Using multiple markers to elucidate the ancient, historical and modern relationships among North American Arctic dog breeds.

Throughout most of the Americas, post-colonial dogs largely erased the genetic signatures of pre-historical dogs. However, the North American Arctic harbors dogs that are potentially descended from pre-historical ancestors, as well as those affected by post-colonial translocations and admixtures. In particular, Inuit dogs from Canada and Greenland are thought to descend from dogs associated with Thule peoples, who relied on them for transportation ca. 1000 years ago. Whether Thule dogs reflected an earlier colonization by Paleoeskimo dogs ca. 4500 years ago is unknown. During the Alaskan Gold Rush, additional sled dogs, possibly of post-colonial derivation, the Alaskan Husky, Malamute and Siberian Husky, were used in the Arctic. The genealogical relationships among and origins of these breeds are unknown. Here we use autosomal, paternal and maternal DNA markers to (1) test the hypothesis that Inuit dogs have retained their indigenous ancestry, (2) characterize their relationship to one another and to other Arctic breeds, and (3) estimate the age of North American indigenous matrilines and patrilines. On the basis of the agreement of all three markers we determined that Inuit dogs have maintained their indigenous nature, and that they likely derive from Thule dogs. In addition, we provide support for previous research that the Inuit dogs from Canada and Greenland dog should not be distinguished as two breeds. The Alaskan Husky displayed evidence of European introgression, in contrast to the Malamute and Siberian Husky, which appear to have maintained most of their ancient Siberian ancestry.

Next-generation sequencing workflow for assembly of nonmodel mitogenomes exemplified with North Pacific albatrosses (Phoebastria spp.).

Use of complete mitochondrial genomes (mitogenomes) can greatly increase the resolution achievable in phylogeographic and historical demographic studies. Using next-generation sequencing methods, it is now feasible to efficiently sequence mitogenomes of large numbers of individuals once a reference mitogenome is available. However, assembling the initial mitogenomes of nonmodel organisms can present challenges, for example, in birds, where mtDNA is often subject to gene rearrangements and duplications. We developed a workflow based on Illumina paired-end, whole-genome shotgun sequencing, which we used to generate complete 19-kilobase mitogenomes for each of three species of North Pacific albatross, a group of birds known to carry a tandem duplication. Although this duplication had been described previously, our procedure did not depend on this prior knowledge, nor did it require a closely related reference mitogenome (e.g. a mammalian mitogenome was sufficient). We employed an iterative process including de novo assembly, reference-guided assembly and gap closing, which enabled us to detect duplications, determine gene order and identify sequence for primer positioning to resolve any mitogenome ambiguity (via minimal targeted Sanger sequencing). We present full mtDNA annotations, including 22 tRNAs, 2 rRNAs, 13 protein-coding genes, a control region and a duplicated feature for all three species. Pairwise comparisons supported previous hypotheses regarding the phylogenetic relationships within this group and occurrence of a shared tandem duplication. The resulting mitogenome sequences will enable rapid, high-throughput NGS mitogenome sequencing of North Pacific albatrosses via direct reference-guided assembly. Moreover, our approach to assembling mitogenomes should be applicable to any taxon.

The effects of dog breed development on genetic diversity and the relative influences of performance and conformation breeding.

Genetic diversity was compared among eight dog breeds selected primarily for conformation (Standard Poodle, Italian Greyhound and show English Setter), conformation and performance (Brittany), predominantly performance (German Shorthaired and Wirehaired Pointers) or solely performance (field English Setter and Red Setter). Modern village dogs, which better reflect ancestral genetic diversity, were used as the standard. Four to seven maternal and one to two Y haplotypes were found per breed, with one usually dominant. Diversity of maternal haplotypes was greatest in village dogs, intermediate in performance breeds and lowest in conformation breeds. Maternal haplotype sharing occurred across all breeds, while Y haplotypes were more breed specific. Almost all paternal haplotypes were identified among village dogs, with the exception of the dominant Y haplotype in Brittanys, which has not been identified heretofore. The highest heterozygosity based on 24 autosomal microsatellites was found in village dogs and the lowest in conformation (show) breeds. Principal coordinate analysis indicated that conformation-type breeds were distinct from breeds heavily used for performance, the latter clustering more closely with village dogs. The Brittany, a well-established dual show and field breed, was also genetically intermediate between the conformation and performance breeds. The number of DLA-DRB1 alleles varied from 3 to 10 per breed with extensive sharing. SNPs across the wider DLA region were more frequently homozygous in all pure breeds than in village dogs. Compared with their village dog relatives, all modern breed dogs exhibit reduced genetic diversity. Genetic diversity was even more reduced among breeds under selection for show/conformation.

