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1.
J Zoo Wildl Med ; 51(2): 334-349, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32549563

RESUMO

There is an unmet need for specific diagnostics of immune perturbations and inflammation in beluga whale (Delphinapterus leucas) clinical care. Quantitative real-time polymerase chain reaction (qPCR) has been used to measure immunomediator gene transcription in beluga whales. The study hypothesis was that a qPCR-based immunomediator assay would supplement routine clinical data with specific and sensitive information on immune status. Two beluga whale clinical cases provided an opportunity to test this hypothesis: a whale with a skin laceration and a whale with gastrointestinal inflammation. Mitogen-stimulated immunomediator gene transcription (MSIGT) was compared between the cases and healthy contact whales. In both case studies, mitogens increased transcription of IL1B, PTGS2 (Cox-2), TNF, HIF1A, and IL2 but decreased IL10 transcription in peripheral blood mononuclear cells (PBMC) from the abnormal whale over the control. Correlations were identified between most immunomediators tested and one or more standard blood clinical values. Considering all 15 immunomediators tested, the whale with gastrointestinal inflammation had a more unique MSIGT signature than the whale with a laceration. These results support further elucidation of beluga whale PBMC cytokine profiles for use as immune biomarkers.


Assuntos
Beluga/genética , Imunomodulação/genética , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transcrição Genética , Animais , Animais de Zoológico/genética , Animais de Zoológico/imunologia , Beluga/imunologia , Feminino , Leucócitos Mononucleares/imunologia , Masculino , Mitógenos
2.
J Zoo Wildl Med ; 51(2): 391-397, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32549570

RESUMO

Systemic isosporosis (formerly atoxoplasmosis), is a protozoal infection that causes death in nestling and fledgling passerine birds impacting ex situ breeding and reintroduction programs. Because current antemortem diagnostic tests lack sensitivity, a qPCR was developed for detection of Isospora spp. using primers and a fluorescent-tagged MGB probe targeting the large subunit (28s) ribosomal RNA gene (assay efficiency = >100%; sensitivity = <1 dsDNA copy). The assay was used to screen postmortem frozen or formalin-fixed paraffin-embedded tissue samples from passerine birds (n = 24; 12 with confirmed systemic isosporosis), whole blood and feces (n = 38) from live passerines, and other tissues infected with phylogenetically similar protozoa. The qPCR identified Isospora sp. DNA in tissues from 21/24 birds including 12/12 birds with cytologically-histologically confirmed infection (100% sensitivity) and 9/12 birds lacking microscopic organisms. The assay also amplified Eimeria sp. DNA; however, sequence analysis ruled out infection in the passerine cases. Blood and/or feces were positive in 30/38 birds, and in only 7/38 birds, blood and feces both contained Isospora sp. DNA. Finally, the qPCR was utilized to screen 30 consecutive daily fecal samples from live passerines (n = 20) to determine optimal sampling protocols. One or more of the daily fecal samples were positive in all 20 birds. In individual birds, the interval between positive qPCR amplification results ranged from 0 to 23 days, with an average of 5.85 days. Simulated application of 13 potential sample collection schedules was used to identify the sensitivity of repeated testing for identification of infected birds. Increased sampling days resulted in higher sensitivity but increased both cost and animal handling requirements. Based on statistical analysis and clinical considerations, the testing recommendation for detection of fecal shedding was collection and assay of five consecutive daily fecal samples, which had an average diagnostic sensitivity of 0.86.


Assuntos
Doenças das Aves/diagnóstico , Isospora/isolamento & purificação , Isosporíase/veterinária , Técnicas de Diagnóstico Molecular/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Aves Canoras , Animais , Doenças das Aves/parasitologia , Sangue/parasitologia , Fezes/parasitologia , Isosporíase/diagnóstico , Isosporíase/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas
3.
Mol Cell Probes ; 53: 101618, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32534013

RESUMO

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 102 copies/µL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.


