scholarly journals Molecular analysis suggests that Namibian cheetahs (Acinonyx jubatus) are definitive hosts of a so far undescribed Besnoitia species

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Gereon Schares ◽  
Maike Joeres ◽  
Franziska Rachel ◽  
Mareen Tuschy ◽  
Gábor Á. Czirják ◽  
...  
Keyword(s):  
Real Time ◽  
North American ◽  
Real Time Pcr ◽  
Sequence Data ◽  
Neospora Caninum ◽  
Didelphis Virginiana ◽  
Intermediate Hosts ◽  
Acinonyx Jubatus ◽  
Two Samples ◽  
Coccidian Parasites

Abstract Background Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the internal transcribed spacer-1 (ITS1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide that are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Also related, but not as closely, is B. besnoiti, the cause of bovine besnoitiosis. Methods Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing. Results One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites, including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti and Neospora caninum, were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but also showed clear divergences. Conclusions Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while prey species of the cheetah may act as intermediate hosts.

scholarly journals Molecular Analysis Revealed that Namibian Cheetahs (Acinonyx Jubatus) are definitive hosts of a so far undescribed Besnoitia species

2021 ◽  
Author(s):  
Gereon Schares ◽  
Maike Joeres ◽  
Franziska Rachel ◽  
Mareen Tuschy ◽  
Gábor Á. Czirják ◽  
...  
Keyword(s):  
Real Time ◽  
North American ◽  
Real Time Pcr ◽  
Sequence Data ◽  
Neospora Caninum ◽  
Didelphis Virginiana ◽  
Intermediate Hosts ◽  
Acinonyx Jubatus ◽  
Two Samples ◽  
Coccidian Parasites

Abstract Background: Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the Internal Transcribed Spacer-1 (ITS-1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide, which are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Related but not closely related to these species is B. besnoiti, the cause of bovine besnoitiosis.Methods: Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy, were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing.Results: One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti, and Neospora caninum were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but showed also clear divergences. Conclusions: Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while a prey species of the cheetah may act as intermediate hosts.

scholarly journals A survey on the occurrence of Brachyspira pilosicoli and Brachyspira hyodysenteriae in growing-finishing pigs

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1702
Author(s):  
Arkadiusz Dors ◽  
Ewelina Czyżewska-Dors ◽  
Grzegorz Woźniakowski
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Finishing Pigs ◽  
Lawsonia Intracellularis ◽  
Fecal Samples ◽  
Brachyspira Pilosicoli ◽  
Brachyspira Hyodysenteriae ◽  
Veterinary Research ◽  
Two Samples ◽  
Intestinal Pathogens

Background: The major pathogenic intestinal spirochetes affecting pigs during the growing- finishing stage of production include Brachyspira hyodysenteriae and Brachyspira pilosicoli. Infections by these pathogens, which affect the economics of pig production, can result in mortality, growth rate losses and substantial antibiotic costs. The aim of this study was to assess the current occurrence of B. hyodysenteriae and B. pilosicoli in Polish pig herds. Moreover, associations between the presence of diarrhea or other intestinal pathogens and occurrence of B. hyodysenteriae and B. pilosicoli in pigs were investigated. Methods: Between January 2017 and August 2019, a total of 401 samples of pig feces from 95 different herds were submitted to the National Veterinary Research Institute of Poland. These samples were obtained from pigs older than 7 weeks. All the received fecal samples were examined for the presence of B. hyodysenteriae, B. pilosicoli and Lawsonia intracellularis by real-time PCR. Results: For B. pilosicoli, 4.5% (95% CI, 2.5–7.0%) of samples and 13.7% (95% CI, 7.5–22.3%) of herds were positive. Out of 12 samples, B. pilosicoli was detected simultaneously with L. intracellularis, B. hyodysenteriae and B. pilosicoli were detected alone in two samples each. In terms of B. hyodysenteriae, 7.0% of samples (95% CI, 4.7–9.9%) from 18.9% of herds (95% CI, 11.6–28.3%) were positive in real time PCR. The presence of B. hyodysenteriae in fecal samples was associated with the presence of diarrhea in pigs. Conclusions: This study confirmed that B. pilosicoli infections occur in Polish pig herds, but the prevalence is at a low level and the presence of B. pilosicoli is not associated with the development of diarrhea in pigs. B. hyodysenteriae is still a common cause of diarrhea among pigs from Polish herds.

