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1.
Res Vet Sci ; 128: 197-204, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31816502

RESUMO

Excretory and secretory products (ESPs) are released by the parasites during Haemonchus contortus (H. contortus) infection. In this study, Tropomyosin (TpMy), one of these ESPs was used to develop western blotting and optimized Enzyme Linked immunosorbent assay (ELISA) for detection of H. contortus during early infection in goat. Microscopic examination was performed parallel for comparison. Recombinant tropomyosin protein was purified successfully. Western blotting results revealed that anti-recombinant H. contortus Tropomyosin (rHc-TpMy) antibodies could recognize the natural proteinand rHc-TpMy antigen did not show any cross-reaction with goat anti-sera of Fasciola hepatica, Trichinella spiralis, and Toxoplasma gondii. Moreover, initial antibodies were detected by both western blotting and indirect ELISA at 14 days post infection (DPI) and persisted till 30 DPI but fecal eggs count couldn't detect the eggs in feces at early stage (7 and 14 DPI). The optimized antigen coating concentration was calculated as 10 µg/ml (P/N Optimum Density450 = 4.165) with optimized dilution of serum (1:50) and secondary antibody (1:2500). Positive and negative cutoff value of the indirect-ELISA assay was calculated as 0.392 and 0.344, respectively. Receiver operating characteristic curve analysis validated the cutoff value (0.392) based on a high specificity and sensitivity. Indirect ELISA showed 90% diagnostic sensitivity and 100% diagnostic specificity. In comparison of serological and conventional method, rHc-TpMy based indirect ELISA showed more positive results (30%; 9/30) than microscopic examination (20%; 6/30). These results demonstrated that rHc-TpMy is a potential immunodiagnostic antigen to detect specific antibodies at early stage of infection in goat and serological methods are more reliable as compared to microscopic examination.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Hemoncose/diagnóstico , Haemonchus , Testes Sorológicos/veterinária , Tropomiosina/imunologia , Animais , Antígenos de Helmintos , Western Blotting/veterinária , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/veterinária , Fezes/parasitologia , Cabras , Haemonchus/isolamento & purificação , Haemonchus/parasitologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade
2.
Vet Parasitol ; 276: 108962, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31704559

RESUMO

Sarcocystis neurona is the major cause of the equine protozoal myeloencephalitis (EPM) in the Americas and has opossums of the genus Didelphis as definitive hosts. Most isolates of Sarcocystis sp. shed by opossums in Brazil differ genetically from the known species of Sarcocystis. These Brazilian isolates behave similarly as Sarcocystis falcatula, which causes sarcocystosis in birds, and for this reason, have been classified as Sarcocystis falcatula-like. Genes coding for the immunodominant surface antigens SAG2, SAG3 and SAG4 of S. falcatula-like are similar to those from S. neurona. It is unknown the Sarcocystis species that causes EPM in Brazil, as S. neurona has never been genetically confirmed in Brazilian horses. All cases associated with EPM in Brazil were diagnosed by immunological tests, which are not specific for S. neurona infection. It is possible that S. falcatula-like may infect horses in Brazil. The aims of the current study were to test the susceptibility of gerbils (Meriones unguiculatus) to experimental infections with S. neurona and S. falcatula-like, and to investigate potential serologic cross-reactivity to these parasites by immunofluorescent antibody test (IFAT) and Western blot (WB). A total of 27 gerbils, distributed in five experimental groups (G1-G5), were employed in this work (G1: 4 negative controls; G2: 6 infected with S. neurona merozoites, G3: 6 infected with S. falcatula-like merozoites; G4 and G5 (5 and 6, respectively, infected with different doses of sporocysts). None of the 17 animals that seroconverted for the parasites in IFAT presented any visualized organism or Sarcocystis DNA in the examined tissues. No serologic cross-reactivity was observed using IFAT. However, sera from animals infected with S. falcatula-like and S. neurona presented the same pattern of antigenic recognition when S. neurona merozoites were used as antigen in WB, including reactivity to proteins of 30 and 16 kDa, regarded as specific markers for S. neurona-infected animals. Gerbils did not sustain infection by these parasites, although produced antibodies after inoculation. These results are suggestive that other animal species that are exposed to S. falcatula-like, including horses, may present serologic cross-reactivity to S. neurona in WB. IFAT was demonstrated to be more specific that WB for the detection of antibodies to S. falcatula-like and S. neurona in the experimental conditions of this study.