Assuntos
Sondas de DNA , Diarreia/veterinária , Doenças do Cão/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Avastrovirus , Coronavirus Canino , Diarreia/diagnóstico , Diarreia/virologia , Doenças do Cão/diagnóstico , Cães , Kobuvirus , Parvovirus Canino , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
4.
PLoS One ; 15(5): e0232571, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32442180

RESUMO

Molecular-based testing of poultry dust has been used as a fast, sensitive and specific way to monitor viruses in chicken flocks but it provides no information on viral viability. Differentiation of viable and nonviable virus would expand the usefulness of PCR-based detection. This study tested three treatments (1. DNAse, 2. propidium monoazide [PMA], 3. immunomagnetic separation [IMS]) applied to dust or virus stock prior to nucleic acid extraction for their ability to exclude nonviable virus from PCR amplification. Infectious laryngotracheitis virus (ILTV) was used as a model. These treatments assume loss of viral viability due to damage to the capsid or to denaturation of epitope proteins. DNAse and PMA assess the integrity of the capsid to penetration by enzyme or intercalating dye, while IMS assesses the integrity of epitope proteins. Treatments were evaluated for their ability to reduce PCR signal, measured as ILTV log10 genomic copies (ILTV GC), of heat and chemically inactivated ILTV in poultry dust and virus stock. Compared to untreated dust samples, there was an overall reduction of 1.7 ILTV GC after IMS treatment (p<0.01), and a reduction of 2.0 ILTV GC after PMA treatment (p<0.0001). DNAse treatment did not reduce ILTV GC in dust (p = 0.68). Compared to untreated virus stocks, there was an overall reduction of 0.5 ILTV GC after DNAse treatment (p = 0.04), a reduction of 1.8 ILTV GC after IMS treatment (p<0.001) and a reduction of 1.4 ILTV GC after PMA treatment (p<0.0001). None of the treatments completely suppressed the detection of inactivated ILTV GC. In conclusion, treatments that use capsid integrity or protein epitope denaturation as markers to assess ILTV infectivity are unsuitable to accurately estimate proportions of viable virus in poultry dust and virus stocks.


Assuntos
DNA Viral/genética , Infecções por Herpesviridae/veterinária , Herpesvirus Galináceo 1/genética , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Embrião de Galinha/virologia , Galinhas/virologia , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Separação Imunomagnética/veterinária , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos
5.
Arch Razi Inst ; 75(1): 17-22, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32291998

RESUMO

Avian influenza viruses (AIV) affect a wide range of birds and mammals, cause severe economic damage to the poultry industry, and pose a serious threat to humans. Highly pathogenic avian influenza viruses (HPAI) H5N1 were first identified in Southeast Asia in 1996 and spread to four continents over the following years. The viruses have caused high mortality in chickens and various bird species and deadly infections in humans. Multiple conventional methods have been so far introduced for the detection and identification of avian influenza viruses. Traditional virus isolation methods are gold standard protocol in AI detection; nonetheless, virus isolation in embryonating chicken eggs (ECE) is not a rapid method for the detection of influenza viruses since it is time-consuming and labor-intensive. Furthermore, the isolation of highly pathogenic viruses, such as H5, needs BSL3 laboratories. Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR) is a sensitive and specific method for the detection of influenza viruses. The application of these nucleic acid-based techniques has increased our ability to identify and perform influenza virus care programs, especially in surveillance programs. The current study aimed to detect H5 subtype of avian influenza (AI) virus using fast, specific, and sensitive TaqMan RRT-PCR. Notably, single step RRT-PCR was used to prevent possible laboratory contamination. The specificity of this test was evaluated using nucleic acid extracted from several poultry pathogenic microorganisms and negative clinical specimens from AI-uninfected birds. The sensitivity analysis of the RRT-PCR assay was performed using in vitro-transcribed RNA copy and 10-fold serial dilution of standard AI virus with specific titer. The results indicated the high sensitivity of this method and the lowest detectable dilution of this method based on RNA copies and 1:10 serial dilutions of the standard virus was 10 1.9 EID50 /100.


Assuntos
Galinhas , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Doenças das Aves Domésticas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H5N2/isolamento & purificação , Vírus da Influenza A Subtipo H5N8/isolamento & purificação , Influenza Aviária/virologia , Óvulo/virologia , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
6.
BMC Vet Res ; 16(1): 114, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32295612

RESUMO

BACKGROUND: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS: A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS: Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.


