[Advances in nanobody screening technology].

Nanobody (Nb) is a novel type of antibody discovered in the serum of Camelidae. It is characterized by its small size, high specificity, stability, and ease of preparation. Nanobodies exhibit the ability to identify hidden epitopes and have diverse applications across various fields. This review aims to introduce three key stages in the screening and optimization of nanobodies, including nanobody library construction, in vitro surface display, and affinity maturation. We provide a brief description of preparation and characteristics of natural, immunological, and semi-synthetic/synthetic libraries. Additionally, we systematically explain eight in vitro display methods, including phage display, yeast display, bacterial display, ribosome display/mRNA display, and eukaryotic cell display. Furthermore, we discuss the application of yeast two-hybrid system high-throughput sequencing and mass spectrometry identification. A thorough analysis of their advantages and limitations is presented in this protocols. Finally, we summarize the platforms for in vitro or computer-aided affinity maturation techniques aimed at enhancing the functional stability of nanobodies. Consequently, this review provides a comprehensive approach to the integrated utilization of various technologies for the rapid development of stable, reliable, and specific nanobody-based drugs or diagnostic agents.

Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display.

BACKGROUND: Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. METHODS: An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. RESULTS: A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M-1 and 2.08 × 107 M-1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. CONCLUSION: The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics.

Determination of hematobiochemical and fore stomach fluid constituents of llama (Lama glama) living in Egypt.

There are no available data regarding the hematology, serum biochemistry, and fore stomach fluid constituents of llama (Lama glama) in Egypt. This study aimed to establish normal reference values for blood and fore stomach fluid constituents of llama and determine the influence of sex and season on these parameters under Egyptian conditions. The study was performed on (n = 38; 22 female, 16 male; 1-7 years) apparently healthy llamas located in the Giza Zoo and private zoo in the Ismailia Governorate. Samples were collected in two seasons and divided into summer and winter samples. Differences in the mean and range values of packed cell volume, serum minerals, fore stomach fluid pH, and total protozoal count in Egypt were recorded. Sex and season had minimal effects on hematology and only erythrocyte count showed a significant (p < 0.05) increase in males compared with females. Regarding serum biochemistry, males showed significant (p < 0.05) increases in alanine transaminase and calcium levels, while globulin significantly (p < 0.05) increased in females. The influence of season on serum biochemistry was evident in alanine transaminase, total protein, albumin, and chloride which increased significantly (p < 0.05) in summer, while urea, bilirubin, and magnesium increased significantly (p < 0.05) in winter. Fore stomach fluid pH and ammonia showed significant (p < 0.05) increases in winter, while the total protozoal count increased significantly (p < 0.05) in summer and in males compared with females. The results obtained in this study can serve as reference values for the hematobiochemical and fore stomach fluid constituents of llama in Egypt.

Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries.

Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, VHHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple VHH-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, bi- and multi-specific, CAR-T, and antibody-drug conjugates. The first approved VHH, a bivalent humanized VHH (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA VHH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based VHH drug (ozoralizumab; Nanozora®) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific VHHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble VHHs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific VHHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated VHHs. To demonstrate the feasibility of the entire strategy, we present examples of VHHs previously isolated and characterized in our laboratory.

Single-domain antibodies applied as antiviral immunotherapeutics.

Viral infections have been the cause of high mortality rates throughout different periods in history. Over the last two decades, outbreaks caused by zoonotic diseases and transmitted by arboviruses have had a significant impact on human health. The emergence of viral infections in different parts of the world encourages the search for new inputs to fight pathologies of viral origin. Antibodies represent the predominant class of new drugs developed in recent years and approved for the treatment of various human diseases, including cancer, autoimmune and infectious diseases. A promising group of antibodies are single-domain antibodies derived from camelid heavy chain immunoglobulins, or VHHs, are biomolecules with nanometric dimensions and unique pharmaceutical and biophysical properties that can be used in the diagnosis and immunotherapy of viral infections. For viral neutralization to occur, VHHs can act in different stages of the viral cycle, including the actual inhibition of infection, to hindering viral replication or assembly. This review article addresses advances involving the use of VHHs in therapeutic propositions aimed to battle different viruses that affect human health.