Assuntos
Antígenos de Protozoários/imunologia , Sarcocystis/imunologia , Sarcocistose/imunologia , Animais , Antígenos de Superfície/imunologia , Western Blotting/veterinária , Linhagem Celular , Galinhas , Chlorocebus aethiops , Reações Cruzadas , Didelphis/parasitologia , Encefalomielite/imunologia , Encefalomielite/parasitologia , Encefalomielite/veterinária , Feminino , Imunofluorescência/veterinária , Gerbillinae , Epitopos Imunodominantes/imunologia , Reação em Cadeia da Polimerase , Sarcocistose/parasitologia , Sarcocistose/patologia , Células Vero
3.
Vet Res ; 50(1): 97, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767033

RESUMO

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.


Assuntos
Western Blotting/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Cabras/classificação , Scrapie/classificação , Animais , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Europa (Continente) , Doenças das Cabras/diagnóstico , Cabras , Scrapie/diagnóstico
4.
BMC Vet Res ; 15(1): 369, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653217

RESUMO

BACKGROUND: Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer's instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. RESULTS: The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. CONCLUSIONS: The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.


Assuntos
Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Mycoplasma/veterinária , Animais , Western Blotting/veterinária , Bovinos , Doenças dos Bovinos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Análise de Classes Latentes , Infecções por Mycoplasma/sangue , Infecções por Mycoplasma/diagnóstico , Mycoplasma bovis/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/veterinária
5.
Exp Parasitol ; 206: 107758, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31521628

RESUMO

The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked immunosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis-infected sera and the lowest OD values with normal bovine sera or B. bigemina-infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis.


Assuntos
Antígenos de Protozoários/imunologia , Babesia bovis/imunologia , Babesiose/parasitologia , Doenças dos Bovinos/parasitologia , Proteínas Recombinantes/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Babesiose/diagnóstico , Babesiose/imunologia , Western Blotting/veterinária , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/imunologia , DNA Complementar/biossíntese , DNA Complementar/imunologia , Egito , Ensaio de Imunoadsorção Enzimática/veterinária , Gana , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , África do Sul , Tailândia
6.
Theriogenology ; 139: 106-112, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31401475

RESUMO

Artificial insemination is the general method of breeding for genetic improvement in offspring. However, almost half of the insemination cases fail to achieve full-term pregnancy, due to male infertility or subfertility. To maximize the success of insemination, accurate semen quality testing is required prior to insemination. Even though basic semen analyses have been used to provide preliminary information, it cannot fully identify the superior or inferior fertility bulls. Therefore, more powerful and easy to use methods for the prediction of male fertility are required, such as proteomic or microarray chips. During past decades, omics approaches have been developed and suggested the numerous fertility-related potential biomarkers. Our previous study identified the fertility related protein markers, enolase1 (ENO1), ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit (ATP5B), voltage-dependent anion channel 2 (VDAC2), phospholipid hydroperoxide glutathione peroxide (GPx4), and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2) in bovine spermatozoa. In the present study, we perform a marker combination assay using the western blot data of ENO1, ATP5B, VDAC2, GPx4, and UQCRC2 from 20 individual bull semen samples. And then, we identified the predictive ability of these markers for normal (non-return rate (NRR) ≥ 70%) and normal fertility (NRR<70%) in bulls. ENO1, a single protein marker, achieved an area under the curve (AUC) of 0.86 and 90% discriminatory power between normal and below-normal fertility bulls, with 90% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Although no meaningful changes existed in overall accuracy (70-85%) to discriminate the normal and below-normal fertility between ENO1 single marker and combined marker panels, multiple marker combination methods using ENO1, VDAC2, GPx4, and UQCRC2 provided absolute sensitivity and NPV, with higher specificity (70%) and PPV (77%). ENO1 can be used as a fertility marker candidate, but there were limitations for providing absolute information about normal and below-normal fertility. Although the combined use of fertility-related markers cannot provide absolute accuracy, it can help in indicating below-normal fertility in bulls. These results may contribute to the maintenance cost in the animal industry, via selection of bulls with inferior fertility.


