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1.
Exp Parasitol ; 205: 107734, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394093

RESUMO

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production around the globe. Although several studies suggest the use of integrated management practices, these parasites have been controlled essentially with synthetic anthelmintic drugs. The resistance mechanism against the imidazothiazole derivative levamisole in Haemonchus contortus has not been fully described. Recently, resistance was associated with a 63bp deletion in the Hco-acr-8b gene that encodes a subunit for a nicotinic acetylcholine receptor. This study aimed to standardize a real time PCR (qPCR) protocol for levamisole resistance diagnosis in H. contortus populations based on this polymorphism and use it to characterize 23 field H. contortus populations obtained from different localities of Ceará State, Northeast Brazil. In addition, two populations of H. contortus were used as a standard of susceptibility and resistance, Inbred Strain Edinburgh (ISE) and Kokstad, respectively. Larval development tests (LDT) were performed on five field isolates and both EC50 and EC95 were estimated. LDT EC95 values provided a wider interval between susceptible and resistant populations than EC50 values (EC95 = 1.96-57.93 µM; EC50 = 0.05-0.39 µM), and were found to be more appropriate for differentiating them. Real time PCR results showed resistance allele frequencies ranged from 20.9 to 76.7%. Our results suggest that levamisole resistance may be present in field populations but it is not as widespread as benzimidazole resistance. This methodology may be useful to monitor levamisole resistance in field populations of H. contortus.


Assuntos
Antinematódeos/farmacologia , Resistência a Medicamentos/genética , Haemonchus/efeitos dos fármacos , Levamisol/farmacologia , Animais , Benzimidazóis/farmacologia , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Frequência do Gene/genética , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/genética , Haemonchus/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores Colinérgicos/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Alinhamento de Sequência/veterinária , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/parasitologia , Tetramizol/farmacologia
2.
Parasit Vectors ; 12(1): 335, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277688

RESUMO

BACKGROUND: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii, a widespread protozoan in the phylum Apicomplexa. In Europe, several studies have demonstrated the presence of the parasite in tissues of wild boars (Sus scrofa), but no data exists on the T. gondii load in tissues which in turn may be an useful way to assess the infection risk for the consumer of wild boar meat. METHODS: We sampled and tested a total of 472 tissue samples of brain, heart and masseter muscle from 177 wild boars from the Campania region of southern Italy by real-time PCR analyses for detection and quantification of T. gondii. The sensitivity and specificity of the method were calculated by ROC analysis curves. RESULTS: PCR analysis revealed the presence of T. gondii in tissue samples of 78 out of 177 (44%) wild boars. In general, the brain presented the highest PCR prevalence (31%), followed by the heart (28.3%) and the masseter muscle (24.2%), with the highest estimated parasite numbers observed in the brain followed by the heart and masseter muscle. The PCR method showed an excellent discriminating ability for each of the examined tissues. According to the ROC analysis curves, the respective sensitivity and specificity were 99 and 100% for masseter muscle, 98 and 98% for brain and 96 and 98% for heart samples. CONCLUSIONS: The high prevalence of infection here detected suggests a widespread distribution of the parasite in the wildlife of the Campania region of southern Italy. The T. gondii burdens detected may potentially represent a source of infection for humans.


Assuntos
Carne Vermelha/parasitologia , Doenças dos Suínos/epidemiologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/epidemiologia , Animais , Humanos , Itália/epidemiologia , Carga Parasitária/veterinária , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sus scrofa , Suínos , Doenças dos Suínos/parasitologia , Toxoplasma/genética , Toxoplasmose Animal/parasitologia , Zoonoses
3.
BMC Vet Res ; 15(1): 214, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238913

