Introduction

Giardiasis is the result of infection with Giardia duodenalis and has become a worldwide public health problem. In 2001 [1], there were a billion cases of giardiasis estimated globally, and in 2004 [2], global prevalence of the infection had reached approximately 30%. The current worldwide prevalence of giardiasis among children under 10 years of age may range from 15% to 20% [2]. Zinc deficiency is another rising public health problem; its global prevalence was estimated to be 31% in 2004, while rates ranged from 4% up to 73% in developing countries [3]. Zinc deficiency occurs not only in developing countries but also in more advanced countries due to inadequate eating habits or medical conditions, such as hepatic or renal failure, diabetes mellitus, or human immunodeficiency virus infection [4, 5]. Zinc deficiency has been associated with growth retardation, neurosensory changes, impaired cognitive abilities, compromised immune functions, and even death [6–8]. Although a high frequency of bacterial, viral, and fungal infections occur in zinc-deficient humans [6], studies of the association between parasitic infection and zinc levels in human or animal hosts have not often been attempted. In 1993 [9], giardiasis was first reported as a risk factor for zinc malabsorption in children. Other authors have also reported this risk [10–12], but the link remains uncertain. One study failed to show the association between giardiasis and zinc malabsorption in children [13], and other studies demonstrated that the association between giardiasis and malabsorption of other nutrients, different to zinc, in mice and children remained unclear [14, 15].

In Mexico, the prevalence of G. duodenalis was estimated at 32% in 1994 [15] and is currently the most important protozoan parasite associated with intestinal infection in northwestern Mexico [16]. Three studies in Mexico have shown evidence of zinc deficiency in women and children, and only one of these studies showed low levels of dietary zinc consumption in 19% to 24% of schoolchildren [17–19].

We hypothesized that giardiasis may be a risk factor for zinc deficiency regardless of dietary zinc intake. This study investigated this issue using the mouse as an experimental model.

Materials and Methods

Animals

Mice that were aged 5–7 weeks (18–30 g) were obtained from the Animal Bioterio Center of the University of Sonora in northwest Mexico to be used in this study. The strain of mice used was C3H/HeJ, which is known to be a strain that is susceptible to G. duodenalis infection [20]. Care and use of the animals were in accordance with the guidelines of the institutional ethical committee [21].

Mice Groups

The mouse cages were sanitized with sodium 10% hypochloride. The temperature in the mouse rooms was maintained between 20°C and 26°C (mean 22 ± 1°C) and humidity between 40% and 70%. There were 10 to 14 air room changes per hour and 12–12-h light/dark cycles. The mice were housed in sanitized stainless steel racks. Biohazard bags (VWR autoclavable bags, 1.5 mil 14220-010, 2005) were used for autoclaving infectious biological materials at the end of the study (urine, fecal material, and carcasses).

Measurements of Weights of the Mice and Their Food

The weights of the prepared diets were measured using a precision (±0.001 kg) electronic balance OHAUS [Model CD-11 Indicator (01010056DB), Florham Park, New Jersey, USA, 1999]. The mice's weights were measured using a precision (±0.01 g) electronic balance OHAUS [(7124331499) Pine Brook, New Jersey, USA, 2003] at baseline and at the time of euthanization.

Preparation of the Diets and Dietary Zinc Content

The standard basal diet (Harlan Teklad Rodent Diet, WI 8604) was comprised of casein (200 g/kg), cornstarch (653 g/kg), corn oil (50 g/kg), vitamin mixture (10 g/kg), mineral mixture (35 g/kg), fiber (50 g/kg), coline (2 g/kg), and zinc. The zinc was added to meet the zinc requirements for the mice groups. The total amount of the study basal diet was estimated using the following calculations: mice number per group × daily food consumption × study days (6 mice × 12 g × 29 days = 2.1 kg per group) and multiplying 6 mice groups × 2.1 kg, calculating 12.6 kg as the total amount. Next, this amount was separated into three portions, mixing each of them with varying zinc concentrations by adding 0, 0.03, and 0.25 g of zinc gluconate per kilogram of basal diet to obtain the low, normal, and high dietary zinc contents, respectively. Dietary zinc concentration was validated by atomic absorption spectrophotometry (Varian-Spectra AA-20) the method 968.08 of the AOAC [22] under qualified technical supervision. One milligram of each dietary regimen, mixed with 5 mL of HNO3 and deionized water, was digested using a microwave (MDS 2000. Falcon, Mexico). A certified sample of bovine liver was used to validate this method. A total of 1.4 mL of HCl and deionized water was added to the digested samples. The concentrations of the final solutions were measured at 213.9 nm using a hollow cathode zinc lamp. A calibration curve build up with 0.5, 1.0 and 2.0 ppm of zinc solutions was used to estimate zinc concentrations. The actual zinc concentrations of the low, normal, and high dietary zinc regimens were 9 (original content of the basal diet), 33, and 288 mg/kg, respectively.

