Dominique Licois

Infectious agents associated with epizootic rabbit enteropathy: Isolation and attempts to reproduce the syndrome

Abstract Epizootic rabbit enteropathy (ERE), a highly lethal (30–80% mortality) disease of broiler rabbits aged 6–14 weeks, first appeared in 1997 in French intensive enclosed rabbitries and is of unknown aetiology. Bacteriological, virological and parasitical examination of the intestinal contents of rabbits that had died either in spontaneous field cases or after experimental reproduction of ERE, were undertaken in an attempt to identify infectious agents that may play a role in the disease. Two bacterial strains, Clostridium perfringens and non-enteropathogenic Escherichia coli were repeatedly isolated at high faecal counts from naturally infected animals. In field cases, a correlation between typical gross lesions of epizootic enteropathy and the presence of the alpha toxin of Cl. perfringens was observed (P <0.0001; Chi-squared test). Although attempts to reproduce the disease by inoculation with different pools of cultivable bacterial strains failed, the disease was successfully reproduced by inoculation with one French and two Belgian samples of caecal contents.

Study of the inter- and intraspecific variation ofEimeria spp. from the rabbit using random amplified polymorphic DNA

A genetic polymorphism study was performed in coccidia from the rabbit. A comparative analysis of the RAPD (random amplified polymorphic DNA)-generated fingerprints, using 11 arbitrary primers, was carried out (1) in nineEimeria species (E. intestinalis, E. magna, E. piriformis, E. flavescens, E. vejdovskyi, E. coecicola, E. perforans, E. exigua, andE. media) and (2) in two strains ofE. intestinalis and four strains ofE. media originating from different geographic areas. For each of these four strains ofE. media, three lines deriving from the multiplication of a single oocyst were compared. All the primers tested yielded about ten amplified fragments. The profiles obtained differed considerably according to the species; thus, it was not possible to establish a phylogeny. On the other hand, species-specific fingerprints were observed, showing that RAPD assays might be useful for diagnosis. InE. media, analysis of the RAPD products showed weak differences between each of the four strains but nevertheless allowed differentiation of the lines deriving from the multiplication of one oocyst. Similar results were obtained with three methods of analysis: correspondence analysis, the hierarchical unweighted pair-group method with arithmetic averages (UP-GMA), and parsimony analysis. RAPD proved to be a useful technique for these intraspecific studies.

Eimeria magna: Pathogenicity, immunogenicity and selection of a precocious line

A precocious line (PrEmag) of Eimeria magna in rabbits was obtained by selecting for early development of oocysts. The prepatent period was shortened by 46 h. The pathogenicity of PrEmag was substantially reduced and its reproductive potential was much lower (500 times) than that of the parent strain. Rabbits given 2500 oocysts of PrEmag were almost totally protected against a challenge with the parent strain. As in other precocious lines of coccidia from the rabbit, PrEmag showed morphological anomalies of the sporulated oocysts. Each sporocyst harboured one large refractile body instead of the two smaller ones in the parent strain.

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus).

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.

Selection and characterization of a precocious line of Eimeria intestinalis, an intestinal rabbit coccidium.

A precocious line ofEimeria intestinalis was obtained by selection for early development of oocysts in rabbits and after six consecutive passages in animals. This line (EiP) was derived from a wild strain (EiO) isolated in 1975 from the caecal content of a rabbit with coccidiosis. The prepatent period of the EiP strain was reduced from 215 h to<144 h, the result being that the oocyst sporulation time was the same for both lines. The excreted and unsporulated oocysts had exactly the same shape, but microscopical examination of the sporulated oocysts showed a marked difference between EiP and EiO strains. A huge refractile globule was located in each of two sporocysts of the precocious line, whereas no refractile globule was seen in the other two. The EiP line had a reproductive potential much lower (1000 times) than that of its parent strain EiO and, as judged by the weight gain, mortality and lesions that also occurred in the jejunum and above all in the ileum, its pathogenicity was substantially reduced.

Eimeria media: Selection and characterization of a precocious line

A precocious line ofEimeria media was obtained by selection for early development of oocysts in rabbits. The prepatent period was reduced from 108 to 72 h. The precocious line was less pathogenic than the original strain, and its multiplication rate was lower. Rabbits given oocysts of the precocious line were totally immune to challenge with the original strain as assessed by change in weight gain but were partially protected as assessed by oocyst output. Selection for precocious development was accompanied by morphological changes in the sporulated oocysts; each sporocyst contained only one large refractile body instead of the two smaller bodies seen in the original strain.

