Camelid Species Research Articles

PLAbDab-nano: a database of camelid and shark nanobodies from patents and literature.

Nanobodies are essential proteins of the adaptive immune systems of camelid and shark species, complementing conventional antibodies. Properties such as their relatively small size, solubility and high thermostability makeVHH (variable heavy domain of theheavy chain) and VNAR (variable new antigen receptor) modalities a promising therapeutic format and a valuable resource for a wide range of biological applications. The volume of academic literature and patents related to nanobodies has risen significantly over the past decade. Here, we present PLAbDab-nano, a nanobody complement to the Patent and Literature Antibody Database (PLAbDab). PLAbDab-nano is a self-updating, searchable repository containing ∼5000 annotated VHH and VNAR sequences. We describe the methods used to curate the entries in PLAbDab-nano, and highlight how PLAbDab-nano could be used to design diverse libraries, as well as find sequences similar to known patented or therapeutic entries. PLAbDab-nano is freely available as a searchable web server (https://opig.stats.ox.ac.uk/webapps/plabdab-nano/).

Resistance to SARS-CoV-2 infection in camelid nasal organoids is associated with lack of ACE2 expression

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects a variety of animal species. Susceptibility to SARS-CoV-2 is primarily determined by the utilization of the viral receptor, ACE2. SARS-CoV-2 can utilize a broad range of animal ACE2 isoforms in vitro, including the ACE2 from various camelid species. However, experimental infection of these animals does not lead to productive infection or seroconversion. In this study, we investigate the susceptibility of camelids to SARS-CoV-2 using novel well-differentiated camelid nasal organoids. We show that camelid nasal organoids are highly susceptible to Middle East respiratory syndrome coronavirus (MERS-CoV) infection, but not to infection with different SARS-CoV-2 variants (614G, BA.1 or EG.5.1.1). All viruses efficiently infected human airway organoids. Immunohistochemistry analysis revealed the absence of ACE2 on camelid nasal organoids and dromedary camel upper respiratory tract. In contrast, DPP4 was expressed in both camelid nasal organoids and the camel upper respiratory tract, which correlates with MERS-CoV infection. This study indicates that the camelid upper respiratory tract lacks expression of ACE2, which is associated with resistance to SARS-CoV-2 infection.

Mitochondrial DNA of the Arabian Camel Camelus dromedarius.

The Camelidae family, ranging from southwest Asia to north Africa, South America, and Australia, includes key domesticated species adapted to diverse environments. Among these, the Arabian camel (Camelus dromedarius) is vital to the cultural and economic landscape of the Arabian Peninsula. This review explores the mitochondrial DNA of the dromedary camel, focusing on the D-loop region to understand its genetic diversity, maternal inheritance, and evolutionary history. We aim to investigate the unique characteristics of Arabian camel mtDNA, analyze the D-loop for genetic diversity and maternal lineage patterns, and explore the implications of mitochondrial genomic studies for camel domestication and adaptation. Key findings on mtDNA structure and variation highlight significant genetic differences and adaptive traits. The D-loop, essential for mtDNA replication and transcription, reveals extensive polymorphisms and haplotypes, providing insights into dromedary camel domestication and breeding history. Comparative analyses with other camelid species reveal unique genetic signatures in the Arabian camel, reflecting its evolutionary and adaptive pathways. Finally, this review integrates recent advancements in mitochondrial genomics, demonstrating camel genetic diversity and potential applications in conservation and breeding programs. Through comprehensive mitochondrial genome analysis, we aim to enhance the understanding of Camelidae genetics and contribute to the preservation and improvement of these vital animals.

Effect of Three Semen Extenders on Sperm Quality and In Vitro Fertilization Rates of Fresh and Cryopreserved Sperm Collected from Llama (Lama glama) Vas Deferens.