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-1ß, 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.

Masticatory myology of the llama (Lama glama, Camelidae) and comparisons with other camelids and euungulates.

Camelids are the only living representatives of the Suborder Tylopoda, and present a unique set of osteo-myological masticatory features, differing from all other extant euungulates. They combine selenodont dentition and rumination with a fused symphysis, and roughly plesiomorphic muscle proportions. Despite its potential relevance as an euungulate model in comparative anatomy studies, the available data is strikingly scarce. The present study represents the first description of the masticatory muscles of a Lamini, analyzing the functional morphology of Lama glama and other camelids in a comparative framework. Both sides of the head of three adult specimens from Argentinean Puna were dissected. Descriptions, illustrations, muscular maps, and weighing of all masticatory muscles were performed. Some facial muscles are also described. The myology of llamas confirms that camelids possess relatively large temporalis muscles, with Lama being less extreme than Camelus. This plesiomorphic feature is also recorded in suines and some basal euungulates. Conversely, the direction of the fibers of the M. temporalis is mainly horizontal, resembling grinding euungulates such as equids, pecorans, and some derived suines. Although the M. masseter of camelids and equids do not reach the particularly modified configuration of pecorans, in which it is rostrally extended and arranged horizontally, the posterior sectors of Mm. masseter superficialis and pterygoideus medialis have acquired relatively horizontal disposition in the former lineages, suitable for protraction. The pterygoidei complex presents several bundles, and its relative size is intermediate between suines and derived grinding euungulates. The whole masticatory muscles are relatively light when compared to jaw weight. The evolution of the masticatory muscles and chewing of camelids implied that grinding abilities were reached with less extreme modifications of the topography and/or proportions than pecoran ruminants and equids. A relatively large M. temporalis recruited as a powerful retractor during the power stroke is a key feature of camelids. The relaxed pressure on chewing derived from the acquisition of rumination explains the slenderer build masticatory musculature of camelids compared to other euungulates except ruminants.

General Trends of the Camelidae Antibody VHHs Domain Dynamics.

Conformational flexibility plays an essential role in antibodies' functional and structural stability. They facilitate and determine the strength of antigen-antibody interactions. Camelidae express an interesting subtype of single-chain antibody, named Heavy Chain only Antibody. They have only one N-terminal Variable domain (VHH) per chain, composed of Frameworks (FRs) and Complementarity Determining regions (CDRs) like their VH and VL counterparts in IgG. Even when expressed independently, VHH domains display excellent solubility and (thermo)stability, which helps them to retain their impressive interaction capabilities. Sequence and structural features of VHH domains contributing to these abilities have already been studied compared to classical antibodies. To have the broadest view and understand the changes in dynamics of these macromolecules, large-scale molecular dynamics simulations for a large number of non-redundant VHH structures have been performed for the first time. This analysis reveals the most prevalent movements in these domains. It reveals the four main classes of VHHs dynamics. Diverse local changes were observed in CDRs with various intensities. Similarly, different types of constraints were observed in CDRs, while FRs close to CDRs were sometimes primarily impacted. This study sheds light on the changes in flexibility in different regions of VHH that may impact their in silico design.

Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

Sarcoptic mange outbreaks in vicuñas (Cetartiodactyla: Camelidae): A scoping review and future prospects.

Sarcoptic mange is considered an emerging disease-causing countless epizootics and significantly affecting wild mammals worldwide. The vicuña (Vicugna vicugna) is a medium-sized South American wild camelid inhabiting Andean ecosystems, where several populations are live-sheared by Andean peasant communities as a way of providing an economic income to the people while promoting vicuña conservation. Institutions and scientists have shown concern for the impact and extent of sarcoptic mange in several vicuña populations across their range, as well as the lack of consistent knowledge about this disease in the species. Here, we perform a review about sarcoptic mange distribution throughout the vicuña's native range, evidence of effects of age and sex, the modes of transmission and the veterinary treatments employed. The review retrieved a few scientific papers, but found several reports and academic studies mostly considered as 'grey literature'. Mange was recorded across the entire native vicuña range (Argentina, Bolivia, Chile and Peru). Mange prevalence varied across vicuña studies (up to 60% prevalence in some populations) and severely affected a number of populations, being an important source of mortality. Mange was reported as more frequent in adults than in offspring. The modes of mange transmission remain unclear, although direct transmission between infected and healthy animals seems to be the most likely, including the transmission between domestic camelids and vicuñas. Regarding the treatments employed, ivermectin was the most frequently used. We further identified several gaps in knowledge and point to future research lines, which seek to promote both species conservation and the maintenance of live-shearing vicuñas under sustainable approaches in low-income Andean peasant communities.

