Studies on Diagnosis of Bovine Brucellosis by Immunocyto-chemistry and Immunohistochemistry

2021 ◽  
Author(s):  
K.S. Lakshmikanth ◽  
N.S. Sharma ◽  
D. Pathak ◽  
Paviter Kaur
Keyword(s):  
Biochemical Analysis ◽  
Placental Tissue ◽  
Diagnostic Techniques ◽  
Clinical Samples ◽  
Bovine Brucellosis ◽  
Reproductive Disorders ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Positive Results

Background: Brucellosis is a major threat to livestock economy and an important zoonotic disease. A rapid and accurate diagnosis is a necessity to curb the spread and progress of the disease. The current study aimed to evaluate sensitivity of Immunocytochemistry and Immunohistochemistry methods for detection of Brucella spp.Methods: A total of 50 samples comprising of fetal stomach content, vaginal discharges and placenta were collected from cattle and buffaloes suffering from abortions and other reproductive disorders in and around Ludhiana, Punjab during the period 2017-2018. All the samples were processed for isolation and confirmed with biochemical analysis and Polymerase chain reaction (PCR). The isolates obtained and 43 clinical samples excluding placental samples were subjected to Immunocytochemistry (ICC). Immunohistochemistry (ICH) was performed on placental samples.Result: A total of four isolates were recovered from the screened samples. The four isolates also yielded positive results in Immunocytochemistry. Among the 43 clinical samples screened by Immunocytochemistry, five were positive, however only 3 isolates were recovered on isolation. A total of seven placental tissue samples were processed and subjected to immunohistochemistry. Of the three placental samples positive by immunohistochemistry, only one sample was isolated on culture. The results suggest that both immunocytochemistry and immunohistochemistry are sensitive diagnostic techniques in comparison to isolation.

scholarly journals Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges

2013 ◽  
Vol 33 (12) ◽  
pp. 1448-1452 ◽  
Author(s):  
Marcelo M. Silveira ◽  
Daphine A.J. Paula ◽  
Maria C. Silva ◽  
Leticia C. Pitchenin ◽  
Raquel A.S. Cruz ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Signs ◽  
High Sensitivity ◽  
Polymerase Chain Reaction Test ◽  
Universal Primers ◽  
Clinical Samples ◽  
Histopathological Analysis ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Polymerase Chain

Conidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analysis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in C. lamprauges that generated products of about 540 bp and 222 bp respectively. The culture was positive in 26.6% of clinical samples. The PCR technique for C. lamprauges showed amplification of DNA from fresh tissues (80%) and paraffin sections (44.4%). In conclusion, the PCR technique described here demonstrated a high sensitivity and specificity for detection of fungal DNA in tissue samples, providing a tool for the rapid diagnosis of C. lamprauges.

scholarly journals Comparison of polymerase chain reaction and Warthin-Starry techniques to detect Leptospira spp. in kidneys of slaughtered cattle

2014 ◽  
Vol 81 (1) ◽  
Author(s):  
Shahrzad Azizi ◽  
Reza Kheirandish ◽  
Elham Rahimi
Keyword(s):  
Polymerase Chain Reaction ◽  
Silver Staining ◽  
Direct Methods ◽  
Clinical Samples ◽  
Post Mortem ◽  
Chain Reaction ◽  
Silver Stain ◽  
Tissue Samples ◽  
White Spots ◽  
Polymerase Chain

