Trichomonas vaginalis is the protozoan flagellate responsible for most common worldwide sexually transmitted diseases (STDs) called trichomoniasis [1] . One-third of T. vaginalis-infected women are asymptomatic [2] , while some clinical manifestations as cervicitis, vulvo-vaginitis and urethritis with complications as infertility [3] and Cancer cervix happen [4] .

Multifactorial mechanisms explain this phenomenon. Many authors suggested the presence of two different strains with different virulence factors [5] - [7] . Others relate this phenomenon to host immune response [8] . Many other factors play important role in parasite virulence e.g. cytoadherence [9] and higher levels of D-lac- tosyl residues on the surface of pathogenic strains [5] . However, virulence factors responsible of wide variation of symptomatology have not been well-defined.

Leukotrienes (LTs) are lipid mediator that are generated via the 5-lipoxygenase (5-LO) pathway in the arachidonic acid metabolism. Leukotriene B₄ (LTB₄) is synthesized from combined action of 5-LO and LTA₄ hydrolase on LTA₄ [10] . Microorganisms interact with phagocytes in vitro and experimental infections in vivo leading to LTs production [11] . LTs play essential roles in both innate and adaptive immune responses [10] [11] . LTB₄ are involved in the defense against protozoan and helminthic infections through modulation both Th1 and Th2 immune responses [12] . Antimicrobial activity [13] antiparasitic activity [14] and antifungal activity of LTB₄ occur through nitric oxide (NO) and cytokine production during infection [15] . Any defect in LTB₄ biosynthesis pathways will decrease the phagocytic and antimicrobial activities against microorganisms including parasites [14] [16] bacteria [17] and fungi [15] .

Many researchers studied the immune responses to T. vaginalis in both murine models and human hosts. Production of Th-1 cytokine such as IL-2 and IFN-γ, IL-4 [18] and IgM, IgA, and IgG subclass [19] responses has also been demonstrated. Others suggested that T. vaginalis-derived secretory lipid mediator LTB₄ induces IL-8 production in mast cells [20] . However, the role of LTB₄ in host defense during T. vaginalis infection has been given less attention. How LTB₄ contributes to host resistance in experimental trichomoniasis is not also well understood and very little information is available in this point. In experimentally infected rats, data showed that infections did not differentiate between symptomatic and asymptomatic human isolates regarding histopathological and immunohistochemical changes. They revealed that interaction between parasite and vaginal epithelium was species specific [21] . However, data about this point is scarce. This issue isn’t clarified using experimentally infected mice.

The present study aims to assess serum and local LTB₄ level and pathological changes in the vagina of mice infected with T. vaginalis isolates from symptomatic (SYM) and asymptomatic (ASYM) women.

The histopathological picture of vagina from mice of uninfected control mice showed normal appearance of posterior vaginal fornix and upper part of the vagina (Figure 1(a)). Different pathological changes were detected among mice infected by T. vaginalis isolates from SYM women. Moderate and severe pathological changes were in mice (30%) and 6 mice (60%) respectively. On the other hand, only one mouse (10%) showed mild pathological changes. No severe pathological changes in vagina of mice inoculated by ASYM isolates. Eight mice (80%) infected by T. vaginalis isolates from ASYM women showed no pathological changes, while mild and moderate pathological changes were observed in only one mouse per each. The differences were statistically insignificant (Table 1).

Mice vagina of group inoculated by SYM T. vaginalis isolates showed destruction of keratin layer and invaded the superficial layer of epithelium with desquamation and sloughing of epithelium (Figure 1(b)), dilated, ruptured vessels with interstitial hemorrhage in subepithelial lamina propria (Figure 1(h)). Mice vagina of group inoculated by SYM T. vaginalis isolates showed also marked increase in number of leukocytic infiltrates and cytoplasmic vacuoles with hydropic degeneration extend all over the fall thickness of stratified squamous epithelium in severe cases (Figures 1(e)-(g)). T. vaginalis parasite could be demonstrated as a round or flattened with a nucleus surrounded by a hallo (Figure 1(b), Figure 1(d)).

Significant increase in mean of LTB₄ levels was observed on the 3^rd to 28^th PI in serum, VWs and vaginal tissue of mice infected with SYM (GIb) in comparison to mice infected with ASYM (GIIb) human isolates (P < 0.001) (Figure 2) and control. Mean LTB₄ concentration in mice infected by SYM and ASYM human isolates increased on the 3^rd PI, reached a peak on the 14^th PI followed by a decrease on the 21^st and 28^th PI. Vaginal tissue showed higher level of LTB₄ as compared with its level in serum and vaginal washes. Mean LTB₄ concen-

GIa: Infected mice with symptomatic isolates (SYM). GIIa: Infected mice with asymptomatic isolates (ASYM). ^*Not significant.

tration in mice (control group) was 13.3 ± 0.5 pg/ml and 13.6 ± 0.6 pg/ml and 14 ± 1 pg/ml in serum, VWs and vaginal tissue respectively.

