life
Article
Parasitism-Induced Changes in Microbial Eukaryotes of
Peruvian Alpaca Gastrointestinal Tract
Diana Sanchez 1 , Celso Zapata 2, Yolanda Romero 3,4 , Nils H. Flores-Huarco 1, Oscar Oros 2 ,
Wigoberto Alvarado 5, Carlos Quilcate 4, Hada M. Guevara-Alvarado 5 , Richard Estrada 3,4,* and Pedro Coila 2,*
1 Unidad de Post Grado de la Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional del
Altiplano de Puno, P.O. Box 291, Puno 21001, Peru; dsanchez@epg.unap.edu.pe (D.S.);
dbionils@gmail.com (N.H.F.-H.)
2 Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional del Altiplano de Puno, P.O. Box 291,
Puno 21001, Peru; czapata@unap.edu.pe (C.Z.); odoros@unap.edu.pe (O.O.)
3 Instituto de Investigación en Bioinformática y Bioestadistica (BIOINFO), Av. Raúl Ferrero 21,
Lima 15024, Peru; yolanda888@hotmail.com
4 Dirección de Desarrollo Tecnológico Agrario, Instituto Nacional de Innovación Agraria (INIA), Av. La Molina
1981, Lima 15024, Peru; promegnacional@inia.gob.pe
5 Facultad de Ingeniería Zootecnista, Agronegocios y Biotecnología, Universidad Nacional Toribio Rodríguez
de Mendoza de Amazonas (UNTRM), Cl. Higos Urco 342, Chachapoyas 01001, Peru;
wigoberto.alvarado@untrm.edu.pe (W.A.); hada.guevara@untrm.edu.pe (H.M.G.-A.)
* Correspondence: genomica@inia.gob.pe (R.E.); pcoila@unap.edu.pe (P.C.)
Abstract: Alpacas, important genetic resources in the Andean region of Peru, are vulnerable to
diarrhea caused by pathogenic parasites such as Eimeria lamae and Giardia sp., which can be fatal,
especially in neonates, due to their physiological immaturity and limited adaptability. The study
investigated the diversity and abundance of intestinal fungi and protists in alpacas infected with
Eimeria lamae and Giardia sp. compared to healthy alpacas. A total of 19 alpacas, aged between
one and two months, were included. They were divided into two groups, one with pathological
Citation: Sanchez, D.; Zapata, C.; conditions (nine) and the other healthy (ten). Parasitological analyses for the detection of parasites
Romero, Y.; Flores-Huarco, N.H.; and subsequent molecular analysis were performed on the collected fecal samples. The results
Oros, O.; Alvarado, W.; Quilcate, C.; revealed a greater diversity and abundance of protists in infected alpacas in comparison with healthy
Guevara-Alvarado, H.M.; Estrada, R.;
alpacas, while the fungal composition did not show significant changes. Therefore, parasitic infections
Coila, P. Parasitism-Induced Changes
affect the protist component of the alpaca gut microbiota. Also, it was observed that Blastocystis was
in Microbial Eukaryotes of Peruvian
Life identified in all healthy alpacas, serving as a possible marker of the health of the intestinal microbiota;Alpaca Gastrointestinal Tract.
2024, 14, 187. https://doi.org/ in addition, Prussia and Pichia are beneficial fungi that help control diseases. This groundbreaking
10.3390/life14020187 study in neonatal alpacas is the first to explore potential changes in the intestinal microbiota during
an infectious state, underscoring the importance of further research to comprehend its effects on
Academic Editors: Xiaoyuan Wei,
alpaca health and immune responses.
Feilong Deng, Jianmin Chai and
Xiaofan Wang
Keywords: alpacas; metabarcoding; microbiota diversity; parasites; biomarkers; NGS
Received: 10 November 2023
Revised: 21 December 2023
Accepted: 29 December 2023
Published: 27 January 2024 1. Introduction
In the Peruvian Andes, alpacas stand as indigenous domestic camelids crucial to the
region’s genetic diversity [1]. These animals, predominantly of the Huacaya and Suri breeds,
Copyright: © 2024 by the authors. contribute significantly to the high-altitude areas of the Andes, with Huacaya prized for
Licensee MDPI, Basel, Switzerland. the quality of its fiber [2–4]. However, diarrhea in alpacas poses a severe threat, particularly
This article is an open access article to newborns (crias), and is often the leading cause of mortality [5]. This condition results
distributed under the terms and in nutrient and water loss, leading to energy deficits, weakness, and increased mortality,
conditions of the Creative Commons especially among crias due to their physiological immaturity [6,7].
Attribution (CC BY) license (https:// The most common infectious pathogens responsible for diarrhea in alpacas include
creativecommons.org/licenses/by/ Salmonella sp., Escherichia coli, Giardia sp., Coccidia, and Cryptosporidium sp. [8]. The vulner-
4.0/). ability to gastrointestinal pathogens emphasizes the significance of comprehending and
Life 2024, 14, 187. https://doi.org/10.3390/life14020187 https://www.mdpi.com/journal/life
Life 2024, 14, 187 2 of 16
addressing diarrhea in alpacas, particularly among the susceptible cria population. In this
context, the diagnosis of coccidiosis often occurs in neonates and juvenile camelids [9].
