First description of sarcoptic mange in a wild coati (Nasua narica), in Ecuador, and cooccurrence of canine distemper virus / Primeira descrição da sarna sarcóptica em um coati selvagem (Nasua narica), no Equador, e coocorrência do vírus distemper canino

Abstract We present a case of Sarcoptes and canine distemper virus (CDV) infection in a white-nosed coati (Nasua narica) that was trapped in the dry tropical forest of Cerro Blanco reserve, located in the coastal region of Ecuador. Sarcoptic mange is a highly contagious and zoonotic disease with worldwide distribution that causes epidemics. Mange is produced by Sarcoptes mites that causes severe epidermal damage. Secondary infections and physiological constrictions without treatment can lead to death of the host. In addition, cooccurrence of canine distemper virus was detected via iiRT-PCR from serum samples. Physical analyses showed that 90% of the skin was affected by severe alopecia due to the sarcoptic mange infection. The presence of mites and histopathological analyses confirmed the diagnosis of infection. This coati was taken to a veterinary clinic and was fed every day, but it died after four days. This is the first report of sarcoptic mange and the first report of CDV in white-nosed coatis in South America. Further studies are needed in this region, to seek out other suspected cases, given the high capacity for disease transmission. Preventive actions to avoid epidemic and zoonotic episodes are needed.

Resumo Apresentamos um caso de Sarcoptes e infecção pelo vírus da cinomose canina (CDV) em um quati-do-nariz-branco (Nasua narica) que ficou preso na floresta tropical seca da reserva de Cerro Blanco, localizada na região costeira do Equador. A sarna sarcóptica é uma doença altamente contagiosa e zoonótica de distribuição mundial que causa epidemias. A sarna é produzida por ácaro do gênero Sarcoptes que causa graves danos epidérmicos. Infecções secundárias e constrições fisiológicas sem tratamento podem levar à morte do organismo. Além disso, a coocorrência do vírus da cinomose canina foi detectada, via iiRT-PCR, a partir de amostras de soro. As análises físicas mostraram que 90% da pele estava afetada por alopecia severa devido à infecção pelo ácaro da sarna sarcóptica. A presença de ácaros e análises histopatológicas confirmaram o diagnóstico de infecção. Esse quati foi levado a uma clínica veterinária e foi alimentado todos os dias, mas morreu após quatro dias. Esse é o primeiro relato de sarna sarcóptica e o primeiro relato de CDV em quatis-de-nariz-branco na América do Sul. São necessários mais estudos nessa região, para buscar outros casos suspeitos, dada a alta capacidade de transmissão da doença. Ações preventivas para evitar episódios epidêmicos e zoonóticos, são necessárias.

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.

Características epizootiológicas da infecção natural pelo vírus da cinomose canina em Belo Horizonte / Natural canine distemper virus infection epizootiology characteristics in Belo Horizonte

O perfil epizootiológico da cinomose canina em Belo Horizonte é desatualizado e não alberga algumas características relevantes. Uma análise recente da distribuição do vírus em relação às características do hospedeiro e do meio ambiente associada aos principais sinais clínicos e achados laboratoriais são importantes para se adotarem medidas estratégicas para o controle da enfermidade. Objetivou-se, assim, determinar as características epizootiológicas da infecção pelo vírus da cinomose canina associada à variedade de sinais clínico-neurológicos e laboratoriais em Belo Horizonte, auxiliando no diagnóstico precoce da infecção e na diminuição das taxas de morbidade e mortalidade da doença. A avaliação do perfil epizootiológico de 90 cães revelou que a doença é mais frequente em animais adultos (um a seis anos de idade) e que não receberam vacinas conforme recomendado pelos protocolos. Os sinais clínicos extraneurais e neurais foram variados, com predomínio para manifestações gastrentérica e respiratória, mioclonia e déficit motor, respectivamente. O exame do fluido cérebro-espinhal demonstrou predomínio de proteinorraquia associada à pleocitose linfocítica. O teste de imunocromatografia para pesquisa de antígeno com amostras do fluido cerebroespinhal foi eficaz para identificar a doença em pacientes com sinais neurológicos, diferentemente das amostras do swab conjuntival, que não devem ser utilizadas.(AU)

