Eosinophilic Granulomatous Myocarditis in Two Pigs.

Eosinophilic myocarditis is a human condition that has been rarely documented in animals. We now report two unrelated porcine cases of idiopathic eosinophilic granulomatous myocarditis that resembled the human disease and which were associated with sudden death. The most relevant gross finding in both cases was marked cardiomegaly, accompanied by raised, multifocal to coalescent small white nodules (1-2 mm) and poorly demarcated multifocal pale areas in the epicardium. Histologically, there were multifocal to coalescent areas of cardiomyocyte loss with replacement by an intense inflammatory infiltrate of eosinophils and epithelioid macrophages, and proliferation of fibrous connective tissue. Immunohistochemistry for porcine circovirus type 2 (PCV2) and Toxoplasma gondii, in-situ hybridization and quantitative polymerase chain reaction tests for PCV2 and porcine circovirus type 3 and aerobic bacterial culture on myocardium samples were negative.

Leishmania hide-and-seek: Parasite amastigotes in the choroid plexus of a dog with neurological signs in an endemic municipality in Brazil.

A female adult mixed-breed stray dog presented with hind limb paraparesis and clinical signs of visceral leishmaniasis. The cerebrospinal fluid presented signs of blood-brain barrier disruption. Both spleen and brain were positive for Leishmania spp. DNA. Besides inflammation, in situ hybridization and immunohistochemistry (IHC) revealed the presence of intracellular amastigotes in the choroid plexus (CP). Despite other studies that revealed parasite DNA, the current study describes the presence of Leishmania within the brain of a naturally infected dog, specifically in CP, with no previous reports in the Americas, and suggests the CP as a possible pathway to parasite entry into the brain.

Chromosomal distribution of the retroelements Rex 1, Rex 3 and Rex 6 in species of the genus Harttia and Hypostomus (Siluriformes: Loricariidae)

The transposable elements (TE) have been widely applied as physical chromosome markers. However, in Loricariidae there are few physical mapping analyses of these elements. Considering the importance of transposable elements for chromosomal evolution and genome organization, this study conducted the physical chromosome mapping of retroelements (RTEs) Rex1, Rex3 and Rex6 in seven species of the genus Harttia and four species of the genus Hypostomus, aiming to better understand the organization and dynamics of genomes of Loricariidae species. The results showed an intense accumulation of RTEs Rex1, Rex3 and Rex6 and dispersed distribution in heterochromatic and euchromatic regions in the genomes of the species studied here. The presence of retroelements in some chromosomal regions suggests their participation in various chromosomal rearrangements. In addition, the intense accumulation of three retroelements in all species of Harttia and Hypostomus, especially in euchromatic regions, can indicate the participation of these elements in the diversification and evolution of these species through the molecular domestication by genomes of hosts, with these sequences being a co-option for new functions.(AU)

Os elementos transponíveis (TE) têm sido amplamente aplicados como marcadores cromossômicos. Contudo, em Loricariidae, há poucas análises de mapeamento físico destes elementos. Considerando a importância de elementos transponíveis para a evolução cromossômica e organização genômica, este trabalho realizou o mapeamento físico cromossômico dos retroelementos (RTEs) Rex1, Rex3 e Rex6 em sete espécies do gênero Harttia e em quatro espécies do gênero Hypostomus, com o intuito de melhor compreender a organização e dinâmica dos genomas das espécies de Loricariidae. Os resultados evidenciaram um intenso acúmulo dos RTEs Rex1, Rex3 e Rex6 e distribuição dispersa em regiões heterocromáticas e eucromáticas no genoma das espécies estudadas. A presença de retroelementos em algumas regiões cromossômicas sugere sua participação em vários rearranjos cromossômicos. Além disso, o intenso acúmulo dos três retroelementos em todas as espécies de Harttia e Hypostomus, especialmente em regiões eucromáticas, pode indicar a participação destes elementos na diversificação e evolução destas espécies através da domesticação molecular pelo genoma dos hospedeiros, com estas sequências sendo co-optadas paras novas funções.(AU)

