scholarly journals Cattle rabies: the effect of clinical evolution, viral genetic lineage, and viral load on the severity of histological lesions

2020 ◽  
Vol 40 (4) ◽  
pp. 227-233
Author(s):  
Claudia S. Wisser ◽  
André Thaler Neto ◽  
Helena B.C.R. Batista ◽  
Enio Mori ◽  
Maria E.R. Chierato ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Fluorescent Antibody ◽  
Inflammatory Lesion ◽  
Genetic Lineage ◽  
Fluorescent Antibody Technique ◽  
Chain Reaction ◽  
Antibody Technique ◽  
Qrt Pcr ◽  
Polymerase Chain

ABSTRACT: Our objective was the characterization and staging of histological lesions in different anatomical sites of the central nervous system (CNS) of rabid cattle. The severity of the lesions was compared with the clinical stages of the disease, the variants of viral isolates, and with the load of virus. Thirty-one spontaneously affected rabid cattle the state of Santa Catarina underwent clinical follow-up and were eventually necropsied. CNS tissues were sampled and submitted to direct fluorescent antibody technique (DFAT), immunohistochemistry (IHC), routine histopathology with hematoxylin and eosin stain (HE), reverse transcriptase polymerase chain reaction (RT-PCR), and polymerase chain reaction in quantitative reverse transcriptase in real time (qRT-PCR). Affected cattle were allotted in four groups according to their clinical stage when euthanized: G1, euthanized while standing; G2, euthanized when in sternal recumbence; G3, euthanized when in lateral recumbence; and G4, affected cattle with natural death. In order to evaluate the degree of severity of the lesions and the presence of Negri bodies (NBs), the brain was sectioned at 9 sites. Additionally, spinal cord and trigeminal ganglion sections were examined. The intensity of the lesions was graded as either absent, mild, moderate, or marked, and the presence or absence of the NBs was noted. Histological lesions were characterized by lymphocytic and monocytic meningoencephalitis with NBs in 28 cases. In all analyzed groups, intensities of histological lesions ranging from mild to severe were observed. Brain regions with the highest inflammatory lesion intensity were the medulla at the level of obex, followed by the colliculus and thalamus. NBs were observed in a higher percentage in the cerebellum, followed by medulla at the obex level, striatum complex, and frontal telencephalon. The duration of the clinical course of the disease did not influence the intensity of the inflammatory lesion, but it did influence the presence of NBs, with a higher percentage of these inclusions in cattle that died naturally than in euthanized cattle. All isolated rhabdovirus included in this study were genetically compatible with samples from hematophagous bats Desmodus rotundus. The evaluation by qRT-PCR did not demonstrate a correlation between lesion intensity and the amount of virus.

scholarly journals EVALUASI KIT DETEKSI CEPAT TERHADAP SAMPEL OTAK ANJING TERINFEKSI VIRUS RABIES

2015 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael Haryadi Wibowo ◽  
Tri Untari ◽  
Sidna Artanto ◽  
Surya Amanu ◽  
AETH. Wahyuni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Fluorescent Antibody ◽  
Rapid Test ◽  
Canine Parvovirus ◽  
Fluorescent Antibody Technique ◽  
Chain Reaction ◽  
Antibody Technique ◽  
Test Kit ◽  
Polymerase Chain

Penelitian ini bertujuan mengetahui potensi kit deteksi cepat Anigen® rapid test kit rabies Ag dalam mendeteksi virus rabies pada sampel otakanjing yang diperoleh dari lapangan yang meliputi batas deteksi, kecepatan reaksi, uji reaksi silang, uji sensitivitas, dan spesifisitas. Batas deteksi ditentukan dengan pengenceran secara serial kontrol positif virus rabies dan selanjutnya diuji dengan rapid test kit sesuai petunjuk produsen. Uji reaksi silang dilakukan dengan canine parvovirus, Escherichia coli, dan Salmonella sp. Uji sensitivitas dan spesifisitas dilakukan terhadap sampel otak yang telah dikonfirmasi positif rabies dengan uji fluorescent antibody technique. Konfirmasi uji rapid test tersebut dilakukan dengan reverse transcriptase polymerase chain reaction. Berdasarkan data yang diperoleh dalam penelitian ini dapat disimpulkan bahwa Anigen® rapid test kit rabies Ag mampu mendeteksi sampel yang mengandung virus rabies dengan titer 0,5 x log 106,5/0,03 ml, dengan rata-rata kecepatan reaksi 1,8 menit 29,35 detik (kurang dari 2 menit). Di samping itu Anigen® rapid test kit rabies menunjukkan tidak terdapat reaksi silang dengan canine parvovirus, Escherichia coli, dan Salmonella sp. serta mempunyai sensitivitas 92,30% dan spesifisitas 97,90%

