste des petits ruminants virus. Indian J. Virol. 8, 53. Chandran, N.D.J., Kumanan, K., Venkatesan, R.A., 1995. Differentiation of Peste des petits ruminants and rinderpest viruses by
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