Seroprevalence of Brucella antibodies in harbor seals in Alaska, USA, with age, regional, and reproductive comparisons.

Populations of harbor seal Phoca vitulina in the Gulf of Alaska have dramatically declined during the past 4 decades. Numbers of seals in Glacier Bay, in southeast Alaska, USA, have also declined despite extensive protection. Causes of the declines and slow recovery are poorly understood. Brucellosis is a zoonotic disease that adversely affects reproduction in many domestic species. We measured the seroprevalence of Brucella antibodies in 554 harbor seals in 3 Alaska locations: Prince William Sound (PWS), Glacier Bay (GB), and Tracy Arm Fords Terror (TAFT) Wilderness Area. Objectives included testing for regional, sex, age, and female reproductive state differences in Brucella antibody seroprevalence, persistence in titers in recaptured seals, and differences in titers between mother seals and their pups. Overall, 52% of adults (AD), 53% of subadults (SA), 77% of yearlings (YRL), and 26% of <5 mo old pups were seropositive. Matched mother-pup samples were consistent with dependent pups acquiring maternal passive immunity to Brucella. Results show higher seroprevalence (64%) for AD and SA seals in the depressed and declining populations in PWS and GB than in TAFT (29%). Lactating females were less likely to be seropositive than other AD females, including pregnant females. Further research is needed to seek evidence of Brucella infection in Alaskan harbor seals, identify effects on neonatal viability, and assess zoonotic implications for Alaska Natives who rely on harbor seals for food.

Precise orientation of a single C60 molecule on the tip of a scanning probe microscope.

We show that the precise orientation of a C(60) molecule which terminates the tip of a scanning probe microscope can be determined with atomic precision from submolecular contrast images of the fullerene cage. A comparison of experimental scanning tunneling microscopy data with images simulated using computationally inexpensive Hückel theory provides a robust method of identifying molecular rotation and tilt at the end of the probe microscope tip. Noncontact atomic force microscopy resolves the atoms of the C(60) cage closest to the surface for a range of molecular orientations at tip-sample separations where the molecule-substrate interaction potential is weakly attractive. Measurements of the C(60)-C(60) pair potential acquired using a fullerene-terminated tip are in excellent agreement with theoretical predictions based on a pairwise summation of the van der Waals interactions between C atoms in each cage, i.e., the Girifalco potential [L. Girifalco, J. Phys. Chem. 95, 5370 (1991)].

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas).

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean +/- SD) 73.0 +/- 12.2, 38.4 +/- 8.8, 88.0 +/- 0.1, and 59.3 +/- 15.7%, respectively. A series of GnRH injections (3 x 250 microg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 +/- 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 +/- 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 +/- 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 +/- 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 +/- 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.

Nutrient digestion and performance by lambs and steers fed thermochemically treated crop residues.

Five studies were conducted to determine nutrient digestibility and performance of lambs and steers fed thermochemically treated crop residues and distillers dried grains with solubles (DDGS) as a corn replacement pellet (CRP; 75% residue:25% DDGS, DM basis). Fifteen Hampshire, Suffolk, or Dorset wethers (BW 33.3 +/- 5.0 kg) were utilized to evaluate nutrient digestibility of the unprocessed native (NAT) and CRP [Exp. 1: wheat straw (WS); Exp. 2: corn stover (CS); Exp. 3: switchgrass (SWG) and corn fiber:wheat chaff (CFWC)] when limit fed (Exp. 1 and 2: 1.8% of BW daily; Exp. 3: 2.5% of BW daily) compared with a 60% corn diet. In Exp. 4, 56 individually fed Dorset-cross wether lambs (BW 32.0 +/- 1.4 kg) were utilized to compare performance and digestibility of WS, wheat chaff (WC), corn fiber (CF), a 3:1 blend of corn fiber:wheat straw (CFWS), a 3:1 blend of CFWC, and SWG-CRP fed for ad libitum intake compared with a 45% corn diet. In Exp. 5, 32 individually fed Holstein steers (BW 185.2 +/- 0.9 kg) were used to evaluate performance and digestibility of diets containing corn, WS-CRP, CFWC-CRP, or NAT-WS fed for ad libitum intake. Crop residues were processed with 5% calcium oxide (DM basis) and 35% water in a double-shaft enclosed mixer (Readco Kurimoto Continuous Processor, York, PA) and subsequently pelleted with DDGS to form CRP. Feeding lambs WS-CRP (Exp. 1) or CS-CRP (Exp. 2) increased digestion of DM, NDF, and ADF compared with NAT (P < 0.05). In Exp. 3, feeding CFWC-CRP increased total tract NDF digestibility and ADF digestibility (P < 0.05). Experiment 4 final BW were greatest for control lambs and least for lambs fed CFWS-CRP or SWG-CRP. Body weight gains for lambs fed CRP averaged 15.9% less than control (P < 0.05). Lambs fed CRP diets had greater (P < 0.05) NDF and ADF intake and output. In Exp. 5, steers fed the corn or 2 CRP diets gained similarly and faster (P < 0.05) than those fed the NAT-WS diet. Steers fed the control corn diet were more efficient (P < 0.05) than steers fed other treatments. Steers fed the corn diet, CFWC-CRP, and WS-CRP had greater (P < 0.01) DM digestibility than NAT. The results confirm benefits for nutrient digestion and subsequent animal performance when crop residues are thermochemically processed. Processed crop residues may be fed in combination with DDGS to partially replace corn in ruminant diets.

