Factors contributing to spontaneous Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected macaques

ORIGINAL ARTICLE

Factors contributing to spontaneous Enterocytozoonbieneusi infection in simian immunodeficiency virus-infected macaquesInderpal Singh1, Wenjun Li2, Margo Woods3, Angela Carville4 & Saul Tzipori1

1 Division of Infectious Diseases, Department of Biomedical Science, Tufts University Cummings School of Veterinary Medicine,

North Grafton, MA, USA

2 University of Massachusetts Medical School, Division of Preventive and Behavioral Medicine, Worcester, MA, USA

3 Department of Public Health and Family Medicine, Tufts University School of Medicine, Boston, MA, USA

4 New England Regional Primate Research Center, Harvard Medical School, Southborough, MA, USA

Introduction

The acquired immunodeficiency syndrome (AIDS) epi-

demic has led to the emergence of infectious agents

previously unrecognized. Several, previously unknown

opportunistic infectious agents have emerged in associ-

ation with chronic diarrhea in persons with HIV/AIDS

[20, 28], who experienced significant weight loss and

shorter survival time [19]. Of all the opportunistic

enteric pathogens identified, the microsporidia Entero-

cytozoon bieneusi is considered the most prevalent

pathogen [12], occurring in up to 30%–50% of

patients with AIDS [45, 46]. Previously classified as

protozoa, the microsporidia are now considered as

degenerate fungi [48, 49].

Enterocytozoon bieneusi is mainly associated with

severe diarrhea, weight loss as well as marked malab-

sorption of vitamins, carbohydrates and fats, and con-

sequently a likely contributor to wasting in HIV/AIDS

patients [2, 19, 21, 22, 45]. In addition to infectious

agents, factors such as poor nutritional intake, malab-

sorption, immune status, and simian immunodeficiency

virus (SIV)/HIV disease progression, also contribute to

wasting in AIDS patients [17]. Individuals with HIV

have lower than optimal levels of selected micronutri-

ents (selenium, vitamins B12, B1, B2, B6, niacin, E

Keywords

Body composition – Enterocytozoon

bieneusi – micronutrient – primate – simian

immunodeficiency virus

Correspondence

Dr Saul Tzipori, Division of Infectious

Diseases, Department of Biomedical

Science, Tufts University Cummings School

of Veterinary Medicine North Grafton,

MA 01536, USA.

Tel.: +1 508 839 7955;

fax: +1 508 839 7911;

e-mail: [email protected]

Accepted May 10, 2006.

Abstract

Background A cohort of SIV-infected macaques had been used to investi-

gate the effect of dietary supplement, immune status, SIV/AIDS disease

progression and serum micronutrients levels on spontaneous acquisition of

Enterocytozoon bieneusi infection in SIV-infected macaques.

Methods Twenty-four SIV-infected macaques were randomized into 2

groups. One group received a vitamin/mineral supplementation and a sec-

ond group received a placebo. Both groups were examined for E. bieneusi

infection.

Results SIV-infected macaques were more prone to acquire E. bieneusi with

the progression of SIV/AIDS, and the increased shedding of infectious

spores was directly associated with decreased CD4 lymphocyte and

increased circulating SIV, in both supplemented and unsupplemented

groups of animals. Dietary supplementation, body composition factors and

serum micronutrients levels however had no association with the acquisi-

tion of E. bieneusi infection in these animals.

Conclusions Acquisition of E. bieneusi infection is related to SIV disease

progression, CD4 counts and viral load but independent of changes in

body composition and serum micronutrient levels.

J Med Primatol doi:10.1111/j.1600-0684.2006.00181.x

J Med Primatol 35 (2006) 352–360 ª 2006 The Authors

352 Journal compilation ª 2006 Blackwell Munksgaard

and A) [3, 5, 7, 29]. Epidemiological and intervention

studies regarding the benefits of micronutrient supple-

ments in persons with HIV/AIDS remains uncertain

[10, 11, 33]. Indeed, studies have shown that vitamin A

supplementation during gestation and lactation of

HIV-infected Tanzanian women significantly increased

the risk of HIV transmission to their children [10, 11].