Eimeria sp. from the rabbit (Oryctolagus cuniculus): pathogenicity and immunogenicity of Eimeria intestinalis.

The pathogenicity and immunogenicity ofEimeria intestinalis was evaluated in SPF rabbits. The antimals were given immunizing doses of 6, 6×102, 6×103, and 6×104 sporulated oocysts and were challenged with 3×103 oocysts. The criteria analysed were the daily weight gain and the occyst output. This study showed thatE. intestinalis had strong immunogenicity, as the inoculation of 6 oocysts was sufficient to minimize the clinical expression of the disease following the challenge and to reduce the oocyst output by about 60%. The immunity towards the excretion of oocysts and the illness was absolute in animals inoculated with 600 or more oocysts. Moreover, this protection seemed to be efficient at least 8 weeks after the challenge. The present results also confirm the pathogenicity ofE. intestinalis, although the occurrence of diarrhoea may be irregular, and emphasize the fact that the capacity of thisEimeria for multiplication is not a criterion for clinical diagnosis of the disease.

Acquired protection of the rabbit (Oryctolagus cuniculus) against coccidiosis using a precocious line of Eimeria magna: Effect of vaccine dose and age at vaccination

Sucklings were vaccinated orally once at 25, 27 or 29 days of age with a precocious line of Eimeria magna. Each group received two doses varying from 3.5 x 10(2) to 3.5 x 10(4) oocysts. At 36 days of age, animals received a challenge inoculation with 10(4) oocysts of the wild strain of E.magna. Vaccination reduced oocyst output 10 to 1000 times after the challenge inoculation and prevented the decrease in the weight gain observed in non vaccinated challenged animals. When the vaccination was performed more than 9 days before challenge, full protection was obtained. An individual oral vaccination performed with 3500 oocysts gave total protection whatever the age at vaccination between 25 and 29 days of age.

Eimeria magna Pérard, 1925: study of the endogenous development of parental and precocious strains

The endogenous development of a parental strain of E. magna and its deriving precocious line was studied after inoculation of coccidia-free rabbits with oocysts or sporocysts directly into the duodenum and using electron microscopy. Four meront generations could be observed mainly in the jejunum and ileum for the parent strain. The first merogony began 24 h post-inoculation (h p.i.). and meronts were matured about 48 h p.i. The second and the third generation were complete at 66 and 84 h p.i. respectively. Thus, each generation needs approximately 16-20 h for maturation. The fourth generation appeared 102 h p.i. and the young gamonts were present 120 h p.i. As in the other rabbit Eimeria, two types of meronts were described: A and B. The morphology of all endogenous stages of the precocious line was similar but refractile bodies were absent in the first generation merozoites and the fourth generation meront was lacking.

Migration of sporozoites and merogony of Eimeria coecicola in gut-associated lymphoid tissue.

The invasive phase ofEimeria coecicola was studied during the first 80 h postinoculation (p.i.). Using a method that synchronized the life cycle, sporozoites were observed in the duodenum and the jejunum until 32 h p.i. They were seen first in the villous epithelial cells or in host cells resembling intraepithelial lymphocytes (IEL). Later they were observed in IEL in the lamina propria. After 48 h p.i., no coccidian stage was identifiable in the mucosa of the small intestine but sporozoites appeared in the lymphoid cells of lymphatic follicles of the gut-associated lymphoid tissue (vermiform appendix, sacculus rotundus, and Peyers patches). The first merogony was observed 64 h p.i. in these lymphoid cells and in membranous epithelial cells (M-cells) but was never seen in the epithelium itself. Morphologically there were two types of meronts, depending on the host cell type, but in both cases the merozoites contained a refractile body and resembled sporozoites. The first meronts of the second generation were observed 80 h p.i. in the villous epithelial cells of the domes of the follicles of the gut-associated lymphoid tissue, where the further development of thisEimeria takes place. This pattern of invasion strongly suggests that sporozoites take an exclusively extraintestinal route to reach the target cells. Moreover, to our knowledge this is the first description of an eimerian merogony that does not take place in epithelial cells.