Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus / 生物工程学报

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

Antigenic and immunogenic properties of the canine distemper virus nucleocapsid protein expressed in Escherichia coli employing codon optimized synthetic gene / Propriedades antigênicas e imunogênicas da proteína do nucleocapsídeo do virus da cinomose canina expressa em Escherichia coli empregando gene sintético com codons otimizados

Despite common occurrence and importance of canine distemper disease the majority of tests currently available for diagnosis are hampered by either low sensitivity or specificity. In this study it was evaluated antigenic and immunogenic characteristics of a conserved region of nucleocapsid protein of canine distemper virus (rCDV NP) expressed in Escherichia coli employing a codon optimized synthetic gene. The expression of rCDVNP in Star strain (mean 300μg/mL, purified) was confirmed by SDS-PAGE and Western blot analysis by using His-Tag monoclonal antibodies. Western blot and ELISA, employing positive and negative control dog sera, demonstrated the rCDVNP antigenicity. The rCDVNP was inoculated in hens and immunoglobulin Y (IgY) was purified from the egg yolk. The mean yield of IgY was 28.55mg/mL. IgY reacted with the recombinant protein as demonstrated by Western blot and ELISA assays. In summary, our findings demonstrated that rCDVNP is antigenic since CDV positive dog sera recognized the protein in vitro. Additionally, the rCDVNP proved to be immunogenic in hens being possible to isolate a high concentration of specific IgY antibodies from the egg yolk. Taken together, these results indicate that the rCDVNP along with the specific IgY could be useful tools for development of the canine distemper immunodiagnostic assays.(AU)

Apesar da ocorrência comum e importância da cinomose canina, a maioria dos testes atualmente disponíveis para diagnóstico são prejudicados pela baixa sensibilidade ou especificidade. Neste estudo foram avaliadas características antigênicas e imunogênicas de uma região conservada da proteína do nucleocapsídeo do virus da cinomose canina (rCDV NP) expressa em Escherichia coli empregando um gene sintético e codons otimizados. A expressão na cepa Star (média de 300μg/mL, purificada) foi confirmada por SDS-PAGE e Western blot utilizando anticorpos monoclonais anti-His-Tag. A antigenicidade da rCDVNP foi demonstrada por western blot e ELISA empregando soros de cães positivos e negativos. A rCDVNP foi inoculada em galinhas e imunoglobulina Y (gY) foi obtida e purificada a partir da gema. A produção média de IgY foi 28.55mg/mL. Anticorpos IgY reagiram com a proteína recombinante, quando analisados por Western blot e ELISA. Em resumo, nossos achados demonstram que a rCDVNP produzida é antigênica, uma vez que os anticorpos de soro de cães positivos para CDV reconheceram a proteína in vitro. Além disso, a rCDVNP foi imunogênica em galinhas, sendo possível isolar anticorpos IgY específicos a partir da gema do ovo em altas concentrações. Tomados em conjunto, estes resultados indicam que a rCDVNP juntamente com a IgY específica podem ser ferramentas úteis para elaborar ensaios de imunodiagnóstico de cinomose canina.(AU)

Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions

Abstract Three dog shelters in Rio Grande do Sul were investigated for associations between the occurrence of respiratory viruses and shelter environmental conditions. Nasal secretions randomly collected during the cold season were tested via PCR, and this data collection was followed by nucleotide sequencing of the amplicons. In shelter #1 (poor sanitary and nutritional conditions, high animal density and constant contact between dogs), 78% (58/74) of the nasal samples were positive, 35% (26/74) of which were in single infections and 44% (32/74) of which were in coinfections. Shelters #2 and #3 had satisfactory sanitary and nutritional conditions, outdoors exercise areas (#2) and animal clustering by groups (#3). In shelter #2, 9% (3/35) of the samples were positive for Canine parainfluenza virus (CPIV), and 6% (2/35) were positive for Canid herpesvirus 1 (CaHV-1). In shelter #3, 9% (7/77) of the samples were positive for Canine adenovirus type 2 (CAdV-2), and 1% (1/77) were positive for Canine distemper virus (CDV). The amplicon sequences (CPIV and CDV nucleoprotein gene; CAdV-2 E3 gene; CaHV-1 glycoprotein B gene) showed 94-100% nucleotide identity with GenBank sequences. Our results demonstrate that CPIV, CAdV-2 and CDV are common in dog shelters and that their frequencies appear to be related with environmental and nutritional conditions. These results indicate the need for control/prevention measures, including vaccination and environmental management, to minimize these infections and improve dog health.