Assuntos
Bovinos , Fertilidade , Animais , Área Sob a Curva , Biomarcadores/metabolismo , Western Blotting/veterinária , Modelos Lineares , Masculino , Análise Multivariada , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/metabolismo , Análise do Sêmen/métodos , Análise do Sêmen/veterinária
7.
Fish Shellfish Immunol ; 91: 216-222, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31121288

RESUMO

In the present study, a monoclonal antibody (mAb) against large yellow croaker IgM was produced by immunizing mice with purified large yellow croaker serum IgM. Western blotting showed that this mAb could specifically react with the heavy chain of large yellow croaker serum IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the resulting mouse anti-IgM mAb could recognize membrane-bound IgM (mIgM) molecules of large yellow croaker. This mouse anti-IgM mAb also can be used for sorting of large yellow croaker IgM+ B cells through the magnetic-activated cell sorting (MACS) method, which was further confirmed by RT-PCR analysis of specific marker genes for B cells. Flow cytometry analysis showed that the percentages of IgM+ B cells in head kidney, spleen and peripheral blood lymphocytes were 29.00 ±â€¯1.58%, 33.00 ±â€¯1.64%, and 16.50 ±â€¯2.39%, respectively. Additionally, the phagocytosis rates of IgM+ B cells for 0.5 µm beads in head kidney, spleen and peripheral blood were calculated to be 7.56 ±â€¯0.58%, 4.053 ±â€¯0.62% and 23.17 ±â€¯2.26%, respectively, while only 2.36 ±â€¯0.23%, 1.16 ±â€¯0.44% and 6.41 ±â€¯0.45 of IgM+ B cells in these three tissues ingested 1 µm beads. Taken together, our data demonstrated that the mouse anti-IgM mAb produced in this study could be used as a tool to characterize IgM+ B cells and to study functions of IgM in large yellow croaker.


Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Imunoglobulina M/imunologia , Perciformes/imunologia , Animais , Western Blotting/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Camundongos , Camundongos Endogâmicos BALB C
8.
J Vet Med Sci ; 81(7): 986-989, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31092762

RESUMO

Atypical scrapie in goats has only been reported in European countries. The present study reports the identification of the first case of atypical scrapie in goats in Japan. The genotype of the animal was ALRQ/ALHQ at codons 136, 141, 154, and 171 in prion protein (PrP). Western blot analysis showed a multiplex proteinase K-resistant prion protein (PrP-res) band pattern with a band <15 kDa that was clearly distinguishable from the triplet PrP-res band pattern observed in classical scrapie cases. Histopathological and immunohistological examination showed mild vacuolation and fine granular to globular immunolabelling of disease-associated PrP in the dorsal horn of cervical spinal cord. Collectively, our results confirmed that this goat was affected by atypical scrapie.


Assuntos
Doenças das Cabras/diagnóstico , Scrapie/diagnóstico , Animais , Western Blotting/veterinária , Feminino , Genótipo , Doenças das Cabras/patologia , Cabras , Japão , Proteínas Priônicas/genética , Scrapie/patologia , Medula Espinal/patologia
9.
Arch Razi Inst ; 74(1): 1-6, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31013002

RESUMO

Brucella bacterium causes Brucellosis, an infectious disease spreading from animals to human. Brucella lumazine synthase (BLS) is a highly immunogenic protein with adjuvant properties, which has been introduced as an effective protein carrier for vaccine development. This protein also plays a significant role in inducing immune system. This study aimed to clone, express, and purify the BLS gene from Brucella melitensis Rev1. The BLS gene was amplified by particular primers with the restriction enzyme sites as a linker and it was inserted into pTZ57R/T vector. Subsequently, it was ligated into pET32(a)+ expression vector. Recombinant expression vector containing coding sequence of BLS was transformed into E. coli BL21 (DE3) host gene expression and stimulated by 0.1mM IPTG. The results of sequencing showed that there were not any mutations in BLS encoding sequence. The expression results were set by sequencing and endorsed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses and western blotting that showed 35 kDa protein band appropriately.