The epizootiology profile of canine distemper in Belo Horizonte is outdated and does not harbor some important characteristics. A recent analysis of the virus distribution in relation to host and environmental characteristics associated with the main clinical signs and laboratory findings are important for adopting strategic measures to control the disease. The aim of this study was to determine the epizootiology characteristics of canine distemper virus infection associated with a variety of clinical and neurologic signs and laboratory findings in Belo Horizonte, helping to detect early infection and reduce morbidity and mortality rates. The evaluation of the epizootiology profile of 90 dogs revealed that the disease is more frequent in adult animals (1-6 years of age) and did not receive vaccines as recommended by the protocols. Extra neural and neural clinical signs were varied, with predominance for gastrointestinal and respiratory manifestations and myoclonus and motor deficit, respectively. Examination of the cerebrospinal fluid of 16 dogs showed a predominance of increase protein associated with lymphocytic pleocytosis. The immunochromatography test for antigen screening with samples of cerebrospinal fluid in 76 animals with neurological signs was effective in identifying the disease, unlike conjunctival swab samples, which should not be used.(AU)

Anatomopathological changes in canine distemper seropositive dogs and virus detection in sinoatrial nodes / Alterações anatomopatológicas em cães soropositivos para cinomose e detecção de vírus em nódulos sinoatriais

Canine distemper is a viral disease that affects several systems on dogs, among them, the cardiovascular system. The aim of this study was to identify canine distemper virus (CDV) in the sinoatrial node (SAN) of dogs serologically positive for distemper by Polymerase Chain Reaction preceded by reverse transcription (RT-PCR), and to analyze gross and microscopic changes of distemper in the heart and other tissues. SAN and tissue fragments were collected from 17 serologically positive dead animals, necropsied from October 2015 to December 2016. In the heart, right heart dilatation was observed in 13 dogs (76.47%) and left concentric hypertrophy in two dogs (11.76%). Microscopically, lymphocytic myocarditis was observed in four (23.53%) dogs and 41.18% presented viral inclusion corpuscles of CDV in the bladder epithelium. Only one (5.88%) dog presented a 319 bp target fragment for distemper virus using primers CDV 1 and CDV 2 at the sinoatrial node. In conclusion, CDV can be located in the sinoatrial node of naturally infected dogs, as demonstrated in this study by the RT-PCR technique, reinforcing the hypothesis that CDV is capable of causing inflammatory lesions in the sinoatrial node of this species. Macroscopic and microscopic cardiac changes are frequently observed in dogs with distemper, mainly cardiac dilatation and myocarditis. Viral inclusions of CDV in bladder epithelial cells are an important microscopic finding for the diagnosis of distemper.

A cinomose canina é uma doença viral que afeta vários sistemas, dentre eles o cardiovascular. Objetivou-se identificar o vírus da cinomose canina no nó sinoatrial (NSA) de cães sorologicamente positivos para cinomose, através da reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR), além de analisar os achados macroscópicos e histológicos da cinomose no coração e outros tecidos. Foram coletados fragmentos de tecidos e do NSA de 17 cães sorologicamente positivos para cinomose que vieram a óbito e foram necropsiados no período de outubro de 2015 a dezembro de 2016. No coração observou-se dilatação cardíaca direita em 76,47% dos cães e hipertrofia concêntrica esquerda em 11,76% dos cães. Microscopicamente observou-se miocardite linfocítica em 23,53% dos cães e 41,18% apresentou corpúsculos de inclusão viral no epitélio vesical. Somente um (5,88%) cão apresentou fragmento alvo de 319 bp para cinomose utilizando os primers VCC1 e VCC2, no nó sinoatrial. Conclui-se que o VCC pode localizar-se no nó sinoatrial de cães naturalmente infectados, como demonstrados neste estudo pela técnica de RT-PCR, reforçando a hipótese de que o VCC é capaz de provocar lesões inflamatórias no nó sinoatrial dessa espécie. Alterações cardíacas macroscópicas e microscópicas, principalmente dilatação cardíaca e miocardite, são frequentemente observadas em cães com cinomose. Inclusões virais nas células epiteliais da bexiga são importantes achados microscópicos para diagnóstico da cinomose.

