4.1. Prevalence of T. gondii antibodies in slaughtered domestic ruminants
The overall occurrence of T. gondii antibodies among slaughtered domestic ruminants was found to be 55.8%. This finding is higher than the reported prevalence of 22.2% from Eastern Ethiopia (Tilahun et al., 2018) and 37% from Tunisia (Lahmar et al., 2015. This discrepancy may be related to the sample size and the age of population studies and methods used. Indeed, previous studies were carried out using an ELISA and DAT technique which are less sensitive than the latex agglutination test used in the present study.
In this study, Toxoplasma gondii antibodies occurrence was significantly higher in females and adults of sheep. Previous reports across the globe (Yibeltal, 2008; Ramzan et al., 2009; Gebremedhin et al., 2013; 2014) also reported a higher occurrence of T. gondii antibodies in females and adults. It might be due to that females are kept for breeding purposes and are lived for a long time and hence, they have a higher chance to harbour the parasite for their life. It is also attributed to the high chance of exposure to the source of infection as age increases and suggests that most sheep acquire the infection postnatal (Andrade et al., 2013; Opsteegh et al., 2016). In cattle, older and cross-breed animals also showed significantly higher T. gondii seropositivity than adult and local breed cattle. Older animals as they lived longer might be more likely to be exposed to the infectious agent from different sources (Jittapalapong et al., 2005; Teshale et al., 2007; Ramzan et al., 2009; Andrade et al., 2013; Opsteegh et al., 2016; Amdouni et al., 2017). According to Furtado et al., (2013), cross-breed cattle also had a higher chance to acquire T. gondii infection than local breeds; this may be ascribed to the genetic and immune status of the host.
4.2. Bioassay in mice
The overall viable T. gondii isolation rate of 38.5% in the current study shows a lower percentage compared to the report of Berhanu (2015) who reported 67.6%. It is also higher than the report by Elfadaly et al. (2017) from Egypt (8.57%) in domestic ruminants (sheep, goats and cow). The isolation rate of viable T. gondii in this study in sheep (50%) is comparable with the report of 57.45% from central Ethiopia (Gebremedhin et al., 2014) and higher than the findings from France with the isolation rates of 26.7% (Dumètre et al., 2006) and from Egypt with isolation rate of 32% (Younis et al., 2015). In contrast, the current result is lower than the report from the USA with the isolation rates of 77.9% (Dubey et al., 2008) and higher than the report from Brazil 19.5% (Ragozo et al., 2008) from MAT seropositive sheep. In goat, isolation rates of viable T. gondii (44.4%) in the current study are in parallel with the reported isolation rate of 45.45% in central Ethiopia (Gebremedhin et al., 2014) and 46.15% in Sao Paolo, Brazil (Ragozo et al., 2009). However, this disagrees with records of Berhanu (2015) 75% from Eastern Ethiopia, (Dubey et al., 2011) 26% from Brazil and (Opsteegh et al., 2011) 62.8% from the USA. The differences between these reports may be due to the density of T. gondii in tissues of sheep and goats, the type of tissue sampled and the strain or genotype of T. gondii.
Attempts to isolate viable T. gondii from cattle tissues have been extremely rare. Only a few successful tissue cyst recoveries have been reported (Scarpelli et al., 2009). This study in cattle on the isolation rate of viable T. gondii (21.43%) via bioassay in mice is comparable with the report of Opsteegh et al. (2016) who demonstrated 3.3% of viable T. gondii from selected European countries. However, the finding of the current study is higher than the reported isolation rate of 0% from Ethiopia in cattle (Berhanu, 2015) 0% from Egypt in cows (Elfadaly et al., 2017) through bioassay in mice and lower than the reported isolation rate of 100% (18/18) in experimentally infected cattle confirmed via bioassay in mice (Scarpelli et al., 2009). The differences between this work and aforesaid reports may be due to the density of T. gondii in tissues of cattle, the type of tissue sampled, the digestion method used and the strain or genotype of T. gondii (Dubey, 2010).