Oocysts cause direct damage to the epithelial mucosa of the small intestine, resulting in the
development of enteritis and diarrhea [10]. The etiology of coccidiosis, an enteric parasitic
disease, manifests through symptoms such as anorexia, the onset of colic, diarrhea, and,
eventually, sudden death, especially in neonates [11]. Moreover, five species of Eimeria
have previously been identified as causative agents of diseases: E. ivitaensis, E. lamae, E.
macusaniensis, and E. punoensis [12,13].
Giardiasis is a prevalent disease in ruminants, manifesting with symptoms like diar-
rhea, weight loss, and malabsorption. However, asymptomatic infections are also frequently
observed. The etiology of giardiasis involves fecal–oral transmission through direct con-
tact with infected humans or animals and the ingestion of water or food contaminated
with cysts, highlighting the importance of proper hygiene and sanitation practices in its
spread [14]. Giardia was initially reported in llamas in 1987 [15]. However, there has been a
limited amount of research concerning the presence of Giardia in alpacas [16–19].
Intestinal parasites, particularly protozoa, can disrupt the structure of the intestinal
microbiota, leading to diseases [20]. Host defense strategies against parasitic infections,
including immune responses, are triggered in response to parasite invasion [21]. However,
the immune system plays a crucial role in regulating the intestinal microbiota and its
relative composition to maintain a mutually beneficial symbiotic relationship between
the host and microorganisms [22]. Species belonging to the Eimeria genus influence host
immune responses to facilitate their invasion and colonization by reducing the production
of inflammatory cytokines [22], thus altering the balance of the intestinal microbiota.
Nevertheless, there are studies suggesting that Giardia may protect against the development
of diarrheal diseases by modulating the immune response, although further research is
needed in this regard [23].
Metagenomics plays a pivotal role in scientific exploration, offering intricate insights
into microbial communities within diverse environments, including the intestinal tract [24].
The use of the 18S rRNA gene in biodiversity and ecology studies is notable for its ubiquity
in eukaryotic organisms and its divergence in different loci. This versatile molecular tool
allows for the acquisition of insightful data on the taxonomy and phylogeny of micro-
bial communities, making a significant contribution to our understanding of ecosystem
structure and function [25].
Although the gut microbiota plays a significant role in ruminants [26], information
about the microbial community in alpacas is limited, especially beyond the stomach,
and a proper understanding is lacking about how this community responds to dietary
imbalances [27]. The main objective of this study is to acquire a greater understanding of the
composition of the microbial community in alpacas and how it is influenced in response to
episodes of diarrhea, taking into account the presence of two pathogens, Eimeria lamae and
Giardia sp. Such research will help further our understanding of the complex interactions
between gut microbiota, dietary factors, and health outcomes in alpacas.
2. Materials and Methods
2.1. Animal and Sample Collection
A total of nineteen alpacas, aged between one and two months, were obtained from
the La Raya Experimental Center, an entity belonging to the National University of the
Altiplano Puno. The selection of these specimens was carried out in compliance with the
Peruvian National Law No. 30407, relating to ‘Animal Protection and Welfare’.
The sample consisted of 9 alpacas with pathological conditions and 10 healthy alpacas,
with an equal distribution of males and females in the group with diarrhea (4 males
and 5 females). The pathological conditions in the affected alpacas were diagnosed by
veterinarians affiliated with the Animal Health Laboratory of the Professional School
of Veterinary Medicine at the National University of San Antonio Abad del Cusco. All
individuals received a diet designed to meet their particular nutritional needs as alpacas.
Life 2024, 14, 187 3 of 16
Hemoglobin levels, leukocyte counts, heart rate, and respiratory rate were assessed in
alpacas overall.
To maintain sample integrity, we separated the alpacas with diarrhea from the healthy
ones a day before sampling, using sterile tools under aseptic conditions. The collected
samples were transferred to sterile 50 mL plastic containers and quickly transported to the
laboratory, where they were initially stored at 5 ◦C for subsequent parasitological analysis
and subsequently at −80 ◦C for subsequent analysis.
2.2. Parasitological Study of Feces
A direct microscopic examination was performed, using parasitological lugol as a
stain [28], for the identification of protozoan cysts (Giardia sp.) and oocysts (Eimeria lamae)
in fecal samples from affected alpacas. Then, qualitative flotation concentration methods
were applied with saturated sucrose solution [29], followed by the implementation of the
modified McMaster quantitative method [30], specifically in fecal samples from alpacas
with pathological conditions.
2.3. DNA Extraction and Sequencing
Genomic DNA was extracted from fecal samples using the PureLink microbiome DNA
purification kit (Invitrogen, Waltham, MA, USA). To assess DNA quality, Qubit® was used
for quantification, and DNA integrity was visualized by running an electrophoretic gel
on 1% agarose. This DNA was prepared for V4 sequencing using primer 528 forward,
5′-GCGGTAATTCCAGCTCCAA-3′ and 706 reverse, 5′-AATCCRAGAATTTCACCTCT-3′
targeting the 18S rRNA gene [31]. The PCR protocol commenced with an initial denatura-
tion step at 94 ◦C for 2 min, followed by an initial set of 5 cycles involving denaturation
at 94 ◦C for 45 s, annealing at 52/54 ◦C for 45 s each, and elongation at 72 ◦C for 1 min.