Leptospirosis is a worldwide zoonotic disease that is caused by Gram-negative spirochaetes, Leptospira species. Affected animals excrete the organism in the urine into the environment and act as a source of infection. Cattle are maintenance hosts for some serovars of leptospirosis and are important in the transmission of the infection to humans. At post mortem examination, affected cattle show white spots in their kidneys but these are not specific for leptospirosis. Sometimes it is necessary that leptospirosis be diagnosed in the carcass. Different direct methods, including polymerase chain reaction (PCR), Warthin-Starry silver stain (WS), immunofluorescence (IF) and immunohistochemistry (IHC) can be used in order to diagnose leptospirosis in the affected tissues, such as kidney. The main advantage of the WS technique is direct visualisation of the bacteria in the tissue samples. Silver staining is useful for retrospective studies on formalin-fixed and paraffin-embedded samples but little information is available on the sensitivity and specificity of the technique. The present study aimed to find a simple and inexpensive method that can be used in any laboratory and that also, if clinical samples are not available, can detect Leptospira in tissue samples post mortem. This study was performed on 19 paraffin-embedded kidneys of slaughtered cows that grossly had focal to multifocal white spots. Leptospirosis was confirmed in these samples with PCR based on the LipL32 gene. Out of 19 PCR positive kidneys, Leptospira was identified in 13 stained samples by WS. The kidneys revealed different grades of interstitial nephritis. No relationship was found between severity of lesions and presence of leptospires in the kidneys. The PCR results on the urine and blood were consistent with matching WS stained kidneys. Out of 13 kidneys that were positive with silver staining, 7 matching blood and 10 matching urine samples were confirmed positive for leptospirosis with PCR. In this study, the WS technique provided fewer positive results than PCR. This may be as a result of a low burden of Leptospira in the kidney, but the sensitivity of WS staining needs more investigation.

Use of Nested Polymerase Chain Reaction Based on Sequence-Based Typing of Clinical Samples to Determine the Source of Infection for Hospital-Acquired Legionnaires' Disease

2011 ◽  
Vol 32 (5) ◽  
pp. 510-512 ◽  
Author(s):  
Maria Scaturro ◽  
Stefano Fontana ◽  
Maria Luisa Ricci
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Tissue Samples ◽  
Source Of Infection ◽  
Disease Case ◽  
Legionnaires Disease ◽  
Polymerase Chain ◽  
Hospital Acquired

The source of infection of a hospital-acquired Legionnaires' disease case was determined for the first time by nested polymerase chain reaction based on sequence-based typing. The typing was performed directly on DNA extracted from tissue samples, allowing a rapid epidemiological correlation with environmental isolates.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Occurrence of abortions induced by Neospora caninum in dairy cattle from Santa Catarina, southern Brazil

2017 ◽  
Vol 26 (3) ◽  
pp. 292-298 ◽  
Author(s):  
Cesar Augusto Barbosa de Macedo ◽  
Madlaine Frigo Silveira Barbosa de Macedo ◽  
Ana Carolina Miura ◽  
Alessandra Taroda ◽  
Sergio Tosi Cardim ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Dairy Cows ◽  
High Frequency ◽  
Neospora Caninum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Southern Brazil ◽  
Chain Reaction ◽  
Santa Catarina ◽  
Tissue Samples ◽  
Polymerase Chain

Abstract The aim of the present study was to investigate the occurrence of N. caninum associated with abortions of dairy cattle from Santa Catarina state, southern Brazil by using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and polymerase chain reaction (PCR). Blood from dairy cows that aborted along with intrathoracic fluid and tissue samples (brain, heart, liver, and lung) from their fetuses were collected and used for serology; PCR, histopathological, and immunohistochemistry (IHC) evaluations were also conducted. Twenty-one cows (51.2%) out of 41, and eight fetuses (26.7%) out of 30 were ELISA (HerdCheck, IDEXX) positive for N. caninum. Dams > 36 months of age had a higher risk of being serum positive than younger animals. PCR and IHC revealed that 38.8% (14/36) and 25.0% (9/36) of the fetuses were positive for N. caninum, respectively for each of the tests. Seropositive cows had a higher frequency of fetuses that were also positive by either intrathoracic fluid, PCR, or IHC. In summary, the present study observed a high frequency of N. caninum in abortions from dairy cows from southern Brazil, with a higher N. caninum prevalence found in cows that were older than 36 months. In addition, serology, PCR, and IHC should be used all together for better diagnosis of neosporosis in cattle.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

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