Molecular detection of feline calicivirus in clinical samples: A study comparing its detection by RT-qPCR directly from swabs and after virus isolation

ABSTRACT: Felid alphaherpesvirus 1 (FeHV-1) and feline calicivirus (FCV) affect cats worldwide. The aim of this study was to evaluate the frequency of occurrence of FeHV-1 and FCV in cats with clinical signs of respiratory, oral and/or ocular disease. Samples were collected from cats cared for in veterinary ambulatory and clinics and submitted to molecular detection and viral isolation. Of the 49 cats evaluated, 45 (92%) were positive for at least one of the viruses; 82% (40/49) were positive for FeHV-1 and 41% (20/49) for FCV. Of these, 31% (15/49) were coinfection cases. For FeHV-1, 45% (18/40) of the cats tested were positive from the collection of eye swab, and the same percentage (9/20) was obtained for the FCV by the oral swab. FeHV-1 and/or FCV were isolated in 35% (17/49) of the samples. The main clinical sign observed was ocular secretion in 71% (35/49) of cats, characterized as mild serous, purulent or serosanguineous, and in some cases associated with ocular injury and marked chemosis. Our findings demonstrate the high occurrence of FeHV-1 and FCV in domestic cats in southern Brazil and indicate that measures should be implemented to improve the diagnostic, prevention and management against of these important diseases.

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Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638715584156 ◽

2015 ◽

Vol 27 (4) ◽

pp. 516-521 ◽

Cited By ~ 10

Author(s):

Katsuhiko Fukai ◽

Kazuki Morioka ◽

Manabu Yamada ◽

Tatsuya Nishi ◽

Kazuo Yoshida ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Kidney Cell ◽

Virus Isolation ◽

Foot And Mouth Disease ◽

Clinical Samples ◽

Porcine Kidney ◽

Kidney Cell Line ◽

Mouth Disease Virus ◽

Porcine Kidney Cell

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-αvβ6 have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-αvβ6 cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-αvβ6 cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same.

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Cell Cultures for Virology: Usability, Advantages, and Prospects

International Journal of Molecular Sciences ◽

10.3390/ijms21217978 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7978

Author(s):

Alexander A. Dolskiy ◽

Irina V. Grishchenko ◽

Dmitry V. Yudkin

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Virus Isolation ◽

Virus Detection ◽

Clinical Samples ◽

Laboratory Setting ◽

Reporter Cell ◽

Isolation And Identification ◽

Reporter Systems ◽

Reporter Proteins

Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.

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Identification of a Continuously Stable and Commercially Available Cell Line for the Identification of Infectious African Swine Fever Virus in Clinical Samples

Viruses ◽

10.3390/v12080820 ◽

2020 ◽

Vol 12 (8) ◽

pp. 820 ◽

Cited By ~ 2

Author(s):

Ayushi Rai ◽

Sarah Pruitt ◽

Elizabeth Ramirez-Medina ◽

Elizabeth A. Vuono ◽

Ediane Silva ◽

...

Keyword(s):

Cell Line ◽

Virus Isolation ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Clinical Samples ◽

Outbreak Strain ◽

Domestic Pigs ◽

Current Outbreak ◽

High Degree

African swine fever virus (ASFV) is causing outbreaks both in domestic pigs and wild boar in Europe and Asia. In 2018, the largest pig producing country, China, reported its first outbreak of African swine fever (ASF). Since then, the disease has quickly spread to all provinces in China and to other countries in southeast Asia, and most recently to India. Outbreaks of the disease occur in Europe as far west as Poland, and one isolated outbreak has been reported in Belgium. The current outbreak strain is highly contagious and can cause a high degree of lethality in domestic pigs, leading to widespread and costly losses to the industry. Currently, detection of infectious ASFV in field clinical samples requires accessibility to primary swine macrophage cultures, which are infrequently available in most regional veterinary diagnostic laboratories. Here, we report the identification of a commercially available cell line, MA-104, as a suitable substrate for virus isolation of African swine fever virus.

