Development of Improved Preventive and Treatment Strategies for Controlling of Toxoplasmosis in Saudi Arabia

Department of Medical Biotechnology, Faculty of Applied Medical Sciences, Taif University, Turrabah, KSA, Saudi Arabia Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig,, Egypt Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, KSA, Saudi Arabia Department of Theriogenology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt


Introduction
Toxoplasmosis is a global zoonotic disease caused by the apicomplexan coccidian Toxoplasma gondii, an obligatory intracellular protozoan that can infect humans and all warm-blooded animals. This wide-ranging host makes it one of the most successful protozoan parasites (Radostits et al., 2007). In human, it is a dangerous disease in immunocompromised and immunosuppressed patients and congenitally infected children (Tenter et al., 2000;Montoya and Liesenfeld, 2004). Primary infection in pregnant seronegative women can cause neonatal death, abortion and severe congenital abnormalities, such as hydrocephalus, blindness, chorioretinitis and mental retardation (Petersen et al., 2001). On the other side, toxoplasmosis in animals has great economic importance worldwide because it can cause stillbirth, abortions and neonatal loss especially in sheep and goats (Buxton, 1998;Buxton et al., 2007). Moreover, viable T. gondii tissue cysts inside raw or undercooked meat of sheep and goats may be important sources of infection to humans and carnivores (Dubey, 2009).
Although several studies of human toxoplasmosis were carried out in Kingdom of Saudi Arabia (Al-Harthi et al., 2006;Tonkal, 2008;Al-Mohammad et al., 2010;Bin Dajem and Almushait, 2012), a little data is available about the T. gondii prevalence in small ruminants under field condition in that country (Sanad and Al-Ghabban, 2007;Al-Mufarrej et al., 2011). Serological diagnosis considers the most commonly approach used to define the stage of infection, whether current, recent, or past (Nagaty et al., 2009).
Despite the seriousness of this disease, there are no effective commercial vaccines to combat it today. Indeed, a live attenuated Toxoplasma tachyzoites S48 vaccine is previously available and it can partially (60%) protect pregnant sheep against toxoplasma infection (Buxton et al., 1991), but now it is difficult to manufacture because it has a short shelf life. Interestingly, we succeeded in development of a potent, successful and effective vaccine against toxoplasmosis that provides highly significant protection against congenital and acquired T. gondii infection in female OF1 mice (more than 96% protection) (Ismael et al., 2006) and in sheep (90% protection) (Mévélec et al., 2010). This vaccine is currently in the approval period and this work was realized in France. The absence of effective commercial vaccine invites the need to develop an improved preventative and treatment strategies for controlling toxoplasmosis.
Chemotherapy as antibiotics and/or sulphonamides are commonly used for treatment of toxoplasmosis in farm animals and human. In addition to antibiotics residues, the antibiotic therapy has numerous complications as direct toxicity, hypersensitivity, superinfections and antibiotic-induced immunosuppression (Adesiyun et al., 2007). This is highlighting the need for a new strategy for non-antibiotic therapy using novel immunomodulators. We succeed in purification and characterization of camel lactoferrin (cLf) from camel milk whey (Ismael et al., 2013). We proved that cLf is a potent natural antimicrobial and novel immunomodulator agent (Ismael et al., 2013). The present study aimed at, (i) determination of the occurrence of toxoplasmosis in small ruminants in studied farm (ii) make confirmatory diagnosis of toxoplasmosis in abortive small ruminants and (iii) comparison of different and new drug regimens and strategies for controlling toxoplasmosis.

History of Animals and Location
This study was performed on sheep and goats farm containing 357 animals (300 sheep and 57 goats) and located in Riyadh region, KSA. This farm had a large numbers of barren ewes, history of abortions (below 4 months), stillbirths, malformations and neonatal losses. The animals had been reared under similar management, feeding systems and environmental conditions.

Clinical Examination
Animals were subjected to general clinical examination including pulse, temperature, respiration, mucous membrane color and others. All managements of the animals and the sample collections were in agreement with the rules for the animal's welfare and those of the concerned ethical authorities.

Samples Blood Samples
The selected animals were subjected to blood collection via Jugular vein into plain vacutainer tubes. The blood samples were centrifuged at 3000 rpm/15 min for separation the serum that stored at -20°C into 1.5 mL Eppendorf tubes until assay.