Assuntos
Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Genótipo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Regiões 5' não Traduzidas/genética , Aerossóis , Microbiologia do Ar , Animais , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 1/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
7.
J Vet Diagn Invest ; 32(3): 356-362, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32310022

RESUMO

Flavobacterium columnare is the causative agent of columnaris disease in a variety of fish hosts. Using modifications to previously established protocols, a quantitative PCR (qPCR) assay was validated for the detection of 2 predominant F. columnare genomovars. The oligonucleotide primer and probe combination was designed to amplify a 203-bp region of the chondroitin AC lyase gene (GenBank AY912281) of F. columnare. There were no significant differences in amplification between genomovars. Comparable quantities of genomic DNA from 10 F. columnare strains, 5 representatives of each genomovar, produced similar results. Serial dilutions of purified PCR product demonstrated the limit of sensitivity for the assay was ~ 10 copies per reaction. The presence of gill and spleen tissue did not significantly affect the sensitivity of the assay. Comparably, bacterial DNA detected from the liver and kidney was less sensitive than pure bacterial DNA. However, detection from these tissues was within one order of magnitude of other tissues, indicating this reduction may have minimal analytic significance. This validated assay was used to approximate the minimum infectious dose for F. columnare isolate 94-081 in channel catfish and assess bacterial loads in gill and kidney tissues 48 h post-infection.


Assuntos
Doenças dos Peixes/diagnóstico , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/isolamento & purificação , Ictaluridae , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/diagnóstico , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/classificação , Flavobacterium/genética , Genótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos
8.
J Dairy Sci ; 103(6): 5431-5439, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32229116

RESUMO

Automatic flushing of milking clusters between milking events is a control measure aimed at reducing transmission of mastitis pathogens from infectious milk to a subsequently milked cow. We evaluated the effect of flushing with cold water and flushing with water containing peracetic acid (PAA) on the concentration of Staphylococcus aureus in teat cup liners. Thirty-two clusters in a swing-over milking parlor (Dairymaster, Causeway, Ireland) were subjected to a simulated milking with S. aureus-contaminated milk. Sixteen clusters were not flushed (controls), whereas 8 clusters were flushed with cold water (966 ± 32 mL) and 8 clusters were flushed with water containing PAA (200 mL/mL). A random teat cup in each cluster was sampled by rinsing with a phosphate buffer. Teat cup samples were cultured on the day following collection on Baird-Parker plates to determine the concentration of S. aureus. In teat cup samples from control clusters, the mean concentration of S. aureus was 2.8 × 105 cfu/mL. The concentration of S. aureus was zero in teat cup samples from clusters flushed with cold water. In teat cup samples from clusters flushed with water containing PAA, the concentration of S. aureus was in general reduced compared with control clusters, but S. aureus was not removed completely. However, the automatic cluster flushing did not function properly when clusters were flushed with water containing PAA; thus, results reflected the effect of inadequate function rather than the effect of adding disinfectant to the flushing water. Before the main study, we conducted a pilot study to evaluate whether teat cup sampling with swabs and sample analysis with quantitative PCR were appropriate methods for the main study. Specifically, we evaluated the effect of swab sample mass on detection of S. aureus by quantitative PCR in the laboratory, Further, we compared PCR and bacterial culture on detection of S. aureus in a suspension following disinfection of the suspension with PAA. We sampled 20 identical S. aureus suspensions for culture and PCR by swabs before and after disinfection with PAA. Swab sample mass was determined by differential weighing and contributed to 46% of the variation observed in detection of S. aureus by PCR. Following disinfection with PAA, S. aureus remained detectable by PCR, although culturability ceased. Based on these results, we sampled teat cups in the main study with a buffer rinse and quantified S. aureus in the samples by bacterial culture. We concluded that automatic cluster flushing with cold water was effective in removing S. aureus from teat cup liners and that addition of PAA was therefore not necessary.