Genetic characterization of healthy and sebaceous adenitis affected Standard Poodles from the United States and the United Kingdom.

The degree of heterogeneity associated with geographic origin and sebaceous adenitis (SA) status in Standard Poodles from the United States (US) and the United Kingdom (UK) was assessed. Healthy and SA-affected Standard Poodles from the US and the UK shared a major mitochondrial DNA (mtDNA) haplotype and a single Y chromosome haplotype. However, minor mtDNA haplotypes and frequencies were somewhat different between US and UK dogs and were significantly less associated with SA than major haplotypes across both populations. The US and UK populations exhibited recent divergence from a common gene pool, based on allele frequencies of 24 highly polymorphic short tandem repeats and principle coordinates and cluster analyses of genotype frequencies. However, there was no differentiation between SA affected and unaffected dogs. Over 90% of US and UK Poodles shared a common dog leukocyte antigen (DLA) class II haplotype, but showed some differentiation in minor haplotype frequency. No difference was observed in haplotype heterozygosity between SA affected and unaffected dogs from the same country and no disease association for SA was found within the DLA region by a high density single nucleotide polymorphism (SNP) scan. Zygosity mapping in the DLA region of Poodles indicated much lower site-specific diversity than in an outbred population of street dogs from Bali, Indonesia, reflecting the degree that breed associated historical bottlenecks have reduced diversity in a polymorphic region of the genome. This study shows possible pitfalls in more extensive genome-wide association studies, such as case and control numbers, population stratification, the involvement of multiple genes, and/or the possibility that SA susceptibility is fixed or nearly fixed within the breed, which can reduce power to detect genetic associations.

Environmental factors associated with Bartonella vinsonii subsp. berkhoffii seropositivity in free-ranging coyotes from northern California.

Bartonella vinsonii subsp. berkhoffii is a newly recognized pathogen of domestic dogs and humans. Coyotes (Canis latrans) are considered an important reservoir of this bacterium in the western United States, but its vectors are still unknown. Our objective was to identify environmental factors associated with Bartonella antibody prevalence in 239 coyotes from northern California, using an enzyme-linked immunosorbent assay. In addition, associations were evaluated between B. v. berkhoffii and two pathogens with known vectors and habitat requirements, Dirofilaria immitis and Anaplasma phagocytophilum. Overall, B. v. berkhoffii seroprevalence was 28% (95% confidence interval [CI], 22.3%, 33.7%) and Bartonella seropositive coyotes were more likely than seronegative coyotes to be positive for Anaplasma phagocytophilum (Odds ratio = 3.3; 95% CI = 1.8, 5.9) and Dirofilaria immitis (Odds ratio = 2.1; 95% CI = 1.2, 3.8). The most likely geographic clusters of Bartonella and Dirofilaria overlapped. Bartonella seropositivity was associated with higher precipitation (p = 0.003) and proximity to the coast (p = 0.007) in univariate analysis. The association with precipitation varied with season, based on a logistic regression model.

Spatial analysis of Yersinia pestis and Bartonella vinsonii subsp. berkhoffii seroprevalence in California coyotes (Canis latrans).

Zoonotic transmission of sylvatic plague caused by Yersinia pestis occurs in California, USA. Human infections with various Bartonella species have been reported recently. Coyotes (Canis latrans) are ubiquitous throughout California and can become infected with both bacterial agents, making the species useful for surveillance purposes. This study examined the geographic distribution of 863 coyotes tested for Y. pestis and Bartonella vinsonii subsp. berkhoffii serologic status to gain insight into the natural history of B. vinsonii subsp. berkhoffii and to characterize the spatial distribution of the two agents. We found 11.7% of specimens positive to Y. pestis and 35.5% positive to B. vinsonii subsp. berkhoffii. The two pathogens had distinct spatial clusters: Y. pestis was more prevalent in eastern portions of the state and B. vinsonii subsp. berkhoffii in coastal regions. Prevalence of Y. pestis increased with increasing elevation, whereas prevalence of B. vinsonii subsp. berkhoffii decreased with increasing elevation. There were differences in the proportions of positive animals on a yearly basis to both pathogens.

Validation for use with coyotes (Canis latrans) of a commercially available enzyme-linked immunosorbent assay for Dirofilaria immitis.