Antibodies against canine distemper virus, parvovirus and Ehrlichia spp. in wild captive carnivores in midwestern Brazil / Anticorpos contra o vírus da cinomose canina, parvovírus e Ehrlichia spp. em carnívoros selvagens cativos no centro-oeste do Brasil

The occurrence of antibodies against canine distemper virus (CDV), parvovirus and Ehrlichia spp. in wild captive carnivores was evaluated in a zoological park in midwestern Brazil. Serum samples were collected between 2007 and 2014 from 45 carnivores. Antibodies were evaluated by virus neutralization assay for CDV, hemagglutination inhibition test for parvovirus, indirect immunofluorescent and Enzyme-linked immunosorbent assay for Ehrlichia spp. Antibodies against CDV and parvovirus were detected in 75% of Canidae and Felidae. Procyonidae were negative for CDV, although one Mustelidae was positive. TwoCanidae presented antibodies reactive to E. canis antigens. The high antibodies rates to CDV and parvovirus suggest the contact with both pathogens, however since no clinical history of disease are registered in the Zoo-UFMT, we can presume that carnivores have responded satisfactorily against the antigens. The low serological rates observed against Ehrlichia spp. may be resulted to the low occurrence of ticks among carnivores.(AU)

A ocorrência de anticorpos contra o vírus da cinomose canina (CDV), parvovírus e Ehrlichia spp. em carnívoros selvagens em cativeiro foi avaliada em um parque zoológico do centro oeste do Brasil. As amostras de soro foram coletadas entre 2007 e 2014 de 45 carnívoros. Os anticorpos foram avaliados por ensaio de neutralização de vírus para CDV, teste de inibição de hemaglutinação para parvovírus, imunofluorescência indireta e ensaio imunoenzimático ligado à enzima para Ehrlichia spp. Anticorpos contra CDV e parvovírus foram detectados em 75% de canídeos e felídeos. Procionídeos foram negativos para CDV, embora um mustelídeo fora positivo. Dois canídeos apresentaram anticorpos reativos aos antígenos de E. canis. As altas taxas de anticorpos para CDV e parvovírus sugerem o contato com ambos os patógenos, entretanto desde que nenhuma história clínica de doença está registrada no Zoo-UFMT, podemos presumir que os carnívoros têm respondido satisfatoriamente contra os antígenos. As baixas taxas serológicas observadas contra Ehrlichia spp. pode ser resultado da baixa ocorrência de carrapatos entre os carnívoros.(AU)

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)

Inhibitory effect of favipiravir on canine distemper virus replication in vitro / 军事医学

Objective To investigate the inhibitory effect of favipiravir (T-705) on canine distemper virus (CDV) replication in Vero cells and DH82 cells.Methods The growth curves of CDV-11 strains from canine and CDV-3 strains from mink in Vero cells and DH82 cells were determined with indirect immunofluorescence assay and 50％ endpoint titration.The viability of Vero and DH82 cells was determined using the Cell Counting Kit-8.CDV inhibition at different concentrations of T-705 at different time points was measured .Results Cytotoxicity data showed that there was a moderate decline of viability in Vero cells after T-705 treatment, but no apparent cytotoxicity in DH 82 cells.T-705 significantly inhibited the replication of CDV-3 and CDV-11 in both Vero cells and DH82 cells in the test range of 2.441-1250 μg/ml. T-705 exhibited effective and stable antiviral activity when given at different time points post virus challenge .Conclusion Our results demonstrate that T-705 has effective antiviral activity and may be a promising anti-CDV drug candidate .

