Toxoplasma gondii infection in slaughtered domestic ruminants in Northwest Ethiopia: seropositivity, bioassay and virulence assessment

This study investigated the seropositivity, isolation and virulence of Toxoplasma gondii in slaughtered domestic ruminants in Gondar city, Northwest Ethiopia. Three hundred thirty-ve blood samples (135 sheep, 50 goats and 150 cattle) were collected from slaughterhouses. Antibodies against T. gondii were assayed using a commercial Toxo-Latex agglutination test. Tissue digestion was also conducted on 39 heart muscles of seropositive animals using the pepsin enzyme. The isolation of viable T. gondii from seropositive ruminants was also performed in white albino mice. The overall seroprevalence of T. gondii infection was found to be 55.8%. The species-wise prevalence of T. gondii seropositivity in cattle, goats and sheep was 59.3%, 58%, and 51.1%, respectively. From observed risk factors, sex (p < 0.033) and age of the sheep (p < 0.006) showed a signicant association with T. gondii seropositivity. Similarly, in cattle, age (p < 0.005) and breed (p < 0.012) showed a statistically signicant association with seropositivity of anti-T. gondii antibodies. In bioassayed mice, the overall viable T. gondii isolates were 38.5% and most of these isolates (87.18%) were avirulent. In conclusion, the high prevalence of T. gondii antibody and a high proportion of viable T. gondii observed in this study indicated the prevalent nature of the parasite and its zoonotic importance in the study areas where slaughtered domestic ruminants serve as an important human protein source. Education of the public about routes of T. gondii transmission and control methods is imperative to prevent T. gondii transmission.


Introduction
Toxoplasma gondii is an obligate intracellular parasite, which can infect all warm-blooded vertebrates including humans, mammals and birds (Schlüter et al., 2014). It infects up to 30% of the human population in the globe (Dubey, 2010). Humans can acquire the infections by ingesting sporulated oocystcontaminated food, vegetables and water; by consuming raw or undercooked meat containing viable tissue cysts of this parasite from infected food animals, and congenitally from an infected mother to the foetus ( Ruminants are considered important in the epidemiology of T. gondii infection worldwide (Tenter, 2009;Dubey, 2010). The ingestion of infected meat from food animals serves as a direct source of infection for humans and felines. In most areas of the world, Toxoplasma infection is prevalent in meat-producing animals (Tenter, 2009 Esubalew et al., 2020). However, little is known about the viability and virulence of T. gondii isolates detected in the meat of such seropositive animals. Therefore, this study was conducted: 1) to estimate the prevalence of T. gondii antibodies in sheep, goat and cattle destined for slaughter in Gondar city, Northwest Ethiopia; 2) to assess the viability and virulence of T. gondii isolate using mice bioassay.

Study Design and Sampling Technique
A cross-sectional study design was employed to collect blood and tissue samples from slaughtered domestic ruminants at Gondar ELFORA abattoir and four local slaughterhouses in Gondar city. A laboratory-based experimental follow up in bioassayed mice was also performed from November 2018 to June 2019. A total of 335 animals (135 sheep, 50 goats and 150 cattle) were sampled for the study. The age of the animals was determined by observing the erupted permanent incisors (Taylor, 1984;Awgichew & Abegaz, 2008). The approximate age of the animals was categorised and recorded as young, adult and old. Sheep and goats ≤ 1 year were considered as young, while those over one year were considered an adult. In the case of cattle, those with ≥ 7 years were considered as old, while those having four to seven years old were considered adults.

Sample collection and transportation
A total of 335 blood samples, each about 5-10 ml whole blood, were collected using plain sterile tubes during exsanguination or intracardially at the slaughter line. Out of the total 335 blood samples, 200 had matched heart tissue samples (75 from cattle, 75 from sheep and 50 from goats) for microscopic examination and bioassay in mice. Each collected tissue sample weighed about 50-60 gram. The samples were labelled and transported with a cold box to the Veterinary Parasitology laboratory, College of Veterinary Medicine and Animal Sciences, University of Gondar.

Serological assay
The blood samples were allowed to clot in a slant position for a few minutes and centrifuged at 4000 rpm for 5 min to separate the sera. Subsequently, sera were decanted into 1.

Tissue digestion and Bioassay in mice
Tissue samples from seropositive animals were digested as described by Dubey (1998). Brie y, tissue samples weighing 50 grams were minced and digested in a pepsin acid solution (pH 1.1-1.2) at 37°C for 1hr. After ltration, neutralisation was executed one time with 1.2% sodium bicarbonate solution (pH = 8.3) and then centrifugation was performed. Thus, the sediment was diluted in 5-10 ml of antibiotic saline solution (1000 IU/ml penicillin and 100µg streptomycin/ml in saline solution). Tissue digest was examined microscopically at 10x and 40x magni cation power. Accordingly, liberated bradyzoites and/or tissue cysts of T. gondii were examined under a microscope from the tissue digests. Then, 39 microscopically positive digested tissue samples were inoculated subcutaneously into mice to assess the viability and virulence of the detected cysts. 5-6 weeks of aged female white albino mice weighing 20-25-gram were used for the experiment. Each microscopically positive digested tissue sample was inoculated into 5 mice (1 ml suspension per mouse). Approximately 5ml aliquots from the suspension were leftover and stored at + 4°C until it was inoculated in the same mice next day (Beltrame et al., 2012).
After inoculation, mice were followed for 49 days for the occurrence of clinical signs and death. During the follow-up, mice were offered pelleted feed and drinking water ad libitum. All survivors were then euthanised on the 49th -day post-inoculation through cervical dislocation after anaesthetising with diethyl ether and blood was collected through cardiac puncture. The blood was allowed to clot for about 3 to 4 hours and then the top part was pipetted and centrifuged at 4000 RPM for 4 minutes, as the serum was not completely clear. The serum was harvested using a disposable pipette into an Eppendorf tube and tested on the same day by LAT. Thus, the sera were examined for T. gondii antibodies using the LAT.
Further, the brain of the mouse was homogenised in 1ml phosphate-buffered saline (pH = 7.2) using a mortar and pestle to detect the cysts microscopically. The number of cysts in the brain of each mouse was determined by converting the sum of cysts in 30 µl to the whole of the brain homogenates (Goodwin et al., 2008;Fritz et al., 2012). The bioassay was considered as positive if at least one T. gondii cyst is detected in any inoculated mouse or if at least one of the mice sera reacts positively by the LAT (Dubey et al., 1995). The virulence of the parasite was classi ed according to Pena et al. (2008) based on mice mortality rate within four weeks of infection (without prior information on infecting dose). Then, we categorized isolates into three groups: virulent (if there were 100% death of mice within four weeks), intermediate virulent (30% to less than 100 % death within four weeks), and non-virulent (< 30 % death within four weeks).