Early embryonic death in equines and camelids.

This paper includes the study of early embryonic death (EED), predisposing factors of EED and treatment. EED refers to the fetal mortality which varies in mare and camelids but most probably not later than 50 days of gestation. This duration may be divided into very early mortality, early mortality and late embryonic mortality. This also varies in mare and camelids. There are different embryonic, maternal, environmental/external, and infectious and noninfectious factors which lead to early embryonic loss. Diagnosis is very difficult as in most of the cases resorption of fetus occurs but it is done by the use of ultrasound. Unfortunately, there is no treatment to avoid early embryonic mortality. However, new reproductive technologies have increased the service rate in a herd, and efforts are still being made to determine the rate and frequency of camel embryonic loss.

INDI-integrated nanobody database for immunoinformatics.

Nanobodies, a subclass of antibodies found in camelids, are versatile molecular binding scaffolds composed of a single polypeptide chain. The small size of nanobodies bestows multiple therapeutic advantages (stability, tumor penetration) with the first therapeutic approval in 2018 cementing the clinical viability of this format. Structured data and sequence information of nanobodies will enable the accelerated clinical development of nanobody-based therapeutics. Though the nanobody sequence and structure data are deposited in the public domain at an accelerating pace, the heterogeneity of sources and lack of standardization hampers reliable harvesting of nanobody information. We address this issue by creating the Integrated Database of Nanobodies for Immunoinformatics (INDI, http://naturalantibody.com/nanobodies). INDI collates nanobodies from all the major public outlets of biological sequences: patents, GenBank, next-generation sequencing repositories, structures and scientific publications. We equip INDI with powerful nanobody-specific sequence and text search facilitating access to >11 million nanobody sequences. INDI should facilitate development of novel nanobody-specific computational protocols helping to deliver on the therapeutic promise of this drug format.

A non-internalised CD38-binding radiolabelled single-domain antibody fragment to monitor and treat multiple myeloma.

BACKGROUND: Antibody-based therapies targeting CD38 are currently used as single agents as well as in combination regimens for multiple myeloma, a malignant plasma cell disorder. In this study, we aimed to develop anti-CD38 single-domain antibodies (sdAbs) that can be used to trace CD38+ tumour cells and subsequently used for targeted radionuclide therapy. SdAbs are derived from Camelidae heavy-chain antibodies and have emerged as promising theranostic agents due to their favourable pharmacological properties. METHODS: Four different anti-CD38 sdAbs were produced, and their binding affinities and potential competition with the monoclonal antibody daratumumab were tested using biolayer interferometry. Their binding kinetics and potential cell internalisation were further studied after radiolabelling with the diagnostic radioisotope Indium-111. The resulting radiotracers were evaluated in vivo for their tumour-targeting potential and biodistribution through single-photon emission computed tomography (SPECT/CT) imaging and serial dissections. Finally, therapeutic efficacy of a lead anti-CD38 sdAb, radiolabelled with the therapeutic radioisotope Lutetium-177, was evaluated in a CD38+ MM xenograft model. RESULTS : We retained anti-CD38 sdAb #2F8 as lead based on its excellent affinity and superior stability, the absence of competition with daratumumab and the lack of receptor-mediated internalisation. When intravenously administered to tumour-xenografted mice, radiolabelled sdAb #2F8 revealed specific and sustained tumour retention with low accumulation in other tissues, except kidneys, resulting in high tumour-to-normal tissue ratios. In a therapeutic setting, myeloma-bearing mice received three consecutive intravenous administrations of a high (18.5 MBq) or a low radioactive dose (9.3 MBq) of 177Lu-DTPA-2F8 or an equal volume of vehicle solution. A dose-dependent tumour regression was observed, which translated into a prolonged median survival from 43 days for vehicle-treated mice, to 62 days (p = 0.027) in mice receiving the low and 65 days in mice receiving the high (p = 0.0007) radioactive dose regimen, respectively. CONCLUSIONS: These results highlight the theranostic potential of radiolabelled anti-CD38 sdAbs for the monitoring and treatment of multiple myeloma.

