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A recent study has shown a close neuroanatomical relationship between the enkephalinergic (methionine-enkephalin) and tachykininergic (substance P) systems in the alpaca diencephalon. In this study, our aim is to show this relationship in the alpaca brainstem. Using an immunohistochemical technique, the distribution of immunoreactive (Ir) fibers and cell bodies containing substance P (SP) or methionine-enkephalin (MET) has been studied in the alpaca brainstem. Five adult males were used; brain tissue was fixed and processed by standard methods. SP- and MET-Ir fibers showed a widespread and similar distribution in the mesencephalon, pons and medulla oblongata. The co-localization of fibers containing SP or MET was found in most of the nuclei/tracts of the alpaca brainstem. This close neuroanatomical relationship suggests multiple physiological interactions between both neuropeptides. The distribution of the cell bodies containing SP was very restricted (cell bodies were only observed in a few nuclei located in the mesencephalon and medulla oblongata), whereas MET-Ir perikarya showed a moderately widespread distribution in the mesencephalon, pons and medulla oblongata. This study increases the knowledge on the neuroanatomical distribution/relationship of the tachykininergic (SP) and enkephalinergic (MET) systems in the alpaca central nervous system.

Forty alpaca kids, comprising 20 huacaya and 20 suri, and 20 llama “chaku” (or woolly type), were chosen for a comparative investigation of the fiber and cuticular attributes based on fiber diameter, number of cuticular scales per 100 µm of fiber length, and scale height. Out of the three parameters investigated, the number of cuticular scales proved to be a valid diagnostic trait in distinguishing suri fleece from both huacaya and llama fleeces. A further characterization of the suri fleece was obtained by considering the frequency distribution of both fiber diameter and number of cuticular scale classes. The suri fleece was clearly different from both the huacaya and llama in that it possessed the highest percentage of fibers with less than eight scales, the lowest percentage of fibers with more than nine scales, along with the lowest percentage of fibers with a diameter of more than 35 µm. The validity of these classification criteria was fully supported by a classification discriminant analysis based on the jointed investigation of the data related to scale and diameter frequency classes, whose output was a 100% correct classification of the suri specimens along with an 85.7% and 71.4% of cases correctly classified in huacaya and llama, respectively.

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called V sub(H)H. These V sub(H)Hs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. V sub(H)Hs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the V sub(H)H-coding sequences were selectively amplified. Three V sub(H)Hs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant V sub(H)Hs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The V sub(H)H was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Alpacas, like all camelids, have elliptical red blood cells (RBCs) in contrast to other mammals. This particular shape is important for increased osmotic resistance and stability. Age-related changes in the RBC count are known in other species, with alterations in both red and white blood cells being described. In alpacas, there are few data on age-related changes, and only a comparison of crias with adult animals. We characterized age-related hematologic changes in a study of 21 female alpacas from a research herd. A total of 87 records of clinically healthy alpacas of different ages were statistically analyzed retrospectively from the hematologic records over a nine-year period. Significant positive correlations of age with hemoglobin (Hb), HCT, MCV, MCH, neutrophils, platelet-to-lymphocyte ratio (PLR), and neutrophil-to-lymphocyte ratio (NLR) were found as well as significant negative correlations of age with lymphocytes in addition to lymphocyte-to-monocyte ratio (LMR). A paired comparison of eight older animals in the herd at three different ages also showed significant differences in the parameters Hb, HCT, MCV, MCH, MCHC, lymphocytes, eosinophils and neutrophils. Similar changes in hematologic parameters have been reported in other species and should be taken into account when interpreting hematologic results in alpacas.

Livestock depredation has implications for conservation and agronomy; it can be costly for farmers and can prompt retaliatory killing of carnivores. Lethal control measures are readily available and are reportedly perceived to be cheaper, more practical and more effective than non-lethal methods. However, the costs and efficacy of lethal vs non-lethal approaches have rarely been compared formally. We conducted a 3-year study on 11 South African livestock farms, examining costs and benefits of lethal and non-lethal conflict mitigation methods. Farmers used existing lethal control in the first year and switched to guardian animals (dogs Canis familiaris and alpacas Lama pacos) or livestock protection collars for the following 2 years. During the first year the mean cost of livestock protection was USD 3.30 per head of stock and the mean cost of depredation was USD 20.11 per head of stock. In the first year of non-lethal control the combined implementation and running costs were similar to those of lethal control (USD 3.08 per head). However, the mean cost of depredation decreased by 69.3%, to USD 6.52 per head. In the second year of non-lethal control the running costs (USD 0.43 per head) were significantly lower than in previous years and depredation costs decreased further, to USD 5.49 per head. Our results suggest that non-lethal methods of human–wildlife conflict mitigation can reduce depredation and can be economically advantageous compared to lethal methods of predator control.

