Hypothalamic-pituitary-gonadal axis response to photoperiod changes in female guanacos (Lama guanicoe).

The guanaco, a wild South American camelid, is renowned for its remarkable resilience to extreme conditions. Despite this, little is known about how reproductive hormones in female camelids are influenced during their seasonal breeding period, which occurs during long photoperiod. To explore this, the study investigated the response of the hypothalamic-pituitary-gonadal axis in female guanacos during short days (10L:14D; July) and long days (16L:8D; December) in the Mediterranean ecosystem (33°38'28″S, 70°34'27″W). Blood samples from 14 adult animals were collected, and measurements of melatonin, 17ß-estradiol, FSH, and LH concentrations were taken. The results showed that melatonin concentration was lower (P < 0.05) during long days than short days, whereas 17ß-estradiol, FSH, and LH concentrations were higher (P < 0.05) during long days compared to short days. Furthermore, the study detected the expression of the melatonin receptor 1A and kisspeptin in the hypothalamus and pituitary, suggesting that the pineal gland of female guanacos is sensitive to seasonal changes in day length. These findings also indicate a seasonal variation in the concentration of reproductive hormones, likely linked to the distinct modulation of the hypothalamic-pituitary-gonadal axis of female guanacos during short and long days.

Type I and III IFNs produced by the nasal epithelia and dimmed inflammation are features of alpacas resolving MERS-CoV infection.

While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.

Mirror-Image Single-Domain Antibody for a Novel Nonimmunogenic Drug Scaffold.

Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (l-anti-GFP VHH and l-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, l-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid (111In-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between l-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.

Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

In brief: Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons. Abstract: Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

MicroRNA-379 mediates pigmentation, migration and proliferation of melanocytes by targeting the insulin-like growth factor 1 receptor.

Melanogenesis, migration and proliferation of melanocytes are important factors that determine the hair colours of mammals. MicroRNAs (miRNAs) have been shown to be closely related to these processes. In melanocytes of alpacas, insulin-like growth factor 1 (IGF1) has been shown to improve melanogenesis through the cyclic AMP (cAMP) pathway. miR-379 was predicted to target insulin-like growth factor (IGF) receptor 1 (IGF1R), which binds to IGF1. Therefore, we hypothesized that miR-379 could mediate melanogenesis, migration and proliferation of melanocytes. Here, we report that miR-379 was highly expressed in alpaca melanocytes. Subsequent overexpression of miR-379 in alpaca melanocytes led to the generation of the phenotype of melanogenesis, proliferation and migration. In addition, the expression of genes related to these phenotypes in melanocytes was detected. Our results showed that miR-379 targets IGF1R in melanocytes. The overexpression of miR-379 stimulated dendrite extension or elongation and limited the perinuclear distribution of melanin, but inhibited melanogenesis via cAMP response element (CRE)-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway. miR-379 attenuated melanocyte migration by downregulating the focal adhesion kinase (FAK) and enhanced melanocyte proliferation by upregulating protein kinase B (AKT). These observations suggest the involvement of miR-379 in the physiological regulation of melanocytes, mediated by targeting IGF1R on insulin receptor (IR) compensation and subsequent crosstalk.

Sedative effects and pharmacokinetics of detomidine when administered intravenously and intravaginally as a gel in alpacas.

OBJECTIVES: To evaluate the sedative effects and pharmacokinetics of detomidine gel administered intravaginally to alpacas in comparison with intravenously (IV) administered detomidine. STUDY DESIGN: Randomized, crossover, blinded experiment. ANIMALS: A group of six healthy adult female Huacaya alpacas (70.3 ± 7.9 kg). METHODS: Alpacas were studied on two occasions separated by ≥5 days. Treatments were IV detomidine hydrochloride (70 µg kg-1; treatment DET-IV) or detomidine gel (200 µg kg-1; treatment DET-VAG) administered intravaginally. Sedation and heart rate (HR) were evaluated at intervals for 240 minutes. Venous blood was collected at intervals for 360 minutes after treatment for analysis of detomidine, carboxydetomidine and hydroxydetomidine using liquid chromatography-tandem mass spectrometry. Measured variables were compared between treatments and over time using mixed model analysis. Data are presented as the mean ± standard error of the mean, and a p value of <0.05 was considered significant. RESULTS: Onset of sedation was faster in treatment DET-IV (1.6 ± 0.2 minutes) than in treatment DET-VAG (13.0 ± 2.5 minutes). Time to maximum sedation was shorter in treatment DET-IV (8.3 ± 1.3 minutes) than in treatment DET-VAG (25 ± 4 minutes). Duration of sedation was not different between treatments. There was a significant linear relationship between sedation score and plasma detomidine concentration. HR was less than baseline for 60 and 125 minutes for treatments DET-IV and DET-VAG, respectively. The maximal decrease in HR occurred at 15 minutes for both treatments. The mean maximum plasma concentration of detomidine, time to maximum concentration and bioavailability for treatment DET-VAG were 39.6 ng mL-1, 19.9 minutes and 20%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Detomidine administration at the doses studied resulted in moderate sedation when administered IV or intravaginally to alpacas.