The overall prevalence of T. gondii antibodies in experimentally infected and surviving mice, from all animal species, was 16.4% (27/165). This finding is in agreement with the report of Gebremedhin et al. (2015) who reported 16.8% seroprevalence in experimentally infected mice with heart homogenates of the pig. However, it is lower than the report of Endrias et al. (2015) who reported 30.58% seroprevalence. The overall prevalence of T. gondii tissue cysts were detected in the brains of experimentally infected mice was 12.7%. This finding is lower than the reported cyst percentage of 17.6% by Gebremedhin et al., (2015) and the reported cyst percentage of 28.82% by Endrias et al., (2015). The mean cyst count per brain of mice in this study was 162.57 (mean ± SE = 162.57 ± 34.84). This finding is comparable with the mean tissue cyst count of 157.2 from central Ethiopia (Gebremedhin et al., 2015). However, it is lower than 277.97 mean cyst count per brain from central Ethiopia (Gebremedhin et al., 2014), tissue cyst counts ranging from 297 to 1380 by Brown et al. (1995) and the mean number of tissue cysts of 600 by Goodwin et al. (2008), but higher than the previous report by Tesfamariam (2013) in free-range chicken from Ada’a Liben, Central Ethiopia with the mean tissue cyst count of 57.4 per brain of mice. Toxoplasma gondii cyst burden in mice is only unassociated with inoculum dose and route, but also by the inoculated parasite strain (Waree et al., 2007) or by the genotype of mice (Brown et al., 1995; Waree et al., 2007). Besides, the number of live bradyzoites in the digested heart tissue of study animals inoculated into mice could contribute to the variation of the counted tissue cysts that are if inoculated bradyzoites are few; few of them reach the brain of mice, there will be fewer numbers of tissue cyst formed (Brown et al., 1995). Thus, microscopic counting of brain cysts in mice may indicate the infectious burden of T. gondii in ruminants (Bourguin et al., 1993).
4.3. Clinical signs and virulence of T. gondii isolates in mice
During the follow-up, most inoculated mice were asymptomatic. However, 29 mice (7 from cattle, 13 from sheep and 9 from goats) died before the 49th day. The observed clinical picture in mice in this study was similar to that observed by Gebremedhin et al. (2014b; 2015), and (Kyan et al., 2012), who observed that clinical conditions varied across seropositive mice and include arched back, ruffled, stiff, depression and forced breathing and most of these symptomatic surviving mice recovered to a normal condition.
The majority (87.2%) of the isolates recovered, based on the observation of mouse bioassay, were avirulent to white Swiss Albino mice. This virulence assessment is in agreement with previous reports in Ethiopia (Gebremedhin et al., 2014; 2015). However, one isolate (sp67) from sheep inoculum was highly virulent killing all inoculated mice (5/5) on the 3rd and 4th day of post-infection. According to Pena et al., (2008), 100% of death within four weeks of post-infection implies high virulence of T. gondii in mice. Besides, 4/5 and 2/5 of mice were killed due to isolates from goat tissue sample inoculum code (Gt6 and Gt 28, respectively), within 4 weeks pi, and 2/5 of mice from each sheep (sp26) and cattle (B65) sample inoculum died within four weeks postinfection. This may be an indication of intermediate virulence of T. gondii strains (Cook et al., 2000; Pena et al., 2008). Alternatively, T. gondii was also isolated from a homogenate of the intestine of the cow (Dubey 1992). According to Dubey et al., (2002; 2007b), T. gondii isolates differ markedly in their virulence to outbred mice. The avirulent strains were defined as having no mortality at any dose, whereas a "low-dose survivability" phenotype was defined by survival time after injection of 100 parasites (Kyan et al., 2012). It has been suggested the T. gondii virulence in mice depends on several factors, including the stage of the parasite, route, dose, types of mice used, host and strain of the parasite (Dardé, 2004; Endrias et al., 2015).
In conclusion, this finding indicates a high overall seroprevalence of T. gondii infection in domestic ruminants slaughtered for human consumption. More importantly, the current result showed a high isolation rate of viable T. gondii from seropositive domestic ruminants slaughtered for human consumption. However, most of the recovered viable isolates were avirulent. Only one isolate from sheep inoculum was found virulent and four intermediate virulent isolates; one from sheep, one from cattle inoculum and two from goat inoculum. Therefore, the findings of high seropositivity, detection of viable T. gondii tissue cysts with some of them a variable degree of virulence coupled with currently increasing trends of beef, mutton and goat meat consumption in the study area signifies the public importance of the disease, particularly in vulnerable groups. Butchers and slaughterhouse workers that handle carcases and organs infected with T. gondii are also at risk of getting an infection with toxoplasmosis. Based on the findings of the study, the education of the public about routes of T. gondii transmission and control methods is imperative to prevent T. gondii transmission to humans. Further advanced diagnostic techniques should be used to identify the genotype and population structure of T. gondii strains.