Study Design

Thirty-five mice were randomly categorized into Giardia-infected (n = 18) and Giardia-free (n = 18) groups. Next, each group was randomly classified into three subgroups to receive low, normal, and high dietary zinc regimens [2, 22]. All of the mouse groups were acclimated to their corresponding experimental diets daily ad libitum for 2 weeks prior to the start of the study. Fresh ultrapure water purified with Milli-Q system (Millipore Corp., Billerica, MA, USA) was also offered ad libitum daily throughout the study. On day 0, the groups to be infected were inoculated with G. duodenalis trophozoites by oral via. All of the mice were maintained on their diets by allowing them to feed ad libitum. On day 14 postinfection, all of the mice were euthanized to obtain the study samples. Blood samples were drawn before the euthanization process. The maximum load of Giardia trophozoites has been found between 9 and 11 days after oral infection in the intestinal tissue of normally nourished NMRI mice [23].

Giardia Inoculation and Confirmation of Infection

G. duodenalis trophozoites (Portland strain) were axenically cultured in a TYI-S-33 medium supplemented with 7.0 mL of 10% bovine serum (Bovine Adult FERUM, SIGMA B2771) using a Purifier Class II Biosafety Cabinet (Delta Series, LABCONCO). For experimental inoculation, actively growing trophozoites (48–72-h-old cultures) were harvested by being chilled in ice for 20 min. Trophozoites were washed with PBS at pH 7.2 (GIBCO) by 10-min centrifugation (800×g) at 4°C and were resuspended in 500 μL of PBS. Dilutions were prepared with PBS (1:15) and 0.4% trypan blue (1:2) (Sigma, Co.) to obtain a suspension of 5 × 106 trophozoites per 200 μL. Before inoculation, a 6- to 9-h fasting period with no water restraint was required to guarantee infection. Using oral administration, the trophozoites were inoculated directly to the duodenum using a syringe fitted with a cannula needle to prevent tissue damage. Separately, two additional inoculated mice consuming low dietary zinc were also sacrificed on day 14 postinfection to confirm qualitatively but not quantitatively the trophozoites in the duodenal tissue.

Euthanization Process

Fourteen days following Giardia inoculation and sustained zinc dietary regimens, all of the mice were anesthetized with chloroform by inhalation. Each mouse was restrained on a styrofoam bed using pins. Abdominal asepsis with alcohol was performed, and sterile surgical equipment was used to dissect the abdominal and thoracic sections of each mouse to obtain the intestinal and blood samples.

Counting Trophozoites

A 5-cm section of the duodenum was removed and longitudinally dissected in the Giardia-free and the Giardia-infected groups. This section was transferred into 5-mL screw cap tubes with 3 mL sterile PBS at 4°C and shaken at 500 rpm for 30 min using a mechanical rotor SOLBAT series 1-86 to release the trophozoites from the tissue. Supernatant was transferred into 1-mL sterile conic tubes and centrifuged at 800×g at 4°C for 10 min. The supernatant was discarded and 0.5 μL of sediment was transferred to a Neubauer chamber. The total number of trophozoites (sum of trophozoites of the four of 1-mm2 squares) was multiplied by 10 mm to estimate the number of trophozoites per milliliter.

Zinc Determination in Serum Samples

One-mL syringes (Plastipak) were used to withdraw blood samples by intracardial puncture. After extraction, the whole blood was centrifuged at 800×g for 10 min and serum (500–600 μL) was separated and transferred into milliliter sterile cryogenic heparinized conic tubes and stored at −20°C until analysis. Hemolyzed samples were rejected [24]. The determination of the serum zinc levels was developed according to D´Haese et al., [25]. Measurements were carried out at 213.9 nm using a hollow cathode zinc lamp with a coefficient of variation of 2.6% and a recovery of 97%. The cutoff point for zinc deficiency in mice was defined as less than 4.65 ± 0.532 μmol/L [26].