Assuntos
Proteínas de Bactérias/imunologia , Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Brucelose/veterinária , Genes Bacterianos , Complexos Multienzimáticos/imunologia , Western Blotting/veterinária , Brucelose/prevenção & controle , Eletroforese em Gel de Poliacrilamida/veterinária , Escherichia coli/genética , Microrganismos Geneticamente Modificados/genética , Proteínas Recombinantes/imunologia , Vacinas de Subunidades/imunologia
10.
Vet Res ; 50(1): 17, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819249

RESUMO

Porcine circovirus-associated disease (PCVAD) is one of the most serious infectious diseases in pigs worldwide. The primary causative agent of PCVAD is porcine circovirus type 2 (PCV2), which can cause lymphoid depletion and immunosuppression in pigs. Our previous study demonstrated that Laiwu (LW) pigs, a Chinese indigenous pig breed, have stronger resistance to PCV2 infection than Yorkshire × Landrace (YL) pigs. In this study, we found that the YL pigs showed more severe lymphocyte apoptosis and higher viral load in the spleen tissue than LW pigs. To illustrate the differential gene expression between healthy and infected spleens, transcriptome profiling of spleen tissues from PCV2-infected and control YL pigs was compared by RNA sequencing. A total of 90 differentially expressed genes (DEGs) was identified, including CD207, RSAD2, OAS1, OAS2, MX2, ADRB3, CXCL13, CCR1, and ADRA2C, which were significantly enriched in gene ontology (GO) terms related to the defense response to virus and cell-cell signaling, and another nine DEGs, KLF11, HGF, PTGES3, MAP3K11, XDH, CYCS, ACTC1, HSPH1, and RYR2, which were enriched in GO terms related to regulation of cell proliferation or apoptosis. Among these DEGs, the CXCL13 gene, which can suppress lymphocyte apoptosis during PCV2 infection, was significantly down-regulated in response to PCV2 infection in YL but not in LW pigs. By analysis of the regulatory elements in the promoter and 3'-untranslated region (3'-UTR) of porcine CXCL13, we found that the single nucleotide polymorphism (SNP) -1014 G (LW) > A (YL) and the Sus scrofa microRNA-296-5p (ssc-miR-296-5p) participated in regulating CXCL13 expression during the response to PCV2 infection.


Assuntos
Apoptose , Quimiocina CXCL13/metabolismo , Infecções por Circoviridae/veterinária , Circovirus , Linfócitos/virologia , Baço/virologia , Doenças dos Suínos/virologia , Animais , Western Blotting/veterinária , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Circovirus/metabolismo , DNA Viral/genética , Citometria de Fluxo/veterinária , Perfilação da Expressão Gênica/veterinária , Regulação Viral da Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de RNA/veterinária , Baço/metabolismo , Baço/patologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Carga Viral/veterinária
11.
Vet Res ; 50(1): 19, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30836990

RESUMO

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen but some extra trigger factors are required for the development of PCV2-associated diseases. By evaluating cap protein expression, viral DNA copies and the number of infected cells, the present study further confirmed that oxidative stress can promote PCV2 replication. The results showed that oxidative stress induced autophagy in PCV2-infected PK15 cells. Blocking autophagy with inhibitor 3-methyladenine or ATG5-specific siRNA significantly inhibited oxidative stress-promoted PCV2 replication. Importantly, autophagy inhibition significantly increased apoptosis in oxidative stress-treated PK15 cells. Suppression of apoptosis by benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone in conditions of autophagy inhibition restored PCV2 replication. Taken together, autophagy protected host cells against potential apoptosis and then contributed to PCV2 replication promotion caused by oxidative stress. Our findings can partly explain the pathogenic mechanism of PCV2 related to the oxidative stress-induced autophagy.