A SNP in the ACT gene associated with astrocytosis and rapid cognitive decline in AD.

There is biochemical and animal model evidence supporting a pathological role of the ACT gene in AD. However, direct genetic evidence remains controversial and has been mostly limited to individual single nucleotide polymorphism (SNP) analysis. To resolve this apparent conflict we have used a high-density ACT SNP map, constructed haplotypes and explored correlations with phenotype. SNPs were identified by sequencing and used to construct haplotypes in 668 AD patients and 419 controls and a case-control association study was performed. Five SNPs, comprising five common haplotypes, represented 93% of ACT gene variation. Although no single SNP or haplotype was associated with AD status, a SNP in intron 2 was associated with later onset and more rapid cognitive decline (P=0.04). This SNP was both individually associated with severe astrocytosis (P=0.004) in AD patients and when combined with the signal sequence SNP (P=0.002). This suggests that astrocytosis may have a protective function for a limited period in some patients. These SNP associations either support a direct role for the ACT gene, in AD pathology or alternatively reflect linkage with polymorphisms in other genes nearby.

Mycobacterium marinum dermatitis and panniculitis with chronic pleuritis in a captive white whale (Delphinapterus leucas) with aortic rupture.

A 16-year-old female white whale, Delphinapterus leucas, died after nearly 18 months of chronic lymphopenia and pyogranulomatous dermatitis. Necropsy revealed rupture of the aorta with hemorrhage into the cranial mediastinum and between fascial planes of the ventral neck musculature. Multiple foci of ulcerative dermatitis and panniculitis were present across the thorax and abdomen and surrounded the genital folds. In addition, there was a chronic proliferative pleuritis with over 20 liters of histiocytic exudate in the thoracic cavity. Acid-fast bacteria consistent with Mycobacterium sp. were identified in sections of skin lesions and in cytospins of pleural exudate. Cultures of pleura and 1 skin lesion collected at necropsy yielded sparse growth of an acid-fast bacillus with colony characteristics and morphology consistent with Mycobacterium marinum. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed the presence of M. marinum DNA in samples of skin. This is the first documented occurrence of mycobacteriosis in a white whale and is a unique presentation of mycobacterial dermatitis and panniculitis with chronic pleuritis in a cetacean. The improved PCR-RFLP protocol utilized in this case unifies techniques from several protocols to differentiate between species of Nocardia and rapidly growing mycobacteria clinically relevant to aquatic animals.

Humoral immune responses of black-footed penguins (Spheniscus demersus) after DNA-mediated immunization with a beta-galactosidase reporter gene.

Humoral immune responses of black-footed penguins (Spheniscus demersus) to DNA-mediated immunization with a beta-galactosidase reporter gene expression plasmid were evaluated. Six male and 6 female adult penguins received either test plasmid, pCMV-beta, containing the beta-galactosidase gene or control plasmid, pCI, lacking a gene for expression. Three birds from each group were used previously in a diluent control group and given one injection of sterile saline. All samples were screened for anti-beta-galactosidase antibodies by indirect enzyme-linked immunosorbent assay with anti-chicken immunoglobulin G as secondary antibody. Antibodies to beta-galactosidase were detected in the sera of pCMV-beta-inoculated penguins, with a peak response on day 21. Antibody titers of the test plasmid group versus both control groups on days 21, 28, and 42 differed significantly. These results demonstrate that black-footed penguins can be safely transfected with the gene encoding beta-galactosidase and will mount a humoral response against the in vivo-expressed protein. Knowledge from this initial study can be applied to the development of DNA-mediated vaccines against specific infectious diseases of penguins.

