Body Weight, Oocyte Elimination and Blood Profile of Rabbit After Challenge Test Using Eimeria Stiedai

The objective of the research was to investigate body weight, oocyte elimination and blood profile of rabbits infected with various doses of Eimeria stiedai isolates. The observed rabbits' blood profile included erythrocyte, hemoglobin, hematocrit, leucocyte, thrombocyte, total protein plasma (TPP) and fibrinogen. Twenty-five male New Zealand White rabbits aged 3 months and weighed approximately 2 kg were provided with pellet and boiled drinking water and Eimeria stiedai isolates. The experiment used Completely Randomized Design to analyze 5 treatments with five replicates. The examined variables included D0: Infection 0 (control of infection without challenge test), D1: Infection 101 with challenge test 103, D2: infection 102 with challenge test 103, D3: infection 103 with challenge test 103, D4: infection 0 with challenge test 103 (control of infection). Data were subject to analysis of variance followed by Honestly Significant Difference Test (HSD). Analysis of Variance result showed that there was no significant difference on body weight, oocyte elimination and blood profile including erythrocyte, hemoglobin, hematocrit, leucocyte, thrombocyte, and fibrinogen. However, total protein plasma (TTP) was significantly different at 5% HSD. It can be concluded that challenge test with Eimeria stiedai has not been used as an alternative in increasing rabbits' body immune against coccidiosis infection.


Introduction
Rabbits are food source that produce white meat high in calorie but low in fat and cholesterol as the alternatives to chicken (Moreki, 2007). Coccidiosis is infection of Eimeria spp. protozoa that causes high mortality which is a major obstacle in rearing rabbits (Mwangi, 2010;Oncel et al, 2011;Nehemiah, 2015). Protozoa Eimeria spp, a parasite that contributes to coccidiosis in rabbits, consists of 11 types, 10 of which infects digestive tracts an one type infects liver (Pakandl, 2009). Eimeria stiedai is a parasite that lives on epithelial cell of bile tract that causes liver damage (Abu-El-Ezz et al., 2012).
A prevention of coccidiosis can be done by providing coccidiostat in the feed and live vaccine. However, regular intake of anticoccidia will induce parasite resistance and drug residue in the meat, therefore harming }v•µu OE•[ health (Pakandl, 2009). Several studies on coccidiosis in poultry informed that oocyte Eimeria spp infection can cause resistance and immunity against coccidiosis when repeatedly infected from the same species (Coudert et al, 1993;Yuwono and Setyawati, 2006). However, the study is comparatively rare to rabbits in Indonesia.
Oocyste fecal excretion is one of the indicators of coccidiosis infection. Blood tests are one of the parameters to determine the physiological conditions of livestock (Indrasanti et al, 2017). Rabbits infected with Eimeria spp. will undergo physiological and blood profile disorders. Therefore, the present study } • OEÀ v u •µOE OE ]š•[ body weight, oocyte elimination and blood profile of rabbits that were infected and challenge-tested with Eimeria stiedai.

Materials and Methods
Twenty-five male New Zealand White rabbits aged 3 months and weighed approximately 2 kg, rabbit pellet and boiled drinking water and Eimeria stiedai isolates. The rabbits were kept in individual iron cage. Pellet without coccidiostat and boiled water were provided ad libitum. Blood sampling was taken from lateral vena auricularis on the 11 th day post-infection using pipette containing EDTA (Ethylene Diamine Tetraacetic Acid).

Preparing experiment rabbit free from coccidia
Coccidiostat was orally administered to the NZW rabbits. Feces examination was conducted in daily basis for 21 days to observe oocyste in the feces. The rabbits were designed as coccidia-free rabbits. Suplement-free feed and boiled water was given ad libitum (Li et al, 2010). Body weight was measured two times a week.

Eimeria stiedai Infection
Infection was in oral administration by taking supernatant from the microtube using micropipette, then placed under the tongue of the rabbit to ease the swallow.

Blood sampling
Two ml srynge was filled with ìUïì …o EDTA and injected through vena aucularis lateral (ears) to take 2 ml blood, then placed in a box filled with ice that had been prepared (Rukayah, 2008;;Indrasanti et al, 2017).