Similarly, persons with low serum selenium levels

reported to have a 20-fold increased risk of death due

to AIDS [3]. Also, zinc supplementation resulted in

poorer survival and significantly increased risk of pro-

gression to AIDS [38], while high intake of antioxi-

dants such as vitamin E resulted in a pro-oxidant

environment [1], which could activate HIV and SIV

viruses [35, 25]. Observations from several studies indi-

cate that some micronutrients, which appear to be

good predictors of slowing the progression of AIDS

include vitamins A, B12 and selenium [38]. There is

only one report on serum micronutrient in SIV-infec-

ted rhesus macaques and it showed a decrease of selen-

ium levels associated with the development of AIDS

[51].

The immune status of the individual appears to

influence whether opportunistic infections including

microsporidia, are cleared, become persistent but

subclinical, or become symptomatic leading to chro-

nic diarrhea and wasting. So do deficiencies of cer-

tain micronutrients, through an adverse effect on the

immune function, can exacerbate HIV infection and

enhance the impact of opportunistic infections. En-

terocytozoon bieneusi infections with profound symp-

toms of diarrhea and wasting were consistently

observed in HIV-infected patients with CD4 counts

below 100/mm3 [2]. What is not clear is whether

chronic diarrhea due to E. bieneusi infection also

contributes indirectly through reduced uptake of

micronutrients.

The SIV-infected rhesus macaque provides a good

model to investigate AIDS-related diseases, including

the role of micronutrients, as the clinical disease asso-

ciated with SIV infection is similar in many ways to

those observed with HIV, except that AIDS is consid-

erably shorter in macaques [50]. We have successfully

established persistent E. bieneusi infections in SIV-

infected macaques [41], demonstrating that E. bieneusi

obtained from macaques was morphologically and

antigenically indistinguishable from E. bieneusi of

human origin [52]. The natural occurrence of E. bien-

eusi infection in macaques, with and without SIV

infection, makes it an ideal model to investigate the

role of this pathogen in AIDS.

In this study, we examined the association, if any, of

naturally infected animals with E. bieneusi with weight

loss, serum micronutrients levels, immune status, SIV/

HIV disease progression (viral load), with a view to

identify early subclinical factors which might be

responsible for the observed wasting and help formu-

late antimicrobial or nutritional interventions.

Materials and Methods

Animals

Thirty-two juvenile rhesus macaques (Macaca mulatta)

were tested for presence of E. bieneusi spores in feces

as evidence of infection. Of them eight SIV-negative

animals were included as the control group. The

remaining 24 were challenged with SIVmac239 (50 ng

of p27 viral-antigen equivalent, intravenously) (pro-

vided by Ronald Desrosiers, New England Regional

Primate Research Center, Harvard Medical School,

Southborough, MA, USA), and were randomized into

two groups. One group of 12 received micronutrient

supplementation in the form of a treat biscuit and a

second group of 12 received certified chow (Purina

No. 5048; Purina Mills, Richmond, IN, USA) as their

usual diet. The animals were monitored for 16 weeks

prior to SIV challenge and for 120 weeks thereafter.

Monthly fecal samples were obtained to determine

the presence of E. bieneusi spores. There was no sig-

nificant baseline difference between the groups for

age and immunological parameters, such as CD4,

CD8 and lymph cell counts as described elsewhere

[16].

All macaques were housed at the New England

Regional Primate Research Center (NEPRC). The

SIV-infected macaques, including supplemented and

unsupplemented, were housed in the same environ-

ment, in a centralized biosecurity level-3 (BSL-3) ani-

mal containment facility while the control group was

maintained in BSL-2 facility, in accordance with the

Guide for the Care and Use of Laboratory Animals.

