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Vascular lesions and their association with porcine circovirus type 2 (PCV2) were evaluated in multiple organs from 10 pigs affected with PCV2-systemic disease (PCV2-SD). Animals had vascular lesions in multiple organs, consisting of lymphohistiocytic lymphangitis and/or phlebitis, mild to severe necrotizing arteritis, and thrombosis within splenic arterioles and choroid plexus capillaries. Variable amounts of PCV2 nucleic acid detected by in situ hybridization were present within endothelial cells, tunica media myocytes, and perivascular and/or intralesional inflammatory cell infiltrates. PCV2 nucleic acid was detected within endothelial cells of both lymphatic and blood vessels without lesions in the associated tissues. Necrotizing arteritis was principally present in lymph nodes and kidney and consisted of degeneration, necrosis, and pyknosis of myocytes, often with intracytoplasmic, brightly eosinophilic inclusion bodies that were strongly positive for PCV2 nucleic acid. Segmental or circumferential fibrinoid necrosis was mainly present in vessels of the lymph node, spleen, and choroid plexus and was variably associated with PCV2 nucleic acid. Severe lymphangitis associated with strong intralesional PCV2 labeling was frequently detected within the mesenteric and mediastinal lymph nodes and the lamina propria of the ileum. In most tissues, medium and large lymphatics and/or veins often had disruption of the intima and mild mononuclear inflammatory cell infiltration that was variably associated with PCV2 nucleic acid. The present study indicates that vasculitis is a frequent finding in natural cases of PCV2-SD and that PCV2 may have a direct cytopathic effect on tunica media myocytes of small- and medium-sized arteries as well as endothelium., (© The Author(s) 2014.)

The degree of apoptosis in the livers of pigs with hepatitis due to naturally-occurring postweaning multisystemic wasting syndrome (PMWS) was evaluated semi-quantitatively by immunohistochemical detection of the apoptotic marker cleaved caspase-3 (CCasp3). The amount and distribution of porcine circovirus type 2 (PCV2) virus in the liver was evaluated using in situ hybridisation. Livers with mild, stage I hepatitis exhibited similar degrees of apoptosis to controls; those with stage II lesions had variable apoptotic rates, ranging from mild to high, and in livers with more severe, stage III hepatitis, high levels of hepatocyte apoptosis was a feature. Statistical analyses indicated a positive association between the rate of apoptosis, the severity of the hepatitis and the amount of PCV2 DNA in the liver. Double immunolabelling for CCasp3 and PCV2 DNA revealed a predominance of cells labelling only for PCV2, followed by fewer cells labelling only for CCasp3, and the least number labelling for both. The findings suggest that apoptosis, possibly triggered by PCV2 infection and/or hepatic inflammation, plays a key role in the pathogenesis of hepatitis in pigs with naturally-occurring PMWS., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Urinary bladder tumours in cattle are caused by chronic ingestion of bracken fern and BPV-1/2 infection. The objective of the present study was to assess if BPV-2 was present in urinary bladder lesions from cattle with chronic enzootic haematuria (CEH) from the Azores archipelago (Portugal), in order to gain further information regarding the epidemiologic distribution of this virus. Samples were analysed using PCR specific primers for BPV-2 DNA and an immunohistochemistry for BPV E5 oncoprotein detection. We found a 28% incidence rate of BPV-2 DNA in different types of tumours and cystitis cases (13 out of 46 samples). Tested positive samples for PCR were also positive for the viral E5 oncoprotein; protein immunolabeling was mainly detected within the cytoplasm of urothelial cells, displaying a juxtanuclear distribution. This is the first report of BPV-2 detection in urinary bladder tumours associated with CEH in cattle from the Azores archipelago., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus (BPV). The aim of the present study was to determine whether there was co-expression of BPV oncoproteins E5 and E7 in such urothelial carcinomas. Fifteen samples were shown by immunohistochemistry to express E7 with labelling of both the cytoplasm and nucleus, in addition to labelling of the urothelial cell membrane. Three of these samples were subsequently investigated by dual-labelling immunofluorescence and co-expression of E5 and E7 was demonstrated. This is the first report of co-expression of these two oncoproteins in bovine urinary bladder carcinomas. The results suggest that the E7 oncogene has a role in urothelial carcinogenesis.

Calodium hepaticum (Syn. Capillaria hepatica) is a zoonotic liver nematode of mammals distributed worldwide. Rodents are believed to be the main reservoirs of this nematode. In this paper, prevalence of the parasite was analyzed in liver histological sections from 51 house mice (Mus musculus) caught in human-inhabited houses, from two localities (Furnas and Rabo de Peixe) on São Miguel island from the Azores archipelago (Portugal). Mean prevalence of infection was 19.6%, with 33.3% prevalence in Furnas and 4.1% in Rabo de Peixe (P=0.07). No significant differences were found between the prevalence of infection and the age, body weight and the sex of mice. Hepatic lesions found were either acute and/or chronic stage and consisted of moderate to severe multifocal pyogranulomatous hepatitis with encapsulated eggs with typical bipolar plugs and moderate to severe necrotizing hepatitis consistent with larva tracks. Periportal inflammatory infiltration, hepatocyte regeneration and bile duct hyperplasia were also noted. In most cases, hepatic lesions occupied more than 50% of the liver, but despite severe lesions, in some mice, no signs of hepatic failure were noticed. The high rate of infection found in the present study suggests that house mice are an important reservoir for this parasite in the Azores and could have a role in human transmission.

The objective of the present study was to evaluate the involvement of apoptosis in the development of post-weaning multisystemic wasting syndrome (PMWS) lymphoid-depletion lesions. Twenty-one pigs that were categorized into three different lesional severity stages (S1, n=5; S2, n=7; S3, n=9) and five healthy control pigs (stage S0) were used. From all pigs, samples of thymus, spleen, tonsil, ileum and superficial inguinal lymph node were processed for histological examination, in situ hybridization for porcine circovirus type 2 (PCV2) detection and cleaved caspase-3 (CCasp3) immunohistochemistry for detection of apoptotic cells. PCV2 was quantified in serum samples by using TaqMan real-time PCR. CCasp3 labelling was measured in the different morphological compartments of all lymphoid tissues, using an automated system for quantification. Differences between each tissue compartment and lesional stage were assessed, as well as the correlation between apoptosis, lesional stage and viral load. Overall, the results indicated that the more intense the lymphoid depletion, the lower the rate of apoptosis. In the thymus, the cortex was the area where differences between PMWS-affected and control animals were more evident; it was found that all PMWS-affected pigs had significantly lower rates of apoptosis than the controls. In the secondary lymphoid organs, B-cell areas presented higher rates of apoptosis; similar apoptotic rates were found in this compartment in control and S1 pigs. In S2 and S3, B-cell areas were lost and the apoptotic pattern observed was a diffusely distributed low rate of positive cells. Significantly lower rates of apoptosis between PMWS-affected pigs and the control group were already evident in S1 for the thymus, spleen, superficial inguinal lymph node and Peyer's patches, but not for the tonsils. Apoptotic rates in lymphoid tissues were correlated inversely with viral load in serum and with severity of lesions. In conclusion, the results indicate that apoptosis is not a remarkable feature in PMWS lymphoid lesion development.

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions., (Copyright 2004 Elsevier B.V.)

Although Toxoplasma gondii infection has been found occasionally in cetaceans, little is known of the prevalence of antibodies to T. gondii in wild dolphins. Antibodies to T. gondii were determined in serum samples from 58 dolphins stranded in the Spanish Mediterranean coast. Modified agglutination test was used to determine T. gondii antibodies, and a titer of 1:25 was considered indicative of T. gondii infection. Antibodies to T. gondii were found in 4 of 36 striped dolphins (Stenella coeruleoalba), in 2 of 4 common dolphins (Delphinus delphis), in 4 of 7 bottlenose dolphins (Tursiops truncatus), and in 1 harbour porpoise (Phocoena phocoena). Antibodies were not found in 9 Risso's dolphins (Grampus griseus) and in 1 long-finned pilot whale (Globicephala melas) surveyed. The results indicate that T. gondii infection is frequent in at least 3 dolphin species from the Mediterranean Sea.

Fatal disseminated toxoplasmosis was diagnosed in a Risso's dolphin (Grampus griseus) dam and its fetus on the basis of pathologic findings, immunohistochemistry, and structure of the parasite. The dolphin was stranded alive on the Spanish Mediterranean coast and died a few hours later. At necropsy the dam was in good condition. From the standpoint of pathology, however, it had generalized lymphadenomegaly and splenomegaly, enlargement of and multifocal hemorrhage in the adrenal glands, diffuse mucosal hemorrhage of the glandular and pyloric stomach, ulcerative glossitis and stomatitis, focal erosions and reddening of the laryngeal appendix, and severe paraotic sinusitis with intralesional nematodes Crassicauda grampicola. The dolphin was pregnant, most probably in the first gestational trimester. The most prominent microscopic lesions were multifocal granulomatous encephalomyelitis, diffuse subacute interstitial pneumonia, mild multifocal necrotizing hepatitis and nonsuppurative cholangiohepatitis, gastritis and adrenalitis, mild lymphoid depletion, medullary sinus and follicular histyocitosis, and systemic hemosiderosis. The fetus had foci of coagulative and lytic necrosis in the kidneys, the lung, and the heart. Most lesions were associated with tachyzoites and tissue cysts of Toxoplasma gondii. The diagnosis was confirmed immunohistochemically. This is the first report on toxoplasmosis in a Risso's dolphin (G. griseus) and on transplacental transmission to an early-stage fetus in any cetaceans.

Objective: To determine the pattern of infection for porcine circovirus type 2 (PCV2) in a herd of pigs with postweaning multisystemic wasting syndrome (PMWS)., Animals: 29 sows and 250 pigs., Procedure: Blood samples were collected from all 3-, 7-, and 12-week old pigs and 59 pigs at 28 weeks of age. Pigs that died during the study were necropsied. Porcine parvovirus and PCV2 antibodies were assayed. A polymerase chain reaction (PCR) was used to detect PCV2 genome in serum of selected pigs., Results: The PMWS started when pigs were 8 weeks old, with a prevalence of 30% in 8- to 10-week-old pigs. Eighty-three pigs died during the period between 3 and 12 weeks of age. Microscopic lesions consistent with PMWS were observed, and PCV2 nucleic acid was detected (50 of 68 pigs). Antibodies to PCV2 decreased from 3 to 7 weeks of age, increased at 12 weeks of age, and were maintained until 28 weeks of age. One sow had a positive result for PCR of serum. Nine, 37 and 8 pigs had PCV2 genome in serum obtained at 7, 12, and 28 weeks of age, respectively., Conclusions and Clinical Relevance: Infection with PCV2 coincided with severe clinical signs; however, infected 28-week-old pigs did not have evidence of disease. Immunity declined over time in young pigs. A long duration of PCV2 viremia was apparent in a high percentage of infected pigs, which may affect transmission and persistence of the virus in a herd.

Fatal hepatic sarcocystosis was diagnosed in a striped dolphin (Stenella coeruleoalba) from the northeastern Spanish Mediterranean coast based on pathologic findings and the microscopic and ultrastructural characteristics of the intralesional parasite. Main gross lesions were icterus, subcutaneous hemorrhages, and hepatic congestion. The most prominent microscopic lesions consisted of severe acute multifocal to coalescing necrotizing hepatitis with cholestasis and intralesional protozoa. There was severe chronic pancreatitis with generalized distension of pancreatic ducts by hyaline plugs and adult trematodes. Only asexual stages of the protozoa were found. The parasite in the liver divided by endopolygeny. Schizonts varied in shape and size. Mature schizonts had merozoites randomly arranged or budding peripherally around a central residual body. Schizonts were up to 22 microm long, and merozoites were up to 6 microm long. Ultrastructurally, merozoites lacked rhoptries. This parasite failed to react by immunohistochemistry with anti-Toxoplasma gondii, anti-Neospora caninum and anti-Sarcocystis neurona antibodies. The microscopic and ultrastructural morphologies of the parasite were consistent with Sarcocystis canis, so far described only from animals in the Unites States. The life cycle and source of S. canis are unknown. The present report of S. canis-like infection in a sea mammal from Spain indicates that the definitive host for this parasite also exists outside of the United States.