A survey of the economic impact of subclinical Eimeria infections in broiler chickens in Norway.

The objective of this work was to examine the impact of subclinical coccidial infection on commercial performance, expressed as a modified European Production Index, in broilers. Performance data, and litter and faecal samples, were collected from two independent observational surveys of Norwegian broilers receiving in-feed narasin during 2000 to 2004. Numbers of oocysts per gram (OPG) of litter collected during rearing (Study 1) or faecal samples collected at slaughter (both studies), and relative frequencies of Eimeria species categories (both studies) were calculated. Polymerase chain reaction-based identification of Eimeria species was performed in Study 2. A definition of flocks at risk of impaired performance associated with coccidia ("risk flock"), using the predominant species and OPG level as criteria, was tested. Coccidia had a significant effect on performance in the first, but not the second study. In Study 1 the following coccidia variables were found to be associated with impaired performance in multivariate models: OPG at slaughter (ordinal), mean OPG during rearing (ordinal) and "risk flock" (binomial). The European Production Index was approximately 9% lower in flocks with infection levels >50 000 OPG at slaughter in Study 1. The composition of coccidial populations shifted between Study 1 and Study 2, from a dominance of medium and large oocysts to a dominance of small oocysts. There was a substantial increase in prevalence of coccidial infection from Study 1 to Study 2, but mean infection levels were similar in the two surveys. The "risk flock" definition was useful as an indicator of coccidia-associated performance loss in Study 1, where subclinical coccidiosis was an important factor. The results suggest that the economic importance of subclinical coccidiosis may vary substantially with time, and they emphasize the need for population studies on the importance and dynamics of specific coccidial infections under different field conditions.

Coccidial infections in commercial broilers: epidemiological aspects and comparison of Eimeria species identification by morphometric and polymerase chain reaction techniques.

The objective of this study was to add to existing knowledge of the epidemiology and the aetiology of coccidial infections in commercial broiler flocks. Polymerase chain reaction (PCR) and morphometric identification of the Eimeria species were compared as means of differentiation in the field samples of faeces and litter. For morphometry, the Eimeria species were categorized into three groups based on lengths of the oocysts. Two random samples of commercial broilers were studied, one during 2000/01 and the other during 2003/04. The prophylactic regime (in-feed narasin), husbandry and methods applied were broadly the same for both subpopulations. Coccidial infection prevalence increased from approximately 45% to approximately 75% during this period, but infection levels (oocysts per gram of faeces) did not significantly change. There were substantial geographical differences in both prevalence and infection levels. A change in Eimeria species profile occurred during the study period. Five Eimeria species were identified at slaughter, by PCR targeting the ITS-1 region of the genome; Eimeria acervulina (100%), Eimeria tenella (77%), Eimeria maxima (25%), Eimeria praecox (10%) and Eimeria necatrix (2%). PCR and morphometric tentative identification were in complete agreement in only 49% of the cases.

A simplified protocol for molecular identification of Eimeria species in field samples.

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.

Counting coccidial oocysts in chicken faeces: a comparative study of a standard McMaster technique and a new rapid method.

For the assessment of coccidial oocyst production by chickens, some modified form of the McMaster counting method is commonly used. The objective of this study was to evaluate a standard method and to compare it to a new, faster method, in which all the preparative stages before counting are carried out in the same container into which the original faecal sample was collected. A stock suspension containing purified oocysts of all seven valid Eimeria species that parasitize chickens was prepared, from which seven concentrations of oocyst suspensions were made. Since the faecal material in a sample influences the ability of oocysts to float up in a McMaster chamber, the new method was tested to establish the optimal amount of faeces in the original sample. Control oocyst suspensions containing no faeces were also tested, and three series of counts using the new method were compared with the standard McMaster method. The results were statistically analysed by agreement analysis. Repeatability and between-operator variation of both methods were also tested by agreement analysis. Counting by the standard McMaster method underestimated the true number of oocysts. The new method gave counts in agreement with the true number of oocysts if using 1 g of faeces per sample. With 2 g of faeces, counts were obtained that agreed with counts by the standard McMaster method. Both methods showed agreement between repeated measurements. The new method used on a sample containing 2 g of faeces provides a convenient alternative to the standard modified McMaster method. A 1-g faecal sample increases agreement with the true numbers of oocysts. Processing of a sample with the new method is about nine times faster than with the standard method.