The advances in Assisted Reproductive Technologies (ARTs) applied in South American camelid species are still scarce. The aim of this study was to compare the effects of three semen extenders, before and after the cryopreservation of spermatozoa obtained from the vas deferens, on sperm quality parameters and in vitro fertilization rates of llama (Lama glama) oocytes. Mature fertile llama males (Lama glama; n = 6; age: 48-60 mo.; BCS: ~2.7) were included in the study. Sperm samples were collected from each male using the surgical technique of the vas deferens deviation. Then, the sperm samples were pooled and diluted with the Tris-EY, Andromed®, or BioxCell® extender in order to subsequently carry out the sperm cryopreservation process. The sperm quality assessment related to each extender was performed before and after cryopreservation with regard to sperm morphological abnormalities, acrosome integrity, sperm viability, membrane permeability, and sperm motility traits. Moreover, in vitro fertilization (IVF) procedures were carried out to evaluate the in vitro fertility of the cryopreserved sperm samples using each extender. Overall, significant differences were observed before and after cryopreservation regarding acrosome integrity, sperm viability, membrane permeability, and sperm motility traits among the extenders used, where Tris-EY and Andromed® were better than BioxCell® (p < 0.05); however, no differences were observed regarding the sperm morphological abnormalities among extenders (p > 0.05). Moreover, multiple differences were observed with regard to the velocity and linearity kinematic parameters obtained by computerized analysis before and after the cryopreservation process, irrespective of the extender used (p < 0.05). Finally, differences were observed regarding the in vitro fertilization rates among the different extender-derived samples (p < 0.05). In conclusion, the sperm quality using Tris-EY and Andromed® was better before and after cryopreservation compared to that using BioxCell®. Although the number of fertilized oocytes obtained after the IVF process between Tris-EY and Andromed® was similar, Andromed®-derived samples showed the best sperm quality results before and after cryopreservation. This indicates that the cryopreservation extender is a determining factor in significantly improving in vitro fertilization rates when using sperm samples obtained from vas deferens in llama (Lama glama) males.

Development and validation of the Axiom-MaruPri SNP chip for genetic analyses of domesticated old world camelids

Camels play a crucial socio-economic role in sustaining the livelihoods of millions in arid and semi-arid regions. They possess remarkable physiological attributes which enable them to thrive in extreme environments, and provide a source of meat, milk and transportation. With their unique traits, camels embody an irreplaceable source of untapped genomic knowledge. This study introduces Axiom-MaruPri, a medium-density SNP chip meticulously designed and validated for both Camelus bactrianus and Camelus dromedarius. Comprising of 182,122 SNP markers, derived from the re-sequenced data of nine Indian dromedary breeds and the double-humped Bactrian camel, this SNP chip offers 34,894 markers that display polymorphism in both species. It achieves an estimated inter-marker distance of 14 Kb, significantly enhancing the coverage of the camel genome. The medium-density chip has been successfully genotyped using 480 camel samples, achieving an impressive 99 % call rate, with 96 % of the 182,122 SNPs being highly reliable for genotyping. Phylogenetic analysis and Discriminant Analysis of Principal Components yield clear distinctions between Bactrian camels and dromedaries. Moreover, the discriminant functions substantially enhance the classification of dromedary camels into different breeds. The clustering of various camel breeds reveals an apparent correlation between geographical and genetic distances. The results affirm the efficacy of this SNP array, demonstrating high genotyping precision and clear differentiation between Bactrian and dromedary camels. With an enhanced genome coverage, accuracy and economic efficiency the Axiom_MaruPri SNP chip is poised to advance genomic breeding research in camels. It holds the potential to serve as an invaluable genetic resource for investigating population structure, genome-wide association studies and implementing genomic selection in domesticated camelid species.

