Comparison of sensitivity of two primer sets for the detection of Toxoplasma gondii DNA in wildlife

Abstract Toxoplasma gondii, a coccidian parasite known to infect almost all warm-blooded animals, is the cause of one of the most common zoonotic parasitic diseases. The aim of the study is to determine whether the 529 bp fragment or the TGR1E gene is more useful target for PCR identification of T. gondii, for common use. The brains of 221 carnivores and omnivores collected between 2013 and 2015 from north-eastern Poland were examined for the presence of this parasite. The DNA was extracted and then amplified using specific primers. Positive results were obtained in 24% of brain samples using the TGR1E target and 19% using the 529 bp sequence. The results demonstrate that both TGR1E and 529 bp repeat element are suitable for detecting T. gondii DNA in wildlife animals, and the combination of two methods is necessary to obtain reliable results.

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PCR amplification of a putative gene for exo-1,3-β-glucanase to identify the pathogenic oomycete Pythium insidiosum

Asian Biomedicine ◽

10.5372/1905-7415.0805.338 ◽

2014 ◽

Vol 8 (5) ◽

pp. 637-644 ◽

Cited By ~ 12

Author(s):

Angsana Keeratijarut ◽

Tassanee Lohnoo ◽

Wanta Yingyong ◽

Umporn Nampoon ◽

Tassanee Lerksuthirat ◽

...

Keyword(s):

Diagnostic Performance ◽

Disease Diagnosis ◽

Detection Sensitivity ◽

Molecular Diagnostic ◽

Molecular Diagnostic Techniques ◽

Specific Primers ◽

Pythium Insidiosum ◽

Putative Gene ◽

Pcr Identification ◽

Primer Sets

AbstractBackground: Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease. Diagnosis of pythiosis is difficult and often delayed. Early diagnosis can lead to prompt treatment, and therefore a better prognosis for patients with pythiosis. Molecular diagnostic techniques are useful if microbiological and immunological assays are not available, or in cases of suspected pythiosis that test negative by other methods. So far, PCR identification of P. insidiosum has been largely relied on amplification of the rDNA region.Objective: To evaluate the diagnostic performance of Dx3 and Dx4 primers specific for a putative gene for exo- 1,3-β-glucanase (PinsEXO1), which encodes a specific immunogen of P. insidiosum, for rapid single-round PCR identification of P. insidiosum, in comparison with the previously-reported rDNA-specific primers, ITSpy1 and ITSpy2.Materials and Methods: Genomic DNA (gDNA) from 35 P. insidiosum isolates and 48 control organisms were prepared to evaluate the diagnostic performance of the PinsEXO1- and rDNA-specific primers.Results: When amplifying the control gDNA by using the Dx3/4 and ITSpy1/2 primer sets, no PCR product was observed, indicating that both primer sets had 100% detection specificity. When amplifying the P. insidiosum gDNA, the Dx3/4 primers provided an expected 550-bp amplicon for all 35 isolates, while the ITSpy1/2 primers provided an expected 230-bp amplicon for only 32 isolates. Thus, detection sensitivity of the Dx3/4 and ITSpy1/2 primer sets were 100% and 91%, respectively.Conclusion: By using the Dx3/4 primers, PinsEXO1 was an alternative, efficient, and novel PCR target for rapid single-round PCR identification of P. insidiosum.

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Descriptive and illustrated diagnosis of Ophiuroidea fauna (Echinodermata) in the shallow waters of North-eastern Brazil

Marine Biodiversity Records ◽

10.1017/s1755267215000652 ◽

2015 ◽

Vol 8 ◽

Author(s):

Fabilene Gomes Paim ◽

Maria Cecília Guerrazzi ◽

Michela Borges

Keyword(s):

Brittle Star ◽

Shallow Waters ◽

Bottom Substrate ◽

Eastern Brazil ◽

North Eastern ◽

Amphipholis Squamata ◽

Biological Substrates ◽

Almost All ◽

First Time ◽

Ophiocoma Echinata

In this study, we present descriptions, illustrations, comments, and bathymetric and geographic distributions of the brittle star species related to the estuary region of Camamu Bay, located in the State of Bahia, Brazil. The brittle star fauna lives on biological substrates, sand bottoms, mud and rubble in the Camamu Bay and comprises 12 species divided into five families. Almost all of them are common in the tropical and subtropical fauna of the regions of shallow water.Ophiophragmus filograneusis reported for the first time in Bahia, and nine other species are recorded for the first time in Camamu Bay:Amphipholis januarii, Amphipholis squamata, Ophiophragmus filograneus, Ophiostigma isocanthum,Ophioderma cinerea, Ophioderma januarii, Ophiactis lymani, Ophiactis savignyi andOphiocoma echinata.The results suggest that the ophiuroid assemblages are strongly affected by marine currents as well as by different kinds of bottom substrate.

