Prevalence of Eimeria spp. (Apicomplexa: Eimeriidae) in Oregon Llamas1

Eimeria alpacae, E. punoensis, E. lamae, and E. macusaniensis were identified in fecal samples from 189 llama (Lama glama (L.)) adults and 50 llama crias (animals less than one year of age of any species in the genus Lama) from central and western Oregon. In both adults and crias, E. alpacae was the most common species found. The least common was E. macusaniensis, which was found in only two adults. Overall prevalence and numbers of animals with mixed infections was approximately twice as high in crias as in adults.     

Prevalence of Eimeria macusaniensis (Apicomplexa: Eimeriidae) in midwestern Lama spp

To compare the prevalence of Eimeria macusaniensis among midwestern llamas (Lama glama), alpacas (Lama pacos), and guanacos (Lama guanicoe), feces were obtained from Lama spp. in 10 states between October 1989 and February 1996. Feces were examined by centrifugal flotation in sugar solution (specific gravity--1.28-1.30), and oocysts were quantified by a modified McMaster method. Data were compared by host species and age classifications. Typical oocysts occurred in samples from 28% of 76 herds and 10.4% of 443 animals including 12% of 301 llamas, 7% of 115 alpacas, and 7.4% of 27 guanacos. Prevalence was significantly greater (P = 0.009) in animals < 1 yr of age in comparison to older animals for llams (22.1 v.s. 8.5%) and for all Lama spp. combined (17.1 vs. 8.4%). Fecal oocyst abundance was significantly greater (P = 0.001) in llamas < 1 yr of age in comparison to older llamas (30 vs. 16 oocysts per g of feces). Fecal oocyst intensities did not differ significantly. Prevalence in both age groups of midwestern llamas was greater than previously reported for llamas in the western United States. Prevalence in midwestern alpacas < 1 yr of age was lower than reported for alpacas of similar age in South America, but oocyst intensities were similar. These results indicate that infection with E. macusaniensis is more common in Lama spp. in North America than previously recognized.     

Experimental infections of Eimeria alpacae and Eimeria punoensis in llamas (Lama glama).

Four llamas (Lama glama) ranging in age from 1.5 yr to 7 yr each were inoculated orally with 10,000 (n = 2) or 50,000 (n = 2) sporulated oocysts of Eimeria alpacae (25%) and Eimeria punoensis (75%). The prepatent period for E. aplacae was 16-18 days, and it was 10 days for E. punoensis. Patent periods for E. alpacae and E. punoensis were approximately 9 days and 24 days, respectively. Although large numbers of oocysts were present in feces, no clinical sign of coccidiosis was observed. Based on ths experiment, E. alpacae and E. punoensis at the numbers given are not likely pathogenic in healthy llamas older than 1 yr.     

Isolation and characterization of 10 polymorphic dinucleotide microsatellite markers for llama and guanaco

We isolated and characterized five polymorphic dinucleotide microsatellite markers from llama (Lama glama) and five from guanaco (Lama guanicoe). All loci were assayed on wild llamas and guanacos from Argentina, as all of the primers were able to amplify in both species.     

Interaction of allosteric effectors with alpha-globin chains and high altitude respiration of mammals. The primary structure of two tylopoda hemoglobins with high oxygen affinity: vicuna (Lama vicugna) and alpaca (Lama pacos)

Polyacrylamide gel electrophoresis and ion-exchange chromatography revealed one hemoglobin component for vicuna (Lama vicugna) and alpaca (Lama pacos). Following chain separation by chromatography on carboxymethyl-cellulose, the amino-acid sequences were elucidated for the alpha- and beta-chains of both hemoglobins using automatic Edman degradation of the chains and the tryptic peptides. Vicuna and alpaca have identical beta-chains showing no substitutions to llama (Lama glama) either. In the alpha-chains alpaca differs from llama by the exchange of one amino-acid residue: alpha 122(H5)Asp----His. The same substitution is present in vicuna too, but in addition we found two more exchanges: alpha 10(A8)Ile----Val and alpha 130(H13)Ala----Thr. The close relationship between llama and alpaca suggests that they both originate from the wild guanaco, and there is no domesticated form of vicuna. The sequence data show that the higher oxygen affinity in vicuna compared to llama and alpaca must be due to the alpha-chains as the beta-chains are identical. The significance of the substitutions in alpha 122(H5), an alpha 1/beta 1-contact, and alpha 130(H13) is discussed.     

Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia.

The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.     