Assuntos
Bovinos , Indústria de Laticínios/métodos , Desinfetantes/farmacologia , Desinfecção/métodos , Glândulas Mamárias Animais/microbiologia , Ácido Peracético/farmacologia , Staphylococcus aureus , Animais , Feminino , Higiene , Irlanda , Leite/microbiologia , Mamilos/microbiologia , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Manejo de Espécimes , Água
9.
Vet Ital ; 56(1)2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-32343095

RESUMO

Cat scratch disease (CSD) is a zoonotic disease, caused predominantly by Bartonella henselae and transmitted to humans through a scratch or bite of the cat. Cat represents the principal reservoir and healthy carrier of Bartonella, which is mainly transmitted, among cats, by the flea Ctenocephalides felis. During 2014, fifty­two samples of whole blood and sera were collected randomly from cats in Abruzzo region and were examined by real-time PCR and IFAT tests, respectively. Seven samples out of fifty­two (13.5%) resulted positive for Bartonella spp. in both tests, while six specimens (11.5%) resulted real-time PCR negative but IgG positive; thirty­nine were instead both real-time PCR and IFAT negative (75%). Sequence analysis of a fragment of DNA identified B. henselae and B. clarridgeiae in four and in two real­time PCR positive samples, respectively.


Assuntos
Bartonella henselae/isolamento & purificação , Doenças do Gato/epidemiologia , Animais , Bartonella henselae/genética , Doenças do Gato/microbiologia , Doença da Arranhadura de Gato/prevenção & controle , Gatos , Ctenocephalides/parasitologia , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Itália/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Zoonoses/prevenção & controle
10.
Parasitol Res ; 119(5): 1683-1690, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32285265

RESUMO

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.


Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Medula Óssea/parasitologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/parasitologia , Linfonodos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Baço/parasitologia
11.
PLoS Negl Trop Dis ; 14(3): e0008129, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203507

RESUMO

BACKGROUND: Schistosomiasis is a neglected tropical parasitic disease associated with severe pathology, mortality and economic loss worldwide. Programs for disease control may benefit from specific and sensitive diagnostic methods to detect Schistosoma trematodes in aquatic environments. Here we report the development of novel environmental DNA (eDNA) qPCR assays for the presence of the human-infecting species Schistosoma mansoni, S. haematobium and S. japonicum. METHODOLOGY/PRINCIPAL FINDINGS: We first tested the specificity of the assays across the three species using genomic DNA preparations which showed successful amplification of target sequences with no cross amplification between the three focal species. In addition, we evaluated the specificity of the assays using synthetic DNA of multiple Schistosoma species, and demonstrated a high overall specificity; however, S. japonicum and S. haematobium assays showed cross-species amplification with very closely-related species. We next tested the effectiveness of the S. mansoni assay using eDNA samples from aquaria containing infected host gastropods, with the target species revealed as present in all infected aquaria. Finally, we evaluated the effectiveness of the S. mansoni and S. haematobium assays using eDNA samples from eight discrete natural freshwater sites in Tanzania, and demonstrated strong correspondence between infection status established using eDNA and conventional assays of parasite prevalence in host snails. CONCLUSIONS/SIGNIFICANCE: Collectively, our results suggest that eDNA monitoring is able to detect schistosomes in freshwater bodies, but refinement of the field sampling, storage and assay methods are likely to optimise its performance. We anticipate that environmental DNA-based approaches will help to inform epidemiological studies and contribute to efforts to control and eliminate schistosomiasis in endemic areas.


Assuntos
DNA Ambiental/isolamento & purificação , Água Doce/parasitologia , Schistosoma/classificação , Schistosoma/genética , Schistosoma/isolamento & purificação , Animais , DNA de Helmintos/isolamento & purificação , Monitoramento Ambiental , Genes de Helmintos/genética , Técnicas de Amplificação de Ácido Nucleico/veterinária , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Schistosoma haematobium/genética , Schistosoma haematobium/isolamento & purificação , Schistosoma japonicum/genética , Schistosoma japonicum/isolamento & purificação , Schistosoma mansoni/genética , Schistosoma mansoni/isolamento & purificação , Esquistossomose/epidemiologia , Esquistossomose/parasitologia , Esquistossomose mansoni/epidemiologia , Esquistossomose mansoni/parasitologia , Caramujos/parasitologia , Especificidade da Espécie , Tanzânia
12.
Poult Sci ; 99(3): 1643-1654, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32115036