All animals were housed separately and clinical proce-

dures were performed under the direction of a veterin-

arian. All possible measures were taken to minimize

discomfort to these animals. Appropriate anesthesia

and analgesics were administered under the direction

of a veterinarian; telazol (5 mg/kg body weight) was

routinely used during handling of macaques. If the vet-

erinary staff considered it to be necessary, animals

were killed in accordance with the recommendations of

the American Veterinary Medical Association Panel on

Euthanasia. All procedures and protocols were

approved by the Institutional Animal Care and Use

Committee at Tufts University and The Harvard Med-

ical Area Standing Committee on Animals.

Singh et al. E. bieneusi in SIV-infected macaques


Journal compilation ª 2006 Blackwell Munksgaard 353

Diet

The animals were maintained on a certified chow diet

(Purina No. 5048) in biscuit form provided by Purina

ad libitum, given twice a day, as described elsewhere

[16]. Briefly, animals in the supplemented group were

given a biscuit daily designed to provide two to three

times the estimated nutritional requirement of

vitamins. The amount of micronutrients in the certified

diet and in the supplemented diet was reported else-

where [16]. Briefly, supplementation contained twice

the amount of vitamin A, three times the amount of

vitamin E, 1.5 times the amount of zinc, and 2.5 times

the amount of selenium, based on a usual food intake

of 225 g/day compared to the un-supplemented certi-

fied diet. Additional information on diets and feeding

procedure have been reported previously [16].

Body composition

Body weight was measured every 4 weeks using a cal-

ibrated scale that measured to the nearest 0.01 g. Body

weight and composition were determined monthly

using methods reported previously [14, 16]. A body-

mass index was calculated as body weight (in kg) divi-

ded by the square of the crown-heel length (in cm).

Measurement of specific body compartment [fat, lean

body mass was performed by dual-energy X-ray

absorptiometry (DEXA) (GE Healthcare Lunar Cor-

poration, Madison, WI, USA)], with software devel-

oped specifically for small primates. The software

provided information on fat tissue and lean tissue,

both in total grams and as a percentage of body

weight.

DNA extraction and polymerase chain reaction

A modified procedure of DNA extraction from fecal

samples and nested polymerase chain reaction (PCR)

were used as described previously [6, 34]. Briefly, the

DNA was extracted using Geneclean III kit (Bio101,

Carlsbad, CA, USA) according to manufacturer’s

instructions. The first round of PCR (primary PCR)

was performed with 1 ll of the DNA preparation des-

cribed above with primers specific for the E. bieneusi

ribosomal internal transcribed spacer (ITS). The nested

PCR was performed with 1 ll of the product from the

primary PCR with primers specific for E. bieneusi ITS-

DNA as described previously [4]. The size of the prod-

uct generated with outer primers (primary PCR) was

435 bp and the size of the product generated with nes-

ted primers was 390 bp [34]. The PCR products were

visualized by the use of ethidium-bromide staining

after electrophoretic separation in 1.5% agarose gels.

Based on PCR analysis, fecal samples were divided

into two groups; positive for nested PCR, or negative

for nested PCR.

Viral load and immunophenotyping

Viral load was determined by quantitative RT-PCR as

previously described [36]. Cell immunophenotyping

was performed to determine peripheral blood CD4+

and CD8+ T cell numbers and ratios. Antibodies used

for immunophenotyping of rhesus lymphocytes inclu-

ded anti-CD3 (6G12), anti-CD4 (PKTA), anti-CD8

(Leu-2a). Cells were stained in the presence of staining

media [phosphate buffered saline (PBS) with 2%

mouse serum]. After antibody staining, the cells were

fixed with fresh 2% paraformaldehyde. Three-color

flow-cytometry analysis of the cells was performed by

use of a FACScan (Becton Dickinson, San Jose, CA,

USA) and Cell Quest Software (version 3.2; Becton

Dickinson). Appropriate isotype controls were used to

establish positive and negative gates. Twenty thousand

events were collected from a live gate to exclude cellu-

lar debris.