Coccidiosis in clinically asymptomatic alpaca (Vicugna pacos) crias from the Peruvian Andes

Parasites of the genus Eimeria are involved in the neonatal diarrhea complex of alpaca (Vicugna pacos) crias, and infection by Eimeria is commonly known as coccidiosis. There are limited reports of these protozoa in clinically asymptomatic crias. In this study, fecal samples from 78 clinically asymptomatic alpaca crias were analyzed to evaluate the prevalence, parasitological load, and diversity of Eimeria species. This study was conducted in the Quenamari community located in the Peruvian Andes (Marangani, Cuzco) at 4500 m above sea level. All fecal samples were examined for parasites using the quantitative McMaster and modified Stoll techniques. Microscopic examination showed the presence of Eimeria oocysts in 68 out of the 78 samples (87.18%). Among the 78 samples we found E. lamae in 67 (85.90%), E. punoensis in 49 (62.82%), E. alpacae in 42 (53.85%), E. macusaniensis in 32 (41.03%), and E. ivitaensis in four (5.13%). Regarding parasitized crias, overall there was a mean parasitological load of 43,920 oocysts per gram of feces (OPG). Eimeria lamae had the highest parasitological load (mean 206,600 OPG). These findings could be due to environmental contamination with oocysts of different Eimeria species. Additional research is needed to determine if this burden of coccidiosis could produce subclinical impacts to the health of alpaca crias.     

Infection of llamas with stored Eimeria macusaniensis oocysts obtained from guanaco and alpaca feces

Oocysts obtained from a guanaco and an alpaca with natural infections were identified as Eimeria macusaniensis and evaluated for host specificity and infectivity over time. In 3 separate trials conducted over 4 yr, 4 adult llamas were fed 500-5,000 sporulated oocysts obtained from guanaco feces stored under laboratory conditions for 41-84 mo. Infections with prepatent periods of 36-41 days and patent periods of 38-55 days developed in 4/4 llamas. In a fourth trial, 3 adult llamas and 1 alpaca were each fed 1,000 sporulated E. macusaniensis oocysts obtained from alpaca feces stored in the laboratory for 3 mo. Infections with prepatent periods of 33-34 days and patent periods of 14-20 days developed in 3/3 llamas. Infection in the alpaca had a prepatent period of 58 days and a patent period of 1 day. Clinical signs associated with infection, if any, were minimal and included increased fecal mucus and occasional soft feces. These results provide evidence that E. macusaniensis is a single species transmissible amongst alpacas, llamas, and guanacos and that oocysts of this species can remain infective for many years.     

Nucleolus organizer regions in the chromosomes of the llama

Silver nitrate (Ag-AS) staining was applied to chromosome preparations from the llama (Lama glama). The NORs were detected on the secondary constrictions of the short arms or the stalks of five small acrocentric chromosome pairs.     

Occurrence of Giardia duodenalis assemblages in alpacas in the Andean region

In this study, 352 fecal samples were analyzed for G. duodenalis from alpaca mothers and crias from three different areas of highland in Peru. The triosephosphate isomerase (TPI) gene of Giardia was amplified using a nested PCR protocol. Forty-six G. duodenalis-PCR positive samples were sequenced. G. duodenalis assemblage A was the most frequent followed by assemblage E. The former was seen in 37 animals whereas the latter was seen in nine. Most of the assemblage A infections were caused by the A1 subtype of sub-assemblage AI, except for three, which were caused by the A2 subtype of sub-assemblage AI. Assemblage A was found in all three geographic regions, while assemblage E was detected in crias from two regions. Among the four alpaca mothers positive for Giardia, three had assemblage AI and one had assemblage AII. Results of this study indicate that possible zoonotic transmission human to alpacas.     

Eimeriid oocysts from archaeological samples in Patagonia, Argentina

Oocysts attributable to E. macusaniensis Guerrero et al. 1971, were found in coprolites and in archaeological sediments dating to the Holocene of Patagonia, Argentina. By means of a non-parametric regression using a generalized additive model, a significant relationship was found between the size of the oocysts and their antiquity. Specifically, a reduction in oocyst size over time was discovered, probably due to a parasite response to host replacement, to an extinct eimeriid species common during the Pliestocene-Holocene transition, or to environmental changes known for the Holocene. Explanations regarding coevolution between parasites, hosts and paleoenvironmental conditions are discussed herein.     