Genetic characterization of the haemagglutinin gene in canine distemper virus strains from naturally infected dogs in Brazil / Caracterização genética do gene da hemaglutinina em vírus da cinomose canina de cães naturalmente infectados no Brasil

Canine distemper is one of the major infectious diseases in dogs and wild animals, resulting in high morbidity and mortality. The H gene has the greatest genetic variability among the genes encoded by the canine distemper virus (CDV) genome, and it has been used to characterise field samples, allowing the identification of specific lineages. Variation in the H gene can allow the virus to evade recognition by vaccine-induced antibodies, resulting in vaccine failure. The purpose of this study was to characterise H gene in CDV strains from naturally infected dogs in the state of São Paulo. The phylogenetic analysis revealed that Brazilian CDV strains were genetically related to the circulating CDV strains in Uruguay, Argentina, and Europe. We found no evidence of South America 2 and 3 CDV lineages circulating in Brazilian dogs. The degree of genetic divergence between wild Brazilian CDV strains and vaccine strains may suggest the possibility of vaccine failures and consequently the occurrence of canine distemper outbreaks.(AU)

A cinomose canina é uma das principais doenças infecciosas em cães e animais selvagens, resultando em alta morbidade e mortalidade. O gene H tem uma das maiores variabilidades genéticas entre os genes codificados pelo vírus da cinomose canina (CDV), e tem sido utilizado para caracterizar as estirpes de CDV, permitindo a identificação de linhagens específicas. A variação no gene H pode permitir que o vírus evite o reconhecimento por anticorpos induzidos pela vacina, resultando em falha vacinal. O objetivo deste estudo foi caracterizar o gene H em estirpes de CDV de cães infectados naturalmente no estado de São Paulo. A análise filogenética revelou que as estirpes de CDV brasileiras estão geneticamente relacionadas as estirpes circulantes no Uruguai, na Argentina e na Europa. Não foi encontrada nenhuma evidência da circulação no estado de São Paulo das linhagens América do Sul 2 e 3. O grau de divergência genética entre linhagens selvagens de CDV brasileiras e as estirpes vacinais podem sugerir a possibilidade de falhas vacinais e consequentemente a ocorrência de surtos de cinomose canina.(AU)

Paper-Based Microfluidics Immunoassay for Detection of Canine Distemper Virus

ABSTRACT Paper-based devices present low-cost and are versatile, making them very attractive for clinical analysis. To manufacture those devices wax patterns are printed on paper surface and upon heating the wax permeates through the entire thickness of the paper, creating hydrophobic barriers that delimit test areas. Antibodies produced in rabbits against canine distemper virus (CDV) were physically adsorbed on the surface of gold nanoparticles (AuNPs) and incubated with CDV viral antigens, forming the immunocomplex. Anti-CDV antibodies were immobilized into the microchannels by physical adsorption, forming the test region. The test solution containing conjugated AuNPs was applied at the bottom of the microchannel and it was eluted with a phosphate buffer solution 0.01 M pH 7.4. When the solution containing the AuNPs reached the test zone the recognition of antigens contained on the immunocomplex occurred with the consequent development of a red line, which represents a positive outcome for the test. This method demonstrated the success of physical immobilization of antibodies on AuNPs and the physical immobilization of antibodies on cellulose's surface. This colorimetric assay brings simplicity and versatility to clinical analyses, presenting potential for CDV diagnosis.