Rabbits treatment
The rabbits were infected with Eimeria stiedai in week one, then subjected to challenge test on week two and treated until week five.
Fecal sampling collection was to analyze oocyst and body weight was measured two times a week. By the end of week five, blood sample was collected (modification from Coudert et al, 1995).

The observed variable
The observed variables were body weight and oocyst calculation using Mc Master method. Blood profile observation used Neubauer chamber to count erythrocyte and leucocyte cell, spectrophotometer for hemoglobin test, microhematocrit for hematocrit, TPP and fibrinogen analysis and blood smear for thrombocyte.

Results and Discussion
The increasing body weight and the eliminated oocyst in rabbits infected with various dose of Eimeria stiedai according to challenge test from week one until week five are presented in Table 1 and 2 and Figure 1 and 2. Result showed that no significant difference was observed across treatments.
Blood profile represents hematologic status of animals, either plasma or blood plasma that essentially contributes to physiological process (Indrasanti et al, 2017). Blood profile of rabbits infected with various doses and subjected to challenge test with 10 3 Eimeria stiedai is presented in Table 3. Hana (2011) declare that the clinical symptoms of coccidiosis-infected rabbits from Eimeria magna includes fever, weight loss, anemia, leukocytosis, lymphocytosis, hyperfibriogenemia and the increased activity of alkaline phosphate (ALP). Anemia is the decrease of erythrocyte and hemoglobin far below the threshold due to infection from bacteria, fungi, virus, protozoa and parasite (Wicaksono and Ardila, 2009).
The result showed that rabbits did not suffer from anemia since the amount of erythrocyte and hemoglobin was within normal range except for D3 and D4 rabbits with erythrocyte slightly above the threshold and D2, D3 and D4 with hemoglobin above normal. Total protein plasma, fibrinogen, hematocrit and leucocyte were normal but thrombocyte in D4 was under the threshold (Table 3).
Some contributing factors to the increased erythrocyte and hemoglobin in rabbits were dehydration and lack of oxygen during treatment. This is probably due to coccidiosis infection, because coccidiosis causes tissue damage, decreases feed intake, poor absorption of nutrients, dehydration, blood loss and death (Handayani and Hariwibowo, 2008;Ola-Fadunsin and Ademola, 2014). Thrombocyte in infected control was under the threshold, likely due to infection of Eimeria stiedai in the liver. Vitamins are mainly produced in the liver, particularly vitamin K that serves as anticoagulant (Supriyanto, 2008 (Peek, 2010). Therefore, the ill condition of liver due to Eimeria stiedai infection could decrease the amount of thrombocyte.
Analysis of variance showed that almost all parameters of blood were not significantly different across treatment except for TPP (P<0, 05). Honestly Significant Difference Test 5% on TPP showed significant difference between D1, D2 and D3 compared to that of control (D0 and D4). Coccidiosis causes stress in rabbit that will decrease plasma serum and eventually mortality (Lester et al, 2005). In rabbits, however, recurring infection resulted in immunity against the infected species only, not in all Eimeria spp (Licois, 2004). In fact, most prevalent coccidiosis in nature is caused by more than one species of Eimeria spp is a common thing (Okumu et al, 2014). So it needs to be reviewed using the combination of Eimeria spp in the test against the incidence of coccidiosis in nature.  10     Most of the parameters examined in this study show the results of an analysis that was not significantly different. Several blood parameters in each treatment showed normal levels, but there was Eimeria spp in rabbit fecal. This was likely because the range of infection doses between treatments was too small so it does not produce significant differences between treatment and control. Rabbit did not show significant clinical symptoms. This result shows that rabbits were infected with subclinical coccidiosis. Sublinical coccidiosis shows mild symptoms where it has a good prognosis because subclinical infection will produce natural immunity (Mayer and Donnelly, 2013). Rabbits infected with subclinical coccidiosis will be better if given herbal formulations in their feed (Nosal et al, 2014). The number of oocysts needed to cause pathogenic effects varies in each type of oocyst (Caudert et al, 1995). In addition, age, condition and whether or not the host is infected with oocysts and the host's immune system also affects the pathogenic effects (Patton et al, 2008;Pakandl, 2009). The use of a higher infection dose can be recommended for subsequent studies so that the results obtained can be seen more clearly.