RESUMO

The physiological roles of thyrotropin-releasing hormone (TRH) are proposed to be mediated by TRH receptors (TRHR), which have been divided into 3 subtypes, namely, TRHR1, TRHR2, and TRHR3, in vertebrates. Although 2 TRH receptors (TRHR1 and TRHR3) have been predicted to exist in birds, it remains unclear whether TRHR3 is a functional TRH receptor similar to TRHR1. Here, we reported the functionality and tissue expression of TRHR3 in chickens. The cloned chicken TRHR3 (cTRHR3) encodes a receptor of 387 amino acids, which shares high-amino-acid identities (63-80%) to TRHR3 of parrots, lizards, Xenopus tropicalis, and tilapia and comparatively lower sequence identities to chicken TRHR1 or mouse TRHR2. Using cell-based luciferase reporter assays and Western blot, we demonstrated that similar to chicken TRHR1 (cTRHR1), cTRHR3 expressed in HEK 293 cells can be potently activated by TRH and that its activation stimulates multiple signaling pathways, indicating both TRH receptors are functional. Quantitative real-time PCR revealed that cTRHR1 and cTRHR3 are widely, but differentially, expressed in chicken tissues, and their expression is likely controlled by promoters located upstream of exon 1, which display strong promoter activities in cultured DF-1 cells. cTRHR1 is highly expressed in the anterior pituitary and testes, while cTRHR3 is highly expressed in the muscle, testes, fat, pituitary, spinal cord, and many brain regions (including hypothalamus). These findings indicate that TRH actions are likely mediated by 2 TRH receptors in chickens. In conclusion, our data provide the first piece of evidence that both cTRHR3 and cTRHR1 are functional TRH receptors, which helps to elucidate the physiological roles of TRH in birds.


Assuntos
Sequência de Aminoácidos , Galinhas/genética , Receptores do Hormônio Liberador da Tireotropina/genética , Animais , Linhagem Celular , Clonagem Molecular , Patos , Feminino , Expressão Gênica , Células HEK293 , Humanos , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores do Hormônio Liberador da Tireotropina/química , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/metabolismo
13.
BMC Genomics ; 21(1): 187, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111155

RESUMO

BACKGROUND: Aohan fine wool sheep (AFWS) is a historically bred fine wool sheep, cultivated in China. The wool has excellent quality and good textile performance. Investigating the molecular mechanisms that regulate wool growth is important to improve wool quality and yield. Circular RNAs (circRNAs) are widely expressed non-coding RNAs that can act as competitive endogenous RNAs (ceRNAs) to bind to miRNAs. Although circRNAs have been studied in many fields, research on their activity in sheep wool follicles is limited. To understand the regulation of circRNAs in the growth of fine wool in sheep, we used RNA-Seq to identify circRNAs in sheep shoulder skin samples at three developmental stages: embryonic day 90 (E90d), embryonic day 120 (E120d), and at birth (Birth). RESULTS: We identified 8753 circRNAs and found that 918 were differentially-expressed. We then analyzed the classification and characteristic of the circRNAs in sheep shoulder skin. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we identified the source genes of circRNAs, which were mainly enriched in cellular component organization, regulation of primary metabolic processes, tight junctions, and the cGMP-PKG and AMPK signaling pathways. In addition, we predicted interactions between 17 circRNAs and eight miRNAs, using miRanda software. Based on the significant pathways, we speculate that circ_0005720, circ_0001754, circ_0008036, circ_0004032, circ_0005174, circ_0005519, and circ_0007826 might play an important role in regulating wool follicle growth in AFWS. Seven circRNAs were randomly selected to validate the RNA-Seq results, using qRT-PCR. CONCLUSION: Our results provide more information about circRNAs regulation of wool follicle development in AFWS, and establish a solid foundation for future research.


Assuntos
RNA Circular/genética , Análise de Sequência de RNA/veterinária , Lã/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Ovinos , Pele , Lã/química
14.
J Vet Diagn Invest ; 32(3): 423-428, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32188352