Establishment and application of a multiplex PCR assay for detection of Aleutian disease parvovirus, enteritis parvovirus and canine distemper virus in the mink / 中国比较医学杂志

Objective Aleutian disease, mink enteritis and canine distemper are the three major diseases affecting health of mink. This study intends to establish a multiplex PCR assay for simultaneously detecting of these three viruses. Methods According to the conservative sequences reported in GenBank, three pairs of specific primers were designed to amplify the DNA templates of Aleutian mink disease parvovirus (ADV), mink enteritis parvovirus (MEV), and RNA templates of canine distemper virus (CDV), and optimized the amplifying conditions. Results The specific objective strips of 601 bp ( ADV) , 205 bp ( MEV) and 451 bp ( CDV) were amplified simultaneously. The sensitivity test showed that the lowest nucleic acid detection limits were 2. 67 × 104 copies perμL for ADV, 3. 02 × 104 copies perμL for MEV, and 1. 72 × 105 copies perμL for CDV. The results of test of the clinical samples showed that the multiple PCR and single PCR assay were consistent. Conclusions The established multiplex PCR assay in this study can be used to rapidly detect the clinical samples of ADV, MEV and CDV single or mixed infections.

Detection of viral infections in wild Korean raccoon dogs (Nyctereutes procyonoides koreensis)

Wild raccoon dogs (Nyctereutes procyonoides koreensis) may play a role transmitting several pathogens to humans and pet animals. Information concerning the incidence of rabies, canine distemper virus (CDV), canine parvovirus (CPV), canine adenovirus type 2 (CAdV-2), canine parainfluenza virus type 5 (CPIV-5), and canine herpesvirus (CHV) is needed in wild raccoon dogs. In total, 62 brain samples of raccoon dogs were examined for rabies virus (RABV) and CDV, and 49 lung samples were screened for CDV, CAdV-2, CPIV-5, and CHV. No RABV, CAdV-2, CPIV-5, or CHV was identified, but nine CDV antigens (8.1%, 9/111) were detected. Moreover, 174 serum samples from wild raccoon dogs were screened for antibodies against the five major viral pathogens. The overall serosurveillance against CDV, CPV, CAdV-2, CPIV-5, and CHV in wild raccoon dogs was 60.3%, 52.9%, 59.8%, 23.6%, and 10.3%, respectively. Comparisons of the sero-surveillance of the five pathogens showed that raccoon dogs of Gyeonggi province have slightly higher sero-positive rates against CDV, CPV, and CHV than those of Gangwon province. These results indicate high incidences of CDV, CPV, and CAdV-2 in wild raccoon dogs of two Korean provinces and a latent risk of pathogen transmission to companion and domestic animals.

Canine distemper virus detection by different methods of One-Step RT-qPCR / Detecção do vírus da cinomose canina por diferentes métodos de One-Step RT-qPCR

ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

RESUMO: Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição seriada de RNA sintético do Vírus da Cinomose Canina ou de uma amostra de urina positiva. Adicionalmente, uma amostra de urina foi avaliada com centrifugação ou ultracentrifugação prévia. Os kits comerciais de One-Step RT-qPCR amplificaram o RNA do vírus da cinomose canina em 10 (100%) amostras de urinas de animais sintomáticos. O kit de One-Step RT-qPCR que apresentou melhor resultado foi utilizado para avaliar a sensibilidade analítica dos sistemas A e B. Na reação da curva padrão com RNA sintético, o limite de detecção do sistema A foi de 11 cópias de RNA µL-1. No sistema B foi de 110 cópias de RNA µL-1 na One-Step RT-qPCR e 11 cópias de RNA µL-1 na NESTED-qPCR. A relação entre os valores de Ct e concentração de RNA foi linear. O RNA extraído das diluições da urina foi detectado nas diluições de 10-3 e10-2 pelos sistemas A e B, respectivamente. A centrifugação prévia da urina aumentou a sensibilidade analítica da análise e mostrou ser importante para a rotina diagnóstica. A reação de One-Step RT-PCR é um método rápido, sensível, específico e aplicável na rotina de diagnóstico molecular da cinomose e em projeto de pesquisa que requer metodologia quantitativa e sensível.