Assuntos
Apoptose , Autofagia , Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Estresse Oxidativo , Doenças dos Suínos/virologia , Replicação Viral , Animais , Western Blotting/veterinária , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Citocinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo , Transfecção
12.
Vet Res ; 50(1): 20, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30841905

RESUMO

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes' function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1ß, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.


Assuntos
Vírus da Leucose Aviária , Leucose Aviária/imunologia , Macrófagos/virologia , Animais , Leucose Aviária/virologia , Vírus da Leucose Aviária/imunologia , Vírus da Leucose Aviária/fisiologia , Western Blotting/veterinária , Galinhas/imunologia , Galinhas/virologia , Feminino , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Análise de Sequência de DNA/veterinária , Replicação Viral
13.
Theriogenology ; 128: 62-73, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30743105

RESUMO

Toll-like receptors (TLRs) are important molecules, which provide protection against infections of the reproductive tract. This study demonstrates for the first time the expression and localization patterns of TLRs in the caput, corpus and cauda segments of the epididymal duct (ED) and the vas deferens (VD) of adult domestic cats using immunohistochemistry and western blotting. While immunoblot analyses revealed relatively similar protein levels for TLRs 2, 4, 5, and 9 in three segments of the ED, the protein levels of TLR2 and TLR4 in the VD were found to be significantly higher than those measured in the ED segments (P < 0.05). On the other hand, immunostaining showed that TLRs exhibited regional- and cell-specific localization patterns. TLR2 and TLR5 were immunolocalized to the nucleus and cytoplasm of the principal cells in all ducts. TLR4 was restricted to the stereocilia, and TLR9 was located in the cytoplasm of the principal cells. Narrow cells displayed positive immunoreactions for TLR4 and TLR5. The basal cells of the different ED segments were positive for all four TLRs. TLR2, TLR5 and TLR9 were detected in the cytoplasmic droplets of the spermatozoa. TLR4 and TLR9 were detected along the entire length of the sperm tail, whilst TLR2 and TLR5 were absent in the midpiece. TLR2 and TLR5 were also detected in the equatorial segment of the sperm head. These results suggest that TLR2, TLR4, TLR5 and TLR9 are important not only for the protection of the ED, VD and spermatozoa but also for the maturation and storage of spermatozoa in the ED and VD, respectively.


Assuntos
Gatos/metabolismo , Epididimo/metabolismo , Receptores Toll-Like/metabolismo , Ducto Deferente/metabolismo , Animais , Western Blotting/veterinária , Gatos/crescimento & desenvolvimento , Imuno-Histoquímica/veterinária , Masculino , Receptor 2 Toll-Like/análise , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/análise , Receptor 4 Toll-Like/metabolismo , Receptor 5 Toll-Like/análise , Receptor 5 Toll-Like/metabolismo , Receptor Toll-Like 9/análise , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/análise
14.
Arq. bras. med. vet. zootec. (Online) ; 71(1): 160-166, jan.-fev. 2019. tab, graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-989358

RESUMO

Brucellosis is an infectious disease caused by bacteria of the genus Brucella spp. with diagnosis based on use of serological techniques. The present study aimed to develop and standardize a western blotting (WB) test for detection of antibodies against B. abortus. Samples from two groups of cattle were analyzed: group I: 60 serum samples from true positive and true negative vaccinated animals (30 positive samples from infected animals according to rose bengal test (RBT), 2-mercaptoethanol, serum agglutination test (SAT) and complement fixation test (CFT) and 30 RBT negatives samples); group II: 383 field samples (90 positive and 293 CFT negative sera). The most reactive band in the western blotting, which properly identified and separated infected from non - infected had a molecular weight of ≤ 20kDa. The sensitivity, specificity and accuracy of the WB compared to RBT was 93%, 99%, 98%, respectively and k= 0.938. When compared to CFT, the sensitivity, specificity and accuracy of the WB was 97%, 98% and 97%, respectively and k= 0.929. The WB developed and standardized in the present study is a serological test with potential use as a confirmatory test for the diagnosis of bovine brucellosis.(AU)