Chromosomal distribution of the retroelements Rex 1, Rex 3 and Rex 6 in species of the genus Harttia and Hypostomus (Siluriformes: Loricariidae)

The transposable elements (TE) have been widely applied as physical chromosome markers. However, in Loricariidae there are few physical mapping analyses of these elements. Considering the importance of transposable elements for chromosomal evolution and genome organization, this study conducted the physical chromosome mapping of retroelements (RTEs) Rex1, Rex3 and Rex6 in seven species of the genus Harttia and four species of the genus Hypostomus, aiming to better understand the organization and dynamics of genomes of Loricariidae species. The results showed an intense accumulation of RTEs Rex1, Rex3 and Rex6 and dispersed distribution in heterochromatic and euchromatic regions in the genomes of the species studied here. The presence of retroelements in some chromosomal regions suggests their participation in various chromosomal rearrangements. In addition, the intense accumulation of three retroelements in all species of Harttia and Hypostomus, especially in euchromatic regions, can indicate the participation of these elements in the diversification and evolution of these species through the molecular domestication by genomes of hosts, with these sequences being a co-option for new functions.(AU)

Os elementos transponíveis (TE) têm sido amplamente aplicados como marcadores cromossômicos. Contudo, em Loricariidae, há poucas análises de mapeamento físico destes elementos. Considerando a importância de elementos transponíveis para a evolução cromossômica e organização genômica, este trabalho realizou o mapeamento físico cromossômico dos retroelementos (RTEs) Rex1, Rex3 e Rex6 em sete espécies do gênero Harttia e em quatro espécies do gênero Hypostomus, com o intuito de melhor compreender a organização e dinâmica dos genomas das espécies de Loricariidae. Os resultados evidenciaram um intenso acúmulo dos RTEs Rex1, Rex3 e Rex6 e distribuição dispersa em regiões heterocromáticas e eucromáticas no genoma das espécies estudadas. A presença de retroelementos em algumas regiões cromossômicas sugere sua participação em vários rearranjos cromossômicos. Além disso, o intenso acúmulo dos três retroelementos em todas as espécies de Harttia e Hypostomus, especialmente em regiões eucromáticas, pode indicar a participação destes elementos na diversificação e evolução destas espécies através da domesticação molecular pelo genoma dos hospedeiros, com estas sequências sendo co-optadas paras novas funções.(AU)

Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares.

To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 ± 3.2 cells), compared to that at 1 dph (11 ± 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species.

HER-2 gene expression in atypical ductal hyperplasia associated with canine mammary carcinomas / Expressão gênica de HER2 em hiperplasias ductais atípicas associadas a carcinomas mamários caninos

O presente estudo apresenta o comportamento do gene HER2, a partir do uso da técnica de hibridização cromogênica in situ, em hiperplasias ductais atípicas associadas a carcinomas mamários caninos positivos para HER2. Aparentemente, uma fraca expressão da proteína HER2 foi observada nas hiperplasias ductais atípicas, bem como uma ausência de amplificação do seu gene codificador nessas hiperplasias e nos carcinomas mamários associados. O comportamento da proteína HER2 e do seu gene em carcinomas mamários caninos é similar ao observado em alguns subtipos histológicos de tumores mamários humanos, e a ausência dessas alterações sugerem que esse gene poderia aparentemente não estar envolvido com os estágios iniciais de proliferação celular atípica...