Molecular detection and phylogenetic relationship of wild-type strains of canine distemper virus in symptomatic dogs from Uberlândia, Minas Gerais / Detecção molecular e relação filogenética de cepas selvagens do vírus da cinomose canina em cães sintomáticos oriundos de Uberlândia, Minas Gerais

This study investigated the occurrence of canine distemper virus (CDV) by evaluating the presence of viral RNA within urine samples of dogs from Uberlândia, MG, with clinical manifestations suggestive of infection by CDV by targeting the CDV N gene. Of the clinical samples collected ( n =33), CDV viruria was detected in 45.5%. Five dogs died spontaneously; all had characteristic CDV-associated histopathological alterations and demonstrated CDV viruria. Statistical analyses revealed that the age, gender, breed, or the organ system of the dog affected had no influence on the occurrence of canine distemper. Myoclonus and motor incoordination were the most significant neurological manifestations observed. A direct association was observed between keratoconjunctivitis and dogs with CDV viruria. These findings suggest that CDV viruria in symptomatic dogs might not be age related, and that symptomatic dogs can demonstrate clinical manifestations attributed to CDV without viruria identified by RT-PCR. Additionally, the results of the sequence identities analysed have suggested that all Brazilian wild-type strains of CDV currently identified are closely related and probably originated from the same lineage of CDV. Nevertheless, phylogenetic analyses suggest that there are different clusters of wild-type strains of CDV circulating within urban canine populations in Brazil.

A presença do ácido nucleico (RNA) do vírus da cinomose canina (CDV) foi avaliada por meio da amplificação parcial do gene N pela técnica RT-PCR realizada em urina de cães provenientes de Uberlândia, Minas Gerais, que apresentavam sinais clínicos sugestivos de cinomose. Das 33 amostras de urina avaliadas, o CDV foi identificado em 45,5%. Em cinco cães que morreram espontaneamente, além da excreção do CDV na urina, foram observadas alterações histopatológicas associadas à infecção por esse vírus. Análises estatísticas demonstraram que a idade, gênero, raça e o sistema orgânico comprometido dos cães avaliados não exerceram influência no diagnóstico da cinomose canina. Mioclonia e incoordenação motora foram as manifestações neurológicas que apresentaram frequência de ocorrência significativa (P<0,05). Uma associação direta foi observada entre a presença de ceratoconjuntivite e a identificação de virúria pelo CDV. Esses achados sugerem que a excreção do CDV pela urina em cães com sinais clínicos compatíveis com cinomose pode não ser relacionada com a idade do animal, e que animais sintomáticos podem apresentar manifestações clínicas atribuídas ao CDV, porém sem a caracterização de virúria por RT-PCR. Adicionalmente, análises filogenéticas sugerem que várias cepas de CDV podem estar circulando em populações caninas de áreas urbanas no Brasil.

Systemic acanthamoebiasis associated with canine distemper in dogs in the semiarid region of Paraíba, Brazil

Infections by free-living amoebae can cause systemic disease in animals and humans. We describe the epidemiological, clinical and pathological aspects of disseminated acanthamoebiasis associated with canine distemper in three dogs of the semiarid region of Paraíba, Northeastern Brazil. Affected dogs developed progressive neurological and respiratory signs that progressed to death within in two to 20 days. Gross lesions were irregular and with yellow-reddish nodules randomly distributed in the lungs, heart, kidneys, spleen, lymph nodes, adrenals, and intestine. One dog had foci of malacia in the parietal cortex and another one in nucleus of brain basis. Histologically, pyogranulomas with areas of necrosis and hemorrhage in all organs affected were observed, associated with myriads of intralesional amoebic trophozoites. All three cases were concomitant canine distemper, that possibly triggered immunosuppression in the dogs. The diagnosis was performed through microscopic findings of infection by free-living amoebae and confirmed Acanthamoeba sp. by immunohistochemistry.