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Performance of 2 Polymerization Protocols Targeting Cloned Toxoplasma Parasites

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2018.400 ◽

2018 ◽

Vol 6 (9) ◽

pp. 1577-1580

Author(s):

Nihal A. Hanafy ◽

Mohamed S. Badr ◽

Ghada M. Nasr

Keyword(s):

Toxoplasma Gondii ◽

Real Time ◽

Parasitic Infection ◽

Clinical Samples ◽

Molecular Assays ◽

Low Concentrations ◽

Target Dna ◽

Primer Sets ◽

Amplification Failure ◽

Reference Samples

BACKGROUND: Toxoplasma gondii is a common parasitic infection of humans. Infection is usually mild. Serious complications can occur in pregnant and immunocompromised patients. AIM: The present study aims to investigate the performance of 2 different PCR protocols; real-time quantitative molecular assays (qPCR) and conventional molecular assays (cPCR), using 2 different sets of primers and by using cloned purified Toxoplasma genomic substances to be evaluated as reference samples. METHODS: The target DNA was provided in 8 different quantities. RESULTS: Amplification failure was reported only with the cPCR in samples of low concentrations using both primer sets. Quantitative PCR detected the 8 different dilutions of the purified Toxoplasma gondii using the 2 sets of primers while cPCR was sensitive to detect only 6 different dilutions. CONCLUSION: Generally real-time quantitative molecular assays, is easy to use method compared to conventional PCR assay and produces more reliable results within only one hour time but still the possible application of qPCRs in routine diagnosis necessitates analysis of a large number of clinical samples in further studies to make the proper choice.

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Detección de Pasteurella multocida, Mannhemia haemolytica, Histophilus somni y Mycoplasma bovis en pulmón de bovinos

Revista Mexicana de Ciencias Pecuarias ◽

10.22319/rmcp.v12i3.5469 ◽

2021 ◽

Vol 12 (3) ◽

pp. 710-720

Author(s):

Seyda Cengiz ◽

M. Cemal Adıgüzel ◽

Gökçen Dinç

Keyword(s):

Immune System ◽

Pasteurella Multocida ◽

Positive Sample ◽

Mycoplasma Bovis ◽

Opportunistic Pathogens ◽

Specific Primers ◽

Histophilus Somni ◽

Healthy Cattle ◽

Positive Results ◽

Molecular Evaluation

In this study, it was aimed to determine of P. multocida, M. haemolytica, H. somni and M. bovis in macroscopically healthy cattle lungs by PCR. The study was carried out on 82 macroscopically healthy cattle lung. DNA extraction was performed to the lung samples. PCR was then performed using all specific primers. By molecular evaluation, positive results were achieved for P. multocida, M. haemolytica, H. somni and M. bovis in 4 (4.8 %), 4 (4.8 %), 6 (7.3 %) and 3 (3.6 %) of the samples, respectively. Mix infections were detected in five samples. Of the samples, two were positive for both P. multocida and M. haemolytica, two were positive for both M. haemolytica and H. somni and one was positive for both P. multocida and H. somni. However, a positive sample, which carried all of pathogens, was not detected. In conclusion, P. multocida, M. haemolytica, H. somni and M. bovis are the important opportunistic pathogens of respiratory tract in cattle and these pathogens have a major role during infections. But multifactorial nature of bovine respiratory disease and immune system affected the formation of the disease. Hence, firstly cattle’s immunity should be strengthened and other conditions should be kept under control.

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Rapid collapse of a population of Dieffenbachia spp., plants used for tadpole-rearing by a poison-dart frog (Oophaga pumilio) in a Costa Rican rain forest

Journal of Tropical Ecology ◽

10.1017/s0266467414000467 ◽

2014 ◽

Vol 30 (6) ◽

pp. 615-619 ◽

Cited By ~ 1

Author(s):

Mark J. McKone ◽

Jonathan W. Moore ◽

Christopher W. Harbison ◽

Ian C. Holmen ◽

Hillary C. Lyons ◽

...