Statistical Analysis

Data were analyzed using the NCSS 2000 software [27]. Descriptive statistics were expressed as the median ± confidence interval. Wilcoxon signed rank was used to test for the differences between the weights of the same groups at baseline and 14 days postinfection. Mann–Whitney U test was used to test for the differences in the final weights between two independent groups. Nonparametric ANOVA (Kruskal–Wallis) test was used determine whether there were any differences in the weights and serum zinc levels among the three independent groups subjected to the different dietary regimens at baseline and 14 days postinfection. In the case of rejecting the null hypothesis in the analysis of variance, Dunnett's method for multiple comparisons was performed to establish which of the groups differed significantly.

The association between the serum zinc levels and the infection was analyzed by multiple regression analysis using the serum zinc levels and the Giardia infection as dependent and independent variables, respectively, as adjusted by the administered dietary zinc regimen and weight. All analyses were considered significant at p ≤ 0.05.

Results

At baseline, no difference was found between the median weights of the mice randomly placed into the Giardia-free groups (21.0 g) and those to be infected with Giardia (22.3 g, p = 0.072). Comparison of the weights at baseline and 14 days postinfection showed a significant increase in the weights of the three Giardia-free groups, while weights remained unchanged in the Giardia-infected groups (Table 1). In addition, a higher final median weight was found in the Giardia-free mice than in the Giardia-infected mice subjected to low dietary zinc regimens (Table 1); however, no differences were found between the final median weights of the Giardia-free groups and the final median weights of the Giardia-infected groups that were maintained on the normal and high dietary zinc regimens (Table 1). In addition, no differences were found between the initial and final median weights of the three Giardia-free groups. The same was found between the initial and final median weights of the three Giardia-infected groups that were maintained on the three different dietary zinc intakes (Table 1).

Table 1 Comparison of weights between the Giardia-free and Giardia-infected groups before the administration of the experimental diet (baseline) and 14 days postinfection
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Fourteen days postinfection, the geometric mean of trophozoites was 3,498 ± 101 (SE) per milliliter in the Giardia-infected group maintained on low dietary zinc. No trophozoites were detected in the other two Giardia-infected groups that were maintained on the normal and high dietary zinc regimens.

The Mann–Whitney U test showed a significantly lower median level of serum zinc in the Giardia-infected mice than in the Giardia-free mice that were maintained on the high dietary zinc regimen (Table 2). However, no difference was found in the serum zinc levels between the Giardia-infected and the Giardia-free groups maintained on the low and normal dietary zinc regimens (Table 2). The negative impact of G. duodenalis on the serum zinc levels was demonstrated by multiple regression analysis (β = −9.29 ± 3.46, p = 0.02), which was adjusted by the zinc dietary regimens and weight (Table 3). Since the reference in the regression analysis was the Giardia-free group, the negative β coefficient value means that the serum zinc level was 9.29 μmol/L lower in the Giardia-infected than in the Giardia-free mice (Table 3).

Table 2 Comparison of serum zinc levels between the Giardia-free and Giardia-infected mice maintained on the three dietary zinc regimens 14 days postinfection
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Table 3 Adjusted association between serum zinc levels and giardiasis in 35 mice maintained on low, normal, and high dietary zinc regimens
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Discussion

Over the course of the study, the mice free of giardiasis had higher median weight gains than did the Giardia-infected mice regardless of the dietary zinc regimens (Table 1). It is well recognized that giardiasis can be asymptomatic or associated with acute and chronic diarrhea with or without malabsorption. Giardia trophozoites may cause intestinal lesions leading to nutrient malabsorption, which may explain the reduced weight gain in infected humans [28] and/or animals [29]. However, this link remains poorly understood [30]. Thomson et al. [31] found a significant impairment of the weight gain in the Giardia muris-infected Swiss albino mice compared to Giardia-free controls from 21 to 8 days of follow-up evaluation. However, Scott et al. [30] found no difference in the mean weights between the Giardia-infected and the Giardia-free mice after 6 to 35 days postinfection. In this study, we found a higher median weight gain in the Giardia-free than in the Giardia-infected group with low zinc intake 14 days postinfection (Table 1). It is well recognized that zinc deficiency in animals is characterized by reduction in growth, diverse reproductive effects and adverse developmental effects, all of which persist after weaning, as well as reduced immunoresponsiveness [32]. In this case, the low dietary zinc exacerbates apparently the effects of the giardiasis through poor physical development [33]. In addition, no difference was found in the final median weights between the Giardia-infected and Giardia-free groups with higher consumptions of zinc (Table 1). It is possible that giardiasis and low zinc intakes are exacerbating the growth retardation and that better dietary zinc intakes replace zinc losses caused by malabsorption more effectively, thereby reducing the growth retardation rate. On the other hand, the trophozoites were only detected in the infected mice that were maintained on low dietary zinc at 14 days postinfection. Low dietary zinc apparently contributed to the longer duration of the Giardia infection, while higher levels of dietary zinc intake may have helped to eradicate giardiasis at that time.

