Molecular analysis of parapoxvirus from a spotted seal Phoca largha in Japan

A spotted seal Phoca largha with nodular and scab lesions on the whole body was brought to an aquarium in Nagoya, Japan. We extracted DNA from the lesions and used the polymerase chain reaction (PCR) method for detecting orthopoxvirus and parapoxvirus DNA. Parapoxvirus but not orthopoxvirus DNA was detected. The partial nucleotide sequence of the envelope gene was determined from the PCR product, and the sequence was seen to be closely related to 2 parapoxvirus strains from spotted seals in Alaska, showing 100% identity at the amino acid level, with one nucleotide substitution. Virus-neutralizing (VN) antibody against canine distemper virus (CDV) was not detected in the serum, indicating that this individual was not infected with CDV or phocine distemper virus (PDV), which both have a high mortality rate for marine mammals. These results suggest that the lesions were caused by infection with pinniped parapoxvirus, and that the viruses spread and are maintained within the habitat range or populations of spotted seals from the Bering Sea to the Japan Sea. This is the first report of molecular analysis of parapoxvirus in marine mammals in Japan.     

Seroepidemiology of parapoxvirus infections in captive and free-ranging California sea lions Zalophus californianus

Cutaneous nodular lesions caused by parapoxvirus infections are commonly observed in stranded pinnipeds following their arrival at rehabilitation facilities. An indirect enzyme-linked immunosorbent assay (ELISA) was developed and validated to determine exposure to parapoxviruses in California sea lions Zalophus californianus in captivity and in the wild. The diagnostic performance of this assay was evaluated using receiver-operating characteristic analysis. At a selected cut-off value, the calculated sensitivity was 100% (95% CI = 86 to 100%) and the specificity was 100% (95% CI = 87 to 100%). Analysis of sera collected from 26 affected sea lions during various stages of the disease revealed anti-parapoxvirus antibodies in all affected sea lions prior to the development of cutaneous pox lesions. This indicated that previous exposure to a parapoxvirus does not confer protection against clinical disease. In at least 7 cases, exposure to the virus occurred during hospitalization. Analysis of paired sera from 74 unaffected sea lions indicated subclinical infections in at least 3 animals. Finally, the prevalence of anti-parapoxviral antibodies in 761 free-ranging California sea lions captured and tested was 91% (95% CI = 89 to 93%). This indicated that infection with a parapoxvirus is a common occurrence in the wild and that the release of captive sea lions infected with parapoxvirus into the wild should not increase the risk of a parapoxvirus outbreak in free-ranging sea lions.     

Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus)

Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.     

Bite Injuries of Grey Seals (Halichoerus grypus) on Harbour Porpoises (Phocoena phocoena)

Bite-like skin lesions on harbour porpoises (Phocoena phocoena) have been suspected to be caused by grey seals (Halichoerus grypus), and a few field observations have been reported. Bite-like skin lesions observed on stranded animals were characterized by two main components: large flaps of loose or missing skin and blubber with frayed edges and puncture lesions. Definitive demonstration of predation by a grey seal was not reported so far in those stranded animals. In this study, five stranded porpoises with bite-like skin lesions were swabbed for genetic investigations. In addition, the head of a recently dead grey seal was used to mimic bite-like skin injuries on a porpoise carcass. Subsequently, the artificial skin injuries were swabbed, along with the gum of the seal used for inflicting them (positive controls). Total DNA was extracted from the swabs and was used to retrieve a fragment of mitochondrial DNA by PCR. Primers were designed to amplify a specific stretch of mitochondrial DNA known to differ between grey seals and porpoises. The amplicon targeted was successfully amplified from the positive control and from two of the stranded porpoises, and grey seal-specific mitochondrial DNA was retrieved from all those samples. We conclude that (1) it is possible to detect grey seal DNA from dead porpoises even after several days in seawater and (2) bite-like skin lesions found on dead porpoises definitively result from grey seals attacks. The attacks are most likely linked with predation although, in a number of cases, scavenging and aggressive behaviour cannot be excluded.     

