Detection of Toxoplasma gondii infection in domestic rabbits by using multiple techniques

Fifty local breed rabbits were purchased from commercial markets in Mosul city for Toxoplasma gondii detection during the period from April 2007 to October 2007, using five techniques. Of these, impression smear made from different body organs were positive in 82% of the tested samples. Trypsin digestion technique was positive in 68% of the samples, while pepsin digestion in 82% of them. Serological techniques (latex agglutination test and modified latex agglutination test) were positive in 86% of the tested samples, with IgG titer of 1/64. Biological assay in mice was performed by intraperitoneal inoculation of 0.1 ml digested organs suspension, (lungs, heart, liver, spleen, kidney, muscles, uterus, testes and brain). Brain tissue shows the highest rate of tissue cysts detection among all other organs examined of scarified mice post one month of inoculation.


Introduction
Infection by Toxoplasma gondii is widely prevalent in humans and many species of warmblooded animals, including domestic rabbits (1,2).Splendore (3) was the first who described toxoplasmosis in domestic rabbits (Oryctolagus cuniculus) in Brazil, since then clinical cases of this disease among rabbits have been reported by many authors in various countries such as England (4) Argentina (5), Italy (6), Scandinavian countries (7,8) and the U.S.A (9).The last author reported fatal toxoplasmosis in three domestic rabbits in USA and the most striking lesions in all three rabbits was necrotic foci in the spleen and liver associated with massive presence of multiplying Toxoplasma gondii tachyzoites.The affected rabbits with toxoplasmosis showing different clinical findings include loss of appetite, fever, anorexia, lethargia, nasolacrimal discharge and respiratory, disorders, tremors, uncoordinated gait, voluminous, hair coat, and diarrhea.The pathological lesions of toxoplasmosis were mainly observed in the liver and spleen (10).
In general, severe acute toxoplasmosis many animals died and most outbreaks occurred in winter months (January and February) and few in summer months (June and July), on the other hand, acute systemic toxoplasmosis is seen especially in young and immunesuppressed animals (1,11).
The positive serological response of rabbits to Toxoplasma antigen is variable from 2.4-8.3% in China, (12,13) to 53% in Germany (14) and 48.4-57.9% in the Czech republic (15).The role of domestic rabbit in epidemiology of toxoplasmosis in humans has not been established in detail, but is probably important although some authors treat this role marginally (1) others place the rabbit among the animal species posing a major source of infection from man (16,17).Ishikawa et al. (18) described the case of cervical toxoplasmosis transmitted from rabbit to man.( 16) found very high liters of anti-Toxoplasma antibodies in rabbit hunters.Sroka et al. (19) also found positive Toxoplasma in workers in rabbit farms, therefore suggested that the rabbits should be considered as a potential source of Toxoplasma infection among agricultural workers.No studies on T. gondii in rabbits are reported in Iraq, therefore the aim of this study was to determine the natural infection with Toxoplasnni gondii in domestic rabbits by using different diagnostic techniques.

Animals
Fifty local breed rabbits were purchased from commercial markets in Mosul city (Iraq) during the period from April 2007 to October 2007.They were 25 females and 25 males, weighted 1000-2500 grams, and were put in cages (1/2 m x 1 m) until scarifying..

Diagnosistic techniques
Toxoplasma gondii was diagnosed by; Impression smears: These were made from various body organs (brain, lungs, heart, liver, spleen, kidney, muscles, uterus and testes) for detection of tissue (20).Trypsin digestion: Tested organs were digested by trypsin as described by (21).Pepsin digestion: Tested organs were digested by pepsin as described by (22).Serological tests: Blood samples (5 ml) were collected from jugular vein of individual animal, and stored under 4°C temperature over night.Sera were isolated by centrifugation of blood samples at a rate of 2500 rpm for 10-15 minutes.Serum samples were tested for reactive animals using latex agglutination test (Biokit, Spain) and Modified agglutination test (2-ME) (23).Biological assay: white Suis-mice (25 gr.) 4-6 weeks age, negative for T. gondii were inoculated intraperitoneally with 0.1 ml suspension of digested organs (lungs, heart, liver spleen, kidneys, muscles, uterus, testes and brain) for confirmation of T.gondii infection of tested rabbits.

Results
Results of different techniques used for detection of T. gondii are presented in Table (1).From this table it is evident that serological examinations (Latex agglutination test) were positive in 86% of the tested rabbits.To a lower extent 82% were the results obtained by using both pepsin digestion and impression smears of the examined rabbits.The lowest result 68% was obtained by using trypsin digestion technique.
The intensity of tissue cysts in different organs of tested rabbits using impression smears are shown in Table (2), which reveled a wide variation of tissue cysts distribution in different organs noticed in examined rabbits, being highest in brain (> 1000) in which severe infection by T.gondii tissue cysts was detected (Figure 1).In the second order were heart, lungs, liver, and spleen, where moderate (500-1000) tissue cysts were observed.In the third order kidneys, testes and uterus with a relatively moderate presence of tissue cysts (100-500).Less than 100 tissue cysts were observed in muscle tissues.The positive male and female rabbits to Toxoplasma gondii are represented in Table (3).It is clear that females were infected more than males with T. gondii with a percentage of 92% for females versus 72% for males and with a ratio of 0.56 for females and 0.43 for males.

