Toxoplasma gondii infection in aborted women and sheep in the governorates of El-Beheira and Alexandria, Egypt: A sero-immunological and molecular study

Introduction

The parasite zoonosis that is most common worldwide is Toxoplasma gondii (T. gondii) (1). Both humans and animals can contract toxoplasmosis from this obligatory intracellular worldwide apicomplexan protozoan parasite (2). All warm-blooded animals, including humans, and birds exist as intermediate hosts for the facultative heteroxenous lifecycle of the Toxoplasma parasite, whereas the definitive hosts are domesticated cats and other feline family members (3). Although T. gondii infections affect over one-third of all people on the planet, toxoplasmosis symptoms are not very prevalent (4). The disease can infect humans if they consume food or water polluted with cat feces that contain sporulated oocyts, or by eating raw or improperly heated meat, that has tissue cysts (bradyzoites) (5). Humans can also contract T. gondii by consuming unpasteurized milk that contains tachyzoites (6). In the majority of immunocompetent, apparently healthy adult individuals, T. gondii infections are not noticeable, however, they could result in clinical symptoms that last for months, including fever, lymphadenitis, influenza-like illness, and muscle aches (7). Conversely, if the mother and female animals’ contracts T. gondii while they are pregnant, the fetuses may have a severe form of toxoplasmosis (8). In such circumstances, the presence of T. gondii infection causes severe clinical manifestations, including retinochoroiditis, microcephaly, stillbirth, hydrocephalus, cerebral calcification, psychosis and may schizophrenic episodes (9) Furthermore, T. gondii infection may linked to neuropsychiatric diseases, encephalitis, meningitis, and neurobehavioral abnormalities in immune compromised individuals, including a cancer patients, organ transplant recipients, HCV and HIV infected individuals (10). One of the most common infectious diseases that inhibits reproduction is sheep toxoplasmosis, since placentitis, stillbirth and miscarriage may each result in large economic losses (11). The main way that sheep contract the disease is by consuming T. gondii oocyts while grazing in contaminated environments (12). Since sheep are more inclined than other farm animals to become infected and that, multiple studies have demonstrated the elevated T. gondii sero-prevalence in sheep from different parts of the world (13-15). Where people frequently eat sheep's mutton, such in Egypt, a public health alert should be issued, it has been noted that the incidence of T. gondii infection in Egyptian sheep fluctuates (16-18). The majority of cases of toxoplasma infection are asymptomatic, even at this point; there are several laboratory methods for diagnosing the infection. The most popular methods for identifying toxoplasmosis in sheep and humans are serological tests that recognize the anti-T. gondii specific immunoglobulin, IgM and IgG, in serum samples (19). The IgG avidity assay has been used to identify the anti-T. gondii specific antibodies in both pregnant and aborted women (20), Indirect Hem Agglutination Test (IHAT) (21) and Latex Agglutination Test (LAT) (16). Furthermore, antibodies against T. gondii were detected in sheep using modified agglutination test (22) and Enzyme Linked Immunosorbent Assay (23). Polymerase Chain Reaction (PCR) in blood samples from aborted women (24) and sheep (25) can molecularly identify T. gondii.

Consequently, it is imperative to collect essential data to have deep understand the effects of toxoplasmosis in Egypt. Thus, the objectives of the current investigation were to apply an ELISA assay followed by the Immunoblotting Technique to ascertain the sero-immunological detection of T. gondii among sheep and women who were aborted in Egypt's El-Beheira and Alexandria Governorates, as well as to molecularly establish.

Material and methods

Ethical approval

Under registration number 19-145, the experiment was conducted in compliance with the institutional norms of the Animal Research Committee of the National Research Center. NRC, Egypt, project number 1201034.

Study area and samples

Between February 2024 and September 2024, an epidemiological study conducted to determine whether toxoplasmosis occurred in apparently healthy sheep and women who reported spontaneous miscarriages. 98 blood samples were drawn from apparently healthy sheep belonging to different herds in the El-Beheira Governorate. In terms of humans, 125 blood samples were taken from women who had abortions and were admitted to the labor and delivery units of El Shatby Hospital for Obstetrics and Gynecology in Alexandria, Egypt. Serum samples separated, placed in 1.5 ml labeled Eppendorf tubes then stored at -20° C until appropriate serological examination and assessment completed.

Antigens preparation

The infectious stages of T. gondii were locally isolated from the pooled flesh sample (heart and diaphragm) obtained from slaughtered sheep in accordance with Shaapan and Ghazy method (26). Both locally isolated and virulent (RH) T. gondii strains kept in Department of Zoonotic Diseases, National Research Centre, by serial passage in mice according to the procedure of Hassan et al. (27). According to Hassan et al. (28), the antigens of T. gondii tachyzoites from the two isolates were prepared by repeatedly freezing and thawing the tachyzoites to break down the parasite wall, sonicating them, centrifuging them for 40 minutes at 4°C at 16,000 rpm, and collecting the supernatants. The protein content of the T. gondii tachyzoites antigens was determined using the Lowry et al. (29) technique.

Sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE)

The RH and regionally isolated strains of T. gondii crude antigen proteins were mixed separately with a reducing buffer containing 5% 2-mercaptoethanol, and they were subsequently electrophoresed on 10% SDS-PAGE using the Laemmli method (30). The relative molecular weights of the bands were ascertained by electrophoresing them on the same gel with pre-stained markers (GeneDirex BLUltra, USA).

Western blot (Immunoblotting)

In order to identify the specific immunogenic bands identified in the crude antigens of RH and isolated T. gondii tachyzoite strains, immunological detection of protein bands on nitrocellulose membranes were carried out using naturally infected human, and negative sheep and human sera, as described by Hassanain et al. (31). To identify immuno-reactive bands, 4-chloro-1-naphthol (Sigma-Aldrich) was utilized as a substrate. Utilizing Bio-Rad's Molecular Imager Gel DocTM XR lab software, the two-nitrocellulose membranes were assessed.

Enzyme Linked Immune-sorbent Assay (ELISA)

Using tachyzoite antigens of the local strain of T. gondii, indirect ELISA was performed to detect the presence of T. gondii IgG in human and sheep serum. The assay was carried out using the procedures outlined by El-Fadaly et al. (32). The optimal concentrations of the antigens, serum samples, and conjugates were determined using checkerboard titration (33). Optical density (OD) values are used to express the results. Three times the standard deviation plus the mean OD values of the negative control sera were used to estimate the cut-off (34). Sero-positive results were defined as OD greater than the cut-off value.

Molecular identification

In accordance with the instructions provided by the DNA gel extraction kit (NORGRN BIOTEK, Cat. No. 13100), the conventional polymerase chain reaction (cPCR), which was employed for the genetic characterization of the Toxoplasma, was utilized to extract genomic DNA from the obtained whole blood samples. At the same time, the tachyzoites of the Toxoplasma gondii RH strain were employed to harvest DNA for use as positive control. The eluted DNA's concentration and purity were assessed using spectrophotometry, and it was stored at -80ºC for subsequent processing. For PCR, two distinct sets of primers based on the B1 genes and the 529 bp repeating region were employed independently (Table 1).

Table 1: The different primer sets used for molecular detection of toxoplasmosis

Results

Electrophoretic profile of the two antigens

Virulent (RH) and locally isolated T. gondii antigens exhibited a complex separation pattern, and the two antigens' electrophoretic similarities were particularly strong in terms of 96, 74, 67, 58, and 41 kDa. However, only virulent (RH) antigens exhibited exclusive molecules, such as 30 kDa, 27 kDa, and 14 kDA (Figure 1, lane A), and 35 kDa, 25 kDa, 21 kDa, and 12 kDa, which were exclusively linked to locally isolated T. gondii antigens (Figure 1, lane B).

Figure 1: Electrophoretic profile showed comparative SDS-PAGE of virulent (RH) antigen (Lane A) and locally isolates of the T. gondii antigen (lane B) and molecular weight standards (Lane St)

Sero-Diagnosis of Human and Sheep Toxoplasmosis

The isolated locally T. gondii antigen demonstrated a higher infection rate of 67.3% (66/98) in sheep (Table 2 and Figure 2), which was higher than the 60.8% (76/125) seen in aborted women serum samples (Figure 3).

Table 2: Sero-Diagnosis of human and sheep toxoplasmosis by Indirect ELISA using locally isolated T. gondii sheep strain antigen

Figure 2: Sheep sera's IgG antibody levels against the locally isolated T. gondii antigen

Figure 3: human sera's IgG antibodies levels against the locally isolated T. gondii antigen

Immunogenic bands of virulent (RH) and locally isolated T. gondii antigens

Two bands with molecular weights of 67 kDa and 58 kDa were found using naturally infected sheep sera, while three immunogenic bands with molecular weights of 67, 58, and 27 KDa were found in virulent (RH) antigen by naturally infected human sera (Figure 4). In the locally isolated T. gondii antigen using naturally infected human and sheep sera three immunogenic bands of 67, 58 and 25 KDa (Figure 5). There are no any immunogenic reactive bands were detected in two antigens when using negative sera of human and sheep (Figures 4 and 5).

Figure 4: Immune-reactive bands identified in virulent (RH) antigen by immunoblot assay using naturally infected human sera (Lane 1), naturally infected sheep sera (Lane 2), negative human sera (Lane 3), negative sheep sera (Lane 4) and molecular weight standards. (Lane St.).

Figure 5: Immune-reactive bands identified in locally isolates of T. gondii antigen by immunoblot assay using naturally infected human sera (Lane 1), naturally infected sheep sera (Lane 2), negative human sera (Lane 3), negative sheep sera (Lane 4) and molecular weight standards (Lane St.).

Molecular identification

Conventional polymerase chain reaction (cPCR) analysis proved the detection of Toxoplasma gondii using B1 gene primers at the expected size is 194 bp (Figure 6). Wherease using Tox-9 and Tox-11 primers, Toxoplasma gondii identified at the expected size of 161 bp (Figure 7).

