Comparative Study of Real-Time PCR (TaqMan Probe and Sybr Green), Serological Techniques (ELISA, IFA and DAT) and Clinical Signs Evaluation, for the Diagnosis of Canine Leishmaniasis in Experimentally Infected Dogs

Canine leishmaniasis (CanL) diagnosis is not fully resolved. Currently, two specific methodologies are in continuous development, the detection of the parasite DNA or RNA in target organs and the detection of specific antibodies against Leishmania sp. For a correct diagnosis, it has been shown that the joint use of this type of test is necessary. In this work, a Sybr Green and a TaqMan Probe based on real time PCRs (qPCR) was performed for the detection of Leishmania sp. in order to correlate the results with clinicopathological and serological evaluations (IFA, ELISA and DAT) to propose an optimal biological sample to be used to detect the parasite in both early and late stages of the infection. A total of four samples were processed: conjunctival swabs, popliteal lymph node aspirates, bone marrow aspirates, and peripheral blood from experimentally infected dogs belonging to a larger study. Our results indicated that a single non-invasive sample (conjunctival swab) and the application of both types of qPCR would be reliable for determining Leishmania infection as well as the disease stage in dogs, thus avoiding bone marrow, lymph node aspirate or blood samples collection.

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Presence of Leishmania amastigotes in peritoneal fluid of a dog with leishmaniasis from Alagoas, Northeast Brazil

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652006000400009 ◽

2006 ◽

Vol 48 (4) ◽

pp. 219-221 ◽

Cited By ~ 5

Author(s):

Filipe Dantas-Torres

Keyword(s):

Bone Marrow ◽

Lymph Node ◽

Peritoneal Fluid ◽

Skin Ulcer ◽

Popliteal Lymph Node ◽

Canine Leishmaniasis ◽

Leishmania Infection ◽

Intracellular Amastigotes ◽

Skin Ulcers ◽

Loss Of Weight

The goal of this short communication is to report the uncommon presence of intracellular amastigotes of Leishmania in peritoneal fluid of a dog with leishmaniasis from Alagoas State, Brazil. Physical examination of an adult male rottweiler suspected to be suffering of leishmaniasis revealed severe loss of weight, ascitis, splenomegaly, moderately enlarged lymph nodes, onychogryphosis, generalized alopecia, skin ulcers on the posterior limbs, and conjunctivitis. Samples of bone marrow, popliteal lymph node, skin ulcer, and peritoneal fluid were collected and smears of each sample were prepared and stained with hematoxylin and eosin. Numerous amastigotes were detected in bone marrow, popliteal lymph node, and skin ulcer smears. Smears of peritoneal fluid revealed the unusual presence of several free and intracellular amastigotes of Leishmania. Future studies are needed to determine whether the cytology of ascitic fluid represents a useful tool for diagnosis Leishmania infection in ascitic dogs, particularly in those living in areas where canine leishmaniasis is enzootic.

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Quantification of Leishmania infantumDNA in the bone marrow, lymph node and spleen of dogs

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612013000300005 ◽

2013 ◽

Vol 22 (3) ◽

pp. 346-350 ◽

Cited By ~ 9

Author(s):

Rafael Antonio Nascimento Ramos ◽

Carlos Alberto do Nascimento Ramos ◽

Edna Michelly de Sá Santos ◽

Flábio Ribeiro de Araújo ◽

Gílcia Aparecida de Carvalho ◽

...

Keyword(s):

Bone Marrow ◽

Lymph Node ◽

Real Time ◽

Real Time Pcr ◽

Biological Samples ◽

Biological Sample ◽

Clinical Signs ◽

Parasite Load ◽

Epidemiological Surveillance ◽

Significant Difference

The aim of the present study was to quantify the parasite load ofLeishmania infantum in dogs using real-time PCR (qPCR). Bone marrow, lymph node and spleen samples were taken from 24 dogs serologically positive for L. infantum that had been put down by the official epidemiological surveillance service. According to the clinical signs the dogs were classified as asymptomatic or symptomatic. After DNA extraction, the samples were subjected to qPCR to detect and quantify L. infantum DNA. Out of the 24 dogs, 12.5% (3/24) were classified as asymptomatic and 87.5% (21/24) as symptomatic. Real-time PCR detected L. infantum DNA in all the animals, in at least one biological sample. In particular, 100% of bone marrow and lymph node scored positive, whereas in spleen, the presence of DNA was detected in 95.9% (23/24). In addition, out of 24 animals, 15 were microscopically positive to amastigote forms of L. infantum in bone marrow. No statistical significant difference was found in the overall mean quantity of DNA among the different biological samples (P = 0.518). Considering each organ separately, there was 100% positivity in bone marrow and lymph nodes, while among the spleen samples, 95.9% (23/24) were positive. Regarding the different clinical groups, the overall mean parasite load varied significantly (P = 0.022). According to the results obtained, it was not possible determine which biological sample was most suitable tissue for the diagnosis, based only on the parasite load. Therefore, other characteristics such as convenience and easily of obtaining samples should be taken into consideration.