RESUMO

BACKGROUND: Per- and polyfluoroalkyl substances (PFASs) are environmentally persistent and bioaccumulative chemicals. Immunomodulation is among the most concerning of toxic effects linked with PFAS exposure in mammalian models. However, no studies had yet shown this to be true in birds. Thus, we designed and conducted the first study to determine if PFASs could cause immunomodulation in birds. Secondly, we wanted to determine the effects on an avian host when exposed not only to immunomodulating chemicals, but also to a viral challenge. The aim, to determine if PFAS mediated immunmodulation functionally affects a pathogen challenge for a host. As innate immune system signalling pathways initiate crucial responses against a pathogen challenge, and are lesser studied than their adaptive counterparts, we focused on these pathways. To provide the first information on this, an in vitro experiment was designed and performed using chicken embryo fibroblasts exposed to perfluorooctane sulfonate (PFOS) (22 ppm) and immune markers characterised before and after being infected with gallid herpesvirus-2 (GaHV-2). RESULTS: The expression of two pro-inflammatory cytokines, namely interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), and the anti-inflammatory cytokine interleukin 4 (IL-4) were investigated in various scenarios. These results showed that exposure to PFOS decreased immune gene expression in chicken fibroblasts from 36 h post-exposure. Next, it was shown that this decrease could be mitigated by infection with gallid herpesvirus-2, which increased gene expression back to the baseline/control levels. CONCLUSIONS: Not only is this the first study to provide the expected evidence that PFOS has immunomodulatory potential in birds, it also provides unexpected data that virus infections can mitigate this negative effect. Thereby, further research, including in vivo and in situ studies, on the impact of PFOS on host-virus interactions is now warranted, as it has been overlooked and might contribute to our understanding of recent disease outbreaks in wildlife. The mechanisms by which gallid herpesvirus mitigates immunomodulation were beyond the scope of this study, but are now of interest for future study.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Fluorcarbonetos/toxicidade , Herpesvirus Galináceo 2/imunologia , Imunomodulação/efeitos dos fármacos , Doença de Marek/imunologia , Viroses/veterinária , Ácidos Alcanossulfônicos/imunologia , Animais , Linhagem Celular , Embrião de Galinha , DNA Complementar , Fibroblastos/efeitos dos fármacos , Fluorcarbonetos/imunologia , Expressão Gênica/efeitos dos fármacos , Imunomodulação/genética , Mediadores da Inflamação/metabolismo , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transdução de Sinais/efeitos dos fármacos , Viroses/imunologia
4.
BMC Vet Res ; 15(1): 192, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182094

RESUMO

BACKGROUND: Degenerative myelopathy (DM) is a progressive neurodegenerative disease frequently found in Pembroke Welsh Corgis (PWCs). Most DM-affected PWCs are homozygous for the mutant superoxide dismutase 1 (SOD1) allele; however, the genetic examination for the SOD1 mutation does not exclusively detect symptomatic dogs. In order to identify novel biomarkers, the plasma microRNA (miRNA) profiles of PWCs with DM were investigated. RESULTS: Quantification of the plasma levels of 277 miRNAs by an RT-qPCR array identified 11 up-regulated miRNAs and 7 down-regulated miRNAs in DM-affected PWCs from those in wild-type SOD1 PWCs. A pathway analysis identified 3 miRNAs: miR-26b, miR-181a, and miR-196a, which potentially regulate several genes associated with SOD1. In order to validate the diagnostic accuracy of the candidate miRNAs in the aged PWC population, candidate miRNAs in plasma were measured by RT-qPCR and a receiver operating characteristic (ROC) curve analysis was performed. miR-26b had the largest area under the ROC curve for distinguishing DM PWCs from healthy PWCs (sensitivity, 66.7%; specificity, 87.0%). The plasma level of miR-26b was significantly higher in the DM group than in the healthy control group. A positive correlation was observed between increases in the plasma level of miR-26b and disease progression. CONCLUSIONS: These results suggest that plasma miR-26b is a potential novel diagnostic biomarker of DM.