Comparison of miRNA-101a-3p and miRNA-144a-3p regulation with the key genes of alpaca melanocyte pigmentation.

BACKGROUND: Many miRNA functions have been revealed to date. Single miRNAs can participate in life processes by regulating more than one target gene, and more than one miRNA can also simultaneously act on one target mRNA. Thus, a complex regulatory network involved in many processes can be formed. Herein, the pigmentation regulation mechanism of miR-101a-3p and miR-144a-3p was studied at the cellular level by the overexpression and equal overexpression of miR-101a-3p and miR-144a-3p. RESULTS: Results revealed that miR-101a-3p and miR-144a-3p directly regulated the expression of microphthalmia-associated transcription factor, thereby affecting melanin synthesis. CONCLUSIONS: The two miRNAs with the same binding site in the same gene independently excreted each other's function. However, the inhibitory effect of miR-144a-3p was stronger than that of miR-101-3p in alpaca melanocytes, although both decreased melanin production.

MicroRNA-5110 regulates pigmentation by cotargeting melanophilin and WNT family member 1.

Mammalian pigmentation requires the production of melanin by melanocytes and its transfer to neighboring keratinocytes. These complex processes are regulated by several molecular pathways. Melanophilin ( MLPH) and WNT family member 1 ( WNT1), known to be involved in melanin transfer and melanin production, respectively, were predicted to be targets of microRNA-5110 using bioinformatics. In the current study, we investigated the effects of microRNA-5110 on pigmentation in alpaca ( Vicugna pacos) melanocytes. In situ hybridization identified high levels of microRNA-5110 in the cytoplasm of alpaca melanocytes. Luciferase activity assays confirmed that MLPH and WNT1 were targeted by microRNA-5110 in these cells. Overexpression and knockdown of microRNA-5110 in alpaca melanocytes downregulated and upregulated MLPH and WNT1 expression at the mRNA and protein levels, respectively. In addition, overexpression and knockdown of microRNA-5110 in alpaca melanocytes decreased and increased, respectively, the mRNA levels of the melanin transfer-related genes, rat sarcoma (RAS)-associated binding ( RAB27a) and myosin 5a ( MYO5a); the mRNA levels of microphthalmia-associated transcription factor ( MITF), tyrosinase ( TYR), and tyrosinase-related protein ( TYRP) 1; and the production of total alkali melanin and pheomelanin. In contrast, overexpression and knockdown of microRNA-5110 increased and decreased the mRNA levels of TYRP2, respectively. Overexpression of microRNA-5110 also increased eumelanin. These results indicate that microRNA-5110 regulates pigmentation in alpaca melanocytes by directly targeting MLPH and WNT1 to affect eumelanin production and transfer.-Yang, S., Liu, B., Ji, K., Fan, R., Dong, C. MicroRNA-5110 regulates pigmentation by cotargeting melanophilin and WNT family member 1.

Expression of ß-NGF and high-affinity NGF receptor (TrKA) in llama (Lama glama) male reproductive tract and spermatozoa.

ß-Nerve growth factor (ß-NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of ß-NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of ß-NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal ß-NGF interacts with ejaculated sperm by localizing ß-NGF and TrKA in epididymal, ejaculated, and acrosome-reacted (AR) sperms and, additionally, by identifying ß-NGF presence in sperm-adsorbed proteins (SAP). Both ß-NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of ß-NGF at prostate (p < 0.05). ß-NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, ß-NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that ß-NGF could have a seminal origin. This was also confirmed by the identification of four ß-NGF isoforms in SAP. This study extends the knowledge about the participation of ß-NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Distribution and morphology of gonadotropin-releasing hormone neurons in the hypothalamus of an induced ovulator - The llama (Lama glama).