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Role of Circulating Immune Complexes in the Pathogenesis of Canine Leishmaniasis: New Players in Vaccine Development

Microorganisms ◽

10.3390/microorganisms9040712 ◽

2021 ◽

Vol 9 (4) ◽

pp. 712

Author(s):

Cristina Cacheiro-Llaguno ◽

Nuria Parody ◽

Marta R. Escutia ◽

Jerónimo Carnés

Keyword(s):

Immune Complexes ◽

Vaccine Development ◽

Correct Diagnosis ◽

Circulating Immune Complexes ◽

Response To Treatment ◽

Canine Leishmaniasis ◽

Leishmania Infection ◽

Serum Samples ◽

Humoral Immune

During canine visceral leishmaniasis (CanL), due to Leishmania infantum (L. infantum), uncontrolled infection leads to a strong humoral immune response. As a consequence of the production of high antibody levels and the prolonged presence of parasite antigens, circulating immune complexes (CIC) are formed, which can be deposited in certain organs and tissues, inducing vasculitis, uveitis, dermatitis and especially glomerulonephritis and renal failure. A method to detect CIC and quantify their levels in serum samples from dogs infected with L. infantum has been recently described. It allowed demonstration of a correlation between CIC levels and disease severity. Thus, CIC measurement may be useful for diagnosis, assessment of disease progression and monitoring response to treatment. This is an interesting finding, considering that there remains an urgent need for identification of novel biomarkers to achieve a correct diagnosis and for optimal disease staging of dogs suffering from Leishmania infection. The objective of the present review is to shed light on the role of CIC in CanL, as well as to highlight their potential use not only as diagnostic and prognostic biomarkers but also as a valuable tool in vaccine development and new immunotherapy strategies to prevent or control disease outcome.

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Detection of Aspergillus species in bone marrow transplant patients

The Journal of Infection in Developing Countries ◽

10.3855/jidc.807 ◽

2010 ◽

Vol 4 (08) ◽

pp. 511-516 ◽

Cited By ~ 12

Author(s):

Parisa Badiee ◽

Abdolvahab Alborzi

Keyword(s):

Bone Marrow ◽

Real Time ◽

Bone Marrow Transplant ◽

Real Time Pcr ◽

Clinical Signs ◽

Marrow Transplant ◽

Aspergillus Species ◽

Pcr Assay ◽

Blood Samples ◽

Transplant Patients

Introduction: Invasive aspergillosis is a severe complication of cytotoxic chemotherapies and bone marrow transplantation (BMT). The aim of this study was to assess the utility of a real-time PCR assay for the early diagnosis of Aspergillus species in blood samples from BMT patients. Methodology: Blood specimens (n = 993) from patients (n = 82) scheduled for BMT were collected prior to transplant and for 100 days post transplantation. The specimens were later tested using an Aspergillus-specific real-time PCR assay. Cultures of clinical samples, along with sonography and computerized tomographic scans, were performed as standard of care. Results: Aspergillus DNA was positive in 94 sequential blood samples from 13 patients with clinical and radiological signs of infection. Samples from three of these patients were PCR-positive for Aspergillus in the first week of admission, prior to transplantation. Four patients with aspergillosis were cured with antifungal agents and nine died. An additional 12 patients without clinical signs of infection were PCR-positive on one occasion each, while two patients with clinical signs of infection were PCR-negative. Compared to routine methods of aspergillosis diagnosis, the respective sensitivity, specificity, negative, and positive predictive values of the PCR method by patient were 86.6%, 82%, 96.5% and 52%. Conclusions: The results show that Aspergillus infections in the blood of bone marrow transplant patients can be dectected by PCR methods. Early detection of Aspergillus infections by PCR has the potential to positively impact patient mortality rate and provide cost savings to hospitals.