RESUMO

Avian coxiellosis is an emerging cause of morbidity and mortality among captive psittacines, and the utility of a rapid detection test using easily obtained samples is paramount in a clinical setting. New sequences were obtained from 3 genes: groEL, dnaK, and rpoB. We developed probe-hybridization quantitative PCR (qPCR) assays using groEL and dnaK genes. Samples, including splenic aspirates, liver aspirates, whole blood, and choanal, conjunctival, and cloacal swabs, were collected from 4 psittacine species including 3 blue-and-gold macaws (Ara ararauna), 2 scarlet-chested parrots (Neophema splendida), 1 Timneh African grey parrot (Psittacus timneh), and 1 yellow-naped Amazon parrot (Amazona auropalliata). Retrospective review of postmortem findings from 3 of these psittacines included splenomegaly, hepatitis, and/or transmission electron microscopy confirmation consistent with previous reports of avian coxiellosis. There was 100% agreement between these assays and consensus PCR with sequencing. A Wilcoxon rank-sum test found a strong correlation between groEL and dnaK cycle threshold values (p < 0.001), validating these assays for detection of this avian Coxiella sp.


Assuntos
Doenças das Aves/microbiologia , Coxiella/isolamento & purificação , Infecções por Bactérias Gram-Negativas/veterinária , Papagaios , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Amazona , Animais , Doenças das Aves/patologia , Coxiella/classificação , Infecções por Bactérias Gram-Negativas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos Retrospectivos
15.
Parasit Vectors ; 13(1): 111, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111232

RESUMO

BACKGROUND: Determination of blood-meal hosts in blood-fed female Anopheles mosquitoes is important for evaluating vectorial capacity of vector populations and assessing effectiveness of vector control measures. Sensitive molecular methods are needed to detect traces of host blood in mosquito samples, to differentiate hosts, and to detect mixed host blood meals. This paper describes a molecular probe-based quantitative PCR for identifying blood-meal hosts in Anopheles malaria vectors from Papua New Guinea. METHODS: TaqMan oligonucleotide probes targeting specific regions of mitochondrial or nuclear DNA of the three primary Anopheles blood-meal hosts, humans, pigs and dogs, were incorporated into a multiplex, quantitative PCR which was optimized for sensitivity and specificity. RESULTS: Amplification of serially diluted DNA showed that the quantitative PCR detected as low as 10-5 ng/µl of host DNA. Application to field-collected, blood-fed Anopheles showed that the quantitative PCR identified the vertebrate hosts for 89% (335/375) of mosquitoes whereas only 55% (104/188) of blood-meal samples tested in a conventional PCR were identified. Of the 104 blood-fed Anopheles that were positive in both PCR methods, 16 (15.4%) were identified as mixed blood meals by the quantitative PCR whereas only 3 (2.9%) were mixed blood meals by the conventional PCR. CONCLUSIONS: The multiplex quantitative PCR described here is sensitive at detecting low DNA concentration and mixed host DNA in samples and useful for blood-meal analysis of field mosquitoes, in particular mixed-host blood meals.


Assuntos
Anopheles/fisiologia , Análise Química do Sangue , Refeições , Mosquitos Vetores/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , DNA/análise , DNA/genética , Cães , Comportamento Alimentar , Feminino , Humanos , Papua Nova Guiné , Suínos , Vertebrados
16.
Vet Res ; 51(1): 46, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209128

RESUMO

Infection with Bordetella bronchiseptica (Bb), a pathogen involved in canine infectious respiratory disease complex, can be confirmed using culture or qPCR. Studies about the canine lung microbiota (LM) are recent, sparse, and only one paper has been published in canine lung infection. In this study, we aimed to compare the LM between Bb infected and healthy dogs, and to correlate sequencing with culture and qPCR results. Twenty Bb infected dogs diagnosed either by qPCR and/or culture and 4 healthy dogs were included. qPCR for Mycoplasma cynos (Mc) were also available in 18 diseased and all healthy dogs. Sequencing results, obtained from bronchoalveolar lavage fluid after DNA extraction, PCR targeting the V1-V3 region of the 16S rDNA and sequencing, showed the presence of Bb in all diseased dogs, about half being co-infected with Mc. In diseased compared with healthy dogs, the ß-diversity changed (P = 0.0024); bacterial richness and α-diversity were lower (P = 0.012 and 0.0061), and bacterial load higher (P = 0.004). Bb qPCR classes and culture results correlated with the abundance of Bb (r = 0.71, P < 0.001 and r = 0.70, P = 0.0022). Mc qPCR classes also correlated with the abundance of Mc (r = 0.73, P < 0.001). Bb infection induced lung dysbiosis, characterized by high bacterial load, low richness and diversity and increased abundance of Bb, compared with healthy dogs. Sequencing results highly correlate with qPCR and culture results showing that sequencing can be reliable to identify microorganisms involved in lung infectious diseases.


