ABSTRACT
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
ABSTRACT
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection. Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The isolate was defined as CPV-2a (JN403045) subtype by sequencing. The VP2 ORF was inserted into pET-32a by T4 ligase and introduced into the E. coli Bal21. VP2 protein was produced by the induction of the E. coli Bal21 containing pET-32a-VP2 with isopropyl-ß-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant VP2 protein (rVP2) fused with His-tag was analyzed by SDS-PAGE and detected by western blotting using anti-His monoclonal antibody. The rVP2 was purified by Ni+-affinity purification chromatography under denature condition and dialyzed against PBS. The concentration of the rVP2 was determined by Bradford method. After immunizing the chickens with rVP2, anti-VP2 IgY was isolated by PEG 6000 precipitation and analyzed by SDS-PAGE. The activity and specificity of the IgY antibody were analyzed by indirect ELISA and western blotting. SDS-PAGE analysis showed that the rVP2 fused with His-tag had a molecular of 85 kDa, accompanied with the low molecular fractions around 50 kDa and 40 kDa. The rVP2 could be recognized be the anti-His monoclonal antibody. The IgY antibody isolated by PEG 6000 method and analyzed by SDS-PAGE showed the IgY mainly contained two parts, 23 kDa and 67 kDa, which corresponded to light and heavy chain, respectively. In additional, some lower bands around 40 kDa were presented on the gel. The anti-VP2 IgY reached to 1:40960 after the fourth immunization. The anti-VP2-IgY could recognize the VP2 specially in Western blotting, while no reaction was seen with the low molecular. Discussion: The emergence of the low molecular proteins in 50 kDa and 40 kDa in the pellets of the bacterial lysates may be due to the degradation of the rVP2 by endogenous proteases in E. coli. The fact that IgY could recognize the entire VP2 fraction suggests the lost of the antigenic sites of the low molecular proteins.
Subject(s)
Humans , Animals , Parvovirus, Canine/genetics , Parvoviridae Infections/veterinary , Dog Diseases/prevention & control , Egg Yolk/immunology , Escherichia coli , FecesABSTRACT
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
Background: Canine parvovirus-2 (CPV-2) disease is highly contagious and often fatal in canine with symptoms of hemorrhagic diarrhea and myocarditis. Three variants, CPV-2a, CPV-2b and CPV-2c, had emerged and replaced the CPV-2 in the past decades worldwide. Emergences of the new variants may increase the difficulty of CPV diagnosis and treatment. CPV capsid consists of 60 copies of a combination of viral coat proteins VP1 (82 kDa), VP2 (67 kDa) and VP3 (63.5 kDa), while about 90% of the capsid proteins are VP2 and most of the B cell epitopes are located on the VP2. IgY technology has been recognized as a promising alternative to generate large amount of qualified high specific antibody for use in immunodiagnostic and in immunotherapy with the advantages of relatively simple, noninvasive method and large-scale production over mammalian antibody. Furthermore, IgY antibody does not react with mammalian IgG nor binding to the rheumatoid factor, which may reduce the false positive results in immunoassays. Anti-VP2 IgY antibody has never been reported before, here we describe a method for the production of anti-VP2 IgY, which could be applied in the diagnosis and treatment for the CPV infection.Materials, Methods & Results: A CPV strain was isolated from a clinical sample. The VP2 ORF (Open reading frame, ORF) was amplified by PCR and inserted into the pMD18-T clone vector. The
ABSTRACT
The exposure of 13 Brazilian free-ranging nondomestic canids (five pampas fox - Pseudalopex gymnocercus and eight crab-eating fox -Cerdocyon thous) from Southern region of Brazil, to Canine distemper virus (CDV), canine parvovirus (CPV) and Canine coronavirus (CCoV) was investigated. Antibodies against CDV were detected in 38.5 percent (5/13) of the samples. There were anti-CDV antibodies in 60 percent (3/5) of P. gymnocercus and in 25 percent (2/8) of C. thous. The frequency was higher among the adults and males. Eleven canids (84.6 percent) presented antibodies against CPV, 80 percent (4/5) were from P. gymnocercus and 87.5 percent (7/8) were from C. thous. There was no difference in positivity rate against CPV between gender and age. Antibodies against CCoV were detected in 38.5 percent (5/13) of the samples, with 60 percent (3/5) of positivity in P. gymnocercus and 25 percent (2/8) in C. thous. The frequency of antibodies against CCoV was higher among the adults and males. The study showed that these canids were exposed to CDV, CPV and CCoV.