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Detection of Listeria spp. and Listeria monocytogenes in biological samples by SYBR Green I and TaqMan probe-based real-time PCRs

Journal of Veterinary Research ◽

10.1515/jvetres-2017-0069 ◽

2017 ◽

Vol 61 (4) ◽

pp. 427-432 ◽

Cited By ~ 2

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Marek Krupa ◽

Anna Szczawińska ◽

Monika Reksa ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Biological Samples ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Taqman Probe ◽

Rdna Gene ◽

Intercalating Dye

AbstractIntroduction:The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene ofListeriaspp. and thehlyA gene ofListeria monocytogenesin biological samples of the liver, brain, and blood.Material and Methods:Five strains ofL. monocytogenesand single strains of each speciesL. ivanovii,L. innocua,L. grayi,L. welshimeri,andL. seeligeriwere used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification thehlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging toListeriaspp. andL. monocytogeneswere conducted.Results:The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA andhlyA genes which confirm their belonging toListeriaspp. andL. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.Conclusion:Both real-time PCR methods for the detection ofListeriaspp. andL. monocytogenesin biological samples demonstrated a significant sensitivity and high specificity.

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EVALUATION OF LYMPH NODE AND BONE MARROW CYTOLOGY IN THE DIAGNOSIS OF CANINE LEISHMANIASIS (LEISHMANIA INFANTUM) IN SYMPTOMATIC AND ASYMPTOMATIC DOGS

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.2005.73.82 ◽

2005 ◽

Vol 73 (1) ◽

pp. 82-86 ◽

Cited By ~ 43

Author(s):

MANOLIS N. SARIDOMICHELAKIS ◽

ALEXANDER F. KOUTINAS ◽

CHARALAMBOS BILLINIS ◽

VASSILIOS I. KONTOS ◽

MATHIOS E. MYLONAKIS ◽

...

Keyword(s):

Bone Marrow ◽

Lymph Node ◽

Leishmania Infantum ◽

Canine Leishmaniasis

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Acute leukemias in dogs: Analysis of 31 cases.

Medycyna Weterynaryjna ◽

10.21521/mw.5635 ◽

2017 ◽

Vol 73 (2) ◽

pp. 118-123

Author(s):

Rafał Sapierzyński ◽

Tomasz Huć ◽

Michał Czopowicz ◽

Urszula Jankowska ◽

Dariusz Jagielski ◽

...

Keyword(s):

Bone Marrow ◽

Acute Leukemia ◽

Current Knowledge ◽

Correct Diagnosis ◽

Clinical Signs ◽

Recurrent Fever ◽

Cytological Examination ◽

Acute Leukemias ◽

Staining Methods ◽

Dog Breed

The aim of the present study was to analyse epidemiological, cytological, laboratory and clinical data from canine acute leukemia (AL) cases. The study was conducted from 2009 to 2015, and included 2384 dogs undergoing cytological examination in two veterinary practices in Warsaw. The analysis included dogs in which bone marrow cytology revealed acute leukemia, regardless of its subtype. Data on breed, age, sex, as well as clinical signs and results of haematological examination were collected for every dog. Breed predisposition to acute leukemia was calculated by statistical methods on the basis of the theoretical distribution of canine breeds in Poland. Acute leukemia was diagnosed in 31 dogs (24.7%) undergoing bone marrow cytology, that is, in 1.3% of all the dogs examined by cytology during the study period. The disease was diagnosed mainly in adults, and a strong predisposition was found particularly in German shepherds and Golden retrievers. The median duration of clinical signs from the onset to diagnosis was 14 days. The clinical signs were mostly non-specific (apathy, recurrent fever, lack of appetite), whereas lymphadenomegaly or/and splenomegaly were observed more seldom. Hematology revealed neoplastic leukocytosis in 75% of dogs, whereas anemia and trombocytopenia were observed in 86% and 85% of patients, respectively. Regardless of the leukemia subtype, prognosis was poor. In conclusion, it can be stated that according to current knowledge on canine acute leukemias, bone marrow cytology based on routine staining methods is sufficient for correct diagnosis in a vast majority of cases.

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A SYBR Green - Based Real Time PCR Method for Detection of Haemopoietic Chimerism in Allogeneic Stem Cell Transplant Recipients.

Blood ◽

10.1182/blood.v106.11.5222.5222 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5222-5222

Author(s):

Lijun Bai ◽

Yi-Mo Deng ◽

Anthony J. Dodds ◽

Sam Milliken ◽

John Moore ◽

...