Assuntos
Carga Bacteriana , Infecções por Bordetella/veterinária , Bordetella bronchiseptica/isolamento & purificação , Doenças do Cão/microbiologia , Pulmão/microbiologia , Infecções Respiratórias/veterinária , Animais , Infecções por Bordetella/microbiologia , Coinfecção/microbiologia , Coinfecção/veterinária , Cães , Microbiota , Mycoplasma/isolamento & purificação , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Infecções Respiratórias/microbiologia
17.
Parasitol Int ; 76: 102069, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32032726

RESUMO

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/µL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.


Assuntos
Citocromos b/análise , Plasmodium/genética , Proteínas de Protozoários/análise , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Mitocondriais/análise , Compostos Orgânicos/química , Plasmodium/classificação , Plasmodium malariae/classificação , Plasmodium malariae/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Especificidade da Espécie
18.
Parasitol Res ; 119(4): 1381-1386, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32107620

RESUMO

The prevalence data of Leishmania infantum infection in cats are characterized by a large variability mainly attributed to the differences in diagnostic techniques. In the absence of consensus about the method of choice for diagnosing feline leishmaniosis, the performance of a new immunofluorescence antibody test (IFAT) was herein analytically described by the comparison with IFAT commonly used for the diagnosis of canine leishmaniosis (i.e., IFAT-OIE) and a laboratory enzyme-linked immunosorbent assay (ELISA). Sera of cats living in visceral leishmaniosis-endemic (n = 105) and visceral leishmaniosis-non-endemic (n = 50) areas were tested by the above methodologies and real-time PCR (qPCR). The most frequent result was represented by triple negativity to the three tests (IFAT-OIE, ELISA, and qPCR) in 42.9% and 80% cats from endemic and non-endemic areas, respectively. Bayes latent class analysis gave an output probability of 34.1% (posterior standard deviation, psd = 5.4%) of true L. infantum cases (TCL) which represent the true estimated prevalence of infection. The sensitivity of each variable contributing to define the TCL was 24% (psd = 6.3%) for qPCR, 78.8% (psd = 8.7%) for ELISA and 91.8% (psd = 5.2%) for IFAT-OIE. The probability to be a TCL was 94.5% for the sample from an endemic area. The cross-validation of the new IFAT by a logistic model correctly identified as positive 80.7% of subjects defined as TCL and negative 89.9% as not TCL, respectively, by the Bayesian model. The study results estimate a good accuracy of the IFAT in predicting cats exposed to L. infantum. Therefore, this procedure may be beneficial for screening cat populations for a better understanding of the epidemiology of feline leishmaniosis.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Gato/diagnóstico , Técnica Direta de Fluorescência para Anticorpo/métodos , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , Teorema de Bayes , Doenças do Gato/epidemiologia , Doenças do Gato/parasitologia , Gatos , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterinária
19.
Pesqui. vet. bras ; 40(2): 82-87, Feb. 2020. tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1098440

RESUMO

The genus Mycoplasma includes more than 200 bacterial species that cause disease in animals. It is responsible for causing mastitis in bovines and may be related to other manifestations, such as arthritis and pneumonia in calves and heifers. The present study aimed to detect Mycoplasma bovis isolated from milk samples of bovine clinical mastitis, and to compare the isolation rates in two culture media: Hayflick and SP4. An initial screening was performed in order to detect the presence of the class Mollicutes in 1166 milk samples from clinical mastitis by the conventional Polymerase Chain Reaction (PCR) technique. According to the 1166 milk samples evaluated, 8.6% (100/1166) were positive to class Mollicutes. Regarding molecular analyses, 1.1% (13/1166) of conventional PCR for positive M. bovis was obtained and 0.9% (11/1166) in real-time PCR. The results of the microbiological culture of the 100 samples previously screened demonstrated that 6% (6/100) of colony growth have been developed when using the Hayflick medium, and 11% (11/100) when using the SP4 medium (including the positive on Hayflick medium). Concerning the 11 isolates obtained in the microbiological culture, conventional PCR confirmed M. bovis in nine of them, and two cultures were negative. In the phylogenetic analysis of the isolates, all of them were grouped in M. bovis and M. agalactiae clusters. The results confirmed the importance of the presence of M. bovis in the etiology of bovine clinical mastitis and reinforced the need for further studies to elucidate other Mycoplasma species that may be involved in bovine clinical mastitis in Brazil.(AU)