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Molecular detection methods and serotyping performed directly on clinical samples improve diagnostic sensitivity and reveal increased incidence of invasive disease by Streptococcus pneumoniae in Italian children

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/000935-0 ◽

2008 ◽

Vol 57 (10) ◽

pp. 1205-1212 ◽

Cited By ~ 59

Author(s):

Chiara Azzari ◽

Maria Moriondo ◽

Giuseppe Indolfi ◽

Cristina Massai ◽

Laura Becciolini ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Molecular Detection ◽

Invasive Disease ◽

Diagnostic Sensitivity ◽

Detection Methods ◽

Clinical Samples ◽

Italian Children

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Isolation, Serotyping and Molecular Detection of Bovine FMD Virus from Outbreak Cases in Aba'ala District of Afar Region

10.21203/rs.3.rs-28928/v2 ◽

2020 ◽

Author(s):

Teshager Dubie Tegegne ◽

Tsedale Amare Mengiste

Keyword(s):

Molecular Detection ◽

Vaccine Development ◽

Universal Primers ◽

Clinical Samples ◽

Effective Vaccine ◽

Rt Pcr ◽

Documentation System ◽

Fmd Virus ◽

Afar Region ◽

Fmdv Serotypes

Abstract Background: Among the top listed economically important transboundary livestock diseases of cattle, foot and mouth disease (FMD) is the leading bottleneck in livestock production and productivity in Ethiopia. On the basis of FMDV active outbreak cases, a cross sectional study was undertaken to collect samples from January, 2019 to March, 2020 intended for isolation, serotyping and molecular detection of FMDV in the study district. Purposive sampling method was applied to select the study area for the reason that the presence of current active FMD outbreak case report during the study period. Totally, 27 FMD suspected clinical samples were collected from clinically affected study population during field outbreak. Out of 27 samples, 18 of them were inoculated on cultured Baby hamster kidney (BHK-21) monolayer cells and all 27 samples were tested using conventional RT-PCR and sets of specific universal primers. Finally, the PCR products were visualized with UV illumination and imaged with gel documentation system. Results: The current study result revealed that out of 18 clinical samples subjected to virus isolation, 72.2%(n=13) of these cultures exhibited FMDV induced cytopathic effect (CPE) and the identified serotype was SAT-2 FMD virus. Out of 27 clinical samples tested by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers. Out of 27 clinical samples detected by conventional reverse transcription polymerase chain reaction (RT-PCR), only 12 FMDV samples were found to be FMDV positive by universal primers.Conclusions: Our study finding indicated FMDV is prevalent in the study area and FMDV serotype SAT-2 was the causality for the outbreaks of the disease in the study area. Hence, region wise regular FMD outbreaks investigation, further phylogenetic analysis and vaccine matching field isolates should be carried out to know in depth data about FMDV serotypes and topotypes involving in afar region of Ethiopia for effective vaccine development and control of the disease.

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Phenotypic and Molecular Detection of Resistance Genes from Multi-drug Resistant Escherichia coli Isolated from Patients Attending Selected Hospitals in Damaturu, Yobe State, Nigeria

International Journal of Research and Review ◽

10.52403/ijrr.20210951 ◽

2021 ◽

Vol 8 (9) ◽

pp. 396-407

Author(s):

