Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

Fs, Carvalho; Aa, Wenceslau; Teixeira, Marcel; Gr, Albuquerque

doi:10.4238/vol10-2gmr1043

Cited by 14 publications

(11 citation statements)

References 20 publications

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“…has traditionally been made on the basis of the morphological characteristics of the sporulated oocysts and host specificity. However, morphological techniques have been described as having relatively low sensitivity, and practical limitations associated with the time (Carvalho et al, 2011a;2011b), labor, and…”

Section: Introductionmentioning

confidence: 99%

Morphological and molecular characterization of three Eimeria species from captured rangeland goats in Western Australia

Al-Habsi

Yang

Ryan

et al. 2017

Veterinary Parasitology: Regional Studies and Reports

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2017)Morphological and molecular characterization of three Eimeria species from captured rangeland goats in Western Australia. Veterinary Parasitology: Regional Studies and Reports, 9. pp. 75-83. ABSTRACT Faecal shedding of Eimeria by captured rangeland goats (Capra hircus) was investigated using a longitudinal observational study. Faecal samples were collected from 125 male goats on four occasions. The first sampling occurred following capture and transport, immediately after arrival at a commercial goat depot (feedlot) in Western Australia, with subsequent 3 sample collections occurring at one month intervals thereafter. Goats were composite breed and aged approximately 9-12 months on arrival at the feedlot. Prevalence and shedding intensity (faecal oocyst concentration) for Eimeria were determined using qPCR. Species were identified from individual oocysts (isolated using micromanipulation) using molecular analysis at two loci, specifically 18S rRNA and mitochondrial cytochrome oxidase gene (COI), and confirmed by microscopy. Longitudinal prevalence (animals positive at least once) for Eimeria spp. by qPCR was 90.4%, with 60% goats shedding Eimeria spp. on more than one occasion. Point prevalence (prevalence at a single sampling occasion) ranged from 2.4% (fourth sampling) to 70.4% (second sampling). Three species were identified at the 18S rRNA locus and confirmed by microscopy: E. christenseni (longitudinal prevalence for single infection 34.4%), E. hirci (17.6%) and E. arloingi (8.8%) over the four sample collections. Mixed infections were identified in 56.8% goats (longitudinal prevalence). 18S rRNA sequences from E. christenseni and E. hirci and were 100% homologous with ovine E. ahsata and E. crandallis respectively, and E. arloingi was 100% similar to caprine E. arloingi. At the COI locus, E. christenseni, E. hirci and E. arloingi grouped separately, and were closely related to ovine E. ahsata, with genetic similarities of 96.5%, 92.6% and 91.4% respectively. This is the first report for molecular characteristics of caprine-derived Eimeria spp. using a combination of 18S rRNA and COI. Molecular techniques can be used to identify Eimeria ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T spp. in goat faecal samples, specifically through characterization at 18S locus and other gene loci when used in parallel. Molecular techniques offer some advantages over microscopy for identification of Eimeria species, particularly with respect to precision.

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Section: Introductionmentioning

confidence: 99%

Morphological and molecular characterization of three Eimeria species from captured rangeland goats in Western Australia

Al-Habsi

Yang

Ryan

et al. 2017

Veterinary Parasitology: Regional Studies and Reports

View full text Add to dashboard Cite

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“…Additionally, the performance of multiplex PCR is directly dependent upon the final concentration of PCR inhibitors and the concentration of DNA of individual infectious agents in the DNA template (Haug et al, 2007). Better results achieved when dividing the multiplex into two tubes in the present study is notable, offering a compromise between sensitivity and utility in agreement with Carvalho et al (2011a). Chi-square analysis of the results obtained from the field samples using each technique identified significant differences between all assays ( p < 0.05), illustrating the importance of selecting and retaining a single, standardised procedure if comparable results are to be generated.…”

Section: Discussionmentioning

confidence: 51%

An optimised protocol for molecular identification of Eimeria from chickens

Kumar

Garg

Moftah

et al. 2014

Veterinary Parasitology

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Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.

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“…Eimeria species in cattle and water buffalo are identified only through morphological characteristics, but to date there are no studies showing that species in cattle are genetically identical to the ones in water buffaloes. For this reason, molecular techniques using the 18S ribosomal RNA (rRNA) region can be used, not only to identify Eimeria species but also to study intra-and inter-genetic variations in cattle and water buffalo species (47,48). The accurate Metaphylactic treatments with toltrazuril was very useful against Eimeria infections in cattle (18,50,53), as well as in water buffaloes (19), showing improved performances in animals (e.g., faster body weight gain, positive influence on the average age at the first birth, increased overall percentage of pregnancies).…”

Section: Discussionmentioning

confidence: 99%

A 10-Year Surveillance of Eimeria spp. in Cattle and Buffaloes in a Mediterranean Area

et al. 2020

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Coccidiosis due to Eimeria spp. are widespread parasitic infections in cattle and water buffaloes and may impair health, welfare, and production of these livestock species. The aims of this study were (i) to investigate the prevalence and seasonal dynamics of eimeriosis and (ii) to characterize the Eimeria species in large ruminants in a Mediterranean area, in order to plan effective control strategies. Parasitological data were obtained from a 10-year surveillance program (2010-2019) on 3,631 farms (2,089 buffalo and 1,542 cattle farms) sampled in central and southern Italy. Pooled fecal samples were analyzed using the FLOTAC technique with an analytic sensitivity of 2 oocysts per gram of feces (OPG) utilizing a saturated sodium chloride flotation solution (specific gravity = 1.200). Eimeria species identification was performed by morphometric analysis after a one week incubation of oocysts in a 2.5% potassium dichromate solution. The results showed high prevalence of Eimeria (up to 100%) in both cattle and buffaloes in the 10 years of surveillance, even if a slight reduction was reported in the last three years. The overall prevalence of eimeriosis was 91.7% (95% confidence interval, 95% CI = 90.2-93.1) in cattle farms and 81.5% (95% CI = 79.8-83.1) in water buffalo farms. The mean OPG value was 66.8 (min = 2; max = 8,065) in cattle and 55.9 (min = 2; max = 15,415) in water buffaloes, but this difference was not statistically significant (p > 0.05). In total, nine species of Eimeria were found in cattle the most prevalent being Eimeria bovis, E. ellipsoidalis, E. cylindrica, and E. zuernii, whereas in water buffaloes eight species of Eimeria were found, the most prevalent being E. ellipsoidalis, E. auburnensis, E. bovis, and E. zuernii. Mixed infections were common in both ruminant species. The seasonal pattern showed a higher prevalence of eimeriosis in cattle in spring (86.9%) whereas in buffalo farms the prevalence was higher in winter (82.3%) and summer (82.4%). In conclusion, the 10-year surveillance program indicates that eimeriosis is common in cattle and water buffaloes and therefore continuous effective control strategies are needed.

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Molecular diagnosis of Eimeria species affecting naturally infected Gallus gallus

Cited by 14 publications

References 20 publications

Morphological and molecular characterization of three Eimeria species from captured rangeland goats in Western Australia

Morphological and molecular characterization of three Eimeria species from captured rangeland goats in Western Australia

An optimised protocol for molecular identification of Eimeria from chickens

A 10-Year Surveillance of Eimeria spp. in Cattle and Buffaloes in a Mediterranean Area

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