Seroprevalence of Bluetongue and Schmallenberg viruses in ruminants in Al-Batinah Governorates, Oman

Bluetongue (BTV) and Schmallenberg viruses (SBV) are the causative agents of Bluetongue and Schmallenberg dis-eases, respectively. Both BTV and SBV are vector-borne viruses that are transmitted mainly by Culicoides (Diptera; Ceratopogo-nidae) and infect do-mestic, wild ruminants, and camelid species. Bluetongue virus be-longs to the genus Orbi-virus, family Reoviridae, whereas Schmallenberg virus belongs to the ge-nus Orthobunyavirus, family Bunyaviridae. In this study, we used en-zyme-linked immuno-sorbent assay (ELISA) to determine the sero-prevalence of both vi-ruses in ruminants in Al-Batinah Gover-norates, Sultanate of Oman. A total of 529 serum samples were randomly collected from 207 sheep, 265 goats, and 57 cattle from five wilayat of Al-Batinah North and Al-Batinah South Governorates. The serum samples were screened for the presence of BTV-specific antibodies against the BTV VP7 protein using ID Screen® Bluetongue Competition (cELISA) and screened for the presence of SBV-specific antibodies against the recombinant SBV nucleoprotein anti-gen using ID Screen® Schmallenberg virus Indirect Mult (iELISA). The overall seropreva-lence of BTV and SBV infections was 69.8% and 44.8%. In cattle, the prevalence of BTV and SBV antibodies was comparatively higher (94.7% and 82.5%) than in goats (83% and 43%) and sheep (45.9% and 36.7%). The high-est BTV seroprevalence was recorded in Nakhal (85.5%), followed by Wadi Al-Maawil (81%), and was lowest in Bar-ka (59.6%). However, the highest seropreva-lence of SBV was ob-served in Wadi Al-Maawil (50%), followed by Barka (48.7%), and lowest in Sohar (34.2%). Overall, the seroprevalence of BTV and SBV in domesticat-ed ruminants was high-er in adults than in young animals. Females showed a higher sero-prevalence of BTV and SBV compared to males. The study pro-vides an update on the epidemiological status of BTV, and to our knowledge, this is the first study on the sero-prevalence of SBV in ruminants in Oman. Future studies are re-quired for the isolation and identification of BTV and SBV along with other potential bio-logical vectors in Oman.

Body measures, growth curves and body weight prevision of alpacas (Vicugna pacos) reared in Italy

Alpacas represent an exotic species for Italy, introduced no more than 20–30 years ago. Nevertheless, they are currently the most widespread camelid species reared in this country, highlighting the growing interest in breeding this species. So, it is useful to assess parameters on their growth in relation to the new environment. Thus, body weight and body measures were recorded on 49 healthy alpacas of Huacaya type (27 females, 22 males), along a period of 27 months. Overall, 111 individual observations were made. Data were analysed by ANOVA, as growth curves were estimated by applying the Gompertz’s model. Multiple regression was finally used to estimate body weight from body measures. Body weight and linear measures, except for the distance between iliac crest and the length of the rump, were influenced by the category (calves, yearlings, adults) and by the age class within category (p < 0.05), while gender and interaction between gender and category were never significant (p > 0.05). Asymptotic weigh of males was 8.25% higher than females (p < 0.05). Moreover, males at maturity had higher withers (+0.96%), longer body (+2.70%) and chest measurements (+3.18%) than females. Asymptotic rump width (both at the ilium and at the ischium) was higher in females (respectively + 6.19%; +12.90%). According to our work, the best equation for the estimate of body weight from body measures was the following: 5.691 + 0.00005624 * chest circumference3 + 0.00002298 * trunk length3 − 1.155 * rump width between ischial tuberosities + 0.00001545 * height at rump3 (R2=0.957; SE of estimate = 4.36 kg), that is important for a better management of the species.