Infecções por amebas de vida livre podem causar doença sistêmica nos animais e no homem. Descrevem-se os aspectos epidemiológicos, clínicos e patológicos de acanthamoebíase disseminada associada com cinomose em três cães na região semiárida da Paraíba, Nordeste do Brasil. Os cães afetados desenvolveram sinais respiratórios e neurológicos progressivos, que evoluíram para a morte em dois a 20 dias. Na necropsia havia áreas nodulares, irregulares e amarelo-avermelhadas distribuídas aleatoriamente em pulmões, coração, rins, fígado, baço, linfonodos, adrenal e intestino. Em um caso havia foco de malácia no córtex parietal e outro em núcleo da base encefálica. Histologicamente, foram observados piogranulomas com áreas de necrose e hemorragia em todos os órgãos afetados, associados a miríades de amebas intralesionais. Nos três casos havia cinomose concomitante, que possivelmente desencadeou imunossupressão nos cães. O diagnóstico foi realizado através dos achados microscópicos de infecção por amebas de vida livre e confirmado Acanthamoeba sp. pela imuno-histoquímica.

An Assessment of Immune Response to Canine Distemper Vaccination in Dogs Experimentally Infected with Ancylostoma and Trypanosome Parasites.

The immunological alteration in vaccinated dogs with single hookworm, Ancylostoma caninum (A. c) and conjunct infection with Trypanosoma congolense (T. c) and Trypanosoma brucei (T. b) was determined. Sixteen dogs grouped into 4 of 4 members each were used. Group 1 was the uninfected control, GPII was infected with A. c, GPIII was infected with A. c /T. c, and GPIV was infected with T. b/A. c. The dogs were first inoculated with canine distemper (CD) vaccine before infection with A. c 4 weeks post vaccination. Two weeks later, both GPIII and GPIV were superposed with trypanosome infection. Prepatent period of A. c was 14 to 16 days in single A. c group and 13 to 14 days in conjunct trypanosome/A. c. The prepatent period of conjunct T. c/A. c was 9.00±1.10 days and 3.00±1.40 days, in conjunct T.bb/A. c. The protective antibody against CDV was considered using haemagglutination inhibition test (HIT) titer >100 as a cut off for seroconversion. At one week post vaccinations, the antibody titer against canine distemper (CDV) and anti-rabies in all the vaccinated groups (GPI, GPII, GPIII, and GPIV) significantly increased (p<0.05) and peaked at 3 weeks post vaccination. Subsequently, there was gradual significant decrease (p<0.05) in all the infected groups (GPII, GPIII and GPIV). The decrease in the conjunct groups (GPIII and GPIV) was higher compared to the single infections (GPII). Treatment with diminazene aceturate and mebendazole in all the groups did not significantly (p<0.05) improve antibody response in the dogs. A secondary vaccination administered at 12 weeks post- primary vaccination significantly increased (p<0.05) the antibody titer with a peak 3 weeks post- secondary vaccination. In conclusion, both trypanosomes and A. c induced primary immune suppression in antibody response to vaccination which improved on secondary vaccination in the infected dogs.

Evaluation of RT-PCR and hemi-nested RT-PCR in brain samples from dogs with neurologic signs compatible with distemper / Avaliação das técnicas de RT-PCR e heminested RT-PCR em cérebros de cães com sinais neurológicos compatíveis com cinomose

The diagnostic value of RT-PCR and hemi-nested RT-PCR (hnRT-PCR) was compared in brain samples of dogs presenting neurological signs compatible with canine distemper. Samples of central nervous system (CNS) were collected from 68 dogs and tested by direct immunofluorescence test (RFID) and, independent of the results, they were stored at -20°C for at least three years. They were submitted to the RT-PCR and hnRT-PCR techniques aiming to determine the gene responsible for the viral nucleoprotein decoding. Fifty-nine samples were positive for RIFD, 40 for RT-PCR (Kappa = 0.358) and 54 for hnRT-PCR (Kappa = 0.740). All nine RIFD negative samples were also negative for RT-PCR and hnRT-PCR. In spite of the storage duration and proper sample conditions, the estimated accordance between hnRT-PCR and RIFD demonstrated that hnRT-PCR technique can be applied in retrospective studies...