Statistical analysis

Generalized linear (logistic) mixed models were used to

evaluate the association of positive readings in E. bien-

eusi nested PCR with clinical indicators, such as CD4

counts, viral load data, serum micronutrient levels,

and body composition data, etc. Presence of E. bien-

eusi spores in feces was coded as negative (0) or posit-

ive (1). The observations from different animals are

assumed to be independent, and observations from the

same animals are assumed to have a first order serial

autocorrelation. In the modeling, we used logit link

and specified a binomial distribution for E. bieneusi

infection. A random intercept was included in the

model for each animal. The effects of clinical indica-

tors of interest were included as fixed effects in the

models. Odds ratio (OR) were used to assess the

strength of associations. Results were considered signi-

ficant when the two-tailed P-value was <0.05.

Results

For comparative analysis, the animals were divided

into three groups; SIV-naı̈ve/un-supplemented, SIV

positive/un-supplemented, and SIV positive/supplemen-

ted. The OR of acquiring E. bieneusi infection was

studied with various clinical predictors of SIV

infection. No natural acquisition or reactivation of

E. bieneusi in SIV-infected macaques Singh et al.



Cryptosporidium parvum was observed in the fecal

sample of any of the macaques throughout this study,

as determined by PCR analysis.

SIV-infection and dietary supplementation

The results showed that fecal samples collected from

SIV-infected animals were more likely to be E. bieneusi

positive by PCR than those collected from SIV naı̈ve

animals (OR ¼ 2.52, P ¼ 0.021), indicating that

SIV-infected animals were more likely to excrete

E. bieneusi spores. The risk of E. bieneusi prevalence

or likelihood for shedding E. bieneusi spores increased

(OR ¼ 1.11/month, P ¼ 0.057) overtime after SIV-

infection of juvenile macaques. Once SIV-infected ani-

mals became E. bieneusi positive, they continued to

excrete spores for the remainder of the monitoring

period. Enterocytozoon bieneusi-infected animals remai-

ned asymptomatic, experiencing neither diarrhea nor

weight loss until the terminal stage of SIV-infection.

Curiously, dietary supplementation was associated with

higher risk for E. bieneusi infection in the SIV-infected

animals (OR ¼ 2.72), but differences between the

groups were only marginly significant (P ¼ 0.082;

Table 1). Whereas in SIV-naı̈ve juvenile animals,

E. bieneusi spore shedding was only observed during

first few months of their age. All SIV-naı̈ve animals

showed transient E. bieneusi infection and the spore

excretion lasted from 3 to 7 months. With increased

age, the probability of E. bieneusi spore excretion

decreased (OR ¼ 0.72/month, P ¼ 0.003; Table 1),

which is attributed to the ability of SIV-naı̈ve animals

to clear the infection.

Body weight change, body mass index and body fat

percentage

As shown in Table 2, no statistically significant associ-

ation was found between risk of the spontaneous

acquisition of E. bieneusi and body weight gain and

body mass index (BMI; kg/m2) in any of the three

groups of animals. Similarly, change in body fat per-

centage after SIV infection did not have any significant

effect on the occurrence of E. bieneusi infection in

Table 1 Odds ratio (OR) of clinical predictors on the acquisition of

E. bieneusi infection was estimated using a single regression

model after all the predictors were mutually adjusted to each other

Clinical predictors Unit OR (95% CI) P-value

SIV status + vs. ) 2.52 (1.15–5.50) 0.021

Weeks since inoculation

(SIV positive)

+1 month 1.11 (1.00–1.24) 0.057

Weeks since inception

(SIV-naive)

+1 month 0.72 (0.58–0.89) 0.003

Supplementation status + vs. ) 2.72 (0.88–8.69) 0.082

Table 2 (A) Effect of clinical predictors on the acquisition of E. bieneusi infection in SIV-infected macaques. Odds ratio (OR) were estimated

using separate generalize estimation equation (GEE) models for each predictor while adjusting for SIV status, age at inoculation and weeks

since inoculation. (B) Odds ratio (OR) of clinical predictors on the acquisition of E. bieneusi infection was estimated using a single regression

model after all the predictors were mutually adjusted to each other

(A)

Predictors Unit

SIV-naı̈ve

unsupplemented (I)

SIV-infected

unsupplemented (II)

SIV-infected

supplemented (III)

Group comparison

(P-value)