Figure 6:

Detection of Toxoplasma genome a using B1 gene primers. Lane 1, 100 bp DNA ladder; lanes 2 & 3, positive human and lanes 4 & 5, positive sheep (194 bp); lane 6, positive control and lanes 7, negative specimen.

Figure 7: Detection of Toxoplasma genome a using Tox-9 and Tox-11 primers. Lane 1, 100 bp DNA ladder; lanes 2 and 3; positive human and lanes 4, positive sheep (161 bp); lane 5, positive control and lanes 6, negative specimen.

Discussion

In the current study, the electrophoretic profile of the two RH and local T. gondii antigens exhibited extensive similarity and when exposed to western blot analysis probed with T. gondii positive sera from human and sheep they identified two common immunogenic bands of 67 and 58 kDa. Numerous investigations have been conducted in recent years to identify the proteins of T. gondii tachyzoites, particularly immunoreactive proteins (35). These research efforts have concentrated on choosing immunoreactive and protective antigens that might be employed in accurate diagnosis and vaccine preparation of toxoplasmosis (17). T. gondii matrix antigens of 67 and 58kDa protein, it is widely expressed in both RH and local T. gondii tachyzoites antigens, were assessed as potential candidates for the development of a diagnostic reagent for ovine toxoplasmosis (36). Human toxoplasmosis was successfully diagnosed using a 65 kDa fraction that was extracted from horse-derived T. gondii tachyzoites (28). Additionally, a 65-kDa fraction of the local camel T. gondii strain tachyzoites that was included in the affinity-isolated fraction was effectively used to diagnose toxoplasmosis in cattle (37). The antisera used in each study may be the reason for the discrepancy between the immunogenic bands of the currently evaluated antigens used and those found in previous studies (38-40).

The quality of the antigen used, the type diagnostic potentials, and the accessibility and affordability of antigen preparation are the primary determinants of the serological diagnosis's accuracy and economy (23,41). The current study uses an easily prepared and relatively low-coasted local isolated T. gondii antigen to detect the sero-prevalence rate of toxoplasmosis, which was 60.8% (76/125) in women who had abortions and 67.3% (66/98) in sheep from El-Beheira and Alexandria Governorates, Egypt. Toxoplasmosis is not the only prevalent cause of women's habitual abortions, even if women infected with T. gondii can have abortions (8). Toxoplasmosis has been detected in many Egyptian sheep and women who had aborted fetuses; the seroprevalence is almost higher in sheep 61.76% and pregnant women 63.79% from the Menoufia Governorate in the Delta (42). In contrast, El-Minya Province pregnant women 22.9% and sheep 39.1% had lower seroprevalence (16). Previous studies found that 46% of sheep and 60.7% of aborted women in the Cairo governorate tested positive for Toxo IgG by ELISA (22,43). Rather than a lower frequency in the urbanized Cairo governorate compared to the rural governorates, the previously observed disparities in seroprevalence may be due to variations in ecological factors, the serological assays utilized, sample size, sheep breed, place, and time of sampling (44).

There have been several published studies on toxoplasmosis in pregnant Egyptian women, however they frequently have tiny sample sizes and do not include information on the populations the researchers were trying to study. Because of the sample size, diagnostic technique employed, and living conditions of the women evaluated, there are few statistics on seroconversion before and during pregnancy, and results cannot be compared across publications (45). The Lower (North) Egyptian governorates and Cairo had higher rates of T. gondii infection than the Upper (South) Governorates; these disparities suggest that socioeconomic and geographic factors may influence T. gondii exposure in Egypt (46). Multivariate regression study shows that eating raw or undercooked food, especially sheep meat, interacting with cats, and working in agriculture are all-important risk factors for T. gondii seropositivity (5,47).

PCR analysis in this study proved that detection of Toxoplasma gondii using B1 gene primers and Tox-9 and Tox-11 gene primers at the expected size 194 bp and 161 bp, respectively. The PCR technique has shown promise in a number of applications, such as allelic discrimination, gene expression, and pathogen detection (48). Numerous PCR-based methods have been created to diagnose toxoplasmosis utilizing a range of clinical specimens, such as tissue biopsies, blood, amniotic fluid, and cerebrospinal fluid (49,50). During the active phase of T. gondii infection, the pregnant mother may test negative, hence preventing detection of the high risk of neonatal toxoplasmosis (20). DNA from T. gondii was found in placentas or other unidentified products of conception in a few of the reports. Once more, strict controls are necessary for PCR accuracy in order to reduce contamination. It is important to highlight that T. gondii DNA in the placenta does not necessarily indicate a prenatal infection (51-53).

Conclusion

The present study suggests that women who experienced several miscarriages and lambs infected with T. gondii had higher levels of IgG. This study highlights the importance of T. gondii blood tests, health education programs, and preventative measures such appropriate meat handling, eating, and cleaning practices in lowering the prevalence of these disorders. Additionally, it recommended that additional comprehensive research be done to validate the results and offer recommendations for public health programs.

Acknowledgments

The authors would like to express their appreciation to their colleagues in the National Research Center's Department of Zoonotic Diseases for helping them finish this work.

Conflict of interest

The authors have disclosed no conflicts of interest.