Borna disease (BD) is a sporadic neurologic disease of horses and sheep caused by mammalian Borna disease virus (BDV). Its unique epidemiological features include: limited occurrence in certain endemic regions of central Europe, yearly varying disease peaks, and a seasonal pattern with higher disease frequencies in spring and a disease nadir in autumn. It is most probably not directly transmitted between horses and sheep. All these features led to the assumption that an indigenous virus reservoir of BDV other than horses and sheep may exist. The search for such a reservoir had been unsuccessful until a few years ago five BDV-infected shrews were found in a BD-endemic area in Switzerland. So far, these data lacked further confirmation. We therefore initiated a study in shrews in endemic areas of Germany. Within five years 107 shrews of five different species were collected. BDV infections were identified in 14 individuals of the species bicolored white-toothed shrew (Crocidura leucodon, HERMANN 1780), all originating from BD-endemic territories. Immunohistological analysis showed widespread distribution of BDV antigen both in the nervous system and in epithelial and mesenchymal tissues without pathological alterations. Large amounts of virus, demonstrated by presence of viral antigen in epithelial cells of the oral cavity and in keratinocytes of the skin, may be a source of infection for natural and spill-over hosts. Genetic analyses reflected a close relationship of the BDV sequences obtained from the shrews with the regional BDV cluster. At one location a high percentage of BDV-positive shrews was identified in four consecutive years, which points towards a self-sustaining infection cycle in bicolored white-toothed shrews. Analyses of behavioral and population features of this shrew species revealed that the bicolored white-toothed shrew may indeed play an important role as an indigenous host of BDV.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20-25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA. Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis. We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

The origins of South America's domestic alpaca and llama remain controversial due to hybridization, near extirpation during the Spanish conquest and difficulties in archaeological interpretation. Traditionally, the ancestry of both forms is attributed to the guanaco, while the vicuña is assumed never to have been domesticated. Recent research has, however, linked the alpaca to the vicuña, dating domestication to 6000-7000 years before present in the Peruvian Andes. Here, we examine in detail the genetic relationships between the South American camelids in order to determine the origins of the domestic forms, using mitochondrial (mt) and microsatellite DNA. MtDNA analysis places 80% of llama and alpaca sequences in the guanaco lineage, with those possessing vicuña mtDNA being nearly all alpaca or alpaca-vicuña hybrids. We also examined four microsatellites in wild known-provenance vicuña and guanaco, including two loci with non-overlapping allele size ranges in the wild species. In contrast to the mtDNA, these markers show high genetic similarity between alpaca and vicuña, and between llama and guanaco, although bidirectional hybridization is also revealed. Finally, combined marker analysis on a subset of samples confirms the microsatellite interpretation and suggests that the alpaca is descended from the vicuña, and should be reclassified as Vicugna pacos. This result has major implications for the future management of wild and domestic camelids in South America.

Genome analysis of the alpaca (Lama pacos, LPA) has progressed slowly compared to other domestic species. Here, we report the development of the first comprehensive whole-genome integrated cytogenetic map for the alpaca using fluorescence in situ hybridization (FISH) and CHORI-246 BAC library clones. The map is comprised of 230 linearly ordered markers distributed among all 36 alpaca autosomes and the sex chromosomes. For the first time, markers were assigned to LPA14, 21, 22, 28, and 36. Additionally, 86 genes from 15 alpaca chromosomes were mapped in the dromedary camel (Camelus dromedarius, CDR), demonstrating exceptional synteny and linkage conservation between the 2 camelid genomes. Cytogenetic mapping of 191 protein-coding genes improved and refined the known Zoo-FISH homologies between camelids and humans: we discovered new homologous synteny blocks (HSBs) corresponding to HSA1-LPA/CDR11, HSA4-LPA/CDR31 and HSA7-LPA/CDR36, and revised the location of breakpoints for others. Overall, gene mapping was in good agreement with the Zoo-FISH and revealed remarkable evolutionary conservation of gene order within many human-camelid HSBs. Most importantly, 91 FISH-mapped markers effectively integrated the alpaca whole-genome sequence and the radiation hybrid maps with physical chromosomes, thus facilitating the improvement of the sequence assembly and the discovery of genes of biological importance.