OR (95% CI) P OR (95% CI) P OR (95% CI) P I vs. II I vs. III II vs. III

Weight gain +100g 0.95(0.81–1.11) 0.527 1.09(0.97–1.23) 0.147 0.95 (0.74–1.20) 0.646 0.147 0.968 0.282

BMI (kg/m2) +1 0.82 (0.54–1.26) 0.367 1.00 (0.66–1.52) 0.996 0.78 (0.37–1.64) 0.519 0.483 0.910 0.548

Body fat % +1 0.92 (0.85–0.99) 0.033 0.98 (0.87–1.08) 0.658 0.97 (0.83–1.13) 0.725 0.296 0.523 0.956

CD4 +100 ct1 – – 0.82 (0.74–0.92) 0.001 0.87 (0.77–0.97) 0.017 – – 0.483

Viral load (log10) +1 – – 1.71 (1.16–2.54) 0.007 2.07 (1.36–3.14) 0.001 – – 0.308

(B)

Clinical predictors Unit OR (95 % CI) P-value

Body weight gain +100 g 0.99 (0.90–1.09) 0.873

Body mass index (BMI) +1 kg/m2 1.01 (0.70–1.45) 0.957

Body fat % +1 0.91 (0.84–0.98) 0.016

CD4 counts ·100 0.91 (0.84–0.97) 0.008

Viral load (log10) +1 in log10scale 1.31 (1.06–1.63) 0.014

1ct ¼ counts.




either the unsupplemented (P ¼ 0.658) or supplemen-

ted (P ¼ 0.725) groups of animals. Although in SIV-

naı̈ve animals, increase in fat mass percentage was

associated with lower risk of E. bieneusi infection

(P ¼ 0.033), difference between the three groups was

not statistically significant (Table 2A).

When the data from the three groups were com-

bined, similar results were obtained (Table 2B). The

results indicated that increase in body weight and BMI

did not have any significant protective effect on the

acquisition of E. bieneusi infection (P ¼ 0.873, 0.957,

respectively). Whereas, increase in fat mass percentage

was associated with slightly lower risk of E. bieneusi

infection (OR ¼ 0.91, P ¼ 0.016; Table 2B).

CD4 counts and viral load

Both SIV/unsupplemented and SIV/supplemented

groups exhibited progressive decrease in CD4 counts

(P < 0.01, 0.025, respectively), and CD4 percentages

(P ¼ 0.006, 0.005, respectively) over time [16]. Changes

in CD4 counts had affected the likelihood of E. bien-

eusi shedding. As shown in Table 2A, higher periph-

eral blood CD4 counts were associated with lower risk

of excretion of E. bieneusi spores in both unsupple-

mented (OR ¼ 0.82, P ¼ 0.001) and supplemented

(OR ¼ 0.87, P ¼ 0.017) groups of SIV-infected ani-

mals. No significant difference was observed between

these two groups (P ¼ 0.483).

In contrast, higher viral load in SIV-infected maca-

ques was associated with higher risk of E. bieneusi

shedding in both SIV/unsupplemented (OR ¼ 1.71 per

log scale, P ¼ 0.007) and SIV/supplemented (OR ¼2.07 per log scale, P ¼ 0.001) groups of juvenile maca-

ques (Table 2A). Again, no significant difference was

observed between these two groups (P ¼ 0.308). Sim-

ilar results for CD4 and viral load were obtained when

the data from both groups of SIV-infected animals

were combined and compared with E. bieneusi spore

excretion (Table 2B).

Serum micronutrient levels

As observed previously, no differences in the levels of

serum Vitamin A, E and selenium were observed

between the three groups of macaques, except for a

significant decrease in serum zinc levels in SIV/unsup-

plemented animals as compared to SIV/supplemented

animals (M. Woods, personal communication). When

the odds ratio of getting E. bieneusi infection were

compared with four serum micronutrients levels, in this

study, the results showed no statistically significant dif-

ference between the association of Vitamin A, E,

Selenium and Zinc levels in the serum with the acquisi-

tion of E. bieneusi infection in all three groups of ani-

mals (Table 3). Although in SIV/supplemented group,

higher serum vitamin A level appeared to have some

protective effect against acquiring E. bieneusi infection

(OR ¼ 0.88 per lg/dl increase, P ¼ 0.010; Table 3),

the difference in OR between groups II and III was

insignificant (Table 3, p ¼ 0.209). Similar results were

obtained when data from all the three groups were

combined (data not shown).