O gênero Mycoplasma inclui mais de 200 espécies que causam doenças nos animais. É responsável por quadros de mastite em bovinos, podendo também estar relacionado à outras manifestações como artrite e pneumonia em bezerros e novilhas. O presente estudo objetivou a detecção de Mycoplasma bovis isolados a partir de amostras de leite de mastite clínica bovina, bem como, a comparação da taxa de isolamento em dois meios de cultura: Hayflick e SP4. Para efeito de triagem amostral, foram avaliadas quanto à presença da classe Mollicutes 1166 amostras de leite de casos de mastite clínica pela técnica de PCR convencional. Das 1166 amostras de leite avaliadas, 8,6% (100/1166) foram positivas à classe. Nas análises moleculares, obteve-se 1,1% (13/1166) de positividade para Mycoplasma bovis na PCR convencional e 0,9% (11/1166) na PCR em tempo real. Os resultados do cultivo microbiológico das 100 amostras triadas previamente demonstraram 6% (6/100) de crescimento de colônias ao se utilizar o meio Hayflick e 11% (11/100) ao se utilizar o meio SP4 (incluindo as positivas ao primeiro). A partir dos 11 isolados obtidos no cultivo microbiológico, a PCR convencional confirmou Mycoplasma bovis em nove deles, e dois foram negativos para o agente. Na análise filogenética dos isolados, todos agruparam no cluster Mycoplasma bovis e Mycoplasma agalactiae. Frente aos resultados, ressalta-se a importância da presença de Mycoplasma bovis na etiologia da mastite clínica bovina e reforça a necessidade de estudos mais aprofundados para a elucidação de outras espécies de micoplasmas que possam estar envolvidas na mastite bovina.(AU)


Assuntos
Animais , Feminino , Bovinos , Mycoplasma bovis/isolamento & purificação , Leite/microbiologia , Mastite Bovina/etiologia , Infecções por Mycoplasma/veterinária , Reação em Cadeia da Polimerase/veterinária , Tenericutes , Mycoplasma agalactiae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária
20.
BMC Vet Res ; 16(1): 16, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937305

RESUMO

BACKGROUND: Parasites of the family Spirorchiidae cause disease and mortality in marine and freshwater turtles; two species, Hapalotrema mistroides and Neospirorchis sp., are reported in the resident population of loggerhead turtles of the Mediterranean Sea, with the first being the most widespread. In vivo diagnosis of spirorchidiasis can represent a challenge in guaranteeing prompt control and treatment of the disease and is currently limited to copromicroscopy. The aim of this study was the development of a real time PCR assay with TaqMan probe for the detection of H. mistroides infection in the blood of live loggerhead turtles, Caretta caretta, hospitalized in rehabilitation centres. Its potential use for in vivo diagnosis is explored. RESULTS: The developed real time PCR successfully detected H. mistroides DNA from both positive controls and experimental blood samples of live loggerhead sea turtles, showing good specificity, sensitivity and good reaction efficiency. Two out of three turtles which had demonstrated positivity at copromicroscopy also tested positive to this blood assay; DNA of H. mistroides was detected within the blood of one sea turtle, which tested negative for copromicroscopy. CONCLUSIONS: This study describes a specific and rapid molecular assay to detect H. mistroides infection from live sea turtles and highlights for the first time the presence of DNA of this species in turtle blood samples. Since this assay is able to detect low amounts of the parasitic free DNA in blood samples, its application could be helpful for in vivo diagnosis of H. mistroides infection as well as for epidemiological purposes.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real/veterinária , Infecções por Trematódeos/veterinária , Tartarugas/parasitologia , Animais , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Mar Mediterrâneo , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Trematódeos/genética , Trematódeos/isolamento & purificação , Infecções por Trematódeos/diagnóstico , Tartarugas/sangue
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