The pathogenesis of trichomoniasis is not clearly understood. Multiple virulence factors are involved in occurrence of symptoms among women. The parasite factors which may be involved in symptomatic infections of trichomoniasis are not clarified. The interaction between the host and T. vaginalis is complex. The existing literature shows little data on the host and parasite factors responsible for symptomatic or asymptomatic infection in women. The existence of two types of T. vaginalis strains, each differing in its morphological features and intrinsic virulence factors has been reported [5] [6] . Previous studies classify T. vaginalis isolates according to isoenzyme patterns [27] , restriction fragment length polymorphism analysis [28] and random amplified polymorphic DNA (RAPD) analysis [29] . However, they show contrasting data.

The murine models had been proven to be valuable for investigating the pathogenesis and treatment of trichomoniasis [30] and can be applied for characterization of this variability [31] .

The present study showed no significant difference in the pathological changes found in the vagina of mice infected with T. vaginalis isolates from symptomatic women as compared with asymptomatic women. Dissimilar result observed by Malla et al. [18] who showed that the mean parasite load significantly increase in mice inoculated with T. vaginalis isolates from symptomatic women versus those inoculated with parasites from asymptomatic women. The presence of two types of T. vaginalis strains with different intrinsic virulence factors has been suggested [32] . High pathogenic index between symptomatic isolates has been observed [33] . Others proved that cytoadherence to vaginal epithelium was higher in isolates from SYM women than ASYM one [34] . T. vaginalis isolated from symptomatic women was able to stimulate strong chemotactic response towards polymorphonuclear leukocytes (PMN) as compared to those isolated from asymptomatic one [35] . In experimental study, a higher inflammatory response also observed in mice infected with symptomatic isolates as compared to those infected with asymptomatic isolates [36] . The presence of chronic non-specific inflammation with sub epithelial infiltration by lymphocytes and neutrophils has been observed. Furthermore, with severe inflammation, deeper layers of the epithelium showed neutrophils [37] . Abundant PMN in vaginal smears with T. vaginalis in

Concentration (pg/ml)

fected women were observed in another clinical study [38] . On the other hand, Helmy et al. [21] showed no difference among rats inoculated with T. vaginalis isolates from symptomatic and asymptomatic women regarding histopathological and immunhistochemical changes. Others failed to find correlation between clinical picture of host and pathogenicity of T. vaginalis in mice [39] . These contradictory findings may be explained by the fact that the effects of virulence factors are genetically initiated and controlled [31] . The diversity of host’s immune response may also affect pathogenicity of T. vaginalis [40] .

Local LTB₄ have been reported in women in previous study. A significant increase in the local LTB₄ was detected in vaginal washes of symptomatic, T. vaginalis-infected women when compared to asymptomatic patients [41] . However, the circulating LTB₄ level in symptomatic and asymptomatic women and local LTB₄ level in vaginal tissue in experimental mice hasn’t been studied. In the present study, higher levels of LTB₄ were detected in the vaginal tissue than in the vaginal wash and serum of the all groups. Other work reported that reactive nitrogen intermediates is higher in vaginal tissue than vaginal washes and blood of infected mice [42] . This can be explained by the fact that vaginal washes and plasma may not be the ideal source of samples. Those samples may fail to identify the level of LTB₄ in the infected individuals exactly.

In the present study, LTB₄ level was highest on 14th p.i.d. and a significant difference was observed in LTB₄ levels in serum, vaginal washes and vaginal tissue of mice infected with T. vaginalis isolated from symptomatic patients as compared with asymptomatic subjects. A significant difference was also observed in LTB₄ in serum and vaginal washes of mice infected with symptomatic isolates as compared to asymptomatic isolates and uninfected mice. These results correlated well with the earlier studies conducted in clinical patients and in vivo studies in trichomoniasis. T. vaginalis releases neutrophil-activating factor known as LTB₄ [43] . Vaginal discharge from symptomatic patients with trichomoniasis showed high concentration of LTB₄ when compared to asymptomatic patients. Authors concluded that LTB₄ is involved in the inflammation and symptoms of trichomoniasis [41] . Whereas, others reported that this factor was extracted from both symptomatic and asymptomatic group of T. vaginalis isolates [44] . In viro studies showed that the supernatant of viable T. vaginalis induced LTB₄ production in neutrophils in an IgG- and complement-(C5-) dependent manner. This effect was decreased by LTB₄ antagonist treatment [45] . T. vaginalis has the ability to communicate directly with immune cells via interaction between LTB₄ and host cell BLT receptors resulting in modulation of the host’s immune responses [20] and Neutrophil activation [46] . Interleukin IL8 production in mast cell and human neutrophils occur via BLT-de- pendent activation of transcription factors NF-κB and CREB [47] . On other hand, LTB₄ down-regulate IL-12, and up-regulate IL-10 by dendritic cells leading to immune tolerance [48] .