Assuntos
Doenças do Cão/genética , MicroRNAs/sangue , Doenças Neurodegenerativas/veterinária , Animais , Biomarcadores/sangue , Progressão da Doença , Doenças do Cão/diagnóstico , Cães , Feminino , Masculino , Mutação , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Superóxido Dismutase-1/genética
5.
J Dairy Sci ; 102(8): 7226-7236, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202648

RESUMO

The mammalian Y chromosome gene families in the ampliconic region are expressed predominantly or exclusively in the testis, and their copy number variations (CNV) are significantly associated with male reproductive traits, suggesting they have important roles in spermatogenesis and testicular development. ZNF280AY (zinc finger protein 280A, Y-linked) is a member of the zinc finger protein family and has been identified as a bovid-specific Y-chromosome gene. The current study applied a reliable quantitative real-time PCR method to estimate the CNV of ZNF280AY in 715 bulls across 21 cattle breeds and to further investigate the association of the CNV of ZNF280AY with bull reproductive traits and ZNF280AY mRNA expression levels in adult testis. The results revealed that the median copy number of ZNF280AY was 47, and the copy number varied from 11 to 154, showing significant CNV between and within the investigated cattle breeds. In addition, all 715 bulls were classified into Y1, Y2, and Y3 lineage groups based on a rapid genotyping method described previously. Pairwise comparisons indicated that bulls belonging to the Y1 lineage had a significantly lower median copy number (40) than bulls belonging to the Y2 (52) and Y3 lineages (57). Association analysis revealed that the CNV of ZNF280AY was correlated negatively with the percentage of normal sperm and sperm concentration in Holstein bulls, whereas no significant correlation was observed with ejaculation volume, total sperm count, sperm motility, postthaw motility (PTM), and scrotal circumference in Holstein and Simmental bulls. Furthermore, no correlation was observed between ZNF280AY copy number and ZNF280AY mRNA expression levels in the testis. The current study suggests that the CNV of the ZNF280AY gene family is associated with male reproductive traits and may serve as a valuable marker for early bull fertility selection in Holstein breeding programs.


Assuntos
Bovinos/genética , Variações do Número de Cópias de DNA , Fertilidade/genética , Regulação da Expressão Gênica , Genes Ligados ao Cromossomo Y/genética , Reprodução/genética , Cromossomo Y/genética , Animais , Cruzamento , Bovinos/fisiologia , Marcadores Genéticos/genética , Genótipo , Masculino , Especificidade de Órgãos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Especificidade da Espécie , Contagem de Espermatozoides/veterinária , Motilidade Espermática/genética , Espermatogênese/genética , Testículo/fisiologia , Dedos de Zinco/genética
6.
Rev Bras Parasitol Vet ; 28(2): 194-202, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31188942

RESUMO

The aim of this study was to compare molecular tests used to diagnose Leishmania spp. in dogs with different stages of infection. Blood and conjunctival swab (CS) samples from dogs classified in four clinical stages were subjected to different PCR protocols (13A/13B, MC1/MC2, LITSR/L5.8S and LEISH-1/LEISH-2 primers). To the study, 22.3% (48/215) of dogs were classified as without clinical signs, 67.5% (145/215) stage I (mild disease), 7.0% (15/215) stage II (moderate disease) and 3.2% (7/215) stage III (severe disease). The results showed that in blood samples, 13A/13B detected a significant higher number of positive dogs in stage I (25/145) and in total (42/215) (p≤0.05). However, when CS samples were tested, no difference was observed (p>0.05). On the other hand, in blood samples, MC1/MC2 detected significantly fewer positive dogs classified as without clinical signs (0/48), in stage I (0/145) and in total (1/215) (p≤0.05). Likewise, in CS samples, this primers showed also lower detection (1/215) (p≤0.05). So than, we can conclude that PCR on blood samples with 13A/13B primers has greater capacity to detect positive dogs, mainly at the initial of clinical disease than do other primers and MC1/MC2 are not a good choice to detect Leishmania infantum infection in dogs.


Assuntos
Doenças do Cão/diagnóstico , Leishmaniose Cutânea/veterinária , Leishmaniose Visceral/veterinária , Animais , Brasil/epidemiologia , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Cães , Leishmania infantum/genética , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Índice de Gravidade de Doença
7.
J Vet Diagn Invest ; 31(4): 568-571, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31046652