Discussion

In the present study, we have examined the association

of different factors on the spontaneous acquisition of

E. bieneusi infection or on E. bieneusi spore shedding

in a population of SIV-infected macaques. Using a

selected group of SIV-infected juvenile macaques, we

have shown that the likelihood of acquiring E. bieneusi

infection from the environment increased after SIV-

infection and the risk increased further with each pass-

ing month post-infection. We have further shown that

this increased probability of acquiring E. bieneusi

infection in SIV-infected macaques can be directly rela-

ted to both [1] the decrease in peripheral blood CD4

counts, indicating that CD4 T cells may be responsible

for protection against this opportunistic infection, and

[2] the increase in viral load in the serum samples

Table 3 Odds ratio1 (OR) of serum micronutrients values as a predictors of E. bieneusi infection in SIV-infected macaques

Predictors Unit

SIV-naı̈ve

unsupplemented (I)

SIV-infected

unsupplemented (II)

SIV-infected

supplemented (III) Group comparison (P-value)

OR (95% CI) P OR (95% CI) P OR (95% CI) P I vs. II I vs. III II vs. III

Vitamin A +1 lg/dl 0.89 (0.78–1.02) 0.095 0.96 (0.90–1.03) 0.247 0.88 (0.79–0.97) 0.010 0.484 0.824 0.209

Vitamin E +100 lg/dl 0.72 (0.40–1.32) 0.291 0.80 (0.58–1.10) 0.168 0.79 (0.54–1.16) 0.230 0.376 0.827 0.748

Selenium +10 lg/dl 1.04 (0.89–1.23) 0.602 1.01 (0.84–1.21) 0.939 0.89 (0.71–1.11) 0.286 0.765 0.263 0.365

Zinc +100 lg/dl 1.07 (0.69–1.65) 0.762 0.90 (0.63–1.28) 0.552 1.21 (0.85–1.71) 0.291 0.103 0.689 0.234

1Odds ratio were estimated using separate generalize estimation equation (GEE) models for each predictor while adjusting for SIV status,

age at inoculation and weeks since inoculation.




during SIV disease progression. This study showed a

high prevalence of E. bieneusi infection in the popula-

tion of SIV-infected macaques. The trend of sponta-

neous acquisition of E. bieneusi infection with

progression of SIV/AIDS in macaques, manifested by

escalating viral load and decrease of peripheral CD4

lymphocyte count, is consistent with observations in

humans with HIV/AIDS in whom chronic diarrhea

and wasting are the prominent features in individuals

with CD4 cell counts below 100/mm3 [2, 8, 9, 22].

Decrease in CD4 cell counts in HIV/SIV infection also

led to the acquisition of other enteric opportunistic

infections such as C. parvum and Mycobacterium avi-

um, but no C. parvum infection was observed in these

groups of animals. Although only peripheral blood

CD4 cell counts were measured in this study, profound

CD4 T cell depletion in the mucosal immune system,

shortly after challenge with SIV [44, 42, 43], probably

plays a major role in the establishment of E. bieneusi

infection. Similarly, several studies have shown that

CD4+ T cells are markedly decreased in intestinal

biopsies of human with AIDS at various stages of

infection [23, 30, 31]. The spontaneous presence of E.

bieneusi in feces could be either due to newly acquired

infection or reactivation of previously established

infection because of SIV-related immunodeficiency.

Infections with E. bieneusi may have occurred early in

macaques, and presumably in humans, but as the

infections are subclinical they are not routinely detec-

ted. But the major clinical signs such as diarrhea was

largely absent in the infected SIV-infected macaque

population, which is consistent with the recent study

reported by Green et al. [18]. Diarrhea and wasting in

both human and macaque species develop at the ter-

minal phase of AIDS, and in the absence of supportive

therapy in macaques, these animals die before symp-

toms of diarrhea due to microsporidiosis begin.

