Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens

Barkway, Christopher P.; Pocock, Rebecca L.; Vrba, Vladimir; Blake, Damer P.

doi:10.1186/1746-6148-7-67

Cited by 33 publications

(28 citation statements)

References 31 publications

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“…Biological identification, which uses molecular methods for protein and amino acid identification, requires specific and costly equipment (BARKWAY et al, 2011). Thus, due to its practicality, morphological characterization utilizing morphometry of sporulated oocysts has been the most common identification method used to differentiate species in many epidemiological studies (AHID et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Epidemiology of Eimeria infections in sheep raised extensively in a semiarid region of Brazil

Souza

Cruz

Neto³

et al. 2015

Rev. Bras. Parasitol. Vet.

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The aim of this study was to identify and determine the prevalence of Eimeria species affecting sheep raised extensively in a semiarid region of Brazil. Fecal samples of native sheep were collected during the rainy and dry seasons. The degree of infection was determined by counting oocysts per gram (OPG) of feces, and the morphometric method was used for species identification. Oocysts were found in all the properties assessed, in which 68.3% of the animals were infected. The prevalence of oocysts was influenced by the season and animal category (P<0.05). It was higher during the rainy season than the dry season (80.2% vs. 55.8%) and highest in young animals than the adults animals (68.2% vs. 39.6%). The OPG was lower during the dry season (1,269 ± 312 vs. 4,400 ± 1,122). Ten species were found; of these, E. ovinoidalis, E. granulosa, E. faurei, and E. crandallis were the most frequent. E. ovinoidalis and E. crandallis were found in all properties, with their prevalences being 19.4% and 13.6% respectively. The high prevalence of pathogenic species shows that eimeriosis is a risk for animals raised extensively in the semiarid region.Keywords: Coccidiosis, morphometry, oocyts, OPG, parasitosis. ResumoObjetivou-se neste estudo identificar e determinar a prevalência de espécies de Eimeria que parasitam ovinos criados extensivamente em região semi-árida. Amostras de fezes de ovinos nativos foram coletados durante as estações chuvosa e seca. O grau de infecção foi determinado pela contagem de oocistos por grama de fezes (OoPG)e o método morfométrico foi utilizado para a identificação das espécies. Foram encontrados oocistos em todas os rebanhos avaliados e observou-se que 68,3% dos animais estavam infectados. A prevalência de oocistos foi influenciada pela estação climática e pela categoria dos animais (P<0,05), sendo mais alta durante a estação chuvosa em relação a estação seca (80,2% vs. 55,8%) e em animais jovens em relação aos animais adultos (68,2% vs. 39,6%). O OoPG foi menor durante a estação seca (1.269 ± 312 vs. 4.400 ± 1.122). Dez espécies foram encontradas sendo a E. ovinoidalis, E. granulosa, E. faurei, e E. crandallis as mais frequentes. E. ovinoidalis e E. crandallis foram encontrados em todas as propriedades, com prevalências de 19,4% e 13,6%, respectivamente. A alta prevalência de espécies patogênicas mostra que eimeriose é um risco para os animais criados extensivamente na região semiárida.

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Section: Introductionmentioning

confidence: 99%

Epidemiology of Eimeria infections in sheep raised extensively in a semiarid region of Brazil

Souza

Cruz

Neto³

et al. 2015

Rev. Bras. Parasitol. Vet.

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“…The amplification method LAMP (Loop-mediated isothermal amplification) is based on a displacement auto cyclic reaction chain using a set of four oligonucleotides (primers) which recognize six sequences within the genomic DNA target region and form a loop structured amplicon, the polymerase that performs this function is Bst polymerase having activity displacement [92,93].…”

Section: Pcr-lampmentioning

confidence: 99%

Toxoplasma gondii in Meat for Human Consumption – A Brief Review of the Most Described Strategies for Its Detection and Quantification

Paredes¹,

Ortega‐Pacheco²,

Rosado-Aguilar³

et al. 2016

Significance, Prevention and Control of Food Related Diseases

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Toxoplasmosis is a parasitic zoonotic disease widely distributed worldwide and is caused by the intracellular parasite Toxoplasma gondii. The definitive host of T. gondii is the domestic cat and the entire cat family, in which the sexual stages of the parasite develop. T. gondii can also infect a wide range of intermediate hosts, affecting most warm-blooded animals including humans. In humans, toxoplasmosis is usually asymptomatic in healthy individuals, but can develop lymphadenopathy and nonspecific symptomatology or even be fatal in infants with congenital toxoplasmosis and in immunocompromised patients. Transmission to humans is mainly through food, especially by eating undercooked meat or meat contaminated with tissue cysts. This has led to various public health organizations worldwide monitoring programs on T. gondii in animals intended for human consumption, especially in meat samples. One of the techniques employed in the laboratory is that based on the polymerase chain reaction and some of its variants, which have proven to be valuable tools for the detection of T. gondii in tissues for human consumption and many other types of biological samples. The development of different strategies for the molecular detection of T. gondii has led to the identification and quantification methodologies varying widely among laboratories. Therefore, this chapter reviews the main methods of extraction, purification, detection and quantification of T. gondii DNA in tissue samples from different species destined for human consumption.

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“…Choose the section (or sections) of intestine to be tested. See Table 1 for a guide to sample site selection and the Eimeria species most likely to be present 18 and Figure 1 for the species-specific range of distribution and location of the sampling sites.…”

Section: Template Preparationmentioning

confidence: 99%

“…Agarose gel electrophoresis was used to resolve each assay and demonstrated absolute species specificity with no host cross-reactivity 18 . Next, a ten-fold serial dilution series prepared using purified Eimeria tenella genomic DNA revealed an assay sensitivity limit of between one and ten genome copies 18 . No upper limit was determined with positive results achieved up to and including the highest concentration (100,000 genome copies) 18 .…”

Section: Assay Validationmentioning

confidence: 99%

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of <em>Eimeria</em> that Infect Chickens

Barkway

Pocock

Vrba

et al. 2015

JoVE

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Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm's anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

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Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens

Cited by 33 publications

References 31 publications

Epidemiology of Eimeria infections in sheep raised extensively in a semiarid region of Brazil

Epidemiology of Eimeria infections in sheep raised extensively in a semiarid region of Brazil

Toxoplasma gondii in Meat for Human Consumption – A Brief Review of the Most Described Strategies for Its Detection and Quantification

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of <em>Eimeria</em> that Infect Chickens

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