RESUMO

The European Community's leukosis key (EC key) is a well-known hematologic method for detecting bovine leukemia virus (BLV) infection in dairy cattle. The key identifies infected cattle with persistent lymphocytosis via a combination of lymphocyte count (LC) and age. Using the EC key to identify BLV-infected Japanese black (JB) cattle is problematic, however, given the inherently lower LCs of JB cattle compared to dairy cattle. We analyzed the LC in BLV-positive and -negative JB cattle and estimated LC cutoff values by age using receiver operating characteristic curve analysis. Among the 716 JB blood samples collected, 452 (63%) JB cattle were confirmed as BLV-positive by an antibody ELISA for ≥1-y-old cattle and by real-time PCR for <1-y-old cattle. The cutoff values for the LC in each age group were calculated as 6.3 × 109/L for <1 y, 5.9 × 109/L for 1 to <2 y, 5.5 × 109/L for 2 to <3 y, 4.5 × 109/L for 3 to <6 y, 4.3 × 109/L for 6 to ≤10 y, and 3.7 × 109/L for >10 y. The sensitivity and specificity of the estimated cutoff values were 0.49 (95% confidence interval: 0.44-0.53) and 0.81 (0.75-0.85), whereas those of the EC key were 0.20 (0.16-0.24) and 0.99 (0.97-1.00). Our LC cutoff values for screening JB cattle for BLV infection appear to be preferable to those of the EC key.


Assuntos
Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/isolamento & purificação , Animais , Bovinos , Leucose Enzoótica Bovina/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Japão/epidemiologia , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade
8.
J Vet Diagn Invest ; 31(4): 604-607, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31046639

RESUMO

Porcine circovirus 3 (PCV-3) is a newly emerging virus that poses a potential threat to the swine industry. We developed a sensitive assay utilizing droplet digital PCR (ddPCR) to detect PCV-3. Specificity of the assay was confirmed by the failure of amplification of DNA of other relevant viruses. The detection limit for ddPCR was 1 copy/µL, 10 times greater sensitivity than TaqMan real-time PCR (rtPCR). Both methods showed a high degree of linearity, although TaqMan rtPCR showed less sensitivity than ddPCR for clinical detection. Our findings indicate that ddPCR might offer faster and improved analytical sensitivity for PCV-3 detection.


Assuntos
Circovirus/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Doenças dos Suínos/virologia , Animais , Circovirus/genética , Limite de Detecção , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Suínos , Doenças dos Suínos/diagnóstico
9.
Fish Shellfish Immunol ; 91: 264-274, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128294

RESUMO

Brown trout are polymorphic salmonid species, and it is of importance to investigate whether hybridization affects disease resistance. In this study, susceptibility of brown trout (Salmo trutta Abant, Anatolian, Black Sea, and Caspius) strains and their hybrids to Lactococcus garvieae and Yersinia ruckeri as well as their immune-related gene expression profiles were studied. Results indicated that reciprocal hybridization did not affect disease resistance in brown trout strains. Purebred Black Sea strain of brown trout was the most resistant group against Y. ruckeri, followed by other Black Sea strain hybrids. On the other hand, purebred Anatolian strain was the most resistant group to L. garvieae, followed by other Anatolian strain hybrids. Expression pattern of target genes differed in families, but the overall gene expression was comparatively high in Y. ruckeri infected families. Upregulations were mainly significant at 7 and 28 d post infection while marginal regulations were observed 8 h after infection. Disease resistance status of strains was supported by high expression of immune-related genes such as major histocompatibility complex class I (MHC-I), immunoglobulin light chain (IgL), and antioxidant- and hemoglobin-related gene expression. Therefore, our findings suggest that Black Sea and Anatolian strains could be used to develop fish stock that are resistant for yersiniosis and lactocaccosis, respectively.