In another investigation in which the rhesus maca-

que model was used to determine the effects of SIV-

infection on growth and body composition, it was

found that the infection caused alteration in body

composition similar to that seen in HIV-infected

humans [14, 13, 26, 27, 32]. After an initial loss of

body weight predominantly from fat tissue, a second

phase of compensatory growth followed. In the final

phase there was a loss from all body compartments

shortly before death [14]. Our study suggests that

changes such as body weight, BMI and percentage

body fat did not significantly impact the spontaneous

acquisition of E. bieneusi infection in SIV-infected

macaques. Although in SIV-naı̈ve animals, increase in

the percentage of fat mass was associated with lower

risk of E. bieneusi infection, the difference between

SIV naı̈ve and SIV-infected groups was statistically

insignificant.

Investigations on micronutrients in some HIV-infec-

ted patients suggested that higher serum values of

selective micronutrients had strong protective effects

on HIV pathogenesis [15, 33, 38], while others demon-

strated deleterious effect [10, 11]. In one study, persons

with low serum selenium levels had a 20-fold increased

risk of death [3] and those with high serum vitamin E

levels had a 30% lower risk of progression to AIDS

[39]. Similarly, SIV-infected macaques showed a signifi-

cant decline in blood selenium levels with the develop-

ment of simian AIDS [51] while others showed

decrease in serum zinc levels, but not in serum vitamin

A and E, during the progression of disease from 0 to

24 weeks in SIV-infected macaques (M. Woods, perso-

nal communication). It appears the spontaneous acqui-

sition of E. bieneusi infection in SIV-infected macaques

was independent of any such changes in serum micro-

nutrient levels (Vitamin A, E, selenium and zinc) meas-

ured. Although animals on dietary supplement,

especially zinc supplementation, displayed a marginally

higher risk of acquiring E. bieneusi infection than non-

supplemented animals, difference between the groups

was inconclusive. This is in agreement with the study

where it was shown that higher levels of zinc intake

was associated with an increased risk of progression to

AIDS in HIV-infected individuals [37, 38]. In a separ-

ate study on the same group of animals we have used,

Goldin et al. [16] reported that micronutrient supple-

mentation at two to three times the estimated nutri-

tional requirement for nonhuman primates is

deleterious in SIV-infected macaques fed an otherwise

healthy diet, with a relative risk of death of 2.4 com-

pared to the non-supplemented group. The mechanism

by which micronutrients may result in increased infec-

tion is not understood, but it has been shown that

vitamin A increased the multiplication and differenti-

ation of lymphoid and myeloid cells which lead to

increased expression of the chemokine receptor CCR5,

that, in turn, increases the progression of HIV-infec-

tion [24, 40, 47]. However, in contrast to this detrimen-

tal effect of Vitamin A supplementation, data shown

in Table 3 indicates that the infected and supplemented

animals, which had higher serum Vitamin A levels,

showed some protection regarding development of

E. bieneusi (p ¼ 0.010). The small number of animals

(n ¼ 12) in the supplemented group needs to be kept

in mind when interpreting these data. It is evident that

further studies in which individual vitamins and miner-

als are added to the diet are needed to evaluate which

component or components contribute to a more-rapid

disease progression.




Our study demonstrated that acquisition of E. bien-

eusi infection is related to SIV disease progression, per-

ipheral blood CD4 counts and viral load in the serum

and may be independent of changes in body composi-

tion and serum micronutrient levels.

Acknowledgments

We gratefully acknowledge the technical assistance of

Amanda Little and Katherine George as well as the

outstanding animal care of the veterinary staff of the

New England Regional Primate center.

This work was supported by grants from National

Institute of Diabetes and Digestive and Kidney Dis-

eases (P01DK55510) and the Primate Center Public

Health Service (P51RR00168–42).

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Factors contributing to spontaneous Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected macaques