Data Analysis
The data obtained were stored in a Microsoft Excel spreadsheet (2010) and analysed using SPSS version 20 (SPSS Inc., Chicago, IL, USA). The data were summarized using descriptive statistics. The association of seropositivity for T. gondii and, age (young, adult and old), sex (male and female), breed (local and cross) and species (cattle, sheep and goat) was tested using the chi-square test. The 95% con dence level was used and statistical analyses were considered signi cant at P < 0.05.

Bioassay in mice
Thirty-nine positive tissue samples containing T. gondii tissue cysts and/or liberated bradyzoites ( Fig. 1) (14 from cattle, 16 from sheep and 9 from goats) were bioassayed in 194 mice. The total viable T. gondii isolated from the three species of animals was 38.5% (15/39) (Table 4). At the species level, viable T. gondii was isolated from 50% (8/16) of sheep, 44.4% (4/9) of goats and 21.43% (3/14) of cattle. However, a sample can be both cyst positive and seropositive, and cyst positive and seronegative or vice versa. The summary of viability data as indicated by serological and cyst positivity in inoculated mice is presented in Table 2 and detailed data for each tested isolate is provided in Table 3.   Table 3: Detailed viability data for each bioassayed isolate as indicated by serological and cyst positivity in the inoculated mice.
The overall seropositivity of T. gondii in experimentally infected and surviving mice from all animal species was found to be 16.4% (27/165). The seropositivity of T. gondii infection in experimentally infected mice using inoculum from sheep, goat and cattle was 18.2% (12/66), 16.7% (6/36) and 14.3% (9/63), respectively. A total of 21 experimentally infected mice were found to harbour T. gondii tissue cysts. Some of the tissue cysts detected in inoculated mice are presented in Fig. 2. The overall mean cyst detected and enumerated from experimentally infected mice was 162.57 ± 34.840 (mean ± SE=) cysts per brain of mice. Overall, higher mean cyst counts in the brains of mice inoculated with heart homogenates from goats were quanti ed compared to those enumerated in the bioassay performed on the sheep and cattle samples (Table 3). Table 4: Mean cyst counts in the brains of mice inoculated with heart homogenates of slaughtered animals.

Clinical signs 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 days. More speci cally, 9 mice (5 from sheep and 4 from goat's inoculum) died on 3rd and 4th days after inoculation while 20 mice (8 from sheep, 7 from cattle and 5 from goats' inoculum) died after the 4th day's postinoculation (pi). The clinical symptoms observed in symptomatic mice were arched back, leg paralysis, tachypnoea, inappetence, rough hair coat and dullness.

Virulence of T. gondii isolates
Two isolates from the sheep with identi cation sp15 killed one mouse on day 8-pi ( Table 2). Most of the recovered isolates (87.18%) in this study were nonvirulent to the mice. One highly virulent isolate (sp67) and four intermediate virulent isolates, namely, sp26, B65, Gt6, and Gt28 were suggested (

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%. However, this study indicates a higher percentage value than the report of (Elfadaly et al., 2017) who reported 8.57% (12/140) (Berhanu, 2015), 0% from Egypt in cow (Elfadaly et al., 2017) through bioassay in mice and lower than the reported isolation rate of 100% (18/18) in experimentally infected cattle con rmed via bioassay in mice (Deksne & Kirjušina, 2013). The differences between this report 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 nding is in agreement with the report of Gebremedhin et al.  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).

Clinical signs 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 days. 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 cowlick, depression and forced breathing and most of these symptomatic surviving mice recovered to a normal condition.

Virulence of T. gondii isolates
The majority (87.2%) of the isolates recovered were avirulent to white Swiss Albino mice. This virulence assessment is in agreement with previous reports in Ethiopia 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 pi implies high virulence of T. gondii in mice. Besides, 80 (4/5) and 40% (2/5) of mice were killed due to isolates from sample code (Gt 6) and (Gt 28), respectively from goat tissue inoculum within 4 weeks pi, and 40% (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 de ned as no mortality at any dose, whereas a "low-dose survivability" phenotype was de ned 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). 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 cowlick, depression and forced breathing and most of these symptomatic surviving mice recovered to a normal condition.
In conclusion, this nding 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 ndings 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 signi es the public importance of the disease, particularly in vulnerable groups. Butchers and slaughterhouse workers that handle carcase and organs infected with T. gondii are also at risk of getting an infection with toxoplasmosis. Based on the ndings 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.

Acknowledgements
We would like to acknowledge the O ce of Vice President for Research and Community Service, the University of Gondar for its nancial support.
Competing interests: The authors declare no competing interests.