scholarly journals Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

2002 ◽  
Vol 22 (4) ◽  
pp. 153-160 ◽  
Author(s):  
Claudio R. Madruga ◽  
Cássia R.B. Leal ◽  
Alda M.T. Ferreira ◽  
Flábio R. Araújo ◽  
Ana L.V. Bonato ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetic Analysis ◽  
Fluorescent Antibody Technique ◽  
Babesia Bigemina ◽  
Chain Reaction ◽  
Antigenic Analysis ◽  
Antibody Technique ◽  
Variable Surface ◽  
Geographical Regions ◽  
Polymerase Chain

A molecular epidemiological study was performed with Babesia bigemina isolates from five geographical regions of Brazil. The genetic analysis was done with random amplification of polymorphic DNA (RAPD), repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) and enterobacterial repetitive intergenic consensus sequences-polymerase chain reaction (ERIC-PCR) that showed genetic polymorphism between these isolates and generated fingerprinting. In RAPD, ILO872 and ILO876 primers were able to detect at least one fingerprinting for each B. bigemina isolate. The amplification of B. bigemina DNA fragments by REP-PCR and ERIC-PCR gave evidence for the presence in this haemoprotozoan of the sequences described previously in microorganisms of the bacterial kingdom. For the first time it was demonstrated that both techniques can be used for genetic analysis of a protozoan parasite, although the ERIC-PCR was more discriminatory than REP-PCR. The dendogram with similarity coefficient among isolates showed two clusters and one subcluster. The Northeastern and Mid-Western isolates showed the greatest genetic diversity, while the Southeastern and Southern isolates were the closest. The antigenic analysis was done through indirect fluorescent antibody technique and Western blotting using a panel of monoclonal antibodies directed against epitopes on the merozoite membrane surface, rhoptries and membrane of infected erythrocytes. As expected, the merozoite variable surface antigens, major surface antigen (MSA)-1 and MSA-2 showed antigenic diversity. However, B cell epitopes on rhoptries and infected erythrocytes were conserved among all isolates studied. In this study it was possible to identify variable and conserved antigens, which had already been described as potential immunogens. Considering that an attenuated Babesia clone used as immunogen selected populations capable of evading the immunity induced by this vaccine, it is necessary to evaluate more deeply the cross-protection conferred by genetically more distant Brazilian B. bigemina isolates and make an evaluation of the polymorphism degree of variable antigens such as MSA-1 and MSA-2.

Influence of euthanasia, the intensity of inflammatory lesion and viral load in the laboratory diagnosis of rabies in cattle

2021 ◽  
Vol 41 ◽  
Author(s):  
Claudia S. Wisser ◽  
Marcélia E.S. Fernandes ◽  
Elaine Melchioretto ◽  
Daiane Ogliari ◽  
Aldo Gava ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Load ◽  
Inflammatory Lesion ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Clinical Evolution ◽  
Early Stages ◽  
Polymerase Chain