Assuntos
Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Positivas/veterinária , Truta/genética , Truta/imunologia , Yersiniose/veterinária , Animais , Suscetibilidade a Doenças/imunologia , Infecções por Bactérias Gram-Positivas/imunologia , Hibridização Genética , Lactococcus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transcriptoma , Yersiniose/imunologia , Yersinia ruckeri/fisiologia
10.
Transbound Emerg Dis ; 66(4): 1789-1795, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077564

RESUMO

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that requires rapid control. Early detection is critical but transportation of samples to laboratory delays testing. Sensitive and specific field-deployable assays are therefore desirable. Real-time reverse transcription polymerase chain reaction (RRT-PCR) and RRT-loop-mediated isothermal amplification assays for FMDV on portable platforms have been described but none of these are handheld. In this report, we have evaluated a handheld Biomeme two3™ Real-Time PCR Thermocycler (two3) as a field-deployable platform for FMDV RRT-PCR targeting the 3D gene segment. Two3's performance was compared with the laboratory-based reference assay on the ABI7500 platform. RNA extraction using a rapid Biomeme proprietary sample prep technology (M1) was compared with MagMax RNA extraction. Two3 successfully detected FMDV isolates for six serotypes (O, A, Asia 1, SAT 1, 2 and 3). Serotype C was excluded since it has not been detected in the field since 2004. The limits of detection for serial 10-fold dilutions of cell culture isolates were equal or one log different between two3 and ABI7500. Furthermore, two3 detected FMDV RNA in multiple sample types including serum, vesicular fluid, tissue suspensions, oral fluid, oral and nasal swabs. Two3 also detected FMDV RNA directly in vesicular fluid and other samples without prior RNA extraction. Comparison of the time to first detection of a positive result in serial samples in MagMax RNA extraction/ABI7500 (MgMx/ABI) system vs. M1 RNA extraction/Two3 system revealed similar or slightly better analytical sensitivity for the MgMx/ABI system. Overall, RNA extraction by M1 yielded good results and FMDV RNA detection on two3 was not significantly different from the ABI7500. Therefore, two3 could potentially enable sensitive penside detection of FMDV within an hour using M1-extracted RNA or direct testing of vesicular fluid and swabs without RNA extraction thereby ensuring prompt implementation of appropriate control measures.


Assuntos
Doenças dos Bovinos/diagnóstico , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Doenças dos Ovinos/diagnóstico , Doenças dos Suínos/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/virologia , Testes Diagnósticos de Rotina/instrumentação , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/veterinária , Febre Aftosa/virologia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Sorogrupo , Ovinos , Doenças dos Ovinos/virologia , Suínos , Doenças dos Suínos/virologia
11.
Prev Vet Med ; 168: 60-65, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31097124

RESUMO

The Surra caused by Trypanosoma evansi (T. evansi) is an economically damaging disease of livestock including camels, horses, and buffaloes. The disease is transmitted by arthropod flies belonging to family tabanidae. The clinical signs of affected animals include recurrent fever, progressive anemia, cachexia, edema, and abortion. In order to determine the point prevalence of Surra in the camel population of north-east of Iran, 152 blood samples from one-humped camels were collected by multiple cluster sampling methods from three provinces, namely, Razavi Khorasan (R.Kh.), Northern Khorasan (N.Kh.), and Southern Khorasan (S.Kh.). The nucleic acid extracted from the buffy coat of each blood sample was analyzed by SYBR green real-time PCR test for the detection of T. evansi in the blood samples. T. evansi was detected in 10 out of 152 camel blood samples (6.5%) with a prevalence rate of 8.6, 9.3, and 1.4 percent in R.Kh., N.Kh., and S.Kh. provinces, respectively. The prevalence of the disease decreased from north to south in the Khorasan provinces. Multivariate logistic regression analyses showed that among risk factors influencing Surra in the camel population, location was the most remarkable risk factor. Different geographical conditions, climate change, and the amount of raining can be considered as the factors affecting Surra vector population from north to south, resulting in a decrease in the rate of the prevalence of Surra from north to south.


Assuntos
Camelus , Tripanossomíase/veterinária , Animais , Camelus/parasitologia , Doença Crônica/veterinária , Feminino , Irã (Geográfico)/epidemiologia , Masculino , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Fatores de Risco , Tripanossomíase/epidemiologia
12.
Vet Res ; 50(1): 32, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046823

RESUMO

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.


