Cryo-EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role.

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo-EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward-open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.

Membrane skeleton hyperstability due to a novel alternatively spliced 4.1R can account for ellipsoidal camelid red cells with decreased deformability.

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to ß-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene.

Genetic variability among and within domestic Old and New World camels at the α-lactalbumin gene (LALBA) reveals new alleles and polymorphisms responsible for differential expression.

α-Lactalbumin (α-LA), which is encoded by the LALBA gene, is a major whey protein that binds to Ca2+ and facilitates lactose synthesis as a regulatory subunit of the synthase enzyme complex. In addition, it has been shown to play central roles in immune modulation, cell-growth regulation, and antimicrobial activity. In this study, a multitechnical approach was used to fully characterize the LALBA gene and its variants in both coding and regulatory regions for domestic camelids (dromedary, Bactrian camel, alpaca, and llama). The gene analysis revealed a conserved structure among the camelids, but a slight difference in size (2,012 bp on average) due to intronic variations. Promoters were characterized for the transcription factor binding sites (11 found in total). Intraspecies sequence comparison showed 36 SNPs in total (2 in the dromedary, none in the Bactrian camel, 22 in the alpaca, and 12 in the llama), whereas interspecies comparison showed 86 additional polymorphic sites. Eight SNPs were identified as trans-specific polymorphisms, and 2 of them (g.112A>G and g.1229A>G) were particularly interesting in the New World camels. The first creates a new binding site for transcription factor SP1. An enhancing effect of the g.112G variant on the expression was demonstrated by 3 independent pGL3 gene reporter assays. The latter is responsible for the p.78Ile>Val AA replacement and represents novel allelic variants (named LALBA A and B). A link to protein variants has been established by isoelectric focusing (IEF), and bioinformatics analysis revealed that carriers of valine (g.1229G) have a higher glycosylation rate. Genotyping methods based on restriction fragment length polymorphism (PCR-RFLP) were set up for both SNPs. Overall, adenine was more frequent (0.54 and 0.76) at both loci. Four haplotypes were found, and the AA and GA were the most common with a frequency of 0.403 and 0.365, respectively. Conversely, a putative biological gain characterizes the haplotype GG. Therefore, opportunities for rapid directional selection can be realized if this haplotype is associated with favorable milk protein properties. This study adds knowledge at the gene and protein level for α-LA (LALBA) in camelids and importantly contributes to a relatively unexplored research area in these species.

Genetic parameters for different types of medullated fibre in Alpacas.

The quality of alpaca textile fibre has great potential, especially if objectionable fibres (coarse and medullated fibres) that cause itching are reduced, considering that objectionable fibres can be identified by diameter and medullation types. The objective of this study was to estimate genetic parameters for medullar types and their respective diameters to evaluate the possibility of incorporating them as selection criteria in alpaca breeding programmes. The research used 3149 alpaca fibre samples collected from 2020 to 2022, from a population of 1626 Huacaya type alpacas. The heritability and correlations of the percentages of non-medullated (NM), fragmented medulle (FM), uncontinuous medullated (UM), continuous medullated (CM), and strongly medullated (SM) fibres were analysed, also the fibre diameter (FD) for each of the medullation types. The heritability estimated for medullation types were 0.25 ± 0.01, 0.18 ± 0.01, 0.10 ± 0.01, 0.20 ± 0.01 and 0.11 ± 0.01 for NM, FM, UM, CM and SM, respectively. The genetic correlations for medullation categories ranged from 0.15 ± 0.03 to 0.66 ± 0.02 (in absolute values). The heritabilility estimated for fibre diameter (FD) of each of the medullation types were 0.29 ± 0.03, 0.27 ± 0.02, 0.35 ± 0.02, 0.30 ± 0.02, 0.25 ± 0.02 and 0.10 ± 0.02 for FD, FD_NM, FD_FM, FD_UM, FD_CM and FD_SM, respectively. The genetic correlations for fibre diameter of the medullation types ranged from 0.04 ± 0.04 to 0.97 ± 0.01. FD, NM and FM are the main traits to be used as selection criteria under a genetic index, since they would reduce fibre diameter, and also increase NM and FM, and, in addition reducing indirectly CM, SM, and SM_FD. Therefore, the quality of alpaca fibre could be improved.