Genetic diversity in leucine-rich repeats (LRR) of toll-like receptor-2 and -4 genes in three species of South American camelids

Toll-like receptors (TLRs) are responsible for the innate immune response to detect and activate pathways against pathogens in animals. Polymorphisms within these genes have been related to susceptibility or resistance to different diseases. A high variability present in these sequences allows the population to develop a better adaptive potential while the opposite will make them more susceptible to new diseases. This variability decrease may occur because of external factors such as bottlenecks or artificial selection. The objective of this study was to verify the genetic diversity in the leucine-rich repeats (LRR) of TLR2 and TLR4 in three species of camelids from the new world. The genetic diversity was evaluated in the LRR of the TLR2 and TLR4 genes of 120 individuals that belong to the population of alpacas (Vicugna pacos), llamas (Lama glama) and vicuñas (Vicugna vicugna). Different polymorphisms were found, as well as the possible parental sequences and haplotype frequencies. Genetic diversity values, the degree of population differentiation, and amino acid variations were analyzed. The tests showed a very low genetic diversity (TLR2, π = 0.00525; TLR4, π = 0.00118) but a medium-high haplotype diversity (TLR2, Hd=0.923; TLR4, Hd=0.476) among the three species analyzed. There are both polymorphisms and haplotypes that are shared between these species and private haplotypes for each species. The demographic fixation index indicates no gene flow between groups of animal populations. Certain amino acid changes detected in the protein structure change its characteristic of it, making this to be a harmful variation. These results suggest that years of interference in the natural development of South American camelids have led to a drastic decrease in the diversity of their TLRs.

General aspects of lama and alpaca nutrition

The llama (Lama glama) and the alpaca (L. pacos) form the group known as domesticated camelids. They belong to the family Camelidae of the order Artiodactyla (ungulates). They are separated from other ruminants in the infraorder Tylopoda (footed) because they differ in gastric morphology (three compartments), with the absence of antlers or horns, and replacement of hooves with callous pads ending in claws (Novoa and Wheeler, 1984). Camellias are tall, robust and the largest of the four camelid species. They are used to transport goods in Central and South America, where their meat is also used. The leather is used for shoes, sandals and bags. Their wool fiber is long and coarse, varying in color from white to black, and is used to make various clothing items. In our country, and in Europe in general, llamas and alpacas are used for leisure purposes and even as pets, in some cases their wool being used to create clothing products.

Quantification of camelid cytokine mRNA expression in PBMCs by microfluidic qPCR technology

Camelids are economically and socially important in several parts of the world and might carry pathogens with epizootic or zoonotic potential. However, biological research in these species is limited due to lack of reagents. Here, we developed RT-qPCR assays to quantify a panel of camelid innate and adaptive immune response genes, which can be monitored in a single run. The assays were validated with PHA, PMA-ionomycin, and Poly I:C-stimulated PBMCs from alpaca, dromedary camel and llama, including normalization by multiple reference genes. Further, comparative gene expression analyses for the different camelid species were performed by a unique microfluidic qPCR assay. Compared to unstimulated controls, PHA and PMA-ionomycin stimulation elicited robust Th1 and Th2 responses in PBMCs from camelid species. Additional activation of type I and type III IFN signalling pathways was described exclusively in PHA-stimulated dromedary lymphocytes, in contrast to those from alpaca and llama. We also found that PolyI:C stimulation induced robust antiviral response genes in alpaca PBMCs. The proposed methodology should be useful for the measurement of immune responses to infection or vaccination in camelid species.

Comparison of the human’s and camel’s red blood cell deformability by optical tweezers and Raman spectroscopy

Red blood cells of vertebrates have undergone evolutionary changes over time, leading to the diversification of morphological and mechanical properties of red blood cells (RBCs). Among the vertebrates, camelids have the most different RBC characteristics. As a result of adaptation to the desert environment, camelid RBCs can expand twice as much of their total volume in the case of rapid hydration yet are almost undeformable under mechanical stress. In this work, the mechanical and chemical differences in the RBC properties of the human and camelid species were examined using optical tweezers and Raman spectroscopy. We measured the deformability of camel and human RBCs at the single-cell level using optical tweezers. We found that the deformability index (DI) of the camel and the human RBCs were 0.024 ± 0.019 and 0.215 ± 0.061, respectively. To investigate the chemical properties of these cells, we measured the Raman spectra of the whole blood samples. The result of our study indicated that some of the Raman peaks observed on the camel’s blood spectrum were absent in the human blood’s spectrum, which further points to the difference in chemical contents of these two species’ RBCs.