Gastric intramural hematoma and hemoperitoneum in a captive northern fur seal.

A 16-yr-old adult male northern fur seal (Callorhinus ursinus) was found dead in its outdoor pool in November 1995. The animal was maintained at Mystic Aquarium (Mystic, Connecticut, USA) from March 1980 to November 1995. Gross necropsy findings included hemoperitoneum and locally extensive gastric intramural hemorrhage that involved the posterior fundic, antral, and pyloric regions and extended into the duodenum. The gastric mural thickening grossly resembled hemangioma, and the gastric serosa was ruptured at the site of maximal mural expansion. In histologic sections of the stomach, a cribiform network of fibrin, which encompassed numerous variably-sized aggregates of closely packed erythrocytes, markedly expanded the submucosa. No vascular endothelium was identified in serial histologic sections of the expanded gastric submucosa stained with hematoxylin and eosin or immunohistochemically with antibodies to vimentin and Factor VIII-related antigen, establishing an absence of hemangioma. Carstairs' and Weigert's histochemical stains confirmed that the framework expanding the submucosa was fibrin. Although the appearance of the gastric wall resembled hemangioma, a population of neoplastic endothelial cells was not identified within the submucosal expansion of hemorrhage and fibrin, and microscopic evidence was most consistent with the diagnosis of gastric intramural hematoma. This lesion is a rare pathologic event that has not been reported in marine mammals, but one that should be included in diagnostic considerations of hemoperitoneum and gastric mural expansion.

Identification of optimal ranges in ribeye area for portion cutting of beef steaks.

Restaurant-ready (21-d aged, needle tenderized) loin steaks (strip, S; and T-bone, TB) representing three portion sizes (227, 284, and 340 g for S; 397, 454, and 510 g for TB) were portioned from 71 low-Choice beef carcasses representing seven ribeye size groups (70.9 cm2 and less to 103.2 cm2 and greater, in 6.3-cm2 increments). Steaks (n = 568) were cooked on a grooved grill to temperatures of either 67 or 77 degrees C and evaluated for cooking characteristics, sensory attributes (n = 284), and shear force (n = 284) to identify optimal ribeye areas for portioned steaks. As expected, thickness of all portioned steaks decreased (P < .05) as ribeye size increased, whereas thickness of steaks increased (P < .05) as portion size increased. The S were significantly thicker than TB. The TB cooked faster (P < .05) than did S for ribeye areas 77.4 to 83.8 cm2 in the average portion sizes and for ribeye areas 71.0 to 96.6 cm2 in the large portion sizes. However, S cooked significantly faster than did TB for ribeye areas less than 70.9 cm2 in the average portion sizes. The S had higher (P < .05) initial tenderness scores and lower (P < .05) shear values than did TB. Initial tenderness scores were lower (P < .05) for ribeye areas greater than 103.2 cm2 compared with ribeye areas less than 83.8 cm2 and 90.3 to 96.6 cm2, with all others being intermediate. Shear force tended (P = .08) to follow the initial tenderness results. The S cooked to both end point temperatures, and TB cooked to 67 degrees C had higher (P < .05) initial tenderness scores than did TB cooked to 77 degrees C. Tenderness was reduced in TB cooked to higher temperatures as well as in S and TB portioned from ribeye areas greater than 103.2 cm2. Steaks portioned from carcasses with ribeye areas between 77.4 to 96.6 cm2 can optimize both cooking time and tenderness for the foodservice sector.

The significance of allelic deletions and aneuploidy in colorectal carcinoma. Results of a 5-year follow-up study.

BACKGROUND: A prospective study was initiated to analyze the prognostic value of both the deletion of candidate tumor suppressor genes and the DNA content in colorectal carcinoma specimens. METHODS: A prospective study was initiated in 1988, into which 104 patients from the Brooklyn VA Medical Center were accrued through March 1992. DNA restriction endonuclease digests, obtained by the Southern blot technique, were examined for allelic deletions by cDNA probes pnm23-H1 (17q21) YNZ 22.1 (17p13), and p15-65 (18q21). DNA content was measured by image analysis cytometry of Feulgen-stained tumor imprints. Median follow-up was 5.5 years (range, 4-8.5 years). RESULTS: Patients with nm23-H1 allelic deletions were 3 times as likely to develop distant metastases as patients without nm23-H1 deletions (relative risk [RR], 3.89; 95% confidence interval [CI], 1.39, 10.89). This connection was even stronger after adjustment for TNM stage and site of primary tumor (RR, 5.27; 95% CI, 1.67, 16.68). No significant association of 17p or 18q deletions or ploidy with either distant metastases or overall survival was noted. In multivariate analysis, clinicopathologic variables associated with decreased survival included intracellular mucin production, nuclear grade, TNM stage, and nerve invasion. CONCLUSIONS: A combination of clinicopathologic and molecular biologic variables may identify patients at high risk for death from disseminated colorectal carcinoma.