This was succeeded by 35 additional cycles with a reduced annealing temperature set at
50/52 ◦C. The process concluded with a final elongation step at 72 ◦C for 10 min. Subse-
quently, this region was individually amplified from each sample using the TruSeq® DNA
sample preparation kit without PCR from Illumina, along with the appropriate primers.
Library quality was evaluated using the Qubit 2.0 fluorometer from Invitrogen and the
fragment analyzer from the Agilent Bioanalyzer 2100 system. Amplicon libraries were then
sequenced using the 2 × 250 paired-end protocol on the Illumina Novaseq 6000 platform
(San Diego, CA, USA).
2.4. Bioinformatics Analysis
During the preparation and analysis of the sequencing data, we utilized the Quantita-
tive Insights Into Microbial Ecology (QIIME) analytical platform [32]. Processed through
the DADA2 v.1.18 protocol [33], paired-end fastq files underwent handling, and ampli-
con sequence variants (ASVs) were generated. In the initial stages of the process, quality
assessment, trimming, and noise reduction were carried out on the forward and reverse
sequences, before their integration into the Amplicon Sequence Variants (ASV), with the
purpose of mitigating the potential for inaccurate ASVs. Exclusion was performed for
unique sequences whose total abundance did not exceed 10 reads in the global set of
samples. The taxonomic categorization of the sequences was carried out by using the naive
Bayes classifier integrated into QIIME2, previously calibrated with the Silva Reference
v.138.1 database, for the identification of protists and fungi. The alignment of high-quality
sequences was carried out using the MAFFT tool [34].
2.5. Statistical Analysis
Rarefaction curves were generated for each sample to assess the sequencing depth.
Alpha diversity of intestinal fungal and protozoan communities was determined based
on the relative abundance distribution of Operational Taxonomic Units (OTUs) in each
sample. Statistical analysis of the data was performed using the R package Phyloseq [35]
in R (v4.1.1) [36], calculating alpha diversity metrics, such as Observe, Pielou, Shannon,
Life 2024, 14, 187 4 of 16
and Simpson, with the MicrobiotaProcess library [37]. Kruskal–Wallis tests were used to
evaluate the distribution of these metrics among the analyzed groups. Beta diversity was
determined using the Bray–Curtis method and visualized through Principal Coordinate
Analysis (PCoA). Differences in fungal and protist communities between groups were
evaluated by PERMANOVA analysis with 9999 permutations [38], using the R Vegan
package [39], considering values of p < 0.05 statistically significant. Additionally, LDA
scores were obtained using the LEfSe algorithm.
3. Results
In this study, the presence of Eimeria lamae oocysts was observed in fecal samples
from two-month-old alpacas, while the presence of Giardia sp. cysts was detected in
samples from one-month-old alpacas. In contrast, no parasites were detected in the control
group of alpacas at 1 month and 2 months of age. Regarding two-month-old Eimeria lamae
samples, an average of 85,750 oocysts per gram of feces (OPG) was observed in samples
with diarrhea, whereas no oocysts were found in samples without diarrhea. Similarly, no
oocysts were detected in one-month-old alpacas (Table S1). In addition, biochemical data
from alpacas in relation to their health status were obtained (Table S2).
For protists, a total of 3,901,824 high-quality reads were obtained, with an average of
52,577 high-quality reads, a maximum of 1,112,183 high-quality reads, and a minimum of
9032 high-quality reads. Similarly, for fungi, a total of 6,733,314 high-quality reads were
generated, with an average of 283,817 high-quality reads, a maximum of 1,219,543 high-
quality reads, and a minimum of 34,082 high-quality reads.
3.1. Alpha Diversity of the Gut Microbiota in the Alpaca Population
To further explore the variations in the intestinal microbiota communities of fungi
(Figure S1) and protists (Figure S2) concerning infection by parasite type, the use of the
rarefaction curve was sought. An expected diversity was obtained in the sampling of fungal
communities (Figure S1) and protists (Figure S2), this curve demonstrated a preference for
optimization. Consequently, the dataset was considered suitable for further analysis.
The results of the alpha diversity index are shown in Figure 1. For protists (Figure 1A),
significant differences were identified between the group of patients diagnosed with Giardia
sp. at one month and the Eimeria lamae negative group, Pielou index (p = 0.0087), Shannon
index (p = 0.0043), and Simpson index (p = 0.0043). Significant differences were also
observed in the two-month patient group diagnosed with Eimeria lamae and the Eimeria
lamae negative group, Pielou index (p = 0.0043), Shannon index (p = 0.0043), and Simpson
index (p = 0.0043). The Shannon and Simpson indices provide a tool to evaluate the
diversity present in the intestinal microbiota. The Pielou index reflects the uniformity
in the distribution of species abundance in the microbial community, which is crucial to
understanding the evenness in the presence of different species in the ecosystem. Greater
richness is observed in the groups of patients infected by Giardia sp. and Eimeira lamae
However, in the context of fungal composition (Figure 1B), no statistically significant
disparities in alpha diversity were discerned.