Serological tests offer a potentially powerful tool for monitoring parasites in wildlife populations. However, such tests must be validated before using them with target wildlife populations. We evaluated in coyotes (Canis latrans) the performance of a commercially available serological test used to detect canine heartworm (Dirofilaria immitis) in domestic dogs. We obtained 265 coyote carcasses and serological specimens from 54 additional coyotes from several regions of California, USA. We necropsied coyotes to determine the adult heartworm infection status. Blood was collected at necropsy on filter paper strips and allowed to dry; it was later eluted in a buffer solution, and the supernatant was tested for heartworm. Receiver operating characteristic (ROC) analysis was used to assess discriminatory power of the test and indicated a 93% probability that a randomly selected infected coyote would exhibit a higher enzyme-linked immunosorbent assay (ELISA) value than a randomly selected uninfected coyote. We estimated specificity at 96% (95% CI: 92-98%) for 165 uninfected coyotes and sensitivity at 85% (77-91%) for 100 infected coyotes, results similar to published values for the commercial serological test used with dog serum or plasma. Test performance was similar for filter paper specimens and supernatant of frozen whole blood collected in EDTA tubes (i.e. hemolyzed plasma). We found no difference in test performance among geographic or demographic coyote groups. Our findings support application of the test to filter paper or standard serological specimens for detection of heartworm in coyote populations.

Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance".

BACKGROUND: Acute renal failure (ARF) leads to secondary adaptive changes that serve to protect proximal tubules from subsequent ischemic or toxic damage [so-called "acquired cytoresistance" (CR)]. A characteristic of CR is increased plasma membrane resistance to attack. Therefore, this study sought to identify potential changes in plasma membrane lipid composition in CR tubules/renal cortex and, if present, to test whether they might mechanistically contribute to the CR state. METHODS: Renal cortices/isolated tubules were obtained from CR mouse kidneys (18-hr postinduction of ischemia reperfusion, myoglobinuria, or ureteral obstruction). Their plasma membrane phospholipid/cholesterol profiles were compared with those observed in either control tissues or tissues obtained one to two hours post-renal damage (that is, prior to emergence of CR). RESULTS: Either no changes or inconsistent changes in phospholipid profiles were observed in CR tissues. Conversely, CR (vs. control) tissues demonstrated a consistent 25 to 50% increase in membrane cholesterol content. To ascertain whether cholesterol impacts tubule susceptibility to injury, its levels were reduced in proximal tubule (HK-2) cells with either (a) mevastatin, (b) a cholesterol "stripping" agent, (c) cholesterol oxidase, or (d) cholesterol esterase. Then cell susceptibility to injury [adenosine 5'-triphosphate (ATP) depletion; Fe-mediated oxidant stress] was assessed. In each instance, cholesterol reductions dramatically sensitized to superimposed injury (for example, a 2 to 3 times increase in the % of lactate dehydrogenase release). When cholesterol levels were restored to normal in CR tubules (with a "stripping" agent), an increased tubule susceptibility to injury resulted. Because cholesterol decreases membrane fluidity, the impact of a membrane-fluidizing agent (A2C) on cell injury was assessed. A2C dramatically sensitized HK-2 cells to superimposed attack. CONCLUSIONS: ARF leads to an up-regulation of proximal tubule cholesterol content. The latter may then contribute to acquired CR, possibly by stabilizing the plasma membrane via its antifluidizing effect.

Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack.

BACKGROUND: Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues. METHODS: Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure. RESULTS: Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data. CONCLUSIONS: (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.

Biochemical composition of human peripheral arteries examined with near-infrared Raman spectroscopy.

PURPOSE: Near-infrared Raman spectroscopy provides an important new means of analyzing the chemical composition of the arterial wall. The objective of this study was to show that Raman spectroscopy can be used to evaluate the lipid and calcium salt contents of human peripheral arteries. The results extend a recently developed Raman-based method for analyzing the chemical composition of coronary arteries. METHODS AND RESULTS: We studied 167 segments of carotid and femoral artery wall in various pathologic states. The Raman spectra from these samples was accurately modeled. The resulting chemical concentrations were compared with the amounts of cholesterol and calcium mineral determined at histologic evaluation by an experienced cardiovascular pathologist. Strong correlations between spectroscopic measurements and morphologic findings were demonstrated and validated the applicability of the method to peripheral arteries. CONCLUSIONS: Raman spectroscopy can provide reliable histochemical information about peripheral and coronary arteries. Such information may help identify rupture-prone plaques before the onset of symptoms and allow aggressive and directed intervention. Accurate knowledge of the chemical composition of a lesion may be useful in selecting the most appropriate treatment.

Increasing prevalence of canine heartworm in coyotes from California.