Gonadotropin-releasing hormone (GnRH) is a decapeptide involved in the regulation of reproduction in all mammals, but the distribution of GnRH neurons within the brain varies widely among species. The objective of the present study was to characterize the number and distribution of GnRH neurons in the hypothalamus and preoptic area of llamas, an induced ovulator. The brains of female llamas (n = 4) were fixed, frozen and sectioned serially every 50 µm in the transverse (coronal) plane. Every 10th section was stained for immunohistochemical detection of GnRH-positive neuron cell bodies and fibers by incubation with 3,3'-diaminobenzidine. The number of counted immunoreactive cells ranged from 222 to 250 (≈241 ±â€¯13 cells in the preoptic area and hypothalamus per animal) and were localized in the medio-basal hypothalamus (44.3%), anterior hypothalamus (27%), preoptic area (14.9%), diagonal band of Broca/medial septum (13.4%), and mammillary area (0.5%). The immunoreactive cells were not localized in specific hypothalamic nuclei, but rather appeared to be distributed diffusely. The highest concentration of immunoreactive neuron fibers was in the median eminence (P < 0.05), but fibers were identified in most of the areas analyzed, including the neurohypophysis. The GnRH neurons within the hypothalamus displayed monopolar (33%), bipolar (39%), and multipolar (28%) morphologies. The bipolar type was most common in the medio-basal region (40%; P < 0.05). We conclude that GnRH neurons and fibers form a network within the anterior and medio-basal hypothalamus of llamas, suggesting the central location of mechanisms controlling reproductive processes in llamas (i.e., induced ovulation).

Pharmacokinetics of intravenous gentamicin in healthy young-adult compared to aged alpacas.

The study objective was to evaluate the effects of age on aminoglycoside pharmacokinetics in eight young-adult (<4 years) and eight aged (≥14 years) healthy alpacas, receiving a single 6.6 mg/kg intravenous gentamicin injection. Heparinized plasma samples were obtained at designated time points following drug administration and frozen at -80°C until assayed by a validated immunoassay (QMS® ). Compartmental and noncompartmental analyses of gentamicin plasma concentrations versus time were performed using WinNonlin (v6.4) software. Baseline physical and hematological parameters were not significantly different between young and old animals with the exception of sex. Data were best fitted to a two-compartment pharmacokinetic model. The peak drug concentration at 30 min after dosing (23.8 ± 2.1 vs. 26.1 ± 2 µg/ml, p = .043) and area under the curve (70.4 ± 10.5 vs. 90.4 ± 17.6 µg hr/ml, p = .015) were significantly lower in young-adult compared to aged alpacas. Accordingly, young alpacas had a significantly greater systemic clearance than older animals (95.5 ± 14.4 and 75.6 ± 16.1 ml hr-1 kg-1 ; p = .018), respectively). In conclusion, a single 6.6 mg/kg intravenous gentamicin injection achieves target blood concentrations of >10 times the MIC of gentamicin-susceptible pathogens with MIC levels ≤2 µg/ml, in both young-adult and geriatric alpacas. However, the observed reduction in gentamicin clearance in aged alpacas may increase their risk for gentamicin-related adverse drug reactions.

SL15: A seminal plasma-derived lectin from the sperm of llama (Lama glama).

The oviductal sperm reservoir of South American camelids is formed when sperm bind to N-acetylgalactosamine (GalNAc) on the surface of oviductal epithelium. The aim of this study was to characterize the GalNAc-binding proteins on llama sperm, and to establish their origin. Sperm-adsorbed proteins were extracted with 0.5 M KCl in Hepes-balanced salts. Sperm-adsorbed and seminal plasma proteins were then subjected to ligand blotting for their GalNAc affinity, and the labeled bands were identified by mass spectrometry. Three proteins were identified in seminal plasma versus only one in the sperm-adsorbed population; SL15, a seminal lectin, was common to both. SL15 is a homologue of Zymogen granule protein 16, homolog B-like, which belongs to the Jacalin-related lectin family. This lectin is likely presented to sperm via seminal plasma since epididymal sperm are not capable of binding GalNAc, whereas ejaculated sperm does, and its transcript was enriched predominantly in the prostate and bulbourethral glands. This is the first report of a seminal lectin in South American camelids that originates in the male reproductive tract, and is probably involved in sperm reservoir formation.

Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos).

The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G-coupled receptors named receptor 1 (OX1 ) and receptor 2 (OX2 ) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX2 by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX2 -IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX2 . Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.

Pharmacokinetics of intravenous and transdermal fentanyl in alpacas.