Tissue Samples
Sections of normal and abnormal placenta, uterus, liver, fetus, lung and trachea tissues were collected just after abortion or death and fixed in 10% formalin buffer for histopathological examination.

Diagnosis of Toxoplasmosis Ultrasonography
The animals were subjected to full ultrasonography reproductive examination using multi-frequency linear trans-rectal probe (Prosound 2, Model UST-660-7.5, Aloka, Japan).

Measurement of Humeral Antibody Responses to Natural
Infection by Enzyme-Linked Immunosorbent Assay (ELISA) The antigen-specific IgG antibodies levels in serum samples were identified pre and post-treatment as previously described (Ismael et al., 2003). The TAg of T. gondii at 10 µg mL −1 was used to coat micro titer plates. The antigen-specific antibody titer is given as the reciprocal of the highest dilution producing an absorbance (OD) that was 2.5-fold greater than that of control serum at the same dilution. Results were expressed as the means of titers ± Standard Deviations (SD).

Histopathological Examinations
A gross necropsy was performed on the placenta and aborted fetus. The fixed tissues were embedded in paraffin then sectioned by microtome. The resulted sections were stained with Hematoxylin-Eosin (HE) stain.

Field Treatment and Synergy Trials
The affected sheep and goats were divided into 7 groups based on the clinical manifestations, 4 groups of 10 sheep and 3 groups of 8 goats (Table 3 and 4). Group I received no treatment and used as a control group. Group II was intramuscularly injected with Pyrimethamine (100 mg/kg/3 days, then 50 mg) + sulfamethazine or sulfadiazine (73 mg/kg for 3 days then 100 mg) (Radostits et al., 2007). Group III was treated with cLf (7%, 10 mL/50 kg), per Os, for 3-5 days of three periods with an interval of 5 days. Group IV received Pyrimethamine, sulfadiazine and cLf combinations regimen. The infected goats received the same therapeutic regimen of groups I, III and IV in sheep. All treated sheep and goats were examined daily for monitoring any clinical improvement. Sera samples were collected several times pre-, during and posttreatment for serological examination. The total treatment period was 25 days. The predisposing factors were monitored and controlled.

Statistical Analysis
The Statistical Products and Service Solutions program was used for all analysis. Comparisons among groups were tested using the using the χ2 (Chi-square) test. A difference was considered to be a statistically significant at p<0.05.

Clinical Findings and Ultrasonography
Clinical summary of the animals before and after treatment was illustrated in Table 1. Clinical reproductive examination of barren ewes revealed normal genital tract and no any abnormalities or pathology in reproductive tract (Fig. 1). Additionally, 15% of animals diagnosed as pregnant at day 23 post insemination ( Fig. 2) was diagnosed non-pregnant (aborted) at day 60 post insemination. Sixty-four out of 357 sheep and goats were clinically affected (Table 1) with 17.9% overall morbidity rate. These animals showed certain clinical symptoms of toxoplasmosis, such as abortions in different stages especially in midgestation (n = 35) (Fig. 3), stillbirth (n = 9) (Fig. 4), malformation (n = 2) (Fig. 5) and neonatal loss. In addition, fever and other systemic signs were observed on 7 out 64 clinically affected animals (10.9%). Two cases (goats) out of 64 were died with 3.13% case fatality rate. The post-mortem findings in dead animals revealed septicemia (Fig. 6). A gross necropsy was performed on the aborted fetus and placenta. The abdominal cavity was usually filled with fluid tinged with blood. The placenta was purple-gray in color and the cotyledons were pale tan and friable, with a gritty texture as though mineralized.

Results of ELISA in Naturally Infected Animals by Toxoplasmosis Pre-and Post-Treatment
Analysis of serum antibody responses in naturally infected sheep and goats elicits serum IgG responses ( Table 2). The IgG responses varied from strong positives, weak positive, suspected cases to negatives cases ( Table  2). The seroprevalence rate was 15.4%. The titers of ELISA were high before treatment in positive cases while after treatment the titers were decreased for certain level (weak) that continued to the end of study (Table 3 and 4).

Histopathological Results
Large numbers of small basophilic to amphophilic bradyzoites were detected in tissue cyst in the heart without prominent inflammatory reaction ( Fig. 7 and 8). Microscopic examination of aborted fetus tissues and placenta indicated sign of necrotic placentitis. Diffuse edema with a population of mononuclear and segmented leukocytes was detected throughout the amnion of the placenta. Necrosis along the chorion and basophilic granular debris, supposed to be mineralization. Histopathological examination of the cotyledon revealed multifocal, mineralized and necrotic lesions.  Table 1.