A brucelose é uma doença infectocontagiosa, causada por bactérias do gênero Brucella spp., com diagnóstico baseado no emprego de técnicas sorológicas. Objetivou-se neste estudo desenvolver e padronizar um teste Western blotting (WB) para detecção de anticorpos contra B. abortus. Foram analisados dois grupos de amostras bovinas: grupo I, com 60 amostras de animais verdadeiros positivos e verdadeiros negativos vacinados (30 amostras positivas de animais infectados e positivos nos testes de antígeno acidificado tamponado (AAT), 2 - mercaptoetanol (2 - ME), soroaglutinação lenta em tubos (SAT) e fixação do complemento e de 30 amostras negativas no AAT); grupo II, com 383 amostras de campo, sendo 90 soropositivas e 293 soronegativas no TFC. O resultado da análise do WB revelou peso molecular ≤20kDa como sendo a área mais reativa e característica para identificação e separação dos animais infectados dos não infectados. A sensibilidade, a especificidade e a acurácia do WB, quando este foi comparado com o AAT, foram, respectivamente, 93%, 99% e 98%, e k= 0,938. Quando comparadas com a TFC, a sensibilidade, a especificidade e a acurácia foram 97%, 98% e 97%, respectivamente, e k= 0,929. O WB padronizado neste estudo mostrou-se um teste sorológico com potencial uso como teste confirmatório no diagnóstico da brucelose bovina.(AU)


Assuntos
Animais , Testes Sorológicos/métodos , Western Blotting/métodos , Western Blotting/veterinária , Brucelose/diagnóstico
15.
Vet Pathol ; 56(3): 369-376, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30612533

RESUMO

Canine urothelial carcinoma (UC) has a poor prognosis and high metastatic rate. Human epidermal growth factor receptor 2 (HER2), a receptor tyrosine kinase involved in cell proliferation and differentiation regulation, has been attracting interest as a therapeutic target molecule for human breast cancer. This study investigated expression of the canine homolog of HER2 (ERBB2) in canine UC, and its association with clinical factors. Since it has been controversial whether commercial anti-human HER2 antibody (Dako A0485) correctly recognizes the canine homolog of HER2, an application of the antibody using a canine UC cell line was validated first. By Western blot, a single band at the appropriate size for canine HER2 (185 kDa) was recognized. Immunohistochemistry for HER2 was performed on 23 samples of UC, 8 samples of polypoid cystitis, and 8 samples of normal urinary bladder, and the results were scored as either 0, 1+, 2+, or 3+ with reference to the evaluation method for human UC. Intense membranous HER2 immunoreactivity was frequently observed in neoplastic cells, especially in grade 2 UC. Minor HER2 expression was found in the epithelial cells of polypoid cystitis and normal bladder. The incidence of HER2 positivity (scores of 2+ or 3+) was 14 of 23 (60.9%) in UC, 3 of 8 (37.5%) in polypoid cystitis, and 0 of 8 (0%) in normal bladder. There was no significant correlation between HER2 positivity and clinical factors. While increased HER2 expression was observed in a subset of urothelial carcinomas, further mechanistic studies are needed to determine its role in the pathogenesis and targeted therapy of this cancer.


Assuntos
Carcinoma de Células de Transição/veterinária , Doenças do Cão/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias da Bexiga Urinária/veterinária , Animais , Anticorpos Antineoplásicos/imunologia , Western Blotting/veterinária , Carcinoma de Células de Transição/diagnóstico , Carcinoma de Células de Transição/metabolismo , Doenças do Cão/diagnóstico , Cães , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Masculino , Prognóstico , Receptor ErbB-2/imunologia , Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/metabolismo
16.
Vet Pathol ; 56(3): 409-417, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30558513