Gastroesophageal-vulvar leiomyomatosis presenting over the course of 20 years.

Gastroesophageal-vulvar leiomyomatosis represents a rare syndrome of diffuse smooth muscle proliferation primarily in the gastroesophagus and vulva. To our knowledge, there are 19 cases reported in the literature, most associated with Alport's syndrome. We recently encountered a case not associated with Alport's syndrome, which had progressed over the course of 20 years. We used immunohistochemistry to further delineate the process. Leiomyomatosis appears to be a systemic disorder, with indolent proliferations of smooth muscle in areas that may not be clinically significant.

Acute gastric dilatation with volvulus in a northern fur seal (Callorhinus ursinus).

Acute gastric dilatation with volvulus occurred in a 4-year-old female northern fur seal (Callorhinus ursinus). The animal had been maintained in an outdoor exhibit at Mystic Marinelife Aquarium (Mystic, Connecticut, USA) from June 1989 to March 1994. On necropsy, we observed a 360 degrees clockwise rotation of the stomach about its mesenteric axis as viewed ventrodorsally. The stomach was markedly distended with gas, fluid, and partially digested fish. The spleen followed the rotation of the stomach 360 degrees and was enlarged and congested with twisted mesentery. This is the first reported case of acute gastric dilatation with volvulus in a pinniped.

Mycotic dermatitis in an Atlantic white-sided dolphin, a pygmy sperm whale, and two harbor seals.

An Atlantic white-sided dolphin (Lagenorhynchus acutus), a pygmy sperm whale (Kogia breviceps), 2 harbor seals (Phoca vitulina) developed raised, firm, erythematous, cutaneous nodules that were most prominent on their heads, trunks, and on the caudal portions of their bodies. Prior to the onset of the condition, all 4 animals may have been stressed by factors such as being stranded on a beach, being transported long distances, or being relocated locally. Microbial culturing of the lesions on multiple media yielded fungal isolates containing conidia characteristic of Fusarium spp. Hyphae consistent with those of an ascomycete were evident on histologic examination of lesions. In each treated animal, the dermatitis resolved 3 to 4 weeks after completing treatment with ketoconazole. Fusarium spp may be opportunistic invaders of the skin of marine mammals that have decreased immunocompetence or integumentary compromise.

Pancreatoblastoma in an older adult.

We present a case of pancreatoblastoma in a 61-year-old man. To our knowledge, this is the oldest patient with this tumor to be reported. Originally regarded as a microadenocarcinoma, the tumor was composed of small cells, sometimes in acinar groups with clusters of squamoid cells. It also showed acinar and neuroendocrine features by immunohistochemistry. Staining for trypsin and several neuroendocrine markers was widespread and intense in the smaller, majority-type cells. There was faint staining for some neuroendocrine markers in the larger squamoid cells. By electron microscopy both exocrine secretory and neurosecretory dense core granules were found. Modern methods of immunohistochemistry and greater understanding of rare tumor types allowed reclassification of this tumor.

Unilateral lymphangiectasis of the kidney: an unusual cause of renal enlargement in an infant.

We report on an infant with unilateral renal lymphangiectasia and the absence of small cysts in the renal parenchyma or renal hilum. We suggest that renal lymphangiectasia be part of the differential diagnosis in children who present with unilateral renal enlargement, especially when contrast enhanced computerized tomography shows preservation of a rim of renal cortex with decreased attenuation of the medulla and/or when ultrasound demonstrates increased echogenicity without displacement of the caliceal system.

Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.