Assuntos
Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Granuloma/veterinária , Rim/metabolismo , Lactococcus , Baço/metabolismo , Esplenopatias/veterinária , Truta/microbiologia , Animais , Doenças dos Peixes/metabolismo , Doenças dos Peixes/patologia , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Granuloma/metabolismo , Granuloma/microbiologia , Granuloma/patologia , Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Baço/patologia , Esplenopatias/metabolismo , Esplenopatias/microbiologia , Esplenopatias/patologia , Transcriptoma , Truta/metabolismo
13.
Vet Immunol Immunopathol ; 211: 25-34, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084890

RESUMO

Red Mark Syndrome (RMS) is a skin disease reported from farmed rainbow trout. Since the turn of the millennium it has been spreading through Europe. RMS is probably a bacterial disease caused by a Midichloria-like organism (MLO). It is non-lethal and causes little obvious changes in appetite or behavior but results in red hyperaemic skin lesions, which may lead to economic losses due to downgrading. Here we transfer RMS to naïve specific pathogen free (SPF) fish by cohabitation with RMS-affected seeder fish. During disease development we characterize local cellular immune responses and regulations of immunologically relevant genes in skin of the cohabitants by immunohistochemistry and qPCR. Skin samples from SPF controls and cohabitants (areas with and without lesions) were taken at 18, 61, 82 and 97 days post-cohabitation. Gene expression results showed that lesions had a Th1-type profile, but with concurrent high expression levels of all three classes of immunoglobulins (IgD, IgM and IgT). The marked local infiltration of IgD + cells in the skin lesions as well as a highly up-regulated expression of the genes encoding sIgD and mIgD indicate that this immunoglobulin class plays an important role in skin immunity in general and in RMS pathology in particular. The co-occurrence of an apparent B cell dominated immune reaction with a Th1-type profile suggests that the local production of antibodies is independent of the classical Th2 pathway.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Humoral/imunologia , Oncorhynchus mykiss/imunologia , Pele/imunologia , Animais , Doenças dos Peixes/microbiologia , Expressão Gênica , Imunoglobulina D/imunologia , Oncorhynchus mykiss/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Pele/metabolismo
14.
Vet Res ; 50(1): 36, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31113477

RESUMO

A longitudinal study was performed in three Danish farrow to grower (30 kilos) herds over a 4-month period to investigate the dynamics and clinical impacts of influenza A virus (IAV) infections. In each herd, four batches consisting of four sows each with five ear-tagged piglets were included. Nasal swabs and/or blood were sampled from the sows and/or the piglets prior to farrowing and at weeks 1, 3, and 5 and at the end of the nursery period. Clinical examinations were performed at each sampling time. The sows and piglets were tested for IAV and IAV antibodies in nasal swabs and blood samples, respectively. The results revealed three enzootically infected herds, where the majority of the pigs were infected during the first 5 weeks after birth. Infected piglets of only 3 days of age were detected in the farrowing unit, where the sows were also shedding virus. In all herds, low to moderate numbers of infected pigs (ranging from 3.6 to 20.7%) were found to be virus positive in nasal swabs at two consecutive sampling times. Furthermore, clinical signs of respiratory disease were associated with IAV detection. The findings of this study documented that IAV can persist in herds and that piglets as young as 3 days can be infected despite the presence of maternally derived antibodies.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Animais , Animais Recém-Nascidos/imunologia , Animais Recém-Nascidos/virologia , Feminino , Estudos Longitudinais , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Suínos , Doenças dos Suínos/imunologia
15.
Vet Parasitol ; 269: 2-6, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31079823

RESUMO

Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.


Assuntos
Aborto Animal/diagnóstico , Doenças dos Bovinos/diagnóstico , Coccidiose/veterinária , Neospora/isolamento & purificação , Complicações Parasitárias na Gravidez/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Feto Abortado/parasitologia , Aborto Animal/parasitologia , Animais , Encéfalo/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/diagnóstico , Coccidiose/parasitologia , Primers do DNA/genética , Sondas de DNA/genética , Feminino , Coração/parasitologia , Neospora/genética , Gravidez , Complicações Parasitárias na Gravidez/diagnóstico , Complicações Parasitárias na Gravidez/parasitologia
16.
BMC Vet Res ; 15(1): 168, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126297

RESUMO

BACKGROUND: Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. RESULTS: Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values. CONCLUSIONS: The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.