These results also correlated well with the earlier reports conducted in experimental animals whereby Increase in LTB₄ in intestine has been demonstrated in other nematode-infected animals [49] [50] and other protozoan parasite as Entamoeba histolytica [51] . Experimental studies conducted on Strongyloides venezuelensis indicated greater LTB₄ release in the lungs, duodena and serum than that seen in the tissue of uninfected animals. Impairment of LTB₄ synthesis, either genetic or pharmacologic, is associated with increased numbers of worms and eggs in infected animals. Altered resistance to parasite in the absence of LTB₄ may explain this increase. LTB₄ levels during S. venezuelensis infection have main role in regulating IL-5 release, IgG and IgE [14] and production of IL-8 [52] . LTB₄ may effectively have a role in the rapid expulsion of T. spiralis and other helminthes by increase the contractility of smooth muscle cells, the permeability of epithelial cells and the production of mucus [53] . In experimental leishmaniasis, treatment with exogenous LTB₄ increase parasite killing by macrophages in BALB/c mice [54] . LTB₄ reduces the parasitic load of macrophages infected with Leishmania amazonensis through production of reactive oxygen species (ROS) and interleukin-1β (IL-1β) [55] . In addition, LTB₄, play a role in immune response to Leishmania infection by promoting leishmanicidal activity and modulation of the immune system in synergism with genetic factors [56] . Genetic variation in LTB₄ synthesis has been suggested and might influence resistance and susceptibility patterns to L. amazonensis infection [54] . In Leishmania infantum in vivo, the macrophage response is characterized by C-type lectin receptors (CLRs) signature. CLRs allow parasite resilience through inhibition of the LTB₄-IL-1β axis. So, CLRs might be a potential targets for treatment and prevention of visceral leishmaniasis [57] . Pharmacologic or genetic inhibition of 5-LO biosynthesis, with subsequent lack of LTs, lead to impaired clearance of bacteria, fungi and parasites with increased mortality. LTs play a role in the control of helminthes and protozoan infections by modulating the immune system and/or through direct cytotoxicity to parasites. In addition, the decreased production of LTB₄ in immunocompromised individuals might have a role in modulating the pathophysiology of helminthic and protozoan infections [12] . In contrast, LTB₄ overproduction in experimental cerebral malaria model lead to increased parasitemia and mortality rate of infected mice [58] . However, some protozoan parasite as T. gondii may inhibit LTB₄ production whereas exogenous LTB₄ promoted intracellular killing of ingested T. gondii in human monocytes [59] . By using of LTB₄ receptor antagonist and genetic approaches (5-LO-deficient mice), researchers have demonstrated increased parasitemia in mice infected with T. cruzi [60] [61] . In Chagas disease, LTB₄ control parasite differentiation and proliferation in the infected host cells allowing the infection to progress to a chronic state. So, LTB₄ initially ensure host survival but later on may cause cardiovascular damage [62] . In acute phase of infection, endogenous LTB₄ are important regulators of NO activity in the heart and therefore, affect cardiac parasite load without a direct action on IL-6 release [61] . Therefore, LTB₄ might represent a new series of targets for therapy with certain precautions [62] .

The present study demonstrates that T. vaginalis infection induces production of LTB₄, which is essential for a protective immunity against parasites. Further study is needed to clarify correlation between pathological changes and LTB₄ in trichomoniasis.

The authors are grateful to doctors in Obstetrics and Gynecology Outpatient Department, Suez Canal University Hospitals and Port Said General Hospital for their valuable help in sample collection.

AM Eida provided the original idea of the research and planned the study design. She shared in the experimental studies, assessment of LTB₄ level, results analysis and wrote the manuscript. OM Eida shared in the study design, the experimental studies, assessment of LTB₄ level and manuscript writing. AS SALEM did the histopathological assessment.