ABSTRACT: This research reports the use of different diagnostic tests in cattle, naturally infected by Rabies lyssavirus (RABV), and correlates the positivity of the tests with the clinical moment of euthanasia, the intensity of the inflammatory lesion and viral load. It also highlights the possibility of euthanasia in early stages of the disease as a way to improve animal welfare. For that, samples of 34 bovine brains were collected for analysis, preserved in 10% buffered formaline and refrigerated with subsequent freezing. The samples were subjected to direct immunofluorescence antibody technique (DFAT) tests, viral isolation in cell culture (VICC), histopathology with hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Shorr stainied neural tissue smears (DSS), Reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction by quantitative reverse transcriptase (qRT-PCR). The areas used for analysis were the cerebellum, parietal telencephalon and thalamus. Samples with Negri bodies (NBs) or immunostaining in at least one of the analyzed areas were considered positive. For the study of the intensity of histological lesions, the lesions were classified into grades 0, 1, 2 and 3 and the positivity of the test in the presence or absence of NBs in one of the three areas analyzed. To verify the influence of the disease clinical evolution, 4-four groups of analysis were created according to the animal’s clinical status at moment of the euthanasia, being: M1 = animal euthanized while standing, M2 = euthanized when in sternal recumbence, M3 = euthanized when in lateral recumbence, M4 = animal with natural death. Of the 34 brains evaluated, IHC was positive in 100% of cases, DFAT was positive in 97.05% of them, and in this negative sample the presence of RABV was confirmed by VICC. NBs ere seen in 88.23% of the cases, and the DSS test was positive in 82.35% of them. All diagnostic techniques showed positive cases in all groups analyzed. Each case was positive in at least two diagnostic methods. All cases that contained NBs were positive for rabies in the other tests. In this study, it was observed that the variables analyzed (intensity of injury and clinical evolution at the moment of euthanasia) had an influence only on HE and DSS techniques, which are based on NB research to form the diagnosis, but did not interfere with the effectiveness of the diagnosis performed by detecting the viral antigen performed by DFAT and IHC. All isolated RABV samples included in the present study have a genetic lineage characteristic of hematophagous bats Desmodus rotundus. The evaluation of qRT-PCR showed that the amount of virus did not interfere in the positivity of the tests. This work shows that IHC and DFAT are safe diagnostic techniques. They are capable of detecting RABV even in euthanized animals in the early stages of clinical evolution with mild intensities of histological lesions.

scholarly journals Rapid, Sensitive, and Specific Severe Acute Respiratory Syndrome Coronavirus 2 Detection: A Multicenter Comparison Between Standard Quantitative Reverse-Transcriptase Polymerase Chain Reaction and CRISPR-Based DETECTR

2020 ◽  
Author(s):  
Eelke Brandsma ◽  
Han J M P Verhagen ◽  
Thijs J W van de Laar ◽  
Eric C J Claas ◽  
Marion Cornelissen ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcriptase ◽  
N Gene ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Ribonucleoprotein Complexes ◽  
Large Patient ◽  
Polymerase Chain

Abstract Background Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. Methods In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. Results These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. Conclusions Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.

scholarly journals Pendeteksian petanda kepuncaan glioblastoma multiforme

2020 ◽  
Vol 3 (1) ◽  
pp. 39-45
Author(s):  
Yohana Yohana
Keyword(s):  
Polymerase Chain Reaction ◽  
Glioblastoma Multiforme ◽  
Western Blot ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Polymerase Chain

Glioblastoma multiforme (GBM) adalah tumor otak ganas yang memiliki populasi sel punca kanker yang dapat  mempertahankan formasi tumor. Peranan sel punca kanker telah banyak dipelajari yang memiliki tanggung jawab terhadap resistensi dan rekurensi terapi GBM seperti radiasi dan kemoterapi. Beberapa petanda kepuncaan dapat dipakai diantaranya CD133, Nestin, A2B5, CD44, SOX2 and OCT4. Pemeriksaan histopatologi terhadap jaringan tumor yang dioperasi menjadi standar baku untuk menentukan derajat, tingkat keganasan, dan prognosis keganasan. Namun di sisi lain, kehadiran populasi sel punca yang memiliki sifat mampu memperbaharui diri dan mampu menginduksi pembentukan tumor memerlukan pemeriksaan yang lebih mendalam mengenai karakteristik biologi sel tumor. Pemeriksaan  sel punca dilakukan menggunakan flowcytometry dan imunohistokimia. Pemeriksaan petanda kepuncaan glioblastoma dilakukan dengan quantitative Reverse Transcriptase Real Time Polymerase Chain Reaction (qRT-PCR), Enzyme Linked Immunoabsorbent Assay (ELISA), Western Blot, Imunohistokimia dan flowcytometry. Petanda CD133 ditemukan ekspresinya meningkat pada berbagai pemeriksaan. CD133 digunakan sebagai prognostik GBM.

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