Keyword(s):

Stem Cell ◽

Real Time ◽

Stem Cell Transplant ◽

Donor Cell ◽

Sybr Green ◽

Taqman Probe ◽

Cell Transplant ◽

Transplant Recipients ◽

Pcr Method ◽

Blood Grouping

Abstract Aim Detection of donor cells (chimerism) in patients post allogeneic stem cell transplantation is for monitoring engraftment, early detection of graft rejection and disease relapse. Current methods include cytogenetics, blood grouping and DNA microsatellite test. Our aim is to develop a reliable and rapid real-time quantitative PCR (Q-PCR) method using SYBR green for detecting chimerism in allogeneic haemopoietic stem cell transplant recipients. Methods Twelve specific nucleotide polymorphisms (NPs) on 11 different chromosomes (including X, Y) were selected. Specific primers and fluorogenic probes were used for Q-PCR analysis. These NPs were screened in pre-transplantation donor and recipient pairs for informative markers. One informative marker was then used for Q-PCR to detect donor cell chimerism post transplantation using either SYBR green or TaqMan probe. Quantitation of donor cell percentage was calculated using a standard amplification curve generated from artificially mixed donor/recipient chimeric DNA samples made with 10 serial dilutions (0.01% ~ 100%), low, medium and high controls were included. Results Thirty-seven donor/recipient pairs including 9 sex-mismatched allograft pre-transplantation DNA samples were examined. In all cases, it is possible to discriminate between recipient and donor genetic profiles. The detection limitation is 0.01%, which is more sensitive than the currently used Microsatellite and FISH methods. The results are highly reproducible with accurate quantitation. In 16 post transplant samples, donor-recipient chimerism were distinguishable using this method. Furthermore this new assay detected similar levels of chimerism compared with either FISH for sex-mismatch donor/recipient pairs or Microsatellite for sex match donor-recipient pairs. We also found that the SYBR green Q-PCR is less expensive and as accurate as the TaqMan probe Q-PCR. Conclusion The new single platform Q-PCR method is capable of detecting haemopoietic chimerism after transplantation and has the potential to replace the three current methods (i.e. cytogenetics, blood grouping and microsatellite).

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Simultaneous Detection and Differentiation of Highly Virulent and Classical Chinese-Type Isolation of PRRSV by Real-Time RT-PCR

Journal of Immunology Research ◽

10.1155/2014/809656 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Shuqi Xiao ◽

Yaosheng Chen ◽

Liangliang Wang ◽

Jintao Gao ◽

Delin Mo ◽

...

Keyword(s):

Real Time ◽

Large Scale ◽

Simultaneous Detection ◽

Sybr Green ◽

Taqman Probe ◽

Economic Losses ◽

Rt Pcr ◽

The Real ◽

Prrs Virus ◽

North American Type

Porcine reproductive and respiratory syndrome (PRRS) is a leading disease in pig industry worldwide and can result in serious economic losses each year. The PRRS epidemic situation in China has been very complicated since the unprecedented large-scale highly pathogenic PRRS (HP-PRRS) outbreaks in 2006. And now the HP-PRRS virus (HP-PRRSV) and classical North American type PRRSV strains have coexisted in China. Rapid differential detection of the two strains of PRRSV is very important for effective PRRS control. The real-time RT-PCR for simultaneous detection and differentiation of HP-PRRSV and PRRSV by using both SYBR Green and TaqMan probes was developed and validated. Both assays can be used for rapid detection and strain-specific identification of HP-PRRSV and PRRSV. However, the TaqMan probe method had the highest detection rate whereas the conventional RT-PCR was the lowest. The real-time RT-PCR developed based on SYBR Green and TaqMan probe could be used for simultaneous detection and differentiation of HP-PRRSV and PRRSV in China, which will benefit much the PRRS control and research.

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Application of SYBR Green I and TaqMan probe-based real-time PCRs for the identification of Listeria spp. and Listeria monocytogenes

Bulletin of the Veterinary Institute in Pulawy ◽

10.1515/bvip-2015-0073 ◽

2015 ◽

Vol 59 (4) ◽

pp. 489-494 ◽

Cited By ~ 2

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Anna Szczawińska ◽

Monika Reksa ◽

Marek Krupa ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Taqman Probe ◽

Rdna Gene ◽

Intercalating Dye ◽

Hlya Gene

Abstract The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

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