Ecological replacement of native red squirrels by invasive greys driven by disease

Although a parapoxvirus harmful to red squirrels is present in UK squirrel populations it has not been considered a major cause of red squirrel decline, and replacement by the introduced grey squirrel, mainly because diseased individuals are rarely observed. By developing a generic model we show that parapoxvirus is likely to have played a crucial role in the red squirrel decline even though the prevalence of infection is low. Conservationists are quite rightly concerned with the invasion of exotic organisms such as the grey squirrel. Our work emphasizes that they, along with other ecologists, should pay particular attention to pathogens, even when they occur at low prevalence.     

Cutaneous and meningeal sporotrichosis in a HIV patient

A male patient with HIV and past history of tuberculosis and suspected neurotoxoplasmosis was admitted to the hospital with vomiting and small nodules through all his body. Few of the nodules were found forming chains of enlarged lymphatic vessels, especially on lesions located on the limbs. Some of the nodules were ulcerated with a serosanguineous discharge. Collected samples from ulcerated and the nodular lesions showed the presence of Sporothrix schenckii in culture. Although all hemocultures were negative, a spinal fluid collected from this patient and cultures from the cutaneous lesions were both positive for S. schenckii. The patient showed improvement after treatment with Amphotericin B. Sadly, he later died of complications not related to the S. schenckii infection. This case of disseminated sporotrichosis is a remainder that in patients with immunological disorders exotic forms of this fungal clinical entity could be expected.     

Parapoxvirus causes a deleterious disease in red squirrels associated with UK population declines

The disease implications of novel pathogens need to be considered when investigating the ecological impact of species translocations on native fauna. Traditional explanations based on competition or predation may often not be the whole story. Evidence suggests that an emerging infectious disease, caused by a parapoxvirus, may be a significant component of the impact that the introduced grey squirrel has had on UK red squirrel populations. Here we validate the potential role of parapoxvirus by proving that the virus is highly pathogenic in the red squirrel while having no detectable effect on grey squirrel health.     

Geographic variation of the major histocompatibility complex in Eastern Atlantic grey seals (Halichoerus grypus)

Pathogen-driven balancing selection maintains high genetic diversity in many vertebrates, particularly in the major histocompatibility complex (MHC) immune system gene family, which is often associated with disease susceptibility. In large natural populations where subpopulations face different pathogen pressures, the MHC should show greater genetic differentiation within a species than neutral markers. We examined genetic diversity at the MHC-DQB locus and nine putatively neutral microsatellite markers in grey seals (Halichoerus grypus) from eight United Kingdom (UK) colonies, the Faeroe Islands and Sable Island, Canada. Five DQB alleles were identified in grey seals, which varied in prevalence across the grey seal range. Among the seal colonies, significant differences in DQB allele and haplotype frequencies and in average DQB heterozygosity were observed. Additionally, the DQB gene exhibited greater differentiation among colonies compared with neutral markers, yet a weaker pattern of isolation by distance (IBD). After correcting for the underlying IBD pattern, subpopulations breeding in similar habitats were more similar to one another in DQB allele frequencies than populations breeding in different habitats, but the same did not hold true for microsatellites, suggesting that habitat-specific pathogen pressure influences MHC evolution. Overall, the data are consistent with selection at MHC-DQB loci in grey seals with both varying selective pressures and geographic population structure appearing to influence the DQB genetic composition of breeding colonies.     

Trophic niche overlap between sympatric harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) at the southern limit of their European range (Eastern English Channel)

Sympatric harbour (Phoca vitulina) and grey seals (Halichoerus grypus) are increasingly considered potential competitors, especially since recent local declines in harbour seal numbers while grey seal numbers remained stable or increased at their European core distributions. A better understanding of the interactions between these species is critical for conservation efforts. This study aimed to identify the trophic niche overlap between harbour and grey seals at the southern limit of their European range, in the Baie de Somme (BDS, Eastern English Channel, France), where numbers of resident harbour seals and visiting grey seals are increasing exponentially. Dietary overlap was identified from scat contents using hierarchical clustering. Isotopic niche overlap was quantified using δ¹³C and δ¹⁵N isotopic values from whiskers of 18 individuals, by estimating isotopic standard ellipses with a novel hierarchical model developed in a Bayesian framework to consider both intraindividual variability and interindividual variability. Foraging areas of these individuals were identified from telemetry data. The three independent approaches provided converging results, revealing a high trophic niche overlap due to consumption of benthic flatfish. Two diet clusters were dominated by either small or large benthic flatfish; these comprised 85.5% [CI95%: 80.3%–90.2%] of harbour seal scats and 46.8% [35.1%–58.4%] of grey seal scats. The narrower isotopic niche of harbour seals was nested within that of grey seals (58.2% [22.7%–100%] overlap). Grey seals with isotopic values similar to harbour seals foraged in coastal waters close to the BDS alike harbour seals did, suggesting the niche overlap may be due to individual grey seal strategies. Our findings therefore provide the basis for potential competition between both species (foraging on benthic flatfish close to the BDS). We suggest that a continued increase in seal numbers and/or a decrease in flatfish supply in this area could cause/amplify competitive interactions and have deleterious effects on harbour seal colonies.     

FNAB thyroid gland: comparison study between pre- and post-operative histological diagnosis

Fine-needle aspiration biopsy is recommended as the first and most important step in the management of nodular thyroid disease. A retrospective study of 520 patients with nodular thyroid disease was done in the north of Jordan, between January 1998 and August 2001. We compared the results of fine-needle aspiration biopsy (FNAB) of thyroid gland with postoperative histological findings. The results are classified into three groups A)-benign, B)-malignant, and C)-suspicious as shown in table II. The results of the FNAB were benign in 96.4% of the cases which include benign colloid nodules (325 cases), multi-nodular goitre (70 cases), diffuse goitre (40 cases), thyroiditis (23 cases) and thyroid cysts (43 cases). A total of 52 patients underwent surgical management, 49 patients found to be accurate with the FNAB. The accuracy of FNAB was 94% (49 patients out of 52), with a specificity of 99% and a sensitivity of 93%. Ultrasound findings showed that 65% (338 patients) had solid nodules, 15% (78 patients) had cystic lesions and 20% (104 patients) had mixed echogenicity lesions. Histological confirmation of malignancy was 10 out of 11 patients with a 91% accuracy rate. FNAB was found to be a highly effective procedure, which can obviate a lot of unnecessary surgery in case of thyroid lesions, and avoid over treatment of benign disease. Surgery was recommended in all suspicious cases. FNAB under ultrasound guidance increases the sensitivity rate which can reach 100%. FNAB accuracy seems to be similar in cold and in hot nodules. Clinically, most of the patients with benign colloid nodules were having goitres suggesting the role played by iodine deficiency is which a prominent feature in that area in Jordan.     

Fine needle aspiration biopsy of hepatocellular carcinoma and hepatocellular nodular lesions: role,controversies and approach to diagnosis

A. Wee
Fine needle aspiration biopsy of hepatocellular carcinoma and hepatocellular nodular lesions: role, controversies and appr oach to diagnosis The role of fine needle aspiration (FNA) biopsy of the liver has evolved. Advances in imaging modalities have obviated the need for tissue confirmation in clinically classic hepatocellular carcinoma (HCC). The risks of needle tract seeding and haematogenous dissemination have been actively debated. Nowadays, cytopathologists are confronted by smaller and smaller nodules, detected due to increased surveillance of high‐risk cirrhotic patients. Tissue characterization of small well‐differentiated hepatocellular nodular lesions (size less than and equal to 2 cm) is extremely challenging and has therapeutic implications. Major issues in the cytodiagnosis of HCC include: (i) distinguishing benign hepatocellular nodular lesions, namely, large regenerative nodules, dysplastic nodules, focal nodular hyperplasia and hepatocellular adenoma from reactive hepatocytes; (ii) distinguishing well‐differentiated HCC from benign hepatocellular nodular lesions; (iii) distinguishing poorly differentiated HCC from intrahepatic cholangiocarcinoma and metastatic carcinomas; (iv) determining the histogenesis of a malignant tumour; and (v) determining the site of origin of a malignant tumour. An overview of the biological evolution and histopathological aspects of dysplastic nodules, small HCCs and ‘nodule‐in‐nodule’ lesions is presented in tandem with clinically relevant nomenclature. An algorithmic approach to FNA diagnosis of HCC and hepatocellular nodular lesions is outlined. Optimal results depend on (i) a dedicated radiologist‐cytopathologist team; (ii) an on‐site cytology service, (iii) a combined cytohistological approach, (iv) immunohistochemistry, and (v) clinicopathological correlation. As we move towards personalized medicine, it is envisaged that hepatic FNA is likely to become a point of care in the management protocol as it takes on the additional role of procurement of tumour and peritumoural tissues for genomic and proteomic profiling to enable targeted molecular therapy.     

Modulating influence of dehydroepiandrosterone administration on the morphology and enzyme phenotype of dimethylaminoazobenzene-induced hepatocellular foci and nodules

The effect o 4 weeks dietary administration of the hormone dehydroepiandrosterone (DHEA) on enzyme and morphological phenotype of focal lesions previously induced by dimethylaminoazobenzene (DAB) treatment was investigated in Sprague-Dawley rats. In contrast to the DAB-alone livers where large numbers of glycogen-storing, mixed cell nodules homogeneously positive for glutathione S-transferase P form (GST-P) were apparent, DHEA treated animals were characterized by significantly fewer, more heterogeneous lesions, in some cases demonstrating increased amphophilia and structured basophilia. The enhanced heterogeneity, in some ways reminiscent of that reported earlier for 'reversibility' or 'remodelling' of rapidly induced nodular lesions, was associated with increased catalase (CAT), acid phosphatase (AP) and glucose-6- phosphatase (G6Pase) and decreased glycogen contents and phosphorylase (PHO) activity in both nodules and background parenchyma. Glucose-6-phosphatase (G6PD) activity was elevated irregularly focal lesions also demonstrating a heterogeneous reaction. The experimental data suggest two separate effects of the hormone treatment the first involving modulation of the usual altered phenotype of preneoplastic lesions with a shift towards 'tigroid' cell character and the second, similar to that reported earlier for rapidly induced nodules, involving enhanced phenotypic instability and leading to reduction in numbers.     

Intraspecific variation in movement patterns: modeling individual behaviour in a large marine predator

In large marine predators, foraging entails movement. Quantitative models reveal how behaviours can mediate individual movement, such that deviations from a random pattern may reveal specific search tactics or behaviour. Using locations for 52 grey seals fitted with satellite-linked recorders on Sable Island; we modeled movement as a correlated random walk (CRW) for individual animals, at two temporal scales. Mean move length, turning angle, and net squared displacement (R²_n: the rate of change in area over time) at successive moves over 3 to 10 months were calculated. The distribution of move lengths of individual animals was compared to a Lévy distribution to determine if grey seals use a Lévy flight search tactic. Grey seals exhibited three types of movement as determined by CRW model fit: directed movers – animals displaying directed long distance travel that were significantly underpredicted by the CRW (23% of animals); residents – animals remaining in the area surrounding Sable Island that were overpredicted by the model (29% of animals); and correlated random walkers – those (48% of animals) in which movement was predicted by the CRW model. Kernel home range size differed significantly among all three movement types, as did travel speed, mean move length, mean R²_n and total distance traveled. Sex and season of deployment were significant predictors of movement type, with directed movers more likely to be male and residents more likely to be female. Only 30% of grey seals fit a Lévy distribution, which suggests that food patches used by the majority of seals are not randomly distributed. Intraspecific variation in movement behaviour is an important characteristic in grey seal foraging ecology, underscoring the need to account for such variability in developing models of habitat use and predation.     

Characterization of parapoxviruses circulating among wild Japanese serows (Capricornis crispus)

We antigenically and molecularly compared 5 parapoxvirus isolates and 7 viral DNA samples from clinical lesions of Japanese serows with 3 viruses from sheep and goats. All isolates from Japanese serows except one, Ishikawa-S, reacted with six monoclonal antibodies to orf virus (ORFV). Restriction endonuclease analysis using amplified viral DNA showed the ORFV-specific pattern in all samples except Ishikawa-S, which showed a bovine papular stomatitis virus (BPSV)-specific pattern. Partial nucleotide sequences of the envelope genes were determined and those of all samples from Japanese serows and sheep except Ishikawa-S were completely identical and also had high identities with the goat virus. These findings suggest that parapoxvirus infection in Japanese serows might be mainly caused by ORFV and accidentally by BPSV. The envelope gene sequenced here seems to be conserved in Japanese ORFVs.     

The growth pattern of transplanted normal and nodular hepatocytes

Overt neoplasia is often the end result of a long biological process beginning with the appearance of focal lesions of altered tissue morphology. While the putative clonal nature of focal lesions has often been emphasized, increasing attention is being devoted to the possible role of an altered growth pattern in the evolution of carcinogenesis. Here we compare the growth patterns of normal and nodular hepatocytes in a transplantation system that allows their selective clonal proliferation in vivo. Rats were pre-treated with retrorsine, which blocks the growth of resident hepatocytes, and were then transplanted with hepatocytes isolated from either normal liver or hepatocyte nodules. Both cell types were able to proliferate extensively in the recipient liver, as expected. However, their growth pattern was remarkably different. Clusters of normal hepatocytes integrated in the host liver, displaying a normal histology; however, transplanted nodular hepatocytes formed new hepatocyte nodules, with altered morphology and sharp demarcation from surrounding host liver. Both the expression and distribution of proteins involved in cell polarity, cell communication, and cell adhesion, including connexin 32, E-cadherin, and matrix metalloproteinase-2, were altered in clusters of nodular hepatocytes. Furthermore, we were able to show that down-regulation of connexin 32 and E-cadherin in nodular hepatocyte clusters was independent of growth rate. These results support the concept that a dominant pathway towards neoplastic disease in several organs involves defect(s) in tissue pattern formation.     

声触诊组织定量技术鉴别诊断甲状腺微小癌的价值

目的：声触诊组织定量技术（VTQ）是一种新的弹性成像方法,能够定量、无创地评价组织硬度信息。本文的研究目的是探讨声触诊组织定量技术在鉴别诊断甲状腺微小癌（TMCs）中的应用价值。方法：应用VTQ 技术对110 例共114 个结节（最大直径≤1 cm）进行检测,获取并分析结节及周边甲状腺组织的横向剪切波速度（SWV）。利用ROC曲线对测量结果进行分析,评价VTQ技术的诊断价值并确定最佳诊断界值。结果：114 个结节中良性结节32 个（30 个为结节性甲状腺肿,2 个为腺瘤）,恶性结节82 个（均为乳头状癌）。甲状腺良性结节及周边甲状腺组织的SWV 平均值分别为（2.30± 0.49） m/s、（2.08± 0.40） m/s,恶性结节及周边甲状腺组织的SWV 平均值分别为（3.12± 0.97） m/s、（2.06± 0.46） m/s。恶性结节的SWV 值明显高于良性结节,两者之间差异具有统计学意义（t=5.911,P＜0.0001）;恶性结节与其周边甲状腺组织比较有显著差异（P＜0.0001）;而良性结节与其周边甲状腺组织无统计学差异（P＞0.05）。ROC曲线下面积为0.833,以2.30 m/s诊断界值点时,敏感度、特异度分别为90.2%、62.5%。结论：甲状腺恶性结节的SWV 值明显高于良性结节。VTQ 技术能够提供组织硬度信息,在鉴别诊断甲状腺微小癌方面具有一定的临床应用价值。     

Molecular methods for differentiating grey seal (Halichoerus grypus) and harbour seal (Phoca vitulina) scat

Pinniped dietary information is crucial for understanding marine ecosystems; however, in the North Atlantic, grey and harbour seals haulout concomitantly and their faeces are visually indistinguishable. Therefore, we developed molecular methods to differentiate the species' scat. PCR primers were created that amplify a portion of mitochondrial 16S ribosomal DNA in grey and harbour seals. The samples were amplified and the resulting products were digested using the restriction enzyme Tsp509I, producing diagnostic banding patterns. These tools provide a mechanism by which separate dietary analysis can be achieved for grey and harbour seals at mixed haulouts in New England.     