Extended Viral Shedding of MERS-CoV Clade B Virus in Llamas Compared with African Clade C Strain.

Middle East respiratory syndrome coronavirus (MERS-CoV) clade B viruses are found in camelids and humans in the Middle East, but clade C viruses are not. We provide experimental evidence for extended shedding of MERS-CoV clade B viruses in llamas, which might explain why they outcompete clade C strains in the Arabian Peninsula.

Non-enzymatic glycation and aggregation of camel immunoglobulins induce breast cancer cell proliferation.

Glycation of biomolecules results in the formation of advanced glycation end products (AGEs). Immunoglobulin G (IgG) has been implicated in the progression of various diseases, including diabetes and cancer. This study purified three IgG subclasses (IgG1, IgG2, and IgG3) from Camelus dromedarius colostrum using ammonium sulfate fractionation and chromatographic procedures. SDS-PAGE was performed to confirm the purity and molecular weight of the IgG subclasses. Several biochemical and biophysical techniques were employed to study the effect of glycation on camel IgG using methylglyoxal (MGO), a dicarbonyl sugar. Early glycation measurement showed an increase in the fructosamine content by ~four-fold in IgG2, ~two-fold in IgG3, and a slight rise in IgG1. AGEs were observed in all classes of IgGs with maximum hyperchromicity (96.6%) in IgG2. Furthermore, glycation-induced oxidation of IgGs led to an increase in carbonyl content and loss of -SH groups. Among subclass, IgG2 showed the highest (39.7%) increase in carbonyl content accompanied by 82.5% decrease in -SH groups. Far UV-CD analysis illustrated perturbation of ß-sheet structure during glycation reaction with MGO. Moreover, glycation of IgG proceeds to various conformational states like aggregation and increased hydrophobicity. In addition, the cytotoxicity assay (MTT) illustrated the proliferation of breast cancer cells (MCF-7) with IgG2 treatment.

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

Targeting the Brain with Single-Domain Antibodies: Greater Potential Than Stated So Far?

Treatments for central nervous system diseases with therapeutic antibodies have been increasingly investigated over the last decades, leading to some approved monoclonal antibodies for brain disease therapies. The detection of biomarkers for diagnosis purposes with non-invasive antibody-based imaging approaches has also been explored in brain cancers. However, antibodies generally display a low capability of reaching the brain, as they do not efficiently cross the blood-brain barrier. As an alternative, recent studies have focused on single-domain antibodies (sdAbs) that correspond to the antigen-binding fragment. While some reports indicate that the brain uptake of these small antibodies is still low, the number of studies reporting brain-penetrating sdAbs is increasing. In this review, we provide an overview of methods used to assess or evaluate brain penetration of sdAbs and discuss the pros and cons that could affect the identification of brain-penetrating sdAbs of therapeutic or diagnostic interest.

Camels' biological fluids contained nanobodies: promising avenue in cancer therapy.

Cancer is a major health concern and accounts for one of the main causes of death worldwide. Innovative strategies are needed to aid in the diagnosis and treatment of different types of cancers. Recently, there has been an evolving interest in utilizing nanobodies of camel origin as therapeutic tools against cancer. Nanotechnology uses nanobodies an emerging attractive field that provides promises to researchers in advancing different scientific sectors including medicine and oncology. Nanobodies are characteristically small-sized biologics featured with the ability for deep tissue penetration and dissemination and harbour high stability at high pH and temperatures. The current review highlights the potential use of nanobodies that are naturally secreted in camels' biological fluids, both milk and urine, in the development of nanotechnology-based therapy for treating different typesQuery of cancers and other diseases. Moreover, the role of nano proteomics in the invention of novel therapeutic agents specifically used for cancer intervention is also illustrated.