Assuntos
Picornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/virologia , Animais , Variação Genética , Picornaviridae/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico
17.
Vet Dermatol ; 30(4): 318-e89, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31135082

RESUMO

BACKGROUND: Detection of Leishmania in cutaneous lesions is possible by visualization of amastigotes. Detection of Leishmania DNA by PCR presents greater sensitivity, and PCR has been used to diagnose cutaneous leishmaniosis in humans using noninvasive clinical specimens. OBJECTIVES: Study I: to determine if Leishmania DNA could be efficiently extracted and amplified from archived Diff-Quik® -stained slides of cytological specimens from canine cutaneous lesions. Study II: to evaluate the diagnostic performance of a Leishmania-quantitative (q)PCR on stained cytological specimens and on filter paper impressions (FPI) obtained from cutaneous lesions suggestive of canine leishmaniosis (CanL). ANIMALS: Samples from cutaneous lesions of 54 dogs. METHODS AND MATERIALS: Study I: Leishmania-qPCR was performed on 19 glass slides (from nine dogs) with cytologically visible amastigotes. Fifteen slides with no visible amastigotes, obtained from 12 dogs seronegative for Leishmania by ELISA, served as controls. Study II: Leishmania-qPCR was performed on glass slides and FPI from cutaneous lesions compatible with clinical leishmaniosis in 33 dogs. RESULTS: Study I: all slides with visible amastigotes had positive qPCR, whereas all control slides yielded negative results. Study II: of 13 dogs definitively diagnosed with clinical leishmaniosis, eight had visible amastigotes on cytology, whereas Leishmania-qPCR was positive on 11 glass slides and 13 FPI. Leishmaniosis was ruled out by standard methods in 20 dogs, four of which yielded positive qPCR on FPI and/or glass slides. CONCLUSIONS AND CLINICAL IMPORTANCE: Leishmania-DNA can be detected efficiently by qPCR from cutaneous cytological specimens and FPI to diagnose Leishmania infection in dogs with cutaneous lesions suggestive of CanL.


Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/isolamento & purificação , Leishmaniose Cutânea/veterinária , Técnicas de Diagnóstico Molecular/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Dermatopatias/veterinária , Animais , DNA de Protozoário/isolamento & purificação , Doenças do Cão/parasitologia , Cães , Feminino , Leishmaniose Cutânea/diagnóstico , Masculino , Papel , Sensibilidade e Especificidade , Dermatopatias/parasitologia
18.
J Dairy Sci ; 102(7): 6485-6494, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31103291

RESUMO

Cattle infected with Mycobacterium avium ssp. paratuberculosis (MAP) shed the bacterium in their feces. This may lead to considerable concentrations of MAP in slurry, which has been postulated to contribute to MAP transmission when this slurry is used as fertilizer. For other bacterial species, anaerobic digestion has been shown to reduce bacterial load and to increase the safety of organic waste. Therefore, the objective of this study was to investigate the effects of anaerobic digestion in biogas plants on MAP survival in slurry from 16 dairy farms with a history of MAP infection. Presence of MAP was determined using MAP culture and a commercial MAP IS900 quantitative PCR (qPCR) applied on untreated slurry samples, slurry samples after primary fermentation, and digestate. Unfermented slurry samples from most enrolled farms tested positive for MAP, via both culture and qPCR. After the fermentation process, MAP could no longer be cultured in most samples, with the exception of 2 samples from farms where high numbers of MAP-shedding cows were kept at the time of sampling. A Bayesian binomial model predicted a probability of 93% for a MAP-negative culture result after fermentation. In most samples, MAP DNA was still detectable when using the IS900 qPCR. The probability of a negative result in qPCR was estimated to be 27%. Results of this study indicate that subjecting MAP-positive slurry to anaerobic digestion in biogas plants leads to a reduction of viable MAP below the detection limit; however, MAP DNA remained detectable. It remains undetermined whether MAP DNA detected in fermentation products is a residue of MAP degradation or belongs to viable MAP below the detection limit or in a dormant state. In conclusion, subjecting MAP-positive slurry to anaerobic mesophilic digestion reduces viable MAP concentration below the detection limit. The use of digestion products as fertilizer on pasture and agricultural soils instead of untreated slurry may therefore reduce the risk of MAP transmission.