Biological assay
Typical tissue cysts were found in the brain of the inoculated mice with suspension of brain and digested organs (brain, heart, lungs, liver, spleen, kidney, muscles, uterus and testes).Others organs found to be infected with tissue cyst but to a lesser extent than brain (Figure3).

Discussion
Diagnosis of toxoplasmosis in rabbit in this study was depended upon organs impression smear, tissue trypsin digestion, pepsin digestion, serological test and mice bioassay.
The serological latex agglutination test was useful in exploring 86% positive exposing rabbits to Toxoplasma gondii infection through either contaminated feed or water with parasite oocysts, or by maternally acquired infection (2,9,19).In Ninevah governorate (north of Iraq) with more rainy seasons, a location that may provide a suitable environmental conditions for Toxoplasma infection, a case which was reported in aborted women (24) and aborted ewes (25) in Ninevah governorate through the recent years.
These results revealed that there was a difference in the ratio of seropositivity in males versus females, this is inconsistent with (26) who reported that there was no effect of age, sex and season on the prevalence of toxoplasmosis.
The positive modified latex agglutination test, means that IgG antibodies superimpose on IgM antibodies and that the infection was a chronic one.This suggestion could be traced by the purchased rabbit, show no symptoms of infection, and so may acquired their infection from their infected does, which pass their IgG antibodies through placenta and suppressing IgM antibody response in their off springs (27).
The moderate titers of antibodies (up to 512) of the tested rabbits may also explain the chronicity of infection.
Our serological results of 86% positivity were higher than those reported in China (2.4-3.8%)(12,13), in Germany (53%) (14) and in the Czeck republic 48.4-57.9%(15).Impression smears taken from different tissue organs, revealed positive results in brain, lungs, heart, liver, spleen, kidneys, muscles, uterus and testes.The presences of T.gondii cysts in these organs also reported by other investigators (9,10,28,29).It is not surprising to find parasitemia to occur spontaneously in rabbits chronically infected with certain strains of Toxoplasma and persist in the presence of high antibody triers (30).
The presence of Toxoplasma cysts in brain tissue reported here was in accordance with (31) who was the first one to describe its presence in rabbits brain tissue and followed by (14).Anyhow, (32) reported no Toxoplasma cysts in histological examination of brain tissue from 51 wild rabbits, while the work of ( 19), confirms the possibility of spontaneous development of T. gondii in rabbits brain.
The absence of gross lesions in necropsed rabbits give an additional confirmation of chronicity of infection (2).
The presence of bradyzoites in different tissue impression smears especially in brain which may be of focal brain lesions of chronic infection (FBL), indeed supports the serological diagnosis (33) and rabbits in the same time is regarded as a good model of toxoplasmosis of the CNS (34).
The presence for example of bradyzoites in brain was not accompanied by posterior paralysis and death of these rabbits.This may be due to the inadvancement of the case to that stage, and even fatal toxoplamosis could occur even without symptoms in rabbits (19).The bioassay positive results in mice through their inoculation with 1 ml suspension of pooled digested organs and the recovery of bradyzoites after 1 month necropsy in different organs, confirms the infectivity of rabbits by T. gondii.The importance of these findings is that these animals could be a major source of infection to human (19,15) especially in those suffering from acquired immunodeficiency syndrome (AIDS) (34), although there is lack of controlled epidemiological studies regarding the degree of correlation between the prevalence of toxoplasmosis in rabbits and in humans in contact with these animals (35).

Conclusion
Toxoplasma gondii was isolated from local rabbit bread in Mosul city-Iraq.Diagnosis was accomplished by different diagnostic methods.Confirmation of results was ascertained by mice bioassay.

Figure 1 :
Figure 1: Cysts of T.gondii in the brain of the natural infected rabbits.

Figure ( 2 )
Figure (2): The antibody titers and the percentage of infection with T.gondii in the infected rabbits.

Figure ( 3 )
Figure (3): Cysts of T.gondii appeared in brain of mice inoculated with suspension of digested organs of rabbits.

Table 1 :
Percentages of Rabbits infected with Toxoplasma gondii by using different diagnostic techniques.

Table 2 :
Scores of Toxoplasma gondii tissue cysts detected in different organs using impression smear method.

Table 3 :
The percentage of infection with T. gondii according to sex by impression smears.