Sheriff Wakil ◽

Mustafa Alhaji Isa ◽

Adam Mustapa

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Resistance Genes ◽

Molecular Detection ◽

Multidrug Resistant ◽

Clinical Samples ◽

E Coli ◽

Resistant Genes ◽

Tract Infections

Multidrug resistance among Escherichia coli causing urinary tract infections (UTIs) and diarrhea are major public health problem worldwide which cause difficulty in treating the infections caused by Escherichia coli due to the high resistances. The study is aimed to determine the phenotypic and molecular detection of multidrug resistant E. coli isolated from clinical samples of patients attending selected Hospitals in Damaturu, Yobe State-Nigeria. Methods: Two hundred (200) clinical samples were collected aseptically from patient diagnosed with (100 stool samples) and UTI’s (100 urine samples) using sterile universal container. The samples were processed using standard microbiological methods for identification of E. coli. Samples were cultured on MacConkey agar (stool) and Cystine lactose electrolyte deficient agar (urine). The resulting colonies of isolates were further subculture on Eosin methylene blue agar for confirmatory and followed by gram stain, biochemical identification at Microbiology laboratory unit of Yobe State Specialist and Yobe State Teaching Hospital respectively. The antimicrobial susceptibility patterns were determined using Kirby-Bauer disc diffusion techniques and the phenotypic expression of extended spectrum beta-lactamases (ESBLs) were determined using modified double disc synergy test (MDDST) and also the three (3) resistance genes (blaTEM, accC1 and qnrA) were detected using polymerase chain reaction. Results: One hundred and twenty-two (122) isolates were resistant to antibiotics. The highest level of resistance was against amoxicillin (90.2%) while the least resistance was against sparfloxacin (24.3%). Thirty-seven (37) E. coli isolates shows MDR; the highest MDR was (24.3%) while least MDR was (5.4%). The PCR amplification of resistant genes (blaTEM, accC1 and qnrA) were detected on E. coli that shows positive ESBL and the bands were separated using agarose gel electrophoresis. Conclusion: The findings of this study show augmentin, ciprofloxacin and sparfloxacin are the most effective antibiotics against E. coli isolated from patients attending the two hospitals in Damaturu; who are diagnose with UTI and diarrheic infection. The resistant genes include; blaTEM, accC1 and qnrA coding for beta-lactam, aminoglycoside and quinolones were present in E. coli isolated from patients attending selected Hospitals in Yobe State, Nigeria. Keywords: Multidrug resistant, Escherichia coli, extended spectrum beta lactamase, resistance-associated genes, urinary tract infections, diarrheic.

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Isolation, Serotyping, and Molecular Detection of Bovine FMD Virus from Outbreak Cases in Abaʼala District of Afar Region, Ethiopia

Veterinary Medicine International ◽

10.1155/2020/8847728 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Teshager Dubie ◽

Tsedale Amare

Keyword(s):

Molecular Detection ◽

Vaccine Development ◽

Universal Primers ◽

Clinical Samples ◽

Effective Vaccine ◽

Rt Pcr ◽

Documentation System ◽

Cross Sectional ◽

Fmd Virus ◽

Study Results

Background. On the basis of FMDV outbreak cases, a cross-sectional study was undertaken to collect samples from January 2019 to March 2020 intended for isolation, serotyping, and molecular detection of FMDV in the study district. The purposive sampling method was applied to select the study area for the reason of the presence of FMD outbreak case report during the study period. Totally, 27 FMD clinical samples were collected from affected study population during field outbreak. Out of 27 samples, 18 of them were inoculated on cultured Baby hamster kidney (BHK-21) monolayer cells, and all 27 samples were tested using conventional RT-PCR and sets of specific universal primers. Finally, the PCR products were visualized with UV illumination and imaged with gel documentation system. Results. The current study results revealed that out of 18 clinical samples subjected to virus isolation, 72.2% (n = 13) of these cultures exhibited FMDV-induced cytopathic effect (CPE) and the identified serotype was SAT-2 FMD virus. Out of 27 clinical samples tested by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers. Out of 27 samples detected by conventional RT-PCR, only 12 FMDV samples were found to be FMDV positive by universal primers. Conclusions. Our study finding indicated that FMDV is prevalent in the study area and FMDV serotype SAT-2 was the causality for the outbreaks of the disease in the study area. Hence, region-wise FMD outbreak investigation, further phylogenetic analysis, and vaccine matching field isolates should be carried out for effective vaccine development to control the disease.

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