Assuntos
Esterco/microbiologia , Mycobacterium avium subsp. paratuberculosis/crescimento & desenvolvimento , Plantas/microbiologia , Anaerobiose , Animais , Teorema de Bayes , Biocombustíveis/análise , Biocombustíveis/microbiologia , Bovinos , Fezes/microbiologia , Esterco/análise , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Plantas/química , Reação em Cadeia da Polimerase em Tempo Real/veterinária
19.
J Vet Sci ; 20(2): e4, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30944527

RESUMO

Foot-and-mouth disease (FMD) is one of the most important livestock diseases in East Africa with outbreaks reported annually that cause severe economic losses. It is possible to control disease using vaccination, but antigenic matching of the vaccine to circulating strains is critical. To determine the relationship between foot-and-mouth disease viruses circulating in districts along the Uganda and Tanzanian border between 2016 and 2017 and currently used vaccines, phylogenetic analysis of the full VP1 virus sequences was carried out on samples collected from both sides of the border. A total of 43 clinical samples were collected from animals exhibiting signs of FMD and VP1 sequences generated from 11 of them. Eight out of the 11 sequences obtained belonged to serotype O and three belonged to serotype A. The serotype O sequences obtained showed limited nucleotide divergence (average of 4.9%) and belonged to topotype East Africa-2, whereas the most common O-type vaccine strain used in the region (O/KEN/77/78) belonged to East Africa-1. The serotype A viruses belonged to topotype Africa-G1 (average nucleotide divergence 7.4%), as did vaccine strain K5/1980. However, vaccine strain K35/1980 belonged to Africa G VII with an average sequence divergence of 20.5% from the study sequences. The genetic distances between current vaccine strains and circulating field strains underscores the crucial need for regular vaccine matching and the importance of collaborative efforts for better control of FMD along this border area.


Assuntos
Vírus da Febre Aftosa/genética , Febre Aftosa/virologia , Animais , Proteínas do Capsídeo/genética , Bovinos/virologia , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/virologia , Febre Aftosa/epidemiologia , Variação Genética/genética , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Análise de Sequência de DNA/veterinária , Sorogrupo , Tanzânia/epidemiologia , Uganda/epidemiologia
20.
Parasit Vectors ; 12(1): 159, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961652

RESUMO

BACKGROUND: The diagnosis of filariasis traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears. This approach has several limitations. We developed a semi-automated microfluidic device to improve and simplify the detection of filarial nematodes. METHODS: The efficiency and repeatability of the microfluidic device was evaluated. Human EDTA blood samples were 'spiked' with B. malayi mf at high, moderate, and low levels, and subsequently tested 10 times. The device was also used for a field survey of feline filariasis in 383 domesticated cats in an area of Narathiwat Province, Thailand, the endemic area of Brugia malayi infection. RESULTS: In the control blood arbitrarily spiked with mf, the high level, moderate level and low level mf-positive controls yielded coefficient variation (CV) values of 4.44, 4.16 and 4.66%, respectively, at the optimized flow rate of 6 µl/min. During the field survey of feline filariasis in Narathiwat Province, the device detected mf in the blood of 34 of 383 cats (8.9%) whereas mf were detected in 28 (7.3%) cats using the blood smear test. Genomic DNA was extracted from mf trapped in the device after which high-resolution melting (HRM) real-time PCR assay was carried out, which enabled the simultaneous diagnosis of filarial species. Among the 34 mf-positive samples, 12 were identified as B. malayi, 15 as Dirofilaria immitis and 7 as| D. repens. CONCLUSIONS: We developed a semi-automated microfluidic device to detect mf of filarial parasites that could be used to diagnose lymphatic filariasis in human populations. This novel device facilitates rapid, higher-throughput detection and identification of infection with filariae in blood samples.


Assuntos
Doenças do Gato/diagnóstico , Filariose/veterinária , Técnicas Analíticas Microfluídicas/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Automação Laboratorial , Gatos , Filariose/diagnóstico , Reprodutibilidade dos Testes
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