Field Treatment
It revealed that the affected sheep and goats treated with cLf and chemotherapy combination regimen significantly reduced the overall severity of toxoplasmosis with reduction in IgG titers better than those treated with chemotherapy alone (Pyrimethamine and sulfamethazine) or cLf alone (Table 3 and 4). The cLf alone was more effective in goats than in sheep (Table 3 and 4).

Discussion
This is the first study reporting the role of camel lactoferrin (cLf) in treatment of ovine and caprine toxoplasmosis in veterinary field. In the present study, the clinical picture of toxoplasmosis was appeared in 64 animals with 17.9% overall morbidity rate and 3.13% case fatality. Approximately similar results were described by Radostits et al. (2007). Abortions, stillbirth, malformation, neonatal loss and fever were observed in infected animals that indicated the appearance of acute phase of infection. Moreover, the systemic disease, with a slightly high case fatality rate occur in young goats (Radostits et al., 2007). Severity of infection is associated with the stage of gestation at which the animals become infected, the earlier in gestation, the more severity of infection (Dubey, 2009). Fetal death and resorption can be resulted from early infection while infection in the latter part of gestation may have no clinical effect with the lambs being born normal but infected and immune (Radostits et al., 2007).
The toxoplasmic infection was confirmed by macroscopic and microscopic pathological lesions. Although DNA detection and serology are useful tools, histopathology is important to establish a cause-effect association, because T. gondii can be passively transmitted transplacentally and fetuses can die of other causes (Dubey, 2009).
The seroprevalence rate was low (15.4%) in our study, as detected by ELISA, that might be attributed to the presence of toxoplasmosis in this region from long time. The furthermost seroprevalence in sheep were found in Brazil (29.41%) (Clementino et al., 2007) and southern Italy (49.9%) (Vesco et al., 2007). The higher seroprevalence in southern Italy might be attributed to farm size and the presence of cats on the farm (Vesco et al., 2007). Various ELISA methods using recombinant, fractionated or crude antigens have been used to detect T. gondii antibodies in ovine sera that gave higher specificity in comparison with other serological tests (Dubey, 2009).
The field treatment revealed that the affected sheep and goats treated with cLf and chemotherapy combination regimen significantly reduced the overall severity of toxoplasmosis better than those treated with chemotherapy alone (Pyrimethamine and sulfamethazine or cLf alone. Moreover, the cLf alone may offer a good alternative treatment. The selection of treated animals was based on clinical findings because the animals in early acute infection showed clinical signs without appearance of antibody titers in serum. We succeed in purification and characterization of camel lactoferrin (cLf) from camel milk whey (Ismael et al. 2013). We proved that cLf is a novel immunomodulator and potent natural antimicrobial agent (Ismael et al. 2013). The role of Lf in sequestering iron from bacterial pathogens is one of the first antimicrobial properties discovered for Lf as in case of S. aureus which is known to be resistant to antimicrobials (Bhimani et al., 1999). This was believed to be the important antimicrobial action of Lf because apolactoferrin possessed antibacterial activity (Yamauchi et al., 1993). Spatial orientation and structural characteristics of the molecule are life-threatening factors in the functionality of an antimicrobial compound. Occurrence in several conditions strongly highlights the significance of the structure-function relationship in the multifunctionality of the Lf (Farnaud and Evans, 2005). It was later established that Lf's antimicrobial function has been attributed to an iron-independent mechanism and direct interaction with microbial surfaces (Farnaud and Evans, 2005;Valenti and Antonini, 2005). The cLf increased lymphocyte transformations and capacity to stimulate the proliferation of allogeneic lymphocyte. Moreover, it increased the production of proinflammatory cytokines (Zimecki et al., 2001;Ismael et al., 2013).

Conclusion
The results of current study record that cLf has a potent natural anti-T. gondii effect. This is the first study reporting the role cLf in treatment of ovine and caprine toxoplasmosis in veterinary field. This study recommended using the cLf in the treatment of various infectious diseases in humans and animals. Nevertheless, a lot of effort is required to obtain a better understanding of cLf activities.