Evaluation of alpaca tracheal explants as an ex vivo model for the study of Middle East respiratory syndrome coronavirus (MERS-CoV) infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to public health. Here, we established an ex vivo alpaca tracheal explant (ATE) model using an air-liquid interface culture system to gain insights into MERS-CoV infection in the camelid lower respiratory tract. ATE can be infected by MERS-CoV, being 103 TCID50/mL the minimum viral dosage required to establish a productive infection. IFNs and antiviral ISGs were not induced in ATE cultures in response to MERS-CoV infection, strongly suggesting that ISGs expression observed in vivo is rather a consequence of the IFN induction occurring in the nasal mucosa of camelids.

Middle East respiratory syndrome coronavirus infection in camelids.

Middle East respiratory syndrome coronavirus (MERS-CoV) is the cause of a severe respiratory disease with a high case fatality rate in humans. Since its emergence in mid-2012, 2578 laboratory-confirmed cases in 27 countries have been reported by the World Health Organization, leading to 888 known deaths due to the disease and related complications. Dromedary camels are considered the major reservoir host for this virus leading to zoonotic infection in humans. Dromedary camels, llamas, and alpacas are susceptible to MERS-CoV, developing a mild-to-moderate upper respiratory tract infection characterized by epithelial hyperplasia as well as infiltration of neutrophils, lymphocytes, and some macrophages within epithelium, lamina propria, in association with abundant viral antigen. The very mild lesions in the lower respiratory tract of these camelids correlate with absence of overt illness following MERS-CoV infection. Unfortunately, there is no approved antiviral treatment or vaccine for MERS-CoV infection in humans. Thus, there is an urgent need to develop intervention strategies in camelids, such as vaccination, to minimize virus spillover to humans. Therefore, the development of camelid models of MERS-CoV infection is key not only to assess vaccine prototypes but also to understand the biologic mechanisms by which the infection can be naturally controlled in these reservoir species. This review summarizes information on virus-induced pathological changes, pathogenesis, viral epidemiology, and control strategies in camelids, as the intermediate hosts and primary source of MERS-CoV infection in humans.

Plasma pharmacokinetics, pulmonary disposition, and safety of subcutaneous gamithromycin in alpacas.

The study objective was to determine the disposition of gamithromycin in plasma, peripheral blood polymorphonuclear cells (PMNs), pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells in alpacas. A single subcutaneous injection of gamithromycin (6.6 mg/kg) was administered to six healthy adult alpacas. At various time points after administration, gamithromycin concentrations were analyzed via LC-MS/MS in plasma, PMNs, PELF, and BAL cells until Day 14 post-injection. Plasma gamithromycin concentrations were measured in all six alpacas; the remaining three body compartments were analyzed in four alpacas. Gamithromycin rapidly concentrated in blood PMNs, BAL cells, and PELF. Shorter Tmax , and lower Cmax, and AUC were observed in plasma than in the other three compartments. Cmax was highest in BAL cells (26001.80 ± 12400.00 ng/ml) and PMNs (2573.00 ± 963.30 ng/ml) compared to PELF (660.80 ± 413.70 ng/ml) and plasma (452.30 ± 196.20 ng/ml). Mean terminal half-lives were 72.60 ± 14.10 h in plasma, 56.60 ± 10.60 h in PELF, 62.80 ± 85.30 h in PMNs, and 93.60 ± 124.80 h in BAL cells. No injection site reactions occurred. One alpaca developed colic but no other adverse reactions were noted. Overall, gamithromycin was highly concentrated in white blood cells and pulmonary fluids/cells. Clinical utilization of gamithromycin in alpacas should be done with caution until further investigation of potential for colic.