Genome-wide scan for runs of homozygosity in South American Camelids

BackgroundAlpaca (Vicugna pacos), llama (Lama glama), vicugna (Vicugna vicugna) and guanaco (Lama guanicoe), are the camelid species distributed over the Andean high-altitude grasslands, the Altiplano, and the Patagonian arid steppes. Despite the wide interest on these animals, most of the loci under selection are still unknown. Using whole-genome sequencing (WGS) data we investigated the occurrence and the distribution of Runs Of Homozygosity (ROHs) across the South American Camelids (SACs) genome to identify the genetic relationship between the four species and the potential signatures of selection.ResultsA total of 37 WGS samples covering the four species was included in the final analysis. The multi-dimensional scaling approach showed a clear separation between the four species; however, admixture analysis suggested a strong genetic introgression from vicugna and llama to alpaca. Conversely, very low genetic admixture of the guanaco with the other SACs was found. The four species did not show significant differences in the number, length of ROHs (100-500 kb) and genomic inbreeding values. Longer ROHs (> 500 kb) were found almost exclusively in alpaca. Seven overlapping ROHs were shared by alpacas, encompassing nine loci (FGF5, LOC107034918, PRDM8, ANTXR2, LOC102534792, BSN, LOC116284892, DAG1 and RIC8B) while nine overlapping ROHs were found in llama with twenty-five loci annotated (ERC2, FZD9, BAZ1B, BCL7B, LOC116284208, TBL2, MLXIPL, PHF20, TRNAD-AUC, LOC116284365, RBM39, ARFGEF2, DCAF5, EXD2, HSPB11, LRRC42, LDLRAD1, TMEM59, LOC107033213, TCEANC2, LOC102545169, LOC116278408, SMIM15, NDUFAF2 and RCOR1). Four overlapping ROHs, with three annotated loci (DLG1, KAT6B and PDE4D) and three overlapping ROHs, with seven annotated genes (ATP6V1E1, BCL2L13, LOC116276952, BID, KAT6B, LOC116282667 and LOC107034552), were detected for vicugna and guanaco, respectively.ConclusionsThe signatures of selection revealed genomic areas potentially selected for production traits as well as for natural adaptation to harsh environment. Alpaca and llama hint a selection driven by environment as well as by farming purpose while vicugna and guanaco showed selection signals for adaptation to harsh environment. Interesting, signatures of selection on KAT6B gene were identified for both vicugna and guanaco, suggesting a positive effect on wild populations fitness. Such information may be of interest to further ecological and animal production studies.

Molecular characterization of toll-like receptor 2 gene in Bikaneri camels (Camelus dromedarius)

Toll-like receptors (TLRs) are an important family of receptors constituting the first line of defense system. Tolllike receptors play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Present investigation was carried out to characterize the coding sequence of TLR2 gene in Indian dromedary camels. Blood was collected from five Bikaneri camels and genomic DNA was isolated. Eight pairs of overlapping primers were designed for amplification of TLR2 gene coding region. All the fragments were successfully amplified using Polymerase chain reaction (PCR). The PCR products were purified and subjected to Sanger’s sequencing. TLR2 gene fragment of 3213 bp consisting of 2287 bp of coding sequence of exon 2 and 926 bp of 3’untranslated region (UTR) was successfully amplified. The sequence generated was submitted to the NCBI with accession number MW557315.1. The encoded amino acids codes for 12 leucine rich repeats (LRR) (Accession no. UBN09134.1). At the nucleotide level, the Bikaneri camels’ TLR2 gene showed highest similarity with camelids (99.30 to 99.59%) followed by vicugna pacos (97.54%). The nucleotide sequence similarity was lowest with Homo sapiens (82.37%). The amino acid sequence based phylogenetic tree revealed close relationship between camelid species followed by pig (86.2%), whereas human, gorilla, horse and donkey were distantly placed on the tree.