Keyword(s):

Secondary Forest ◽

Costa Rican ◽

Primary Forest ◽

Rapid Decline ◽

Field Station ◽

La Selva ◽

Oophaga Pumilio ◽

North Eastern ◽

Pecari Tajacu ◽

Almost All

Abstract:Amphibian populations have been declining worldwide, with multiple potential causes. At La Selva field station in north-eastern Costa Rica, previous work has shown that populations of many amphibians have decreased significantly since the 1970s, especially in primary forest. Starting in 1998, we investigated one of the most common frog species at La Selva, the poison-dart frog Oophaga pumilio (= Dendrobates pumilio). In a survey of 50 plots of 100 m2 in 1998, adult frogs were 4.6 times more abundant in secondary forest than in primary forest. Tadpoles were found only in secondary-forest plots. Almost all (89%) of the tadpoles were found in leaf axils of Dieffenbachia spp., which were much more abundant in secondary-forest than in primary-forest plots. The greater abundance of Dieffenbachia spp. in secondary forest was confirmed in a broad survey of ~11 km of trails within La Selva in 2002. When the same trails were resampled in 2012, Dieffenbachia spp. had been extirpated from 72% of the 50-m segments where plants were present in 2002; abundance was greatly reduced in the few trail segments where any Dieffenbachia spp. remained in 2012. The loss of Dieffenbachia spp., especially in secondary forest, removed the species most often used by O. pumilio for tadpole rearing. Based on counts of calling frogs in 2010, there was no difference in O. pumilio abundance in primary versus secondary forest, in striking contrast to multiple earlier surveys that found much greater frog abundance in secondary forest. We propose that the reason for the rapid decline in Dieffenbachia spp. is herbivory by the collared peccary (Pecari tajacu), which has increased in abundance at La Selva in recent years. A likely consequence is continued reduction in O. pumilio populations.

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Effect In Vitro of Antiparasitic Drugs on Microbial Inhibitor Test Responses for Screening Antibiotic Residues in Goat's Milk

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-020 ◽

2015 ◽

Vol 78 (9) ◽

pp. 1756-1759 ◽

Cited By ~ 5

Author(s):

T. ROMERO ◽

M. C. BELTRÁN ◽

W. REYBROECK ◽

M. P. MOLINA

Keyword(s):

Antibiotic Residues ◽

Goat’S Milk ◽

Drug Concentrations ◽

Test Microorganism ◽

Antiparasitic Drug ◽

Goat's Milk ◽

Antiparasitic Drugs ◽

Almost All ◽

Positive Results

Microbial inhibitor tests are widely used to screen antibiotic residues in milk; however, these tests are nonspecific and may be affected by various substances capable of inhibiting the growth of the test microorganism. The objective of this study was to determine the effect of antiparasitic drugs in goat's milk on the microbial inhibitor test response. Raw antibiotic-free milk from Murciano-Granadina goats was supplemented with eight concentrations of seven antiparasitic substances (albendazole, 10 to 170 mg/kg; closantel, 1 to 140 mg/kg; diclazuril, 8 to 45 mg/kg; febendazole, 10 to 140 mg/kg; levamisole, 40 to 440 mg/kg; diazinon, 8 to 45 mg/kg; and ivermectin, 40 to 200 mg/kg). Twelve replicates for each concentration were analyzed with three microbial inhibitor tests: BRT MRL, Delvotest SP-NT MSC, and Eclipse 100. The results were interpreted visually (negative or positive). Using a logistic regression model, the concentrations of the antiparasitic drugs producing 5% (IC5), 10% (IC10), and 50% (IC50) positive results were determined. In general, the Eclipse 100 test was less sensitive to the effect of antiparasitic substances; the inhibitory concentrations of almost all the drugs assayed were higher than those for other tests. Conversely, the BRT MRL test was most affected, with high levels of interference at lower antiparasitic drug concentrations. Closantel and diazinon interfered with all microbial tests at lower concentrations than did other drugs (IC5 = 1 to 26 and 12 to 20 mg/kg, respectively), and higher concentrations of levamisole and diclazuril (IC5 = 30 to 240 and 50 to 117 mg/kg, respectively) were required to produce 5% positive results. These findings indicate that microbial inhibitor tests can be affected by elevated concentrations of antiparasitic drugs in goat's milk.