3.2. Beta Diversity of the Gut Microbiota in the Alpaca Population
Principal Coordinate Analysis (PCoA) was conducted (Figure 2). When comparing the
similarity of protist composition (Figure 2A), a notable convergence was observed in both
healthy and diseased groups concerning the influence of parasites such as Eimeria lamae and
Giardia sp., resulting in wider distance dispersion. The Adonis test further validated this
observation, a statistical analysis highlighting the influence of health status. Health status
demonstrated statistical significance with a p-value of 0.0003 (Table 1), further emphasizing
the influential role of health status in the composition of intestinal protist microbiota.
Life 2023, 13, x FOR PEER REVIEW 5 of 17 
 Life 2024, 14, 187 5 of 16
Figure 1. Alpha diversity (Observe, Pielou, Shannon, and Simpson) gut microbiota indices betw een
Figuhre a1l.t hAclponhdai dtioivnesrwsitityh (IOllbnseessrvGei,a Prdiiealospu.,, SIlhlnaensnsoEnim, aenrida  lSaimape,sGoina)r dgiua ts pm. incerogabtiiovtea aindiEciems ebreiatwlameeane 
healtnhe cgoantidveit.io(Ans)  Pwriotthis Itllanlpehssa Gdiiavredrisai tsypg.,u Itllmneicsrso Ebiimoteariian dlaemx.ae(,B G) iGarudtima sipcr.o nbeiogtaatiivned eaxndof Efiumnegraial  alalpmhaae 
negadtiivvee.r s(Aity). Protist alpha diversity gut microbiota index. (B) Gut microbiota index of fungal alpha 
diversity. 
3.2. Beta Diversity of the Gut Microbiota in the Alpaca Population 
Principal Coordinate Analysis (PCoA) was conducted (Figure 2). When comparing 
the similarity of protist composition (Figure 2A), a notable convergence was observed in 
both healthy and diseased groups concerning the influence of parasites such as Eimeria 
 
Life 2023, 13, x FOR PEER REVIEW 6 of 17 
 
lamae and Giardia sp., resulting in wider distance dispersion. The Adonis test further val-
idated this observation, a statistical analysis highlighting the influence of health status. 
Health status demonstrated statistical significance with a p-value of 0.0003 (Table 1), fur-
ther emphasizing the influential role of health status in the composition of intestinal pro-
tist microbiota. 
Similarly, the analysis was conducted for the fungal group (Figure 2B), revealing a 
slight separation in the dispersion of groups based on health status. This finding was cor-
roborated by the Adonis test, yielding a significant p-value of 0.0092 (Table 1), thus em-
Life 2024, 14, 187 phasizing the influence of health status on the composition of the intestinal funga6l omf i1c6ro-
biota. 
 
Figure 2. PriFnicgiuprael 2C. oPorrindciinpaatle CAonoardlyinsiaste( PACnoaAly)spisl o(PtCoofAb)e tpalodti voef rbseitay dbiavseerdsitoyn bBasready –oCn uBrrtaisy–dCisutratnisc deis-
derived fromtasnecqeu deenricvinedg fdroamta .seTqhueesnacmingp ldeastaa.r eThree psaremspenletse dareb yrecporelosersntaendd byd icvoildoersd ainndto dtiwvidoe: dh einatloth tywo: 
(blue) and illhneeaslsth(yre (db)luger)o aunpds i.llTnhesesr e(riesda) lgsroouapssh. aTpheerree pisr easlseon ata sthioanpef orerpirnefseecnttiaotniobny foGr iianrfdeicatisopn. b(cyi rGcilaer)dia 
sp. (circle) and Eimeria lamae (triangle). (A) Beta diversity of protists. (B) Beta diversity of fungi. 
and Eimeria lamae (triangle). (A) Beta diversity of protists. (B) Beta diversity of fungi.
Table 1. Two-way PERMANOVA of the Bray–Curtis distance between the age and health status of
 the alpacas.
Items Df SumOfSqs R2 F Pr (>F)
Protists
Influenced by parasites 1 0.3052 0.04171 0.8475 0.7089
Health status 1 1.1938 0.16313 3.3146 0.0003 ***
Influenced by parasites:
Health status 1 0.4165 0.05691 1.1564 0.2005
Fungi
Influenced by parasites 1 0.17297 0.07116 1.537 0.11
Health status 1 0.27745 0.11414 2.4654 0.0092 **
Influenced by parasites:
Health status 1 0.17985 0.07399 1.5982 0.0906
(**) and (***) implicit significance (p < 0.01 and p < 0.001, respectively).
Life 2024, 14, 187 7 of 16
Similarly, the analysis was conducted for the fungal group (Figure 2B), revealing a
slight separation in the dispersion of groups based on health status. This finding was
Life 2023, 13, x FOR PEER REVIEW corroborated by the Adonis test, yielding a significant p-value of 0.0092 (Table 17), otfh u17s 
 emphasizing the influence of health status on the composition of the intestinal fungal
microbiota.