RESUMO

Scrapie is a fatal neurodegenerative disease of sheep resulting from the accumulation of a misfolded form of the prion protein (PrPSc). Polymorphisms in the host prion protein gene ( PRNP) can affect susceptibility to the scrapie agent. Lysine (K) at codon 171 of PRNP is an inadequately characterized, naturally occurring polymorphism in sheep. We inoculated Barbado sheep with PRNP genotypes QQ171, QK171, or KK171 by either the intracranial (IC, n = 2-7 per genotype) or oronasal (ON, n = 5 per genotype) routes with a scrapie isolate to investigate the effect of lysine at codon 171 on susceptibility. When neurologic signs were observed or at the end of the experiment (70 months postinoculation [MPI]), sheep were necropsied and tissue collected for histopathologic, immunohistochemical, enzyme immunoassay and Western blot examination for PrPSc. All genotypes of sheep developed scrapie after IC inoculation. After ON inoculation, sheep with the QK171 genotype had prolonged incubation periods compared to the QQ genotype. During the experiment, 2 of 5 of the ON-inoculated QK genotype sheep developed neurologic signs and had PrPSc in the brain. The other 3 of 5 sheep were asymptomatic at 70 MPI but had detectable PrPSc in peripheral tissues. None of the ON-inoculated sheep of the KK171 genotype developed signs or had detectable PrPSc. Our experiments demonstrate that sheep with the KK171 genotype are resistant to scrapie via oronasal exposure and that sheep with the QK171 genotype have prolonged incubation relative to QQ171 sheep. The K171 prion protein allele may be useful to enhance scrapie resistance in certain breeds of sheep.


Assuntos
Imunização/veterinária , Proteínas Priônicas/genética , Scrapie/imunologia , Administração Intranasal/veterinária , Animais , Western Blotting/veterinária , Resistência à Doença/imunologia , Feminino , Genótipo , Imunização/métodos , Técnicas Imunoenzimáticas/veterinária , Masculino , Polimorfismo Genético , Proteínas Priônicas/administração & dosagem , Proteínas Priônicas/imunologia , Scrapie/prevenção & controle , Ovinos
17.
J Vet Med Sci ; 81(2): 314-320, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30584200

RESUMO

Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50=10-2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.


Assuntos
Adenoviridae/imunologia , Patos/virologia , Infecções por Flavivirus/veterinária , Flavivirus/imunologia , Doenças das Aves Domésticas/virologia , Vacinas Sintéticas/genética , Vacinas Virais/genética , Adenoviridae/genética , Animais , Western Blotting/veterinária , Patos/imunologia , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/virologia , Imunofluorescência/veterinária , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Testes de Neutralização/veterinária , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/virologia , Vacinas Virais/imunologia
18.
BMC Vet Res ; 14(1): 337, 2018 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-30419898

RESUMO

BACKGROUND: Avian coccidiosis is often caused by co-infection with several species of Eimeria worldwide. Developing a multivalent vaccine with an antigen common to multiple Eimeria species is a promising strategy for controlling clinical common co-infection of Eimeria. In the previous study, 14-3-3 was identified as one of the immunogenic common antigen in E. tenella, E. acervulina and E. maxima. The aim of the present study was to evaluate the immunogenicity and protective efficacy of Ea14-3-3 in the form of DNA vaccine against infection with three species of Eimeria both individually and simultaneously. RESULTS: After vaccination with pVAX-Ea14-3-3, the Ea14-3-3 gene was transcribed and expressed in the injected muscles. Vaccination with pVAX-Ea14-3-3 significantly increased the proportion of CD4+ and CD8+ T lymphocytes and produced a strong IgY response in immunized chickens. Similarly, pVAX-Ea14-3-3 stimulated the chicken's splenocytes to produce high levels of Th1-type (IFN-γ, IL-2) and Th2-type (IL-4) cytokines. The vaccine-induced immune response was responsible to increase weight gain, decreased the oocyst output, and alleviated enteric lesions significantly in immunized chickens as compared to control group, in addition to induce moderate anti-coccidial index (ACI). CONCLUSION: These results indicate that Ea14-3-3 is highly immunogenic and capable to induce significant immune responses. Furthermore, Ea14-3-3 antigen can provide effective protection against infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima both individually and in combination with three Eimeria species. Significant outcomes of our study provide an effective candidate antigen for developing a multivalent Eimeria vaccine against mixed infection with various Eimeria species under natural conditions.