Foi comparado o valor diagnóstico das técnicas de RT-PCR e heminested RT-PCR (hnRT-PCR) em amostras de cérebro de cães com sintomatologia nervosa compatível com cinomose. Fragmentos do sistema nervoso central (SNC) colhidos de 68 animais foram testados pela Imunofluorescência direta (IFD) e, independentemente do resultado, foram armazenados a -20°C por pelo menos três anos. Após esse período, foram submetidos a RT-PCR e a hnRT-PCR com oligonucleotídeos iniciadores direcionados ao gene codificador da nucleoproteína N. As proporções de resultados positivos/examinados foram: 59/68 para a IFD, 40/68 para a RT-PCR (Kappa = 0,358) e 54/68 quando associada à heminested PCR (Kappa = 0,740). Houve nove resultados negativos nas três técnicas empregadas. Os resultados do coeficiente Kappa entre a IFD e hnRT-PCR demonstram que apesar das condições de armazenamento, a hnRT-PCR pode ser utilizada em estudos retrospectivos...

Establishment of canine kidney cell line for canine distemper virus replication / 동물의과학연구지

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

Efeitos colaterais do uso da ribavirina, prednisona e DMSO em cães naturalmente infectados pelo vírus da cinomose / The side effects of ribavirin, prednisone and DMSO in dogs naturally infected by canine distemper virus

O estudo tem o objetivo de identificar efeitos indesejáveis da ribavirina, prednisona e DMSO em cães naturalmente infectados com o vírus da cinomose. Foram utilizados 60 cães apresentando quadro neurológico da cinomose com evolução de 10 dias. Os animais foram internados e receberam tratamento de suporte; foram avaliados diariamente e realizados hemograma, dosagem bioquímica e exame de urina tipo I. Os grupos 1 e 2 foram tratados com ribavirina e sua associação com DMSO; os grupos 3 e 4 com DMSO e prednisona e o grupos 5 com ribavirina e prednisona e o grupo 6 com ribavirina, prednisona e DMSO. Os animais foram anestesiados para a colheita de líquor, medula óssea e sangue, antes do tratamento para diagnóstico através da RT-PCR. As amostras negativas foram analisadas pela técnica de hn-PCR. Todos os animais apresentaram resultado positivo em pelo menos uma das duas reações. O efeito adverso da ribavirina e a sua associação com a prednisona foi a anemia hemolítica, que foi confirmada pela observação de bilirrubina na urina apenas dos cães tratados com ribavirina.

The present study aims at the identification of undesirable effects of ribavirin, predinisone and DMSO in dogs naturally infected by canine distemper virus. The research analyzed 60 dogs with clinical neurological signs and 10 days of evolution. The animals were hospitalized for the appropriate support treatment; were daily observed, and complete blood cells count, biochemical analysis, and urine exam type I were conducted. Groups 1 and 2 were treated with ribavirin and its combination with DMSO; Groups 3 and 4 treated with prednisone and DMSO, Group 5 treated with ribavirin and prednisone, while Group 6 with ribavirin, prednisone and DMSO. Before the treatment, animals were anesthetized for the cerebrospinal fluid, bone marrow and blood samples collection for the diagnosis based on RT-PCR. The negative samples were analyzed using the hn-PCR technique. All the animals presented positive results in at least one of the 2 tests. The adverse result of ribavirin and its association with prednisone was characterized by haemolytic anemia, confirmed by the evaluation of bilirrubin occurrence only in the urine of dogs treated with ribavirin.

Use of hydrophilic extra-viral domain of canine distemper virus H protein for enzyme-linked immunosorbent assay development

Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.

Public survey of knowledge concerning canine distemper and protective measures

To help understand why there is a prevalence of canine distemper in Brazil, we interviewed 404 owners of dogs that were in clinics or veterinary hospitals about distemper. Our results showed that 80.4% of respondents knew about distemper and 67.6% knew that it is a severe and fatal disease. Approximately 50.9% knew that distemper can be prevented by the use of the vaccine, but 60.9% did not know about distemper vaccines. Our results show that the lack of knowledge and, consequently, the lack of vaccination against distemper may be the most important factors in the endemic presence of distemper in Brazil.