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iinntteennssiittyy aanndd ddoott ssiizzee aarree prropoorrttiioonnaall ttoo tthhee ccoorrrreellaattiioonn vvaalluueess wiitthhiinn eeaacchh ccoorrrreellaattiioonn ggrroouupp. .HH: :
Heemoogllobiin;; L: LLeeuukkooccyyttee;;H HRR: H: HeaeratrRt aRtea;teB;F B: RF:e sRpeirsaptiorarytoRrya teR.aAtelp. hAalpdhivae rdsivtyerisnidtyic eins:dOicbese: rOvebd- ,
sCerhvaeod1,, CAhCaEo,1S, hAaCnEn,o Snh, aSnimnopnso, nSi,manpdsoPni,e alonud. Pielou. 
T3a.4b.leC 1o.m Tpwoosi-twioany TPaExRoMnoAmNicOoVf Ath eofG tuhet  MBriacyr–oCbiuorttais distance between the age and health status of 
the alpFaigcausr. e 4 presents the composition at the phylum level in fungi and at the class level in
protists. The results indicate that concerning fungi (Figure 4A), in the intestinal microbiota
of the IllnesIsteGmiasr dia sp., IllnessDEfim eriaSluamaOe,fGSiqarsd ia sp. nReg2a tive and EiFm eria lamaPern (e>gFa)t ive
groups, the phyla Ascomycota (25%, 21.88%, 20%, and 31.94%) and Basidiomycota (41.44%,
Protists      
Influenced by parasites 1 0.3052 0.04171 0.8475 0.7089 
Health status 1 1.1938 0.16313 3.3146 0.0003 *** 
 
Life 2024, 14, 187 8 of 16
Life 2023, 13, x FOR PEER REVIEW 9 of 17 
 
19.25%, 20.17%, and 18.90%) were the predominant phyla, representing approximately 97%
of the total fungal composition.
Figure 4. Relative abundances were observed in 1-month disease groups infected with G iardia sp.
Figaunred 42.- Rmeolanttihvei lalnbeusnsdgarnocueps swinerfee cotebdsewrvietdh Eini m1-emriaonlatmh adei,sienasceo mgrpoaurpiss oinnfwecittehdG wiaitrhd iGa isapr.dinae sgpa.t ive and
andE 2im-meroinatlha milalneenses ggartoiuvepsg irnofuepctse.dO wniltyh tEhiemmeroias tlarmepaer,e isne cnotemdptaarxisaoanr ewpitrhe sGeinarteddia. s(pA. )nRegealatitvivee aanbdu ndance
Eimoefritah leamaoe sntepgraetidvoem girnoaunpts.p Ohnylya itnhef umnogsit. r(eBp)reRselnatteidv etaxbau anrdea pnrceeseonftethde. (mAo) sRtealabtuivned abnutnc-lasses in
danpcreo otifs tthse.  most predominant phyla in fungi. (B) Relative abundance of the most abundant classes 
in protists. 
Regarding class abundance in protists (Figure 4B), it was observed that in the intesti-
3.5.n Daletmecitciorno bofi oMtaodoiffictahteionIlsl nine stsheG Tiaaxrodniaomspic. ,CIollmnpeosssitEioinm Leirniakelda mtoa teh,eG Iniaflrudeinacse po.f Negative, and
ParEaismiteesr ia lamae negative groups, the classes Stramenopiles (12.16%, 15.98%, 28%, and 43.61%),
AlLvienoelaart aD(is4c5r.i3m%in, a1n3t.6 A%n,a1l2y.s6is5 %(L,DaAnd) w28a%s u),tialnizdedR thoi zaasrsieass( 2e6ff.6e%ct ,s5iz1e.9 a%nd, 1 d7e.3li%ne,aatne d 3.5%)
diffaecrceonucenst eind tfhoer b9a0c%teroifatl hceomprpootsisittiocno mbeptwoseietino nsicink athlpeaaclapsa icnafemctiecdr owbiitoht aG.iardia sp. and 
Eimeria lamae, and Giardia sp. negative and Eimeria lamae negative groups. 
 
Life 2024, 14, 187 9 of 16
Life 2023, 13, x FOR PEER REVIEW 3.5. Detection of Modifications in the Taxonomic Composition Linked to the Influe1n0 coef o1f7 Parasites
 
Linear Discriminant Analysis (LDA) was utilized to assess effect size and delineate
differences in the bacterial composition between sick alpacas infected with Giardia sp. and
EimeIrnia Fliagmuraee 5, ,a pnrdotGisitasr wdieares pid.ennetigfiaetdiv. Iena tnhed GEiiamrdeirai aspl.a-minafeecnteedg gartiovuepg, trhoeu pprse.sence of 
MischIoncoFcigcauleres w5,aps roobtsisetrsvewde (rFeigidureen 5tiAfi)e. dF.uIrnthtehrme Goriea,r gdeianesrpa. -siuncfhe catse dCegrcroomuopn,atsh, eHpetr-esence of
eMroimscitha,o Gcoycmcnaoledsinwipahsyoicbidseaerv, Beadci(lFlairgiouprehy5tAin)a., FCuerrcthozeoram, Sopreir,ogtreicnheeraa, asnudch GalisssComeroconma-onas, Het-
deriodma iwtae,rGe iydmenntiofdieidn iinp hsiycikc iadlpaaec,aBsa (cFiilglaurrieo 5pBh).y Ftiinnaal,lyC, etrhceo Eziomae,rSiap liarmoatre-iicnhfeeact,eadn gdroGulpis somona-
edxihdiabiwteder ethied epnrteisfieendcei nofs iHcketaerlopmaictaa,s C(Feirgcoumroen5aBs, ).BFaicnilalallryio, pthheytEiniam, eCriearclaomzoaae,- iGnfyemct-ed group
nodiniphyicidae, Coccidia, RT5iin19, and Eocercomonas sp. HFCC 909 (Figure 5C). 
exhibited the presence of Heteromita, Cercomonas, Bacillariophytina, Cercozoa, Gymnodini-
phyicidae, Coccidia, RT5iin19, and Eocercomonas sp. HFCC 909 (Figure 5C).
Figure 5. Differences in the intestinal protist microbiota among various health states in alpacas. Bar
 
chart of Linear Discriminant Analysis (LDA) for differentially abundant genera. (A) Alpacas positives
Figure 5. Differences in the intestinal protist microbiota among various health states in alpacas. Bar 
with Giardia sp. versus Giardia sp. negative group. (B) All unhealthy alpacas versus negative (control).
chart of Linear Discriminant Analysis (LDA) for differentially abundant genera. (A) Alpacas 
(C) Alpacas positives with Eimeria lamae versus Eimeria lamae negative group.
 
Life 2023, 13, x FOR PEER REVIEW 11 of 17 
 
positives with Giardia sp. versus Giardia sp. negative group. (B) All unhealthy alpacas versus nega-
Life 2024, 14, 187 tive (control). (C) Alpacas positives with Eimeria lamae versus Eimeria lamae negative group. 10 of 16
In comparison to the Giardia sp. negative group, various taxa were identified, includ-
ing BlastocyIsnticso smp.p Saurbistoynpeto 1t0h Ce GAi6a,r Bdila stpo.cynsetgisa stipv.e Sgurbotuyp,ev 1a4r iCouows t1a,x Eanwtaemreoeidbae natnidfi eBdl,aisn-cluding
tocystisB lsaps.t oScuybsttiyspsep .1S4u Mbtoyupfelo1n0 (CFAig6u,rBel a5sAto)c. yAstdisdsitpio. nSualblyty, pthee1 4coCnotrwo1l ,gErnotuapm soheboawaend tBhlea stocystis
presenscpe. oSfu Bbltaysptoecy1s4tiMs sopu. flsounbt(yFpigeu 1r0e C5A)6., ABdladstioticoynstaisll,y B, ltahsetoccoynstirso slpg.r soubptyspheo w14e dCothwe1p, resence
and EnotfamBloaesbtao c(yFsigtiusrsep 5.Bs).u Tbhtyep Eeim10eriCa Ala6m,aBe lnasetgoactyisvteis g, rBoluapst odciyssptliasysepd. Bsluasbttoycypseti1s,4 BClaos-w1, and
tocystisE nspta. msuobebtyap(Fei 1g4u rCeo5wB1)., Tanhde EOicmherroiamloamn ade anlegs a(tFiivgeugrero 5uCp).d isplayed Blastocystis, Blastocystis
Ins pF.igsurbet y6p, feu1n4giC wower1e, iadnedntOificehdr.o Imn tohnea gdraoleusp( iFnifgeucrted5 Cw)i.th Giardia sp., the presence 
of LeotiomyInceFteigs uwraes6 o, bfusenrgviedw (eFrieguidren 6tAifi)e. dA.dIdnittiohneagllryo,u inp sinicfke catlepdacwasi,t hgeGniearrad isauscph. ,asth e pres-
Candidena-cLeoodfdLereomtioymceys,c Leteeostiwomasycoebtsees,r avned C(Fyisgtoufrileob6aAsi)d.iuAmd wdietrioe nidaellnyt,ifiineds i(cFkigaulprea 6cBas)., genera
Finallys,u tchhe EasimCeraina dlaidmaa-eL-iondfedceterodm gyrocuesp, eLxehoibtiiotemdy tcheet epsr,esaenndceC oyfs tSoyfimlombaestridosiupmoraw (Feirgeuirdee ntified
6C). (Figure 6B). Finally, the Eimeria lamae-infected group exhibited the presence of Symmetro-
spora (Figure 6C).
 
Figure 6. Differences in the intestinal fungal microbiota among different health states in alpacas.
Linear Discriminant Analysis (LDA) bar chart displaying differentially abundant genera. (A) Alpacas
 positives with Giardia sp. versus Giardia sp. negative group. (B) All unhealthy alpacas versus negative
(control). (C) Alpacas positives with Eimeria lamae versus Eimeria lamae negative group.
Life 2024, 14, 187 11 of 16
In comparison to the Giardia sp. group, Thelebolus and Hanseniaspora were identified
(Figure 6A). Additionally, the control group displayed the presence of LKM11, Thelebolus,
Dothideomycetes, Hanseniaspora, Preussia, and Sordariales (Figure 6B). Eimeria lamae nega-
tive group showed LKM11, Dothideomycetes, Sordariales, Paramicrosporidium, Thelebolus,
Ustilaginaceae, and Pichia (Figure 6C).
4. Discussion
This study conducts the first analysis of fungi and protists in the intestinal micro-
biota of alpacas, comparing those affected by Eimeria lamae and Giardia sp. with healthy
counterparts, filling a gap in existing research. Fungi and protists coexist and interact
with other microorganisms in the intestinal tract of mammals [40]. Additionally, certain
protozoa belonging to the protist group are recognized as pathogens, and their presence
can influence the modulation of the intestinal microbiota composition in ruminants [41]
and other mammals [42].
In the analysis of alpha diversity of ASVs corresponding to protist communities, signif-
icant differences were identified, with higher richness observed in sick alpacas with Giardia
sp. and Eimeria lamae (Figures 1A and 2A). On the other hand, in fungal communities
(Figures 1B and 2B), richness remained constant. In the context of a clinical presentation of
diarrhea, a significant imbalance in the composition of the intestinal microbiota becomes
evident, leading to an increase in opportunistic microorganisms [43]. These opportunis-
tic microorganisms are predominantly parasitic in nature [44]. A comprehensive study
has revealed a pronounced richness of protozoan parasites in the intestinal microbiota
of mammals affected by this pathological condition [42,45]. Our finding unequivocally
demonstrates that in the context of a condition such as diarrhea, there is a significant in-
crease in the population of protists, particularly protozoans, in individuals afflicted by this
physiological disturbance. This increase in protists is noteworthy due to their role in modu-
lating bacterial and fungal communities through predatory activity [46]. They play a pivotal
role in regulating changes in the structure and dynamics of these microorganisms [47], thus
exerting a substantial influence on the intestinal microbiota of alpacas.
The influence of parasites on the composition of the microbiota is significantly related
to the health status, with no discernible differentiated effect attributable to the type of
parasite. Research conducted with pandas has emphasized the pivotal role of protists in
shaping the intestinal microbiota, highlighting their predatory activities [48]. Furthermore,
a significant presence of protists has been documented in non-human primates [49]. In con-
trast, prior investigations have not assigned significant importance to eukaryotes in human
individuals experiencing diarrhea as a result of Clostridioides difficile infection [43,50]. It is
worth noting that variability in the composition of protists is associated with factors that can
influence resource availability and interactions among protists and other microorganisms
in the intestinal microbiome, thereby affecting the structure of the protist community [48].
In light of this, the need for further similar studies to assess the impact of parasites on the
beta diversity of ruminants and other mammals is underscored.
A significant inverse correlation was observed between alpha diversity indices
(Figure 3), specifically Shannon, Simpson, and Pielou indices, and the biochemical pa-
rameter hemoglobin. Giardia sp. does not directly impact hemoglobin levels [51], in
the same way that Eimeria lamae does not affect hemoglobin levels [52]. However, both
pathogens induce diarrheal conditions, diarrhea can lead to a series of perturbations in
the immune system. These immune disruptions can lead to a cascade of effects, such as
increased susceptibility to infections and reduced immune surveillance. Ultimately, this
immune dysregulation translates into a complex biological response that directly affects
hemoglobin levels [53].
Furthermore, considering the specific pathological manifestations associated with
Giardia sp. and Eimeria lamae in alpacas expands upon the impact of these parasites on the
health of the host. Lesions observed in the epithelium of the jejunum and ileum villi, such
as eosinophil infiltration, hyperemia, and epithelial denudation, underline the pathological
Life 2024, 14, 187 12 of 16
impact of these infections [54,55]. These specific manifestations signify a direct effect on the
intestinal health of alpacas, potentially influencing the immune response and overall health,
which might correlate with alterations in the microbiota, warranting further investigation.
The results obtained regarding the taxonomy of fungi in alpacas (Figure 4A) show a
high degree of concordance with previous studies on ruminants [56–61]. Various studies
have identified Ascomycota and Basidiomycota as the most predominant fungal phyla
in mammals and ruminants, as seen in cows [56–58], buffaloes [59], and goats [60,61].
Similarly, the classes identified in alpaca protists in our study (Figure 4B) have been reported
in previous research on mammals, including humans [43,50], pandas [48], primates [49],
and cows [57]. However, there is limited research on the gut microbiota of fungi and
protists in alpacas.
The presence of Coccidia has been observed In individuals affected by Eimeria lamae
(Figure 5C) [16]. This parasite is commonly found in newborn alpacas, leading to severe
gastrointestinal manifestations, including diarrhea in approximately 80% of cases, and, in
more critical situations, the death of crias aged between 1 to 2 months [62]. The severity of
the infection can be particularly pronounced in captive camelids. This is because, in wild
environments, camelids less frequently exhibit noticeable clinical signs, possibly due to a
greater capacity to excrete a more substantial number of oocysts in various environments,
thereby reducing the likelihood of subsequent infection or a higher parasitic burden in the
organism [63].
In control groups (Figure 5), irrespective of parasite, the presence of various Blastocystis
subtypes was detected. While Blastocystis is known to potentially induce gastrointesti-
nal symptoms, such as diarrhea, constipation, abdominal pain, and flatulence [64], it
has also been reported in several studies to be present in alpacas without any clinical
signs of disease [58,65]. Additionally, this parasite has been documented in captive wild
animals [66,67]. Therefore, it is suggested that the colonization of Blastocystis may serve as a
reasonable marker for the assessment of gastrointestinal health [60]. Similarly, the presence
of the pathogen Entamoeba was observed in alpacas from the control group (Figure 5A,B).
Notably, the presence of this pathogen has been reported in healthy alpacas [68] and in ani-
mals from zoological collections [69,70]. This parallels the situation with Blastocystis, where
no clinical symptoms are evident in these healthy alpacas, despite the pathogen’s presence.
Genus Preussia was identified in the total control group of alpacas (Figure 6B), and
the presence of Genus Pichia was also noted in the two-month-old control group of alpacas
(Figure 6C). This occurrence has similarly been documented in yaks [71]. The presence of
these fungal genera in healthy alpacas can be attributed to the strong antibacterial capacity
of Preussia [72] and its antioxidative properties [73], which are heightened as they age.
These characteristics contribute to their resilience against diseases, bolstering their immune
system and enhancing their environmental adaptability. In the case of Pichia, it effectively
inhibits Candida infection [74].
Therefore, this is the first study of alpacas infected with parasites Eimeria lamae and
Giardia sp., assessing the changes in the fungal and protist intestinal microbiota. Hence, fur-
ther in-depth research is required to fully comprehend the implications of these infections
on alpaca health and microbiota balance, as well as their potential impact on the immune
response and adaptation to various environments.
5. Conclusions
In this study, an investigation was conducted into the diversity and composition of
intestinal fungi and protists in alpacas affected by Eimeria lamae and Giardia sp., with a
comparison made with healthy alpacas. The results showed increased protist diversity
and abundance in infected alpacas, while fungal richness remained largely consistent.
Regarding the composition of the intestinal microbiota, it was observed that both fungi and
protists exhibited variations influenced by their health status. This study provides valuable
insights into the impact of these parasites on alpaca intestinal microbiota, emphasizing the
need for further research to understand their effects on health and immune responses in
Life 2024, 14, 187 13 of 16
alpacas. However, it is crucial to acknowledge certain limitations inherent in the study.
Illumina sequencing may introduce bias in capturing microbial diversity, suggesting that
future studies incorporating PacBio-HiFi sequencing could offer a more comprehensive
assessment. Additionally, the inclusion of further biochemical variables in subsequent
research would enrich our understanding of the intricate dynamics within the alpaca
intestinal microbiota.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/life14020187/s1, Figure S1: species richness rarefaction curves
show the sequencing depth of 18S data obtained from fungi from gut samples. Figure S2: species
richness rarefaction curves show the sequencing depth of 18S data obtained from protists from gut
samples. Table S1: ooquistes per gram of feces (OPG) in 2-month-old alpacas with and without
diarrhea infected with Eimeria lamae. Table S2: biochemical parameters of alpacas about their health
status. H: hemoglobin; L: leukocyte; HR: heart rate; BF: respiratory rate.
Author Contributions: Conceptualization, D.S., C.Z., O.O., N.H.F.-H. and P.C.; methodology, D.S.,
C.Z., Y.R., O.O., N.H.F.-H., R.E. and P.C.; software, R.E., and Y.R.; validation, D.S., R.E. and Y.R.;
formal analysis, D.S., C.Z. and P.C.; investigation, D.S., C.Z., O.O., N.H.F.-H., W.A., C.Q., H.M.G.-A.,
R.E. and P.C.; resources, D.S., C.Z., O.O., N.H.F.-H., W.A., C.Q., H.M.G.-A. and P.C.; data curation,
R.E. and Y.R.; writing—original draft preparation, D.S., C.Z., Y.R., R.E. and P.C.; writing—review and
editing, D.S., C.Z., Y.R., O.O., N.H.F.-H., R.E. and P.C.; visualization, D.S., C.Z., Y.R., W.A., C.Q. and
H.M.G.-A.; supervision, R.E. and P.C.; project administration, D.S., and P.C.; funding acquisition,
D.S., W.A., C.Q., H.M.G.-A. and P.C. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by “Isolation and Molecular Identification of Anaerobic Bacteria
from Compartment 1 of the Alpaca”, project code: 377-2019, FONDECYT.
Institutional Review Board Statement: The sample collection from the cattle specimen was con-
ducted in accordance with the Peruvian National Law No. 30407: “Animal Protection and Welfare”.
Our institution has followed the guidelines of the “Committee for Scientific Research Ethics” (Resolu-
tion No. 0345-2018-CU-UNALM) for scientific research at the National Agrarian La Molina University
(UNALM). The undersigned declares that the protocol executed for the collection of fecal and blood
samples is of conventional application and is internationally recognized, under the requirements of
National Law No. 30407, “Animal Protection and Welfare Law”, in effect in Peru since 7 January 2016.
Resolution No. 0345-2018-CU-UNALM.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Acknowledgments: We thank Ivan Ucharima for image editing. Also, we thank Linda Arteaga for
her collaboration in the laboratory.
Conflicts of Interest: The authors declare no conflicts of interest.
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