Assuntos
Antígenos de Protozoários/imunologia , Coccidiose/veterinária , Eimeria tenella/imunologia , Eimeria/imunologia , Doenças das Aves Domésticas/parasitologia , Vacinas Protozoárias/uso terapêutico , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Western Blotting/veterinária , Galinhas/imunologia , Galinhas/parasitologia , Coccidiose/imunologia , Coccidiose/parasitologia , Coccidiose/prevenção & controle , Eimeria/genética , Eimeria tenella/genética , Citometria de Fluxo/veterinária , Genes de Protozoários/genética , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas Protozoárias/imunologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Análise de Sequência de DNA , Subpopulações de Linfócitos T/imunologia
19.
Vet Res ; 49(1): 114, 2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30454073

RESUMO

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein-protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.


Assuntos
Aderência Bacteriana , Frutose-Bifosfato Aldolase/fisiologia , Mycoplasma hyopneumoniae/enzimologia , Animais , Aderência Bacteriana/fisiologia , Western Blotting/veterinária , Eletroforese em Gel Bidimensional/veterinária , Citometria de Fluxo/veterinária , Frutose-Bifosfato Aldolase/genética , Genoma Bacteriano/genética , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/patogenicidade , Pneumonia Suína Micoplasmática/microbiologia , Proteômica , Mucosa Respiratória/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Suínos/microbiologia , Traqueia/microbiologia , Virulência
20.
Vet Clin Pathol ; 47(4): 682-687, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30358180

RESUMO

BACKGROUND: Lymphoma is an important disease of pet guinea pigs, although validation of immunophenotyping techniques based on cytologic or hematologic samples has not been reported. OBJECTIVE: To describe an immunocytochemical method for immunophenotyping of lymphoma (as either T- or B-cell) in guinea pigs, and to validate antibodies for this purpose. METHODS: Blood and tissues were obtained at the time of necropsy from laboratory guinea pigs and a privately owned dog (control) euthanized for reasons unrelated to lymphoproliferative disease. Fine-needle aspirates of enlarged peripheral lymph nodes were obtained from a case of spontaneous lymphoma in a pet guinea pig. Anti-CD3 and anti-Pax5 antibodies were validated by a combination of western blotting performed on splenic lysates of both the dog and guinea pigs, immunohistochemical studies on normal guinea pig tissues, and immunocytochemistry on normal guinea pig peripheral blood and splenic impression smears. RESULTS: The antibodies bound to antigens of an appropriate size in both the dog and guinea pig splenic lysates by Western blot analysis. Immunohistochemistry and immunocytochemistry demonstrated the expected distribution of putative T- and B-lymphocytes in normal tissues, peripheral blood, and splenic impression smears. As a proof-of-principle for its clinical utility, this immunocytochemical assay was used to diagnose a B-cell phenotype in a spontaneous lymphoma case in a pet guinea pig. CONCLUSIONS: Here, we validated an immunocytochemical method for immunophenotyping of lymphoma in guinea pigs as either a T- or B-cell phenotype. This enables future research into the clinical attributes of these subtypes and may ultimately improve both prognostication and therapy of lymphoma in guinea pigs.


Assuntos
Cobaias/anatomia & histologia , Imunofenotipagem/veterinária , Linfoma/veterinária , Animais , Anticorpos Antineoplásicos/imunologia , Western Blotting/veterinária , Complexo CD3/imunologia , Cães , Feminino , Imuno-Histoquímica/métodos , Imuno-Histoquímica/veterinária , Imunofenotipagem/métodos , Linfonodos/patologia , Linfoma/diagnóstico , Linfoma/imunologia , Linfoma/patologia , Linfoma de Células B/diagnóstico , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma de Células B/veterinária , Linfoma de Células T/diagnóstico , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Linfoma de Células T/veterinária , Reprodutibilidade dos Testes
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