Molecular characterization of toll-like receptor 2 gene in Bikaneri camels (Camelus dromedarius)

Toll-like receptors (TLRs) are an important family of receptors constituting the first line of defense system. Tolllike receptors play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Present investigation was carried out to characterize the coding sequence of TLR2 gene in Indian dromedary camels. Blood was collected from five Bikaneri camels and genomic DNA was isolated. Eight pairs of overlapping primers were designed for amplification of TLR2 gene coding region. All the fragments were successfully amplified using Polymerase chain reaction (PCR). The PCR products were purified and subjected to Sanger’s sequencing. TLR2 gene fragment of 3213 bp consisting of 2287 bp of coding sequence of exon 2 and 926 bp of 3’untranslated region (UTR) was successfully amplified. The sequence generated was submitted to the NCBI with accession number MW557315.1. The encoded amino acids codes for 12 leucine rich repeats (LRR) (Accession no. UBN09134.1). At the nucleotide level, the Bikaneri camels’ TLR2 gene showed highest similarity with camelids (99.30 to 99.59%) followed by vicugna pacos (97.54%). The nucleotide sequence similarity was lowest with Homo sapiens (82.37%). The amino acid sequence based phylogenetic tree revealed close relationship between camelid species followed by pig (86.2%), whereas human, gorilla, horse and donkey were distantly placed on the tree.

Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive cellular immune responses in camelid reservoirs.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease.

Potential Promises and Perils of Human Biological Treatments for Immunotherapy in Veterinary Oncology.

The emergence of immunotherapy for the treatment of human cancers has heralded a new era in oncology, one that is making its way into the veterinary clinic. As the immune system of many animal species commonly seen by veterinarians is similar to humans, there is great hope for the translation of human therapies into veterinary oncology. The simplest approach for veterinarians would be to adopt existing reagents that have been developed for human medicine, due to the potential of reduced cost and the time it takes to develop a new drug. However, this strategy may not always prove to be effective and safe with regard to certain drug platforms. Here, we review current therapeutic strategies that could exploit human reagents in veterinary medicine and also those therapies which may prove detrimental when human-specific biological molecules are used in veterinary oncology. In keeping with a One Health framework, we also discuss the potential use of single-domain antibodies (sdAbs) derived from camelid species (also known as Nanobodies™) for therapies targeting multiple veterinary animal patients without the need for species-specific reformulation. Such reagents would not only benefit the health of our veterinary species but could also guide human medicine by studying the effects of outbred animals that develop spontaneous tumors, a more relevant model of human diseases compared to traditional laboratory rodent models.

Single-Chain B cell receptor mice as a novel tool for antibody generation and exploring B cell signaling

Abstract Conventional antibodies consist of a pair of heavy and light dimers, while heavy-chain only antibodies (HCAbs), like those found in camelid and shark species, lack a domain critical for light chain binding and develop without a light chain. HCAbs have unique physiochemical properties that confer advantages: they are smaller, have greater accessibility to their target antigens, higher hydrophilicity, higher tissue penetrance, and greater conformational stability under environmental stresses compared to conventional Abs. They are also highly amendable to biochemical engineering. We have generated a mouse model, that can produce HCAbs from the endogenous murine heavy chain locus. We have shown that these mice undergo efficient B cell development in the bone marrow and generate a diverse repertoire of B cells that populate all peripheral immune compartments. Single-cell sequencing of B cells revealed some biases in VH and JH usage, but highlighted the tremendous diversity of rearrangements that give rise to B cells bearing HCAbs. With these mice, we have been able to generate antigen specific HCAbs to the SARS-CoV2 spike protein that have a K Din the nM range, measured via bio-layer interferometry. Further, the IgG3dCH1 mice provide us with a novel model to understand how single domain B cell receptors (BCRs) function in comparison to conventional heavy/light chain BCRs. We have observed single-chain BCRs have distinct transcriptional profiles by single-cell sequencing and support higher plasma cell differentiation in vitro and in vivo. These data highlight the utility of our small animal platform for the generation of clinically relevant HCAbs, and as a model to study the biology of HCAb encoding B cells which remain poorly understood. Bernard Levine Postdoctoral Fellowship, R43AI136141