Serologic Survey of Rabies Virus, Canine Distemper Virus and Parvovirus in Wild Raccoon Dogs (Nyctereutes procyonoides koreensis) in Korea

Oral rabies vaccination (ORV) program for the wild animals in rabies risk regions of Korea has been conducted since 2000. Evaluation of ORV program under field condition and information concerning the incidence of exposure to canine distemper and canine parvovirus (CPV) are needed in wild raccoon dogs (Nyctereutes procyonoides koreensis). Ninety four sera of wild raccoon dogs were screened for antibodies against rabies, canine distemper virus (CDV) and CPV in Korea. The overall prevalence of antibodies against rabies virus (RABV), CDV and CPV in wild raccoon dogs was 35.1%, 89.4% and 24.5%, respectively. Comparisons of sero-prevalences of RABV, CDV and CPV were assayed in two regions (Gyeonggi-do and Gangwon-do). The Gyeonggi-do (36.4%) showed higher sero-positive rate against CPV than Gangwon-do (20.8%). In contrast, Gangwon-do (41.7% and 97.2%) showed higher sero-positive rates against RABV and CDV than Gyeonggi-do (13.6% and 63.6%). These results indicate that there was severe circulation of CDV and CPV among wild raccoon dogs in the two regions of Korea. Furthermore, raccoon dogs showing a protective antibody titer (0.5 IU/ml) were 15.9%, suggesting that new rabies control program such as trap-vaccination-release (TVR) should be launched urgently in rabies risk regions.

Canine distemper virus: detection of viral RNA by Nested RT-PCR in dogs with clinical diagnosis / Vírus da cinomose canina: detecção do RNA viral pelo Nested RT-PCR em cães com diagnóstico clínico

Canine distemper virus (CDV) is a pathogen which affects dogs and causes severe disease leading to death. Dogs infected with CDV can be diagnosed by RNA detection by Nested PCR technique. The following study proposed to evaluate CDV RNA in blood, urine and saliva samples. The Nested-PCR technique was able to detect CDV RNA in different types of biologic samples. The higher number of positive results was obtained in urine samples.

vírus da cinomose canina (CDV) é um patógeno que afeta cães, causando doença grave e que pode levar a morte. Os cães infectados pelo CDV podem ser diagnosticados pela detecção do RNA utilizando-se a técnica de Nested-PCR. O presente estudo teve como objetivo avaliar o RNA do CDV no sangue, urina e saliva em cães com diagnóstico clínico de cinomose. A técnica de Nested-PCR foi capaz de detectar o RNA em diferentes tipos de amostras biológicas. Obteve-se um maior número de resultados positivos em amostras de urina.

Canine Distemper Virus Utilizes Different Receptors to Infect Chicken Embryo Fibroblasts and Vero cells / 中国病毒学·英文版

Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF)is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV)also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PERTM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

Exposure of pampas fox (Pseudalopex gymnocercus) and crab-eating fox (Cerdocyon thous) from the Southern region of Brazil to Canine distemper virus (CDV), Canine parvovirus (CPV) and Canine coronavirus (CCoV)

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.

Seroprevalence of susceptibility to morbillivirus infection on giant panda in captivity / 中国兽医学报

Canine distemper virus (CDV) neutralizing antibody (NT) titer was examined against the sera from 7 giant pandas aged between 8 to 21 years housed at Chengdu Research Base of Giant Panda Breeding,China.Anti-CDV NT titer against the Onderstepoort strain showed a wide range from × 2 to×256 (median=16),even though the ani-mals had been receiving an attenuated live vaccine made from an anonymous domestic CDV strain twice a year since 2003.A single administration of attenuated morbillivirus antigen often be enough to give corresponding host a steady immunogenicity.Anti-CDV-NT variation in the giant panda suggests some deficiency in the relationship between the vaccine and the host.