Modulating influence of dehydroepiandrosterone administration on the morphology and enzyme phenotype of dimethylaminoazobenzene-induced hepatocellular foci and nodules

The effect of 4 weeks dietary administration of the hormone dehydroepiandrosterone (DHEA) on enzyme and morphological phenotype of focal lesions previously induced by dimethylaminoazobenzene (DAB) treatment was investigated in Sprague-Dawley rats. In contrast to the DAB-alone livers where large numbers of glycogen-storing, mixed cell nodules homogeneously positive for glutathione S-transferase P form (GST-P) were apparent, DHEA treated animals were characterized by significantly fewer, more heterogeneous lesions, in some cases demonstrating increased amphophilia and structured basophilia. The enhanced heterogeneity, in some ways reminiscent of that reported earlier for ‘reversibility’ or ‘remodelling’ of rapidly induced nodular lesions, was associated with increased catalase (CAT), acid phosphatase (AP) and glucose-6-phosphatase (G6Pase) and decreased glycogen content and phosphorylase (PHO) activity in both nodules and background parenchyma. Glucose-6-phosphate dehydrogenase (G6PD) activity was elevated irregularly, focal lesions also demonstrating a heterogeneous reaction. The experimental data suggest two separate effects of the hormone treatment, the first involving modulation of the usual altered phenotype of preneoplastic lesions with a shift towards ‘tigroid’ cell character and the second, similar to that reported earlier for rapidly induced nodules, involving enhanced phenotypic instability and leading to reduction in numbers.     

Movements, diving and foraging behaviour of grey seals (Halichoerus grypus)

This study presents the first direct observations of the movements and behaviour of free-ranging grey seals at sea. Radio and ultrasonic transmitters were attached to three sub-adult male grey seals which were then tracked from a suitable vessel. Behaviour at sea fell into one of three categories: travelling between haul-outs, short duration trips, and resting adjacent to haul-out sites. Travelling was characterized by direct, relatively fast horizontal movement and by V-shaped dive profiles. During short duration trips the seals swam slower and invariably exhibited square-wave dive profiles, spending approximately 60% of total dive duration at the maximum depth. Resting involved shallow dives close to haul-out sites and an absence of directed lateral movement.
The excellent navigational abilities of grey seals are illustrated by the rapid, direct swimming between distant haul-out sites. It is proposed that short duration trips are specifically for foraging because of their association with other piscivores, and because swimming was slow and mostly on or near the sea bed (grey seals are known to feed almost exclusively on demersal and benthic fish). These trips accounted for only 14% of the nine days that seal 1 was tracked. It is also proposed that the habit of diving to the sea bed whilst travelling between distant haul-out sites is to allow opportunistic foraging with only a small increase in total swimming distance.     

The pathology of the nodular goiter

Pathologic studies of thyroid nodules indicate that a definite percentage of nodular goiters are malignant, and that an even higher percentage are true neoplasms. Malignant tumors may arise from adenomas, involutionary nodules or possibly from nodules of the unclassified type. Some carcinomas of the thyroid gland are undoubtedly malignant processes from the start. Because of the high incidence of neoplasms, benign and malignant, in a group of single nodules of the thyroid gland, the presence of such nodules should warrant radical resections of the involved lobe and adjacent isthmus. The high incidence of epithelial growth activity in nodular involutionary goiters may revise the accepted practice of removing non-toxic nodular goiters only when clinical evidence of growth or local pressure is present.