scholarly journals ORAL HIV-ASSOCIATED KAPOSI SARCOMA: A COMPARISON BETWEEN IMMUNOHISTOCHEMISTRY AND qPCR TECHNIQUES FOR DETECTION OF HHV8

2015 ◽  
Vol 21 (1) ◽  
pp. 29
Author(s):  
Priscila Lie Tobouti ◽  
Juliana Seo ◽  
Michella Bezerra Lima ◽  
Bruno Tavares Sedassari ◽  
Norberto Nobuo Sugaya ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Kaposi Sarcoma ◽  
Positive Staining ◽  
Extraction Protocol ◽  
Simple Phenol ◽  
Two Samples ◽  
Hematoxylin And Eosin ◽  
The Mean

<p><strong>Objective: </strong>To compare the diagnostic accuracy of immunohistochemistry compared to real-time PCR (using a simple phenol-chloroform DNA extraction protocol) in the detection of HHV8 in small oral biopsies of Kaposi sarcoma. Also to validate the use of this DNA extraction protocol to extract HHV8 DNA.</p><p><strong>Material and methods:</strong> Seventeen cases of oral KS were examined. Data including gender, age, and anatomic location were obtained from patient´s records and histological sections stained with hematoxylin and eosin (H&amp;E) were reviewed. Immunohistochemistry was used to detect HHV8 in lesions of interest, as well as real-time PCR.</p><p><strong>Results: </strong>All the patients were HIV positive, the mean age was 35.5 years old, and the affected oral sites were palate (47%), gingiva (29.4%), tongue (11.8%), lip (5.9%), and oral floor (5.9%). Fifteen samples showed positive staining for HHV8 and only two samples were negative. The same results were observed using real-time PCR HHV8-DNA detection.</p><p><strong>Relevance: </strong>Our findings suggest that immunohistochemistry is faster and cheaper to perform than real-time PCR and was shown to have similar levels of sensitivity and accuracy for detection of HHV8 even in small biopsies. Additionally DNA extraction using a non-commercial kit, as done in this study can further reduce the costs to a pathology service.</p>

scholarly journals Paired real-time PCR assays for detection of Borrelia miyamotoi in North American Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae)

2016 ◽  
Vol 7 (6) ◽  
pp. 1230-1235 ◽  
Author(s):  
Christine B. Graham ◽  
Mark A. Pilgard ◽  
Sarah E. Maes ◽  
Andrias Hojgaard ◽  
Rebecca J. Eisen
Keyword(s):  
Real Time ◽  
North American ◽  
Real Time Pcr ◽  
Ixodes Scapularis ◽  
Borrelia Miyamotoi ◽  
Ixodes Pacificus ◽  
Pcr Assays

scholarly journals Prevalence of Bovine viral diarrhea virus in cattle herds from Basrah and Nassirya Provinces by direct and indirect Elisa and Real time qPCR

2012 ◽  
Vol 11 (2) ◽  
pp. 1
Author(s):  
B. A. Jarullah, J. Aed Gati, and A. Saleh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Indirect Elisa ◽  
Positive Sample ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Virus Rna ◽  
Two Samples ◽  
Cattle Herds

The current study was conducted to investigate the prevalence of BVD virus in Basrah and Nassirya city by using ELISA and RT-PCR. Two hundreds and eighty two samples of non vaccinated cattle sera samples collected from two regions of Iraq (188 samples from Nassirya city and 92 samples from Basrah city). Samples tested by Enzyme Linked Immunosorbent Assay (ELISA) antigen capture. Positive results were 20 samples ( 8 sample in Thi-Qar and 12 positive samples from Basrah). All samples submitted to indirect ELISA(IDEXX HerdCheck ELISA )for detect BVDV antibodies .Genotyping of all 20 positive samples to antigen detection were tested by Real time PCR, using Cador BVDV ½ kit, after extraction of virus RNA by QIAamp mini kit. The results revealed that there were 20 positive sample according to direct ELISA(Ag detection), while 66 sample were positive to indirect ELISA, as well as, the result of RT-PCR showed that there were two sample positive to BVDV type-1 (one sample form each city).Key words: BVDV, Genotype, ELISA, Iraq, Real time PCR.

scholarly journals A Real-time Quantitative Polymerase Chain Reaction for the Specific Detection of Hammondia Hammondi and Its Differentiation from Toxoplasma Gondii

2020 ◽  
Author(s):  
Gereon Schares ◽  
Majda Globokar Vrhovec ◽  
Mareen Tuschy ◽  
Maike Joeres ◽  
Andrea Bärwald ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Post Infection ◽  
In Silico ◽  
Repetitive Element ◽  
Sequence Data ◽  
Quantitative Polymerase Chain Reaction ◽  
Analytical Sensitivity ◽  
Biological Studies

Abstract Introduction: Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic . Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529 bp repetitive element (TgREP-529) is of utmost diagnostic importance for PCR diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529).Material and Methods: Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species were used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon knockout (GKO) mice at varying time points post infection. Results: Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than 1 oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent suggesting 100- to 10.000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post infection. Discussion: The use of the 529 bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay because this repeat probably exists about 200-times in the genome of a single organism, like its counterpart in T. gondii. Although there was enough sequence data available, only few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529 bp repetitive element of H. hammondi.Conclusions: The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell-biological studies on this and related parasites.

scholarly journals Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR

2019 ◽  
Vol 5 (4) ◽  
pp. 105
Author(s):  
Jan Springer ◽  
Grit Walther ◽  
Volker Rickerts ◽  
Axel Hamprecht ◽  
Birgit Willinger ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Contact Lenses ◽  
Fusarium Species ◽  
Invasive Fungal Disease ◽  
Clinical Samples ◽  
Fusarium Spp ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Two Samples

The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.

The first detection ofToxoplasma gondiiDNA in environmental air samples using gelatine filters, real-time PCR and loop-mediated isothermal (LAMP) assays: qualitative and quantitative analysis

Parasitology ◽  
2017 ◽  
Vol 144 (13) ◽  
pp. 1791-1801 ◽  
Author(s):  
ANNA LASS ◽  
BEATA SZOSTAKOWSKA ◽  
KRZYSZTOF KORZENIEWSKI ◽  
PANAGIOTIS KARANIS
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Microscopic Analysis ◽  
Detection Methods ◽  
Type I ◽  
Air Samples ◽  
Two Samples ◽  
Environmental Air ◽  
Tract Infections

SUMMARYToxoplasma gondiiinfections are acquired through the ingestion of oocysts present in the environment. However, there is no data about their occurrence in the air or about airborne transmission of these infections. In the present paper, we report on the identification ofT. gondiiusing rapid molecular detection methods, supported by microscopic analysis, in environmental air samples. A total of 71 samples were collected, using gelatine filters, from kitchen gardens, recreational areas and sandpits located in northern and north-eastern Poland. Material recovered from the filters was analysed using real-time PCR and loop-mediated isothermal assays targeting theT. gondiiB1 gene.Toxoplasma gondiiDNA was found in two samples, as confirmed by both molecular assays. Genotyping at the SAG2 locus showedToxoplasmaSAG2 type I. Moreover, the presence ofT. gondiioocysts was confirmed in one of the positive samples with the use of microscopy. The results showed thatT. gondiimay be present in environmental air samples and that respiratory tract infections may play a role in the high prevalence of toxoplasmosis in humans and animals. To the best of our knowledge, this is the first epidemiological evidence that oro-fecal and foodborne toxoplasmosis may be traceable to an airborne respiratory origin and that this may represent a new, previously unknown transmission route for this disease.

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