The purpose of the study was to determine pharmacokinetics of fentanyl after intravenous (i.v.) and transdermal (t.d.) administration to six adult alpacas. Fentanyl was administered i.v. (2 µg/kg) or t.d. (nominal dose: 2 µg kg-1  hr-1 ). Plasma concentrations were determined using liquid chromatography-mass spectrometry. Heart rate and respiratory rate were assessed. Extrapolated, zero-time plasma fentanyl concentrations were 6.0 ng/ml (1.7-14.6 ng/ml) after i.v. administration, total plasma clearance was 1.10 L hr-1  kg-1 (0.75-1.40 L hr-1  kg-1 ), volumes of distribution were 0.30 L/kg (0.10-0.99 L/kg), 1.10 L/kg (0.70-2.96 L/kg) and 1.5 L/kg (0.8-3.5 L/kg) for V1 , V2 , and Vss , respectively. Elimination half-life was 1.2 hr (0.5-4.3 hr). Mean residence time (range) after i.v. dosing was 1.30 hr (0.65-4.00 hr). After t.d. fentanyl administration, maximum plasma fentanyl concentration was 1.20 ng/ml (0.72-3.00 ng/ml), which occurred at 25 hr (8-48 hr) after patch placement. The area under the plasma fentanyl concentration-vs-time curve (extrapolated to infinity) after t.d. fentanyl was 61 ng*hr/ml (49-93 ng*hr/ml). The dose-normalized bioavailability of fentanyl from t.d. fentanyl in alpacas was 35.5% (27-64%). Fentanyl absorption from the t.d. fentanyl patch into the central compartment occurred at a rate of approximately 50 µg/hr (29-81 µg/hr) between 8 and 72 hr after patch placement.

The nerve of ovulation-inducing factor in semen.

A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of ß nerve growth factor (ß-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as ß-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC(12)) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of ß-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is ß-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.

Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa.

The aim of this study was to determine the effect of two equilibration temperatures (5 °C and room temperature) and two cryoprotectants (glycerol and dimethylformamide, both at 7%) on llama sperm cryopreservation. Llama ejaculates were divided into four aliquots. A lactose-EDTA-egg yolk (LEEY) extender with either 7% glycerol (LEEY-G) or 7% dimethylformamide (LEEY-DMF) was added to two of the aliquots, which were equilibrated for 20 min at room temperature and subsequently frozen. The other two aliquots were extended in LEEY, cooled to 5 °C, then LEEY-G or LEEY-DMF was added, equilibrated for 20 min at 5 °C and frozen. No significant differences (P > 0.05) were observed in membrane function and chromatin condensation between any of the freeze-thawing protocols. Post-thaw motility was greater (P < 0.05) in LEEY-DMF than LEEY-G. DNA fragmentation was not different between raw and frozen semen with LEEY-DMF but was high in all samples with glycerol. Our results indicate that 7% glycerol would be detrimental for llama spermatozoa, but further studies are needed to evaluate effectiveness if used at lower concentrations. Dimethylformamide preserved motility and DNA integrity of frozen-thawed llama spermatozoa and could be used to replace glycerol at the concentrations used in this study.

The alpaca melanocortin 1 receptor: gene mutations, transcripts, and relative levels of expression in ventral skin biopsies.

The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5'-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3'UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation.

Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol.

A phage-displayed library of variable domain of heavy chain of the heavy chain antibody (VHH) or nanobody (Nb) was constructed after immunizing an alpaca with aflatoxin B1 (AFB1) conjugated with bovine serum albumin (AFB1-BSA). Two AFB1-specific nanobodies were selected. The obtained nanobodies were compared to an aflatoxin-specific monoclonal antibody B5 with respect to stability under organic solvents and high temperature. The two nanobodies could bind antigen specifically after exposure to temperatures as high as 95 °C. Besides, the nanobodies showed better or similar tolerance to organic solvents. A competitive ELISA with nanobody Nb26 was developed for the analysis of AFB1, exhibiting an IC50 value of 0.754 ng/mL (2.4 µM), linear range from 0.117 to 5.676 ng/mL. Due to the high tolerance to methanol, sample extracts were analyzed by nanobody-based ELISA without dilution. The recovery from spiked peanut, rice, corn and feedstuff ranged from 80 to 115%. In conclusion, the isolated nanobodies are excellent candidates for immunoassay application in aflatoxin determination.

Middle East respiratory coronavirus (MERS-CoV) internalized by llama alveolar macrophages does not result in virus replication or induction of pro-inflammatory cytokines.

Severe Middle East respiratory syndrome (MERS) is characterized by massive infiltration of immune cells in lungs. MERS-coronavirus (MERS-CoV) replicates in vitro in human macrophages, inducing high pro-inflammatory responses. In contrast, camelids, the main reservoir for MERS-CoV, are asymptomatic carriers. Although limited infiltration of leukocytes has been observed in the lower respiratory tract of camelids, their role during infection remains unknown. Here we studied whether llama alveolar macrophages (LAMs) are susceptible to MERS-CoV infection and can elicit pro-inflammatory responses. MERS-CoV did not replicate in LAMs; however, they effectively capture and degrade viral particles. Moreover, transcriptomic analyses showed that LAMs do not induce pro-inflammatory cytokines upon MERS-CoV sensing.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.