Background
Cryptosporidium is a protozoan parasite of public health and veterinary significance that causes gastroenteritis in a range of vertebrate hosts. The majority of human infections are attributed to
C. hominis, which humans as the only natural hosts and
C. parvum which infects mostly mammals, including humans [
1]. The infection is transmitted mainly by the fecal–oral route, and is initiated when sporozoites are released from oocysts present in water or food or by direct contact with an infected person or animal [
2].
Cryptosporidium infection ultimately results is epithelial cell death thus leading to villous atrophy, malabsorption and distorted intestinal permeability, all of which contributes to the occurrence of watery diarrhea [
3]. The host’s immune state has a key role in determining susceptibility to infection as well as the severity of cryptosporidiosis. In healthy people, cryptosporidiosis is self-limiting and gastrointestinal symptoms usually resolve spontaneously within 1–2 weeks, although asymptomatic carriage might occur. Nevertheless, in immunocompromised patients, severe watery diarrhea can develop which can lead even to death [
4]. In tropical countries,
Cryptosporidium caused diarrhea can result in impaired childhood development [
5]. Numerous human cryptosporidiosis outbreaks have been identified on a global scale. The most extensive outbreak was recorded in 1993 in Milwaukee, U.S.A., during which approximately 400,000 people were infected with
Cryptosporidium oocysts by drinking contaminated water. An increase in
Cryptosporidium infections was also reported in Germany, United Kingdom and the Netherlands in 2012 [
6]. In Asia and Africa, cryptosporidiosis is now considered the second most common cause of diarrhea [
7]. Even though promising chemotherapeutic treatments against cryptosporidiosis are beginning to appear, no consistently effective treatments exist for humans and animals [
8,
9]. Paromomycin and Nitazoxanide have been used but results showed limited efficacy against cryptosporidiosis and sometimes relapse of diarrhea has been observe [
10,
11]. Halofuginone lactate is another anticoccidial agent used against cryptosporidiosis and although it diminishes oocyst shedding and the severity of the diseases, in calves, it does not result in a complete cure [
12,
13].
Considering the potential side effects of and resistance to many drugs, attention has shifted towards plant extracts. Plants represent an excellent source of bioactive compounds and have a long history in the prevention as well as treatment of a range of human and animal diseases [
14]. Therefore, interest in natural products with antiparasitic properties has increased in recent years. Curcumin has showed promising effects against
C. parvum in vitro [
15]. Also, the ethanolic extract from olive pomace has been shown to effectively inhibit
C. parvum development [
14]. Therefore, there is a great need to develop new anti-cryptosporidial agents, in order to provide an alternative method to control cryptosporidiosis. The present study aimed at investigating the antiparasitic effectiveness of the experimental natural feed supplement Auranta 3001 in the prevention
C. homins and C. parvum infections in human and primary bovine cell models.
Discussion
In order to tackle the issue of the lack of effective therapeutic agents against Cryptosporidiosis, we explored the efficiency of Auranta 3001, a natural feed supplement, against C. hominis and C. parvum in vitro. Preliminary results revealed that the feed supplement was toxic to HCT-8 bovine cells at concentrations higher than 1%. Lower concentrations had insignificant effects on both of the cell models and thus concentrations up to 1% were investigated.
It has been reported that different
Cryptosporidium species utilise different mechanisms to infect cells of different origins [
17] therefore anti-
Cryptosporidium agents able to effectively prevent invasion in different host cells would be of great benefit. In the present study,
C. hominis and
C. parvum is able to effectively invade both HCT-8 and primary bovine cells. To evaluate the effect on parasite invasiveness, the feed supplement was applied at different concentrations to
C. hominis and
C. parvum oocysts for 1 h before infection. Pre-treatment of the oocysts resulted in statistically significant reductions (
p < 0.01–0.001) in the invasiveness for both HCT-8 and bovine cells with the reduction being more pronounced with increasing supplement concentrations. It is noteworthy that although the invasiveness of
C. hominis and
C. parvum was similar for the HCT-8 cells, it showed differences in the bovine cells with
C. parvum invasiveness being significantly lower compared to the HCT-8 cell line (Figs.
1a,
2a).
Potentially the novel feed supplement can also interfere with host cell metabolic pathways, which subsequently affect parasite infection and growth. To address this possibility, HCT-8 cells and bovine cells were pre-treated with the feed supplement for 1 h and then infected with
C. hominis or
C. parvum in the absence of the supplement. The pre-treatment of host cells with increasing concentrations of Auranta 3001 (0.1–1%), lead to a significant inhibition of invasiveness for both parasites (Figs.