The complete mitochondrial DNA sequence of the guanaco (<Emphasis Type="Italic">Lama guanicoe)</Emphasis>: comparative analysis with the vicuña (<Emphasis Type="Italic">Vicugna vicugna)</Emphasis> genome

South American camelids comprise the guanaco (Lama guanicoe) and the vicuña (Vicugna vicugna), which are wild species, and the domestic llama (Lama glama) and alpaca (Lama pacos). This paper presents the first complete mitochondrial (mt) genome of the guanaco and the mt coding sequence of the vicuña. The guanaco mtDNA is 16,649 nt long and its composition and organization are similar to the mitochondrial genome of other mammals. Excluding the control region, comparison of the complete guanaco and vicuña mtDNA showed 4.4% sequence divergence. Nucleotide differences in peptide coding genes varied from 1.9% in ATP6 to 6.4% in Cyt b. These values are compatible with the close relatedness of both species identified by other authors. Based on the differences between the control region sequence here reported and that previously described, we also discuss the occurrence of NUMTs in the genome of South American camelids.     

Traditional livestock activities modify the spatial behavior of small wildcats in the high Andes

Human activities lead to declines in species’ abundance and diversity, as well as loss of habitat quality from overexploitation. The effects of livestock grazing increase on small felids are poorly understood, primarily due to the low detectability of these species. The grazing of domestic camelids, such as the llama (Lama glama) and the alpaca (Vicugna pacos), is the main traditional source of income for the inhabitants of the Andes, where the Andean cat (Leopardus jacobita) and Pampas cat (L. colocolo) are also present. In this study, we employ camera trap data to evaluate the association of llama and alpaca grazing with occupancy by the Andean and Pampas cat in Sajama National Park. In each of 36 sampling stations, we installed a camera trap, a grid of traps for rodents and two habitat transects along a gradient of intensity of livestock grazing. Occupancy models showed that negative effect of camelid abundance was stronger for the Andean cat than the Pampas cat. For both species, occupancy was also associated positively to the abundance of prey, proximity to water bodies, and vegetation. Given that livestock activity is a way of life for human populations in the Andes, we suggest that mechanisms should be implemented to compensate for the negative effects of livestock on Andean and Pampas cat populations.     

Prediction of gestational age by ultrasonic fetometry in llamas (Lama glama) and alpacas (Lama pacos)

Fetal biparietal diameter (BPD) and thorax height (TH) were measured by ultrasound during intrauterine growth in pregnant llamas (Lama glama) and alpacas (Lama pacos). The goal was to establish representative curves that allows estimation of gestational age (GA) from real-time ultrasonic measurements of these fetal structures at any stage of gestation. Llamas and alpacas were mated under controlled conditions. Ultrasound exams were conducted to determine pregnancy status 1 month later. Measurements of fetal BPD and TH were conducted from the second month of pregnancy until term. Observation and assessment of fetal TH was difficult during the last 3 months of pregnancy, specially in llamas. Regression curves were calculated from the data as a function of GA, with the best fit represented by the following equations: llama GA=(BPD-0.002399)43.02293,r=0.98,P<0.001; llama GA=(TH-0.07137)46.94485, r=0.95,P<0.001; alpaca GA=(BPD-0.11376)47.23287, r=0.98,P<0.001; alpaca GA=(TH-0.36436)52.87663, r=0.96,P<0.001, where GA was measured in days and BPD and TH in centimeters. Results indicate that ultrasonic measurement of these fetal biometric variables constitute a valuable tool to estimate GA at any stage of pregnancy in these domestic South American camelids.     

Comparison of the effect of ovulation-inducing factor (OIF) in the seminal plasma of llamas, alpacas, and bulls

We have recently reported the presence of an ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas-species characterized as induced ovulators. The study was designed to test the hypothesis that the seminal plasma of bulls will induce ovulation in llamas, and to compare the ovulation-inducing effect of seminal plasma of conspecific versus hetero-specific males. The seminal plasma of alpacas, a closely related induced ovulator (Lama pacos), and cattle, a distantly related ruminant species (Bos taurus) considered to be spontaneous ovulators, were compared with that of the llama (Lama glama). Ovulation and maximum corpus luteum diameter were compared by ultrasonography among female llamas (n=19 per group) treated intramuscularly with 2 mL of phosphate buffered saline (PBS, negative control) and those treated with 2 mL of seminal plasma of bulls, alpacas, or llamas (conspecific control). The diameter of the preovulatory follicle did not differ among groups at the time of treatment. Bull seminal plasma induced ovulations in 26% (5/19) of llamas compared to 0% (0/19) in PBS group (P<0.001). The proportion of females that ovulated was lower (P<0.01) in bull seminal plasma group compared to the groups treated with alpaca or llama seminal plasma (100%). A corpus luteum was detected on Day 8 (Day 0=treatment) in all llamas in which ovulation was detected earlier (Day 2) by ultrasonography. The diameter of the CL did not differ among groups. Results document the presence of an ovulation-inducing factor in the seminal plasma of B. taurus. The interspecies effects of seminal plasma on ovulation and luteal development provide rationale for the hypothesis that OIF is conserved among both spontaneous and induced ovulating species.     