Numerous animal and human studies have indicated that zinc deficiency decreases resistance to infectious diseases. Zinc-deficient animals have suppressed immune responses and are more susceptible to a diverse range of infectious agents [34]. Zinc-deficient mice infected with Trypansoma musculi harbored three times more parasites than uninfected mice because of a delay in the production of protective antibodies [35]. Further, 40% of moderately zinc-deficient mice succumbed to a normally nonlethal infection of Plasmodium yoelii 17XNL malaria [36] because of delayed protection of immunoglobulin G antibodies. In human studies, Junqueira and Queiroz [37] and Al-Mekhafi et al. [38] reported that severely malnourished individuals had more exacerbated cases of giardiasis and a more impaired immune response than those with moderately deficient nutrition. Shukla and Sidhu [39] published that malnourished Giardia-infected mice became Giardia-free 19 days later (48 days postinoculation) than those normally nourished Giardia-infected mice (29 days postinoculation). Finally, in our study, a median value of serum zinc levels was higher in the Giardia-free mice than in the Giardia-infected mice consuming high dietary zinc regimen 14 days postinfection (Table 2). However, no differences were found in the serum zinc levels between the Giardia-infected and the Giardia-free groups consuming low and normal dietary zinc regimens. The serum zinc levels probably were negatively impacted by both the giardiasis and poor and normal zinc intakes in the Giardia-infected and the Giardia-free groups, respectively (Table 2). Alternatively, a high dietary zinc consumption is not only probably reducing the growth retardation but is also contributing to the better serum zinc levels in the Giardia-free group in comparison to the Giardia-infected group (Tables 2 and 3). How zinc metabolism is compromised by G. duodenalis is not well known, but it is likely that zinc deficiency, as assessed by serum analysis, is not a sensible method for measuring the redistribution of zinc from serum to other tissues [40], the reduction of zinc transporter proteins, nor the rise in the production of metallothionein during the course of giardiasis infection [41].

Although the association between malabsorption and giardiasis is well documented, little is known about the giardiasis–zinc interaction both in humans and animals. A recent follow-up study in schoolchildren [42] found significantly higher serum zinc values in Giardia-infected children than in Giardia-free children 6 months after treatment, even adjusting for dietary zinc intake. Other studies from Turkey [9, 12] showed that Giardia-infected groups of children ranging from 2 to 14 years old had lower mean serum zinc levels than the Giardia-free control group (10.3 vs. 22.2 μmol/L and 16.7 vs. 20.8 μmol/L, respectively). Another study from Turkey [4] published similar findings in 20 Giardia-infected children from ages 3 months to 14 years. In contrast, a Spanish study [13] found no change in the mean serum zinc levels before (14.1 μmol/L) and 3 months after treatment (14.1 μmol/L) in 25 Giardia-infected children, ages 6 to 9 years old. Unfortunately, these studies did not include dietary zinc records. In reference to this current study, the following limitations can be identified: (a) the low number of mice analyzed, (b) no dietary zinc intakes recorded per mouse, and (c) the failure to include an infected group at baseline to estimate the serum zinc levels changes before and after infection, which would have ensured an important step in the interpretation of the mechanism of giardiasis–zinc interaction. However, results from this study show that giardiasis may be a risk factor for zinc deficiency in mice regardless of the dietary intakes. Further studies should be developed to elucidate the possible alterations in zinc metabolism observed over the course of giardiasis in mice. Such a study would help to redesign parasitic control strategies, micronutrient supplementation, and food fortification programs in order to improve the quality of life of human populations vulnerable to parasitic infections.