Cooled storage of semen from livestock animals (Part II): Camelids, goats, and sheep.

This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques in cooled storage of semen from livestock animals such as camelids, goats, and sheep. Facing worldwide environmental changes and a trend towards more conscious and healthy eating behaviors, the development of a stable animal breeding industry is a significant challenge for the near future. In the present review, factors influencing semen handling in camelids, goats and sheep are described and relevant methods as well as current trends to improve liquid-storage of cooled semen are discussed, including extenders, additives, cooling rates, and storage temperatures. The species-specific physiology and resulting challenges are taken into consideration. While the main problem for camelid semen processing is the relatively greater viscosity as compared with that of some other animals, the deciding factor for successful artificial insemination (AI) in goats and sheep is the site (i.e., cervical or vaginal) of semen placement in the reproductive tract. Due to the type of cervical anatomy, the penetration of the cervix when using AI instruments is rather difficult. Furthermore, the seminal plasma of small ruminants affects the interaction with milk-based extenders and egg yolk which results in species-specific regimens for cooled liquid-preservation. Comparing all three species, the greatest pregnancy rates were obtained by AI with goat semen after cooled liquid-storage for several days.

Transportation of Single-Domain Antibodies through the Blood-Brain Barrier.

Single-domain antibodies derive from the heavy-chain-only antibodies of Camelidae (camel, dromedary, llama, alpaca, vicuñas, and guananos; i.e., nanobodies) and cartilaginous fishes (i.e., VNARs). Their small size, antigen specificity, plasticity, and potential to recognize unique conformational epitopes represent a diagnostic and therapeutic opportunity for many central nervous system (CNS) pathologies. However, the blood-brain barrier (BBB) poses a challenge for their delivery into the brain parenchyma. Nevertheless, numerous neurological diseases and brain pathologies, including cancer, result in BBB leakiness favoring single-domain antibodies uptake into the CNS. Some single-domain antibodies have been reported to naturally cross the BBB. In addition, different strategies and methods to deliver both nanobodies and VNARs into the brain parenchyma can be exploited when the BBB is intact. These include device-based and physicochemical disruption of the BBB, receptor and adsorptive-mediated transcytosis, somatic gene transfer, and the use of carriers/shuttles such as cell-penetrating peptides, liposomes, extracellular vesicles, and nanoparticles. Approaches based on single-domain antibodies are reaching the clinic for other diseases. Several tailoring methods can be followed to favor the transport of nanobodies and VNARs to the CNS, avoiding the limitations imposed by the BBB to fulfill their therapeutic, diagnostic, and theragnostic promises for the benefit of patients suffering from CNS pathologies.

[Diagnosis of heptic diseases in ruminants and camelids with a special focus on sonographic examination]. / Diagnostik von Lebererkrankungen beim Wiederkäuer und Kameliden unter besonderer Berücksichtigung der sonographischen Untersuchung.

INTRODUCTION: Clinical symptoms are mostly unspecific in hepatic diseases, thus most clinical examinations are of limited benefit in the diagnosis of hepatic diseases and often only general statements can be made about parenchymal damage or loss of function. In contrast, sonography of the liver can provide information about the type of lesion and thus also about the prognosis for the animal. A systematic assessment of organ size, parenchymal, vascular and bile duct system structures can facilitate to make possible findings. Sonography is also used in further diagnostics such as liver biopsy or chole-cystocentesis. This review describes the basic procedure for the sonographic examination in ruminants and camelids on the basis of selected findings relevant to the clinical veterinarian. In addition, potential and limits of additional diagnostics are discussed.