Foi investigada a ocorrência de exposição em 13 canídeos não domésticos de vida livre (cinco graxains-do-campo - Pseudalopex gymnocercus e oito graxains-do-mato - Cerdocyon thous) da região sul do Brasil ao vírus da cinomose canina (CDV), parvovírus canino (CPV) e coronavírus canino (CCoV). Anticorpos contra o CDV foram detectados em 38,5 por cento (5/13) das amostras. Haviam anticorpos anti-CDV em 60 por cento (3/5) dos P. gymnocercus e em 25 por cento (2/8) dos C. thous. A freqüência foi maior entre machos e adultos. Para CPV, 11 canídeos (84,6 por cento) apresentaram anticorpos, 80 por cento (4/5) eram da espécie P. gymnocercus e 87,5 por cento (7/8) eram C. thous. Não houve diferença de positividade para o CPV entre sexos e idades. Anticorpos contra o CCoV foram detectados em 38,5 por cento (5/13) das amostras, sendo 60 por cento (3/5) de positividade entre os P. gymnocercus e 25 por cento (2/8) entre os C. thous. A freqüência de anticorpos para CCoV foi maior entre os machos e adultos. O estudo revelou que estes canídeos foram expostos ao CDV, CPV e CCoV.
ABSTRACT
This paper relates the clinical and epidemiological aspects of canine parvovirus infection (CPV) in the State of Rio de Janeiro from April 1995 to March 2004. A total of 341 fecal samples were collected from up to 6-months-old puppies with gastroenteritis. The diagnosis of CPV infection was confirmed by hemagglutination/ hemagglutination inhibition tests, enzyme immunoassay, virus isolation in cell culture or polymerase chain reaction. One hundred and fifty-seven samples (46 percent) were positive for CPV. No correlation among sex, breed or age and the occurrence of CPV infection was observed. The classical signs of parvoviral enteritis (anorexia, lethargy, vomiting and hemorrhagic fluid diarrhea) were observed in 70 percent of CPV-positive and in 60 percent of CPV-negative puppies. Although CPV could be detected throughout the studied period, its occurrence was significantly higher from June to September and November to December. These results show that CPV is still circulating in the State of Rio de Janeiro.(AU)
Este trabalho relata os aspectos clínicos e epidemiológicos da infecção pelo CPV no Estado do Rio de Janeiro, no período de abril de 1995 a março de 2004. Coletaram-se 341 amostras fecais de cães com até seis meses de idade que apresentavam gastrenterite. O diagnóstico da infecção pelo CPV foi confirmado através dos testes de hemaglutinação/inibição da hemaglutinação, ensaio imunoenzimático, isolamento viral em cultura de células ou reação em cadeia pela polimerase. Cento e cinqüenta e sete amostras (46 por cento) foram consideradas positivas para CPV. Não foi observada correlação entre sexo, raça ou idade e a ocorrência da infecção por CPV. Os sinais clínicos clássicos de parvovirose (vômito, anorexia, apatia e diarréia líquida hemorrágica) foram observados em 70 por cento dos animais positivos e 60 por cento dos animais negativos para CPV. O CPV foi detectado ao longo do período estudado, entretanto observou-se um aumento do número de casos positivos nos períodos de junho a setembro e novembro a dezembro. Estes resultados mostram que o CPV ainda circula no Estado do Rio de Janeiro.(AU)
Subject(s)
Animals , Enteritis/diagnosis , Enteritis/epidemiology , Enteritis/prevention & control , Parvovirus, Canine/isolation & purification , Coliforms/analysis , DogsABSTRACT
We evaluated the genetic diversity in the VP1/VP2 gene of CPV type 2b isolates from symptomatic dogs in Brazil. A total of 21 isolates collected from 1990 through 1995 previously typed as CPV2b by PCR assay were studied. Overall we found a high degree of similarity among sequences from different CPV clinical isolates collected. Genetic analysis of this selected region gave no indication of a specific Brazilian parvovirus lineage.
Neste estudo foi avaliada a diversidade genética no gene VP1/VP2 do parvovírus canino tipo 2b a partir de amostras isoladas de cães sintomáticos no Brasil. Foram estudadas 21 amostras coletadas no período de 1990 à 1995, previamente caracterizadas como CPV 2b pela técnica de PCR. Observou-se alto grau de similaridade entre as seqüências estudadas e a análise genética da região selecionada não indicou a presença de uma linhagem brasileira específica.