Oxidatively modified lipoproteins have been implicated in atherogenesis, but the mechanisms that promote oxidation in vivo have not been identified. Myeloperoxidase, a heme protein secreted by activated macrophages, generates reactive intermediates that oxidize lipoproteins in vitro. To explore the potential role of myeloperoxidase in the development of atherosclerosis, we determined whether the enzyme was present in surgically excised human vascular tissue. In detergent extracts of atherosclerotic arteries subjected to Western blotting, a rabbit polyclonal antibody monospecific for myeloperoxidase detected a 56-kD protein, the predicted molecular mass of the heavy subunit. Both the immunoreactive protein and authentic myeloperoxidase bound to a lectin-affinity column; after elution with methyl mannoside their apparent molecular masses were indistinguishable by nondenaturing size-exclusion chromatography. Peroxidase activity in detergent extracts of atherosclerotic lesions likewise bound to a lectin column and eluted with methyl mannoside. Moreover, eluted peroxidase generated the cytotoxic oxidant hypochlorous acid (HOCl), indicating that enzymatically active myeloperoxidase was present in lesions. Patterns of immunostaining of arterial tissue with antihuman myeloperoxidase antibodies were similar to those produced by an antimacrophage antibody, and were especially prominent in the shoulder region of transitional lesions. Intense foci of myeloperoxidase immunostaining also appeared adjacent to cholesterol clefts in lipid-rich regions of advanced atherosclerotic lesions. These findings identify myeloperoxidase as a component of human vascular lesions. Because this heme protein can generate reactive species that damage lipids and proteins, myeloperoxidase may contribute to atherogenesis by catalyzing oxidative reactions in the vascular wall.

Herpes-like viral dermatitis in a beluga whale (Delphinapterus leucas).

Approximately 3.5 mo following its capture, a beluga whale (Delphinapterus leucas) developed focal pale gray skin lesions. These lesions persisted for at least 8 mo. A biopsy from one of these sites revealed epithelial intranuclear inclusions. Herpes-like viral particles were seen by transmission electron microscopy. The eventual regression of skin lesions and lack of other clinical signs suggests the virus was only mildly pathogenic in this animal.

Detection of hepatotoxicity associated with methotrexate therapy for psoriasis.

To determine whether liver function tests and clinical and demographic information would predict methotrexate-associated hepatotoxicity, we identified 78 patients who had undergone 147 liver biopsies associated with methotrexate therapy for psoriasis. The joint sensitivity of aspartate aminotransferase, alkaline phosphatase, and total bilirubin values in detecting abnormal results from a biopsy specimen obtained after treatment was .86; the predictive value of negative test results was .93. A logistic regression model significantly predicted the presence of abnormal (grade III or higher) liver biopsy specimen results. The concordance index was .92 (perfect, 1.0). Regression coefficients may be used along with information from a specific patient to calculate the predicted probability of an abnormal result from a liver biopsy specimen after treatment. We conclude that this multivariate risk estimation model significantly predicts the likelihood of positive findings from liver biopsy specimens in this patient population. The clinical use of this model awaits further validation.

Gastric carcinoid tumors, endocrine cell hyperplasia, and associated intestinal metaplasia. Histologic, histochemical, and immunohistochemical findings.

Eleven cases of gastric carcinoid tumor have been studied to review their clinical and pathologic spectrum, to identify any relationship to pernicious anemia, and to evaluate the accompanying gastric mucosal changes, with particular reference to the endocrine cell population. Seven patients were male and four female; ages ranged from 26 to 83 years. Two male patients had documented pernicious anemia and one female patient had unconfirmed pernicious anemia. All patients had marked gastric intestinal metaplasia (atrophic gastritis), which was predominantly fundal (Type A) in three patients with suspected/proven pernicious anemia and antral (Type B) in the other eight. In seven patients, the tumors were typical carcinoids, whereas in 4 patients the carcinoids were "atypical"; one carcinoid was completely polypoid. All cases were argyrophilic, and focal mucin positivity was present in four. Focal somatostatin immunoreactivity was present in four cases, serotonin in three cases, vasoactive intestinal polypeptide (VIP) in two cases, and gastrin (G) in one case. Endocrine cell hyperplasia was identified in the gastric mucosa of eight of 11 patients, including all cases with pernicious anemia; in three of eight cases, G-cell hyperplasia was evident. Numbers of serotonin-positive cells were increased in areas of intestinal metaplasia in all cases. In two patients, there was marked endocrine-cell hyperplasia with multiple small carcinoid tumorlets; the tumorlets stained for G in one. Gastric intestinal metaplasia includes intestinal-like endocrine cells. An association exists between atrophic gastritis and gastric carcinoids, and there is a histogenetic link between atrophic gastritis and some cases of gastric carcinoid tumor.