P. Coudert

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.

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.

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 coecicola Cheissin 1947 : endogenous development in gut-associated lymphoid tissue

Abstract Coccidia-free rabbits were inoculated with different doses of a pure strain of Eimeria coecicola and samples of gut were taken at 80, 96, 112, 128, 144, and 160 h postinoculation. The use of a very low infective dose (2–20 oocysts) was sufficient to study the last merogony. The number of merozoites in meronts increased when the infective dose decreased. Only the first merogony of this coccidium in lymphocytes or M-cells of gut-associated lymphoid tissue (GALT) has previously been described. Three other generations of meronts are described herein. All these endogenous stages were observed in the epithelium of the vermiform appendix, sacculus rotundus, and Peyer’s patches, especially at the bases of the domes. However, in heavily infected tissues the gamonts were seen throughout the epithelium of the GALT. The third- and fourth-generation meronts were of two types. As in other eimerian species of the rabbit, type A meronts produced thick polynucleated merozoites, whereas type B meronts gave rise to large numbers of thin merozoites with one nucleus. Microgamonts were polynucleated and less numerous than macrogamonts. Type A meronts were also polynucleated and less numerous at the end of the merogony. Therefore, types A and B could correspond to a sexual phenotype differentiation occuring during the two asexual phases of multiplication.

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.

Characterization ofEimeria species

ZusammenfassungDie sogenannten kleinen Coccidien sind morphologisch schwierig zu unterscheiden.Um einen reinen Stamm von Coccidien zu erhalten, müssen absolut coccidienfreie Kaninchen zur Verfügung stehen. Solche werden am INRA in Tours durch vielseitige Einrichtungen und Maßnahmen gehalten. E. perforans wurde charakterisiert durch seine Zykluslänge von 5 Tagen, einer Sporulationszeit von 21 h bei 26°C und den typischen morphologischen Kriterien der sporulierten Oocysten. Von einem Stamm, der diesen Erforderungen entsprach, wurden nach einer statistischen Verteilung der Länge und Breite der Oocysten aus dem mittleren Bereich 12 Oocysten einzeln isoliert und je in einem SPF Kaninchen vermehrt.Der so neu gewonnene Stamm, dessen Größenverteilung dem Mutterstamm am meisten entsprach, wurde als rein bezeichnet. Zur Prüfung der Pathogenität wurden 3 verschiedene Mengen verabreicht. Es traten keine Todesfälle auf, bei hohen Mengen zeigte sich der Caecuminhalt 2 Tage nach Inokulation leicht verflüssigt. Alle Tiere (alle Mengen) zeigten zwischen dem 2. und 4. Tag kleinere Gewichtszunahmen als die Kontrolltiere. Parallel dazu war auch die Futteraufnahme entsprechend vermindert. Mit diesen Befunden wirdE. perforans als praktisch apathogen bezichnet.SummaryEimeria perforans was isolated in pure strain by utilizing specific biological characters and specific-pathogen free rabbits. The purity of this strain was demonstrated by measurements of oocysts, derived from a line, started by infection from a single oocyst. The parasite, when inoculated in large numbers, causes a mild disease response for only about two days.Eimeria perforans was isolated in pure strain by utilizing specific biological characters and specific-pathogen free rabbits. The purity of this strain was demonstrated by measurements of oocysts, derived from a line, started by infection from a single oocyst. The parasite, when inoculated in large numbers, causes a mild disease response for only about two days.

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.

Tissues and cells involved in the invasion of the rabbit intestinal tract by sporozoites of Eimeria coecicola.

Abstract This study was designed to identify an extra-intestinal route of migration of Eimeria coecicola sporozoites and the types of cell harbouring the parasite during the invasion of the intestine. The presence of E. coecicola in blood, spleen and mesenteric lymph nodes of infected donor rabbits was demonstrated by immunohistology on donor organs and measurement of oocyst excretion by coccidia-free recipient rabbits injected with whole-cell suspensions prepared from donor tissues. Two types of donor lymphocyte, B (IgM+) and T (CD5+), were labelled using a two-colour immunofluorescence-labelling technique and separated with a cell-sorter (FACStarplus). The presence of parasites in the sorted cells was assessed by direct examination and by using the same in vivo test after intravenous injection of IgM+ B or CD5+ T lymphocytes collected from donors at different times after inoculation. This test provided evidence that the parasites were alive and still infectious within the sorted lymphocytes. It was demonstrated that both B and T lymphocytes were infected.

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.