Thirty-seven subadult and adult coyotes (Canis latrans), collected August 1992 through December 1996 from a coastal foothill area in northern California (USA), were examined for adult heartworm (Dirofilaria immitis). During 1992 through 1993, at the end of a 6 yr drought, none of four coyotes examined were infected with heartworms. However, during 1994 through 1996, after the drought had ended, prevalences were 91% in 23 adult coyotes and 40% in 10 subadult coyotes. Heartworm intensity did not differ by sex of coyote, and averaged (+/- SE) 19.4 +/- 3.8 among adults; one subadult had > 238 heartworms. The prevalence and intensity of heartworm infection in coyotes reported here for 1994 through 1996 are the highest reported anywhere in the United States.

Noninvasive vascular imaging in the diagnosis and treatment of adventitial cystic disease of the popliteal artery.

This brief case report describes the successful outcome after surgical excision of multiple adventitial cysts of the popliteal artery in a 75-year-old man with rapidly worsening claudication. It highlights several unsettled points concerning the diagnosis, cause, and management of cystic adventitial disease of the popliteal artery and compares duplex ultrasound, computed tomography, and magnetic resonance angiography in the noninvasive diagnosis and treatment of this condition.

Symmetry of the femoral notch width index.

A small femoral notch width index has been reported as a predictive factor for anterior cruciate ligament injury and implicated in the higher incidence of anterior cruciate ligament injuries in female athletes. Notch-plasty has been recommended for the unaffected knees of patients who have torn one anterior cruciate ligament and whose notch width index falls one standard deviation below "normal". However, the symmetry of the notch width index has not been specifically studied. We compared the notch width index in both knees of 40 male and 40 female patients. Half of the patients in each group had anterior cruciate ligament injuries, all from a noncontact mechanism. We found that the notch width indexes of the right and left knees of the same patient are essentially symmetrical, regardless of sex or anterior cruciate ligament status. Although the female patients tended to have smaller notch width indexes than the male patients, the difference was not statistically significant. Moreover, the ranges of notch width indexes in male and female patients overlapped considerably. Finally, there was no difference in notch width index between patients with and without anterior cruciate ligament tears. These findings suggest that the notch width index alone is not the critical etiologic factor in the patient with a unilateral anterior cruciate ligament tear. Furthermore, the increased incidence of anterior cruciate ligament tears in female patients compared with male patients in the same sports cannot be attributed to notch width index alone.

Strategies to increase the use of autogenous arteriovenous fistula in end-stage renal disease.

The purpose of this retrospective study was to review our experience with a consecutive group of end-stage renal disease (ESRD) patients using simple strategies to increase the use of autogenous vascular access, and determine whether the current trend of using synthetic bridge-graft fistula (BGF) rather than autogenous arteriovenous fistula (AVF), could be reversed, despite an aging population and broadening criteria for hemodialysis. All patients for vascular access surgery had careful preoperative clinical examination of the arm veins with outflow occlusion to determine the venous anatomy and continuity. Where no veins were apparent or their continuity in doubt, selective preoperative venography was performed. Where veins were unsatisfactory for forearm AVF, new or modified surgical procedures to use both the basilic and cephalic veins in the upper arm were performed. Intraoperative angioscopy was used to monitor vein quality and surgical technique. Ninety-eight primary vascular access procedures were performed in 76 patients, 75 (76.5%) AVF (forearm, n = 41; upper arm, n = 34) and 23 (23.5%) BGF. Forty-one of 76 (54%) had already had at least one previous access procedure prior to this study. More than one access procedure was needed in 16 patients. Preoperative venography was performed in 22 (22.4%) and intraoperative angioscopy in 45 (45.9%) of the 98 procedures. The number of revisions required to maintain patency was significantly higher for BGF (37 revisions in 14/23) than AVF (16 revisions in 13/75) (p < 0.0001, Poisson test) with an annualized secondary revision rate of 1.168 for BGF and 0.173 for AVF (p < 0.0001, Poisson test). AVF had both longer primary (p = 0.0001, log rank test) and secondary patency (p = 0.038, log rank test) than BGF. AVF as the primary vascular access can be significantly increased and the current trend of using BGF reversed with the use of simple clinical strategies to evaluate the suitability of the arm veins for vascular access.

Diagnosis of parathyroid adenomas: efficacy of measuring parathormone levels in needle aspirates of cervical masses.