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Infection with Toxoplasma gondii Bradyzoites Has a Diminished Impact on Host Transcript Levels Relative to Tachyzoite Infection

Infection and Immunity ◽

10.1128/iai.01228-06 ◽

2006 ◽

Vol 75 (2) ◽

pp. 634-642 ◽

Cited By ~ 21

Author(s):

A. E. Fouts ◽

J. C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Transcript Level ◽

Infected Host ◽

Host Immune System ◽

Cell Microarrays ◽

Macrophage Colony ◽

Lower Magnitude ◽

Almost All

ABSTRACT Toxoplasma gondii, an intracellular pathogen, has the potential to infect nearly every warm-blooded animal but rarely causes morbidity. The ability for the parasite to convert to the bradyzoite stage and live inside slow-growing cysts that can go unnoticed by the host immune system allows for parasite persistence for the life of the infected host. This intracellular survival likely necessitates host cell modulation, and tachyzoites are known to modify a number of signaling cascades within the host to promote parasite survival. Little is known, however, about how bradyzoites manipulate their host cell. Microarrays were used to profile the host transcriptional changes caused by bradyzoite infection and compared to those of tachyzoite-infected and uninfected hosts cells 2 days postinfection in vitro. Infection resulted in chemokine, cytokine, extracellular matrix, and growth factor transcript level changes. A small group of genes were specifically induced by tachyzoite infection, including granulocyte-macrophage colony-stimulating factor, BCL2-related protein A1, and interleukin-24. Bradyzoite infection yielded only about half the changes seen with tachyzoite infection, and those changes that did occur were almost all of lower magnitude than those induced by tachyzoites. These results suggest that bradyzoites lead a more stealthy existence within the infected host cell.

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Biology and epidemiology ofToxoplasma gondiiin man and animals

Animal Health Research Reviews ◽

10.1079/ahr2005100 ◽

2005 ◽

Vol 6 (1) ◽

pp. 41-61 ◽

Cited By ~ 270

Author(s):

Dolores E. Hill ◽

Sreekumar Chirukandoth ◽

J. P. Dubey

Keyword(s):

Toxoplasma Gondii ◽

Intermediate Host ◽

Host Range ◽

Marine Mammals ◽

Parasitic Infections ◽

Sea Otter ◽

Coccidian Parasite ◽

Wild Carnivores ◽

Southern Sea Otter

AbstractToxoplasma gondiiis a coccidian parasite which utilizes felids as definitive hosts, and which has an unusually wide intermediate host range. The parasite was initially described by Nicolle and Manceaux in 1908 from the rodent,Ctenodactylus gundi. Infection withT. gondiiis one of the most common parasitic infections of man and other warm-blooded animals. It has been found worldwide from Alaska to Australia. Nearly one-third of humanity has been exposed to this parasite; serologic surveys indicate thatT. gondiiinfections are common in wild carnivores, including pigs, bears, felids, fox, raccoons, and skunks. Clinical and subclinical toxoplasmosis has been reported from wild cervids, ungulates, marsupials, monkeys, and marine mammals. Southern sea otter populations have been severely impacted byToxoplasmainfections.

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Sex-dependent toxoplasmosis-associated differences in testosterone concentration in humans

Parasitology ◽

10.1017/s0031182007004064 ◽

2008 ◽

Vol 135 (4) ◽

pp. 427-431 ◽

Cited By ~ 86

Author(s):

J. FLEGR ◽

J. LINDOVÁ ◽

P. KODYM

Keyword(s):

Toxoplasma Gondii ◽

Opposite Direction ◽

Direct Evidence ◽

Latent Infection ◽

Indirect Evidence ◽

Male Students ◽

Coccidian Parasite ◽

Testosterone Concentration ◽

Gender Specificity

SUMMARYSeveral lines of indirect evidence suggest that subjects with latent infection of the coccidian parasiteToxoplasma gondiihave a higher concentration of testosterone than uninfected controls. Here, we searched for direct evidence of latent toxoplasmosis-associated differences in testosterone concentration among a population of 174 female and 91 male students screened forToxoplasmainfection. We have foundToxoplasma-infected men to have a higher concentration of testosterone andToxoplasma-infected women to have a lower concentration of testosterone thanToxoplasma-free controls. The opposite direction of the testosterone shift in men compared to women can explain the observed gender specificity of behavioural shifts inToxoplasma-infected subjects.

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