1b,
2b). In this case too,
C. parvum invasiveness in bovine cells was significantly lower compared to the HCT-8 cells.
Natural extracts have been employed in other studies against Cryptosporidium. Garlic juice (
Allium sativum) has been shown to significantly reduce
Cryptosporidium oocysts from stool of immunosuppressed mice without affecting intestinal architecture [
10]. Curcumin, a natural polyphenolic compound, although does not reduce viability of
C. parvum it can attenuate invasiveness in HCT-8 cells by 65% at a concentration of 200 μM [
15]. Pomegranate extract (3.75%) supplemented in milk has also shown to reduce fecal oocyst count and diarrhea intensity and duration in neonatal calves [
20]. Although
C. hominis and
C. parvum possess different pathways to infect different host cells [
17], the results presented here revealed that the natural feed supplement is a potent inhibitor of
C. hominis and
C. parvum invasiveness in both human and primary bovine cells.
The cytokine profile of HCT-8 and primary bovine cells was also determined in order to investigate the host immune response to the natural feed supplement. The immune defence mechanism of an immunocompetent host against
Cryptosporidium spp. used to recover from
Cryptosporidium spp. infection involves both functional cellular and humoral immunities, but the actual mechanism is still unknown. Studies have shown that IL-8 is expressed after
Cryptosporidium spp. in vitro infection of intestinal cell lines and human intestinal xenographs [
21]. In the present study, exposure to Auranta 3001 significantly decreased the IL-8 levels, which suggests that Auranta 3001 is not recognized as a pathogenic signal by both HCT-8 cells and primary bovine cells indicating the anti-inflammatory role for Auranta 3001 in the gastro-intestinal tract inflammation during
Cryptosporidium spp. infection. Also, IFN-γ results showed that Auranta 3001 did not have any effect on IFN-γ production in HCT-8 cells, keeping concentrations at similar levels with the controls, whereas in bovine primary cells, the levels of IFN-γ were significantly increased, showing its effectiveness in mediating the immune response in this type of cells when infected with
C. hominis and
C. parvum. Studies have shown that the immune response towards
Cryptosporidium in humans differs from that in animals. IFN-γ production in mice seems to be linked with the innate and primary immune responses [
22], whereas in humans it is probably related with the memory response towards the parasite. Even though IFN-γ has been shown to play an important role in both the innate and adaptive immune responses to
C. hominis and
C. parvum, the mechanisms of resistance mediated by this cytokine alone are not completely understood yet [
21]. Interleukin-10 plays a major role in the resolution of inflammation during sepsis and infection but is also involved in persistence of pathogens by interfering with innate and adaptive immunity [
23]. The Auranta 3001 results on HCT-8 cells, revealed its anti-inflammatory properties. The levels of IL-10 in HCT-8 cells increased significantly during infection with
C. hominis and
C. parvum. On the other hand, the supplement did not have any effect on IL-10 production in primary bovine cells, with the levels of this cytokine remaining at similar concentrations with the controls.
Parasite proteases are of particular importance since they are involved in the proteolysis of surface and apical proteins that mediate invasion [
24,
25]. A number of these proteins are processed by serine proteases. Evidence from the use of protease inhibitors has suggested that serine proteases might be essential for host cell infection by
C. parvum infection in vitro [
26,
27].
Genes expressing serine proteases have been identified for
Cryptosporidium [
28,
29]. In order to better understand the antiparasitic mechanism of the natural feed supplement, in this study, we focused on the subtilisin-like serine protease CpSUB1 involved in the proteolytic cleavage of
Cryptosporidium gp40/15 to produce the surface glycopeptides gp40 and gp15 which are involved in infection [
27]. In the present study, qPCR results showed that the CpSUB1 gene in
C. parvum is expressed throughout infection which is consistent with the study of Wanyiri et al. [
16] in which a semi-quantitative determination was employed. Specific quantification of CpSUB1 gene revealed that when infection of both HCT-8 and primary bovine cells took place in the presence of different concentrations of the natural supplement expression was significantly reduced at all the concentrations used and at all time points investigated during infection (Fig.
5a, b). These results in combination with the fact that the natural supplement significantly inhibited
C. parvum infection of HCT-8 cells in a dose-dependent manner compared to untreated controls shows that subtilisin-like serine protease CpSUB1 is targeted by the natural feed supplement leading to decreased invasion. However, it is possible that the natural supplement might exert an effect on other targets as well. Although, the expression of subtilase protease gene for
C. hominis (ChSUB1) was not studied here, it has been shown that ChSUB1 and CpSUB1 are 98% identical with the alignment of the CpSUB1 and ChSUB1 nucleotide sequences identifying primarily silent substitutions [
28]. Therefore, based on the significantly reduced invasiveness of
C. hominis observed, we hypothesised that ChSUB1 is a target for the natural feed supplement as well.
Disruption of cell tight junctions is known to contribute to intestinal diseases by enteric pathogens [
30]. TEER values are considered strong indicators of cellular barrier integrity [
31]. In vitro studies have shown a rapid decrease in TEER as one of the characteristics of Cryptosporidiosis [
32,
33]. In this study, infection with
C. hominis and
C. parvum resulted in low TEER values for both HCT-8 and bovine cells which corresponds to disrupted tight junction architecture and barrier function. However, in the presence of the novel supplement significantly higher TEER values were observed showing the protective effect of Auranta 3001 on both cell monolayers (Figs.
1d,
2d). Protein kinace C (PKC) mediates calcium-induced tight junction assembly and its inhibition has been found to stop the normal distribution of tight junction-associated proteins such as ZO-1 and cingulin [
34]. A possible explanation of the reduced invasiveness observed is that exposure of HCT-8 and primary bovine cells to the feed supplement during infection caused stimulation of tight junctions in the host cells by inhibiting PKC although further studies are needed to confirm this.
Authors’ contributions
Conceived design and performed the experiments AS, FS, CK, ML, LP, GP and LS. Analysed the data GP, IP and TI. Contributed reagents/materials/analysis tools NC, PW, GP. Wrote the paper NC, AS and FS. All authors read and approved the final manuscript.