HER-2 gene expression in atypical ductal hyperplasia associated with canine mammary carcinomas / Expressão gênica de HER2 em hiperplasias ductais atípicas associadas a carcinomas mamários caninos

O presente estudo apresenta o comportamento do gene HER2, a partir do uso da técnica de hibridização cromogênica in situ, em hiperplasias ductais atípicas associadas a carcinomas mamários caninos positivos para HER2. Aparentemente, uma fraca expressão da proteína HER2 foi observada nas hiperplasias ductais atípicas, bem como uma ausência de amplificação do seu gene codificador nessas hiperplasias e nos carcinomas mamários associados. O comportamento da proteína HER2 e do seu gene em carcinomas mamários caninos é similar ao observado em alguns subtipos histológicos de tumores mamários humanos, e a ausência dessas alterações sugerem que esse gene poderia aparentemente não estar envolvido com os estágios iniciais de proliferação celular atípica.(AU)

Interaction between IGF1 and IGFBPs in bovine cystic ovarian disease.

Cystic ovarian disease (COD) is one of the main factors responsible for reproductive disorders in cattle. Although the pathogenesis and mechanism of cyst formation are not fully understood, it has been proposed that the IGF system could play an essential role, as it is a key intraovarian regulator. The aim of the present study was to determine whether the altered levels in IGF1 detected in bovines with COD are associated with changes at mRNA level or with differential modulation by IGFBPs. The mRNA levels of the IGF components studied were analyzed by real time PCR and in situ hybridization, and IGFBP expression and activity were assayed by immunohistochemistry and ligand blot, respectively. Results showed a decreased IGF1 mRNA level due to a lower granulosa cell gene expression in cystic follicles (P<0.05). Results also showed variations in IGFBP expression in the intraovarian cellular compartment and concentration in follicular fluid, and suggest that IGFBP3 is a key regulator of intrafollicular IGF1 in animals with COD.

Growth hormone expression in stromal and non-stromal cells in the bursa of Fabricius during bursal development and involution: Causal relationships?

Growth hormone (GH) is expressed in the chicken bursa of Fabricius (BF), an organ that undergoes three distinct developmental stages: rapid growth (late embryogenesis until 6-8 weeks of age [w]), plateaued growth (between 10 and 15w), and involution (after 18-20w). The distribution and abundance of GH-immunoreactivity (GH-IR) and GH mRNA expression in stromal and non-stromal bursal cells during development, as well as the potential anti-apoptotic effect of GH in bursal cell survival were the focus of this study. GH mRNA expression was mainly in the epithelial layer and in epithelial buds at embryonic day (ED) 15; at 2w it was widely distributed within the follicle and in the interfollicular epithelium (IFE); at 10w it clearly diminished in the epithelium; whereas at 20w it occurred in only a few cortical cells and in the connective tissue. Parallel changes in the relative proportion of GH mRNA expression (12, 21, 13, 1%) and GH-IR (19, 18, 11, <3%) were observed at ED 15, 2w, 10w, and 20w, respectively. During embryogenesis, GH-IR co-localized considerably with IgM-IR, but scarcely with IgG-IR, whereas the opposite was observed after hatching. Significant differences in bursal cell death occurred during development, with 9.3% of cells being apoptotic at ED 15, 0.4% at 2w, 0.23% at 10w, and 21.1% at 20w. Addition of GH increased cultured cell survival by a mechanism that involved suppression (up to 41%) of caspase-3 activity. Results suggest that autocrine/paracrine actions of bursal GH are involved in the differentiation and proliferation of B lymphocytes and in BF growth and cell survival in embryonic and neonatal chicks, whereas diminished GH expression in adults may result in bursal involution.

Comparison between two in situ methods for Mycobacterium avium subsp. paratuberculosis detection in tissue samples from infected cattle.

Paratuberculosis or Johne's disease is a chronic infectious disorder caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease produces diarrhea and weight loss in cattle and other animal species, and it is characterized by granulomatous enteritis and lymphadenitis. Histopathology and in situ techniques can be used as a diagnostic test, but the performance of these methods was not previously compared. The aim of this paper was to evaluate the ability of immunohistochemistry and in situ hybridization to detect Map in formalin-fixed tissue samples from infected cattle. Samples (ileum or ileocecal lymph node) from four animals that had positive Map culturing, lesions and detectable acid fast bacilli, as well as