Evaluación de l retrogradación del almidón en harina de yuca precocdda / Evaluatin of staarch retrogradation in cassava flour from precooked parenchyma / Avaliação a retrrogradação do amido em farinha de mandioca precozida

Las modificaciones del almidón, que ocurren durante el proceso de elaboración de harina de yuca precocida, se evaluaron utilizando técnicas como calorimetría diferencial de barrido (CDB), difracción de rayos X, comportamiento al empastamiento y capacidad de formación de complejo con yodo. La harina precocida se obtuvo a partir de trozos de parénquima de yuca cocinados en vapor o en agua a ebullición, los cuales fueron posteriormente almacenados a 5 ºC o a -20 ºC por 24 h. La temperatura utilizada durante el periodo de almacenamiento del parénquima cocinado no es un factor significativo en los resulta dos de retrogradación del almidón. La entalpía de fusión y cristalinidad del almidón retrogradado de la harina proveniente del parénquima cocinado en vapor fue ligeramente mayor, comparado con la elaborada a partir de parénquima cocinado en agua a ebullición para el periodo de almacenamiento a 5 ºC. Por otra parte, en el periodo de almacenamiento a -20ºC, el método de cocción no tuvo efecto significativo sobre la entalpía de fusión del almidón retrogradado, la cristalinidad y el índice del valor azul.

Starch modifications during the processing of precooked cassava flour was monitored using techniques as differential scanning calorimetry (DSC), pasting behaviour, wide angle X-ray diffraction and iodine binding capacity. Cassava flour was obtained from parenchyma pieces cooked either in steam or in boiling water and then stored either at 5 ºC or at -20 ºC for 24 h. The temperature during the rest period of the cooked parenchyma was not a significant factor in the starch retrogradation results. For a rest period at 5 ºC, flour from parenchyma cooked in steam presented a slightly higher melting enthalpy of retrograded starch and crystallinity as compared to that from parenchyma cooked in boiling water. Whereas for conditioning period at -20 ºC, the cooking method had no significant effect on the enthalpy of retrogradation, crystallinity and blue value index.

As modificações do amido durante o processo de elaboração de farinha de mandioca precozida, foram avaliadas utilizando as técnicas de calorimetria diferencial de varredura (CDV), difração de raios X, comportamento no empastamento e capacidade de formação de complexo de iodo. A farinha precozida obteve-se de troços do parênquima da mandioca cozidos com vapor o em água em ebulição, os quais foram armazenados depois a 5 ºC ou -20 ºC por 24 h . A temperatura do armazenamento do parênquima cozido no foi um factor significativo nos resulta dos de retrogradação do amido. A entalpia de fusão e cristalinidade do amido retrogradado da farinha proveniente do parênquima cozido no vapor foi levemente maior, do que aquela elaborada do parênquima cozido em água em ebulição e armazenada a 5 ºC. Além disso, o método de cozimento não teve efeito significativo na entalpia de fusão do amido retrogradado, na cristalinidade o no índice de valor azul (IVA).

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

Preparation of canine multivalence serum and its application / 中国免疫学杂志

Objective:To prepare the canine multivalence serum for emergent prevation and theatmentor canines infection disease.Methods:Selecting 20 healthy Kunming dog,which is more than one years old.The canines were immuned by regulary with Canine distemper virus(CDV),Canine parvovirus virus(CPV),Infectious canine hepatitis virus(ICHV),Canine coronavirus(CCV),Canine parainflu-enza virus(CPIV).The blood were draw in freedom germ from neck's artery at canines and then blood was centrifugate for isolating serum.To check antibody's tite, micro serum neutrolization(SN) and hemagglutination inhibition(HI) were used.Results:Canine multivalence serum is successfully preparation and good curative effects in clinic.Conclusion:Canine multivalence serum may be of great value in economics and society and contribute to curative disease.