INTRODUCTION: Dans les maladies hépatiques, les symptômes cliniques sont pour la plupart non spécifiques et la plupart des examens cliniques sont d'un bénéfice limité pour le diagnostic; souvent seules des considérations générales peuvent être faites sur les lésions parenchymateuses ou la perte de fonction. En revanche, l'échographie du foie peut fournir des informations sur le type de lésion et donc également sur le pronostic pour l'animal. Une évaluation systématique de la taille de l'organes, des structures des systèmes parenchymateux, vasculaire et biliaire peut faciliter le ciblage des résultats possibles. L'échographie est également utilisée dans d'autres diagnostics tels que la biopsie hépatique ou la cholécystocentèse. Cette revue explique la procédure de base de l'examen échographique chez les ruminants et les camélidés sur la base de résultats sélectionnés, pertinents pour le vétérinaire praticien. De plus, le potentiel et les limites des méthodes de diagnostic supplémentaires sont discutés.

Unique aspects of adaptive immunity in camelids and their applications.

Members of the Camelidae have unique adaptive immunological features that are not widely observed in other species. All camelids are known to have three distinct IgG isotypes - IgG1, IgG2 and IgG3. While IgG1 has a conventional antibody structure, both IgG2 and IgG3 are devoid of light chains and instead possess hypervariable regions in their heavy chain (VHH), while lacking the typical CH1 domain found in heavy chains. VHH domains are increasingly being utilized as "next generation" antibodies, as they have unique biochemical and structural properties including high pH stability as well as a lower molecular weight allowing for increased tissue penetration. These features of VHH domains offer a number of advantages for both biotechnology and clinical applications and are commonly termed "nanobodies". A second unique aspect of the camelid adaptive response is involves T cell-mediated immunity. Characterization of gamma delta (ꝩδ) T cells in camelid species has found they use somatic hypermutation in their T cell receptor gamma (TRG) and delta (TRD) loci to increase the structural stability of their ꝩδ T receptor. The use of somatic hyper mutation to increase the diversity of their T cell repertoire, is a feature that has not been observed in other mammalian species. In addition, in alpacas there is a unique subset of ꝩδ T cells called Vꝩ9Vδ2 T cells. Activation of these cells is dependent upon phosphoantigen (PAg)-mediated interaction with B7-like butyrophilin molecules (BTN-3). This makes alpacas the first species outside of primates to be identified with this unique subset and activation mechanism. Here we review some fundamentals of camelid adaptive immunity that make them distinct from other vertebrate species and their potential applications to human therapies.

A Deadly Cargo: Gene Repertoire of Cytotoxic Effector Proteins in the Camelidae.

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

Global seroprevalence of Toxoplasma gondii in Camelidae: A systematic review and meta-analysis.

PURPOSE: The present study aimed to analyze data available of the seroprevalence of Toxoplasma gondii (T. gondii) among camelids around the world. METHODS: The search was performed using seven international databases including Scopus, PubMed, Google Scholar, ProQuest, ScienceDirect, Web of Science, and EMBASE up to 11 October 2018. Random effects model was used to determine the pooled seroprevalence of T. gondii infection with 95% confidence intervals (CI) and analyzed data from four continents. Subgroup and meta-regression analyses were also performed according to continent and gender. RESULTS: In total, 42 studies out of 3517 published articles involving 14,542 camels from 17 countries were included for the final analyses. The global pooled seroprevalence of T. gondii infection in the Camelidae family was 28.16% (95% CI 23.64-32.68%). Besides, the highest seroprevalence rate was in Europe (49.64%) followed by Africa (37.63%), America (21.76%), and Asia (17.58%). Moreover, the overall seroprevalence rates of T. gondii infection were 22% (95% CI 10-33%) and 15% (95% CI 9-22%) for the females and males, respectively. CONCLUSION: This meta-analysis showed a high seroprevalence of T. gondii infection in camelids as these animals play an important role in the transmission cycle of this zoonotic disease.

Nanobody-Based Indirect Competitive ELISA for Sensitive Detection of 19-Nortestosterone in Animal Urine.

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples.