Seroprevalence of antibodies to Encephalitozoon cuniculi in domestic rabbits in Nigeria

OKEWOLE, E.A. 2008. Seroprevalence of antibodies to Encephalitozoon cuniculi in domestic rabbits in Nigeria. Onderstepoort Journal of Veterinary Research, 75:33–38 Serum samples from 237 randomized rabbits from the five ecological zones of Nigeria, i.e. Northwest (NW), Northeast (NE), Southeast (SE), Southwest (SW) and Northcentral (NC), were evaluated for the presence of antibodies to Encephalitozoon cuniculi by the indirect immunofluorescent antibodies test. A titre of 10 or more was taken as positive. Thirty-nine (16.5 %) of the 237 samples were positive with 11, 10, 8, 6 and 4 seropositive rabbits occurring in the NW, NE, SE, SW and NC zones of Nigeria, respectively. Age, sex, live mass and access to grass as a feed supplement were not statistically (P > 0.05) associated with seropositivity, but cage type (single-versus multi-rabbit type), contact with free-range rats and previous illness were strongly (P < 0.05) associated with it. The practice of selling unscreened and untreated 5 to 10-week-old weaners to prospective buyers as foundation stock, use of multi-rabbit communal cages, occasional release of rabbits in runs and contact with free-range house rats should be discouraged. Regular prophylactic and curative treatments, occasional serological screening to remove carriers, and the practice of a high level of hygiene in rabbit colonies are effective control measures.


INTRODUCTION
Encephalitozoonosis in animals and humans is caused by the obligate intracellular protozoan, Encephalitozoon cuniculi, a member of the Phylum Micro spora.More than 1 000 species of microsporida exist which are classified into about 100 genera and which infect insects and vertebrate animals.(Canning & Lom 1986).To date, several species of the genus Encephalitozoon have been described with three strains of E. cuniculi being indentified (Cali, Kotler & Orenstein 1993;Didier, Orenstein, Aldras, Bertucci, Rogers & Janney 1995) most of which are opportunistic pathogens in immunocom-promised contact animals and humans (Harcourt-Brown 2002).
Infection of most mammalian hosts with E. cuniculi occurs by ingestion or inhalation of spores from contaminated urine and faeces from infected hosts (Canning & Lom 1986).Once in the susceptible hosts, the spores invade by a germination process and undergo schizogony, an asexual cell division, followed by sporogony and maturation.Infections, characterized by muscular weakness, emaciation, urinary incontinence, vestibular signs such as head tilt, seizures, ataxia, posterior paralysis and uveitis, were first reported in rabbits by Kimman & Akker manns (1987).Infections have also been reported in mice, guinea pigs, hamsters, dogs, cats, monkeys, and humans but systemic disease is rare in these species except in athymic or immunocompromised mice and neonatal dogs or foxes (Gannon 1980).Encephalitozoon cuniculi has been recognized as an important opportunistic pathogen in human patients suffering from the acquired immunodeficiency syndrome (AIDS) in which it causes interstitial pneumonitis, chronic diarrhoea, renal disease and wasting (Cali, Kotler & Orenstein 1993;Van Gool, Snijders, Reiss, Eeftinck Schattenkert, Van den Bergh Weerman, Bartelsman, Bruins, Canning & Dankert 1993;Didier, Visvesvara, Baker, Rogers, Bertucci, De Groote & Voss Brinck 1996).As the prevalence of encephalitozoonosis in a country may be higher than is realized (Keeble & Shaw 2006) which can have a negative effect on the results of experiments in which rabbits are used (Wilson 1979), the screening of a nation's rabbit colonies may be a worthwhile exercise.
The objectives of this survey were to determine the seroprevalence of encephalitozoonosis in domestic rabbits in different ecological zones of Nigeria in order to formulate appropriate cause-based control measures that would promote public health and produce rabbits free of the infection for experimental use in scientific studies.

Place and time of study
The assay was conducted at the Experimental An imal Unit of the University of Ibadan, Ibadan, Nigeria from May to August 2003 on 314 serum samples obtained from rabbit colonies in the five ecological zones of Nigeria, viz.Northwest (NW), Northeast (NE), Southeast (SE), Southwest (SW) and Northcentral (NC) zones.

Inclusion criteria for animals
Rabbits of both sexes of all breeds and health status, in all husbandry systems, diets and preventive medicine routines that were over 4 weeks of age were included.

Study design
The survey targeted a randomized sample of 50 rabbits from each secondary school located in the capital cities of the ecological zones.(Federal authority mandated the keeping of rabbits in all federal schools for use in agricultural science and biology practicals.) The selected schools were requested to complete questionnaires in order to collect information such as the history (origin) of rabbit colony, breed, age and sex of the rabbits in the colonies, previous and current clinical observations, feed and water consumption, routine treatments, weekly morbidity and mortality rates, housing types, urine and faecal/disposal schemes, access to wild birds or animals, and access to grass as a feed supplement.

Serum samples
Approximately 2 mℓ of blood were collected either from an incision made in the margin of the ear or the jugular vein of each of the rabbits into sterile gel tubes and allowed to clot.Centrifugation was done after the clot had retracted in order to separate serum from clot.The serum samples were stored frozen at -20 °C before testing.

Serology
A commercial heat-fixed E. cuniculi antigen stored at 4 °C (Sigma-Aldrich Co, England) was used.Sera were tested by the indirect immunofluorescent antibody technique (IFAT) as described by Cox & Pye (1975) for the presence of antibodies to E. cuniculi at a dilution of 1:10.The conjugate used was a fluorescein-isothiocyanate (FITC)-conjugated sheep IgG anti-rabbit immunoglobulin (Sigma-Aldrich Co, England).Those serum samples showing any evidence of reactivity were subsequently titrated by doubling dilutions commencing at a dilution of 1:10.The slides were examined at a magnification of 630 under a Leitz fluorescence microscope.Titres were expressed as the highest serum dilution giving a bright staining of the spore body.Titres of 10 or higher were regarded as positive, while those below 10 were regarded as negative.

Statistical analysis
The rabbits' ages and live masses as possible influencing factors on seropositivity to E. cuniculi were tested by the standard 2-sample t-tests (Snedecor & Cochran 1967), while other possible influencing factors such as sex, breed and health status were analyzed by the chi-squared test.In all tests, values of P < 0.05 were taken as significant.

RESULTS
The results of the serological screening at the five zones are summarized in Table 1, while the statistical analyses of possible influencing factors are given in Table 2. Of the 237 rabbits sampled, 39 (16.5 %) were seropositive.These 39 rabbits were According to their histories, 31 (79.5 %) of the seropositive 39 had manifested clinical signs such as head tilt (13), hind leg weakness (7), ocular lesions (3) and urinary incontinence (8).All had responded positively to therapy with enrofloxacin (Baytril®, Bayer) or fenbendazole (Panacur®, Hoechst).Twen tythree of the rabbits that had previously manifested clinical signs were more than 36 weeks old.However, the eight that had incontinence were relatively young.
Only eight (20.5 %) of the 39 seropositive rabbits had not shown any clinical signs suggestive of encephalitozoonosis at least 5 months before sampling, but they were in contact with feed and water troughs and other seropositive cage mates in their multi-rabbit cages.Thirty-five (87.7 %) of the 39 seropositive rabbits were from multi-rabbit communal cages, housing 3-10 rabbits with open feed troughs, but inverted water bottles (Table 1).Only 4 (10.3 %) of the seropositive rabbits were from single-rabbit cages but had occasionally been released to run on urine-contaminated floors before being re-caged (Table 1).
The 5 to 10-week-old rabbits were under-represented in the randomized zonal samples as a result of their regular sales to other schools as foundation stock.
There were no significant associations between the number of rabbits that were seropositive and the age, sex, live mass and access to grass as feed supplement, (Table 2).However, significant associations were observed between seropositivity and multi-rabbit housing, contact with other rabbits when occasionally released into runs and previous illnesses (Table 2).

DISCUSSION
The results of this survey have confirmed that E. cuniculi is present in a sizable proportion of apparently clinically healthy rabbits in Nigeria.In addition, they also show that age, live mass and sex of the rabbits had no influence on their seropositivity.These findings are consistent with the earlier report of Keeble & Shaw (2006) from the United Kingdom.However, other factors such as contact with wild rabbits or rats that were scored low on the chisquared analysis might be epidemiologically important.Recently Thomas, Finn, Twigg, Deplazes & Thompson (1997) demonstrated exposure to E. cuniculi in wild Australian rabbits, which conflicts with the earlier negative report of Cox, Pye, Edmonds & Shepard (1980).Muller-Doblies, Herzog, Tanner, Mathis & Deplazes (2002) have also demonstrated the presence of the same parasite in the free-ranging rats, Rattus norvegicus.The potential danger posed to caged rabbits by wild, free-ranging rats that are sustained principally by the few food pellets dropped on the floors of the colonies require further investigation.It is also feasible that their urine and faeces contaminate feed of rabbits in open troughs as they climb on cages seeking possible entry in order to obtain the feed.
The most likely mode of infection of caged rabbits is the ingestion of spores in urine-contaminated feed in open feed troughs, since spores have been reported to survive in the environment for long periods of time (Kucerova-Pospisilova, Carr, Leitch, Scanlon & Visvesvara 1999).This possibility is supported by the fact that more seropositive rabbits were observed in colonies containing a higher number of communal multi-rabbit cages, each housing 3-10 rabbits.Another contributing factor is possibly the policy of selling weaner rabbits of 5-10 weeks of age to other schools as foundation stock, without any prophylactic treatment.According to Lyngset (1980), young weaner rabbits suffering from an active infection of E. cuniculi seroconvert at 8-10 weeks of age.Such infections could spread by contact with the doe or buck, while the former may also transmit transplacentally to their offspring in the new colony.Test imony to this possibility is the fact that the NW, NE and NC stocks were all purchased in Zaria city (Table 1).In order to prevent the introduction of infection in this way it is suggested that all weaners be serologically tested before introducing them into colonies.Positive animals should be either treated aggressively or euthanazed.
The clinical signs of head tilt, ocular lesions and hind leg paresis in 23 of the seropositive rabbits that were observed about 5 months prior to the sampling in the NE, NW and SE zones are suggestive of the nervous manifestations of encephalitozoonosis that occur particularly in older rabbits.This again is corroborated by the observation of higher number of seropositive rabbits from this age group by Harcourt-Brown & Holloway (2003).
The IFAT, though rated as suitable and sensitive as the ELIZA and carbon immuno-assays for the immunodiagnosis of encephalitozoonosis (Boots 2002), could be made more useful and informative, if sampling is repeated 4 weeks later to evaluate seroconversion, or to detect the presence of both IgM + IgE with appropriate conjugates.This, if it had been done, would have assisted in predicting the age of infection, and the different status of the seropositive rabbits, which are needed for epidemiological control.The high cost of repeated screening, logistical problems and poor knowledge of the transmission of E. cuniculi are some of the constraints in the control of the disease.
While a national seroprevalence rate of 16.5 % may be considered relatively low, it probably points to a small rabbit industry in Nigeria.
In view of current husbandry policies, which, it seems, promote a very high prevalence of encephalitozoonosis in weaners, the following steps to control the disease are suggested: weaner rabbits should be screened before sale and infected ones removed, design and use hygienic single-rabbit cages with raised feed troughs and inverted water bottles, pro-phylactic treatment of animals that have been in contact with infected ones, thorough disinfection of all implements and strict hygiene practices.Rabbits for investigational studies should first be serologically tested and certified free of encephalitozoonosis.

TABLE 1
Summary of the anamnestic, management, clinical and serological results of the seroprevalence survey for encephalitozoonosis in rabbits in Nigeria

TABLE 2
Statistical analysis of the information returned from the zonal survey of antibodies to E. cuniculi by rabbits in Nigeria