The interaction between phosphate and protein, and the respiration of the llama, the human fetus and the horse (author's transl)

The sequence analysis of llama (Lama glama, Camelidae) hemoglobin is described. The chains were separated, cleaved by trypsin as previously described, quantitatively characterized and sequenced in the sequenator. The llama hemoglobin differs from the human hemoglobin in that it has 25 different amino acids in the alpha chain and 24 different amino acids in the beta chain. The interaction between protein and phosphate is discussed. The earlier finding that the O2 affinity of the llama hemoglobin is dependent on its content of 2, 3-bisphosphoglycerate is interpreted here as a mutation of the 2, 3-bisphosphoglycerate contact position beta2 His in human hemoglobin to beta2 Asn in llama hemoglobin, whereby one of the four 2, 3-bisphosphoglycerate contact points is interrupted. This interruption gives rise to a diminished reduction of intrinsic oxygen affinity in the hemoglobin molecule and explains, on a molecular basis, the increased oxygen affinity of the llama hemoglobin, and consequently, the high-altitude respiration of the llama. By analogy, the increased O2 affinity of human fetal hemoglobin is interpreted according to previous physiological investigations on blood and fetal hemoglobin by the inactivation of the phosphoglycerate contact point beta143 His in the adult hemoglobin by mutation to gamma 143 Ser in the fetal hemoglobin. With respect to respiration in horses (2, 3-bisphosphoglycerate contact beta2 Gln), measurement of atomic parameters show that the amido group of the glutamine is situated close enough to the 2, 3-bisphosphoglycerate oxygen to build a hydrogen bond with the phosphate. Consequently, the explanation of the low-altitude respiration of the horse lies in the fact that glutamine and histidine fulfill sterochemically an identical function.     

Y‐chromosome and mtDNA variation confirms independent domestications and directional hybridization in South American camelids

Investigations of genetic diversity and domestication in South American camelids (SAC) have relied on autosomal microsatellite and maternally‐inherited mitochondrial data. We present the first integrated analysis of domestic and wild SAC combining male and female sex‐specific markers (male specific Y‐chromosome and female‐specific mtDNA sequence variation) to assess: (i) hypotheses about the origin of domestic camelids, (ii) directionality of introgression among domestic and/or wild taxa as evidence of hybridization and (iii) currently recognized subspecies patterns. Three male‐specific Y‐chromosome markers and control region sequences of mitochondrial DNA are studied here. Although no sequence variation was found in SRY and ZFY, there were seven variable sites in DBY generating five haplotypes on the Y‐chromosome. The haplotype network showed clear separation between haplogroups of guanaco–llama and vicuña–alpaca, indicating two genetically distinct patrilineages with near absence of shared haplotypes between guanacos and vicuñas. Although we document some examples of directional hybridization, the patterns strongly support the hypothesis that llama (Lama glama) is derived from guanaco (Lama guanicoe) and the alpaca (Vicugna pacos) from vicuña (Vicugna vicugna). Within male guanacos we identified a haplogroup formed by three haplotypes with different geographical distributions, the northernmost of which (Peru and northern Chile) was also observed in llamas, supporting the commonly held hypothesis that llamas were domesticated from the northernmost populations of guanacos (L. g. cacilensis). Southern guanacos shared the other two haplotypes. A second haplogroup, consisting of two haplotypes, was mostly present in vicuñas and alpacas. However, Y‐chromosome variation did not distinguish the two subspecies of vicuñas.     

Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae

Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.     

Fine structure and development of Sarcocystis aucheniae in llamas

Isolated cysts of Sarcocystis aucheniae of the llama (Lama glama) were fed to one dog and one cat. Only the dog excreted sporocysts, measuring 13.1-15.7 (15.0 +/- 0.54) X 9.0-11.3 (10.4 +/- 0.36) micron after 11 days for 21 days. A second cat, which had ingested meat of a llama containing macrocysts of S. aucheniae as well as sarcosporidial cysts visible only under a microscope also did not excrete sporocysts. The cysts of S. aucheniae are surrounded by a folded primary cyst wall forming cauliflower-like protrusions into the muscle fibre. The protrusions contain numerous microfilaments. In addition, the primary cyst wall forms numerous tiny vesicles. The parasitized muscle fibre is located in a large cavity within the normal muscle tissue. The cyst wall of S. aucheniae is similarly structured to that of S. gigantea of the sheep.     

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.