Camelid Single-Domain Antibodies: Promises and Challenges as Lifesaving Treatments.

Since the discovery of camelid heavy-chain antibodies in 1993, there has been tremendous excitement for these antibody domains (VHHs/sdAbs/nanobodies) as research tools, diagnostics, and therapeutics. Commercially, several patents were granted to pioneering research groups in Belgium and the Netherlands between 1996-2001. Ablynx was established in 2001 with the aim of exploring the therapeutic applications and development of nanobody drugs. Extensive efforts over two decades at Ablynx led to the first approved nanobody drug, caplacizumab (Cablivi) by the EMA and FDA (2018-2019) for the treatment of rare blood clotting disorders in adults with acquired thrombotic thrombocytopenic purpura (TPP). The relatively long development time between camelid sdAb discovery and their entry into the market reflects the novelty of the approach, together with intellectual property restrictions and freedom-to-operate issues. The approval of the first sdAb drug, together with the expiration of key patents, may open a new horizon for the emergence of camelid sdAbs as mainstream biotherapeutics in the years to come. It remains to be seen if nanobody-based drugs will be cheaper than traditional antibodies. In this review, I provide critical perspectives on camelid sdAbs and present the promises and challenges to their widespread adoption as diagnostic and therapeutic agents.

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA.

The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.

Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody.

Since their discovery, the matrix metalloproteinase (MMP) family proteases have been considered as therapeutic targets in numerous diseases and disorders. Unfortunately, clinical trials with MMP inhibitors have failed to yield any clinical benefits of these inhibitors. These failures were largely due to a lack of MMP-selective agents; accordingly, it has become important to identify a platform with which high selectivity can be achieved. To this end, we propose using MMP-targeting antibodies that can achieve high specificity in interactions with their targets. Using a scaffold of single-domain antibodies, here we raised a panel of MMP10-selective antibodies through immunization of llamas, a member of the camelid family, whose members generate conventional heavy/light-chain antibodies and also smaller antibodies lacking light-chain and CH1 domains. We report the generation of a highly selective and tightly binding MMP10 inhibitor (Ki < 2 nm). Using bio-layer interferometry-based binding assays, we found that this antibody interacts with the MMP10 active site. Activity assays demonstrated that the antibody selectively inhibits MMP10 over its closest relative, MMP3. The ability of a single-domain antibody to discriminate between the most conserved MMP pair via an active site-directed mechanism of inhibition reported here supports the potential of this antibody as a broadly applicable scaffold for the development of selective, tightly binding MMP inhibitors.

Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria.

Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from Escherichia coli after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+ also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing E. coli spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant S. aureus strains that express efflux transporters such as NorC in hospital and community settings.

Comparative analysis of transposable elements provides insights into genome evolution in the genus Camelus.

BACKGROUND: Transposable elements (TEs) are common features in eukaryotic genomes that are known to affect genome evolution critically and to play roles in gene regulation. Vertebrate genomes are dominated by TEs, which can reach copy numbers in the hundreds of thousands. To date, details regarding the presence and characteristics of TEs in camelid genomes have not been made available. RESULTS: We conducted a genome-wide comparative analysis of camelid TEs, focusing on the identification of TEs and elucidation of transposition histories in four species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. Our TE library was created using both de novo structure-based and homology-based searching strategies ( https://github.com/kacst-bioinfo-lab/TE_ideintification_pipeline ). Annotation results indicated a similar proportion of each genomes comprising TEs (35-36%). Class I LTR retrotransposons comprised 16-20% of genomes, and mostly consisted of the endogenous retroviruses (ERVs) groups ERVL, ERVL-MaLR, ERV_classI, and ERV_classII. Non-LTR elements comprised about 12% of genomes and consisted of SINEs (MIRs) and the LINE superfamilies LINE1, LINE2, L3/CR1, and RTE clades. Least represented were the Class II DNA transposons (2%), consisting of hAT-Charlie, TcMar-Tigger, and Helitron elements and comprising about 1-2% of each genome. CONCLUSIONS: The findings of the present study revealed that the distribution of transposable elements across camelid genomes is approximately similar. This investigation presents a characterization of TE content in four camelid to contribute to developing a better understanding of camelid genome architecture and evolution.

Acute fasciolosis in an alpaca: a case report.

BACKGROUND: The popularity of new world camelids, particularly alpacas, is growing rapidly in Ireland, presenting a clinical challenge to veterinary practitioners who may not have worked with these species previously. To the authors' knowledge, the clinical course of a case of acute fasciolosis in an alpaca has not previously been reported, and fasciolosis has not been reported at all in alpacas in Ireland, making this case report a valuable addition to the current literature. CASE PRESENTATION: A three-year-old male castrated huacaya alpaca was admitted to UCD Veterinary Hospital with a two-day history of colic and tenesmus. He had been treated with albendazole, dexamethasone and potentiated amoxycillin by the referring veterinary practitioner with no response. On initial clinical exam, sensitivity to abdominal palpation was the only abnormality. However, the alpaca proceeded to show abnormal lying positions, tenesmus and reduced faecal output over the next 24 h. A general blood panel demonstrated moderate anaemia, marked hyperglobulinaemia and moderately increased hepatocellular and hepatobiliary enzyme activity. Abdominal radiography revealed enlargement of the first forestomach compartment without evidence of gastrointestinal obstruction or peritonitis. An abdominal ultrasound exam revealed an elongated, heterogenous mass in the caudoventral abdomen that appeared to be contiguous with the liver. FNA of this mass revealed that it was in fact a liver lobe with biliary stasis and inflammation. Faecal sedimentation demonstrated Fasciola hepatica eggs. In spite of treatment with triclabendazole and supportive treatment including blood transfusion, the alpaca's condition continued to deteriorate and he was euthanised. On post-mortem exam, acute fasciolosis was diagnosed. CONCLUSIONS: The clinical presentation and course of a case of acute fasciolosis in an individual alpaca is described, including the results of a range of diagnostic tests that were carried out. The final diagnosis is supported by a description of post-mortem findings. This information will serve as a resource for veterinary practitioners involved in the diagnosis and treatment of similar cases.

Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents.

Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called "nanobodies," derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody "RANbodies." They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.

Preliminary evidence for a forestomach washing mechanism in llamas (Lama glama).

Dust and grit are ingested by herbivores in their natural habitats along with the plants that represent their selected diet. Among the functions of the rumen, a washing of ingesta from adhering dust and grit has recently been demonstrated. The putative consequence is a less strenuous wear on ruminant teeth by external abrasives during rumination. The same function should theoretically apply to camelids, but has not been investigated so far. We fed six llamas (Lama glama) a diet of grass hay and a lucerne-based pelleted food in which fine sand had been included at about 8% of ingredients, for ad libitum consumption for 6 weeks. Subsequently, animals were slaughtered and content of the different sections of the gastrointestinal tract was sampled for the analysis of dry matter (DM), total ash, and acid detergent insoluble ash (ADIA, a measure for silica). Additionally, two of the animals were subjected to whole-body computer tomography (CT) after death in the natural sternal resting position. No clinical problems or macroscopic changes in the faeces were observed during the experimental period. The results indicate an accumulation of ADIA in the C3 compartment of the stomach complex, in particular in the posterior portion that is the equivalent of the abomasum in ruminants. By contrast, contents of the C1, from which material is recruited for regurgitation and rumination, were depleted of ADIA, indicating that the contents had largely been washed free of sand. The washing effect is an unavoidable side effect of the flotation- and sedimentation-based sorting mechanisms in the ruminant and the camelid forestomachs. In theory, this should allow ruminants and camelids to live in similar habitats as nonruminant herbivores at lower degrees of hypsodonty.