OBJECTIVE: The purpose of this study was to assess the value of measuring parathormone levels in percutaneous needle aspirates of suspicious cervical lesions in patients with hyperparathyroidism to confirm whether the lesion represents abnormal parathyroid tissue. SUBJECTS AND METHODS: The study group consisted of 66 patients with hyperparathyroidism in whom 80 cervical lesions were aspirated and levels of parathormone in the aspirates were measured. CT guidance was used for two patients and sonographic guidance for the remainder. The lesions selected for aspiration were demonstrated on either sonography or CT and had either an unusual position (separate from the thyroid gland or were intrathyroidal) or configuration (irregular shape or atypical heterogeneous sonographic texture). In 15 patients, an indeterminate, posteriorly located intrathyroidal mass was detected and felt most likely to represent a thyroid nodule by sonographic criteria. These masses were aspirated to rule out atypical parathyroid adenomas. In patients who had been previously explored for hyperparathyroidism and presented with persistent or recurrent hypercalcemia, all indeterminate, cervical, potentially parathyroid masses were aspirated for parathormone determination. The level of parathormone in each aspirate was measured by using an immunoradiometric assay. RESULTS: Levels of parathormone were increased in the aspirates in 37 of the 45 patients in whom sonography showed classic lesions suggestive of parathyroid adenoma. This included the 25 patients who had previously undergone exploratory surgery. At surgery, all 37 had parathyroid adenomas in the indicated locations, for a specificity of 100%. In six patients, the results of the aspiration were false-negative. Parathyroid adenomas were suspected on sonograms and confirmed at surgery, but no parathormone was detected in the aspirate. Results of aspiration of indeterminate lesions were true-negative in two patients who had both characteristic and indeterminate lesions on sonography and in the 15 patients who had indeterminate lesions that were felt to be of thyroid origin. Aspirates contained no parathormone, and surgical findings confirmed the lesions were not of parathyroid origin. CONCLUSION: Our results show that increased levels of parathormone in percutaneous needle aspirates of cervical masses in patients with hyperparathyroidism confirm the mass is a parathyroid adenoma. Although absence or low levels of parathormone in the aspirates usually excludes a parathyroid adenoma, this is not absolute as sometimes the needle may miss the mass, rendering the parathormone value invalid (false-negative).

Influence of freezing method on thaw drip and protein loss of low-voltage electrically stimulated and non-stimulated sheeps' muscle.

South African Mutton Merino wethers (n = 32) were slaughtered, yielding carcasses with a mean weight of 22·18 ± 2·11 kg. Sixteen carcasses were electrically stimulated (ES) (21 V, 60 Hz, 120 s) immediately and all carcasses were chilled at room temperature (16°C) for 3 h and then overnight at 4°C, 95% RH. Both left and right Mm. longissimus lumborum et thoracis were excised and cut into six portions (77 g ± 7·8 g), each placed separately in a polyethylene bag and randomly allocated to five freezing treatments. These were: (1) cryogenic, -65°C; (2) cryogenic, -90°C; (3) walk-in-freezer, -21°C; (4) blast freezer, -21°C; (5) domestic freezer, -25°C. The respective freezing rates were 4·4, 6·4, 0·55, 0·35 and 0·51 cm h(-1) to -2·2°C at core depth of 1 cm below the surface. Samples were frozen to core temperatures of -20°C, removed and placed in a storage freezer (-20°C) for 48 h and 2·5 months. Samples were then suspended in perforated bags in a chiller (4°C) to thaw, and drip was collected in outer bags over the periods 0-24, 24-48, 48-72 and 72-96 h and expressed as g (100 g)(-1). Freezing methods had significant (P < 0·01) influences on drip loss in both ES and NES samples. Following storage for 48 h post-freezing at -20°C, total drip (g (100 g)(-1)) over 96 h of both ES and NES samples for the five freezing treatments were respectively: (1) 7·61 and 4·61; (2) 7·35 and 3·29; (3) 9·44 and 4·68; (4) 9·07 and 5·43; (5) 10·58 and 5·15. Following storage for 2·5 months, the total ES and NES drip were respectively, (1) 11·25 and 9·38; (2) 10·36 and 9·15; (3) 13·72 and 12·65; (4) 13·70 and 12·26; (5) 11·92 and 11·29. Total protein in the drip did not differ between freezing treatments. Differences between ES and NES samples did occur in the 48 h storage group. It is concluded that cryogenic freezing results in less thaw drip than the vapour compression systems. This advantage of cryogenic freezing disappears if meat is stored for long periods at -20°C. Electrical stimulation increases the drip loss in samples frozen for 48 h, but the differences are not significant after 2·5 months frozen storage. Protein losses parallel the drip.