Detection and characterization of nuclear mitochondrial DNA (NUMTs) in the alpaca genome

NUMTs are mitochondrial DNA sequences that are inserted into the nuclear genome of almost all eukaryotic organisms, although high interspecific variability regarding abundance, size, and location is observed. In particular, the NUMT amount is highly dependent on the detection method utilized. The aim of this study was to provide the first comprehensive description and annotated collection of NUMTs for the alpaca. We have examined different detection strategies and obtained 267 hits, representing 0.011% of the genome (233,411 bp aligned). By merging these hits, we have identified and characterized 158 NUMTs across the alpaca genome. We found copies from all the mtDNA with variable identity values and two particularly large NUMTs that covered 72–83% of the mitogenome with a mean identity of 80%. Results showed that NUMTs were preferentially inserted in the non-coding region of the nuclear genome and were flanked by repetitive elements. Besides, the main mechanism for NUMT generation in this species seems to be independent insertion events, rather than duplication. Furthermore, we have selected and validated by PCR the presence of four NUMTs in the alpaca genome and proved their presence in the other three South American Camelid species. The results generated by this work represent a useful resource for curating databases and can be used to avoid co-amplification of NUMTs in studies involving mtDNA, like phylogenetics, populations or evolution studies.

A novel method to analyse DART TOFMS spectra based on Convolutional Neural Networks: A case study on methanol extracts of wool fibres from endangered camelids

Monitoring the illegal trade of wool fibres of wild vicuña (Vicugna vicugna) and guanaco (Lama guanicoe) is highly desirable. The high market value of fleece from these camelid species poses a threat to their wild populations. A previous study showed that direct analysis in real time time-of-flight mass spectrometry (DART-TOFMS) effectively identifies wool fibres to species. Producing high-resolution data in a short period of time makes DART-TOFMS a reliable identification tool, even though data analysis can still be improved. The present study proposes a novel data analysing pipeline based on Convolutional Neural Networks (CNN), applicable to any kind of DART-TOF MS data. We tested our proposed method on keratin fibres of four camelid species (Vicugna vicugna: n = 19; Vicugna pacos: n = 20; Lama guanicoe: n = 20, and Lama glama: n = 20). Analyses showed that selecting 512 ions with the highest relative intensity provides the best resolution and yields 100% accuracy for species identification.

Extract-Shaped Immune Repertoires as Source for Nanobody-Based Human IgE in Grass Pollen Allergy.

The presence of allergen-specific IgE in serum is a biomarker for allergic disease. Specific IgE antibodies for research and diagnostics, however, remain scarce. In contrast to prototypic antibodies, camelid species have evolved single domains as moiety for antigen recognition. These so-called nanobodies represent a versatile platform for the development of diagnostic and therapeutic approaches. In this study, we aimed for generating nanobodies and derived IgE formats from an extract-shaped immune repertoire. Timothy grass pollen represents a complex, but well-defined mixture of individual allergens. Therefore, a repertoire library from a timothy grass pollen extract immunised llama was established. The selection by phage display yielded 3 nanobodies with immunoreactivity to the extract. IgE-like nanobody-based human IgE (nb-hIgE) antibodies were produced in mammalian cells and assessed in different immunoassays and commercial platforms. Immunoblotting and diagnostic ImmunoCap analysis of single timothy grass pollen allergens identified the major allergens Phl p 6 and Phl p 4 as targets. Assessment of immunoreactivity further documented significant molecular cross-reactivity with pollen extract of different grass species and variant presence of allergens within extracts of Pooideae grasses. In summary, our study shows that extract-based immunisation enables the generation of allergen-specific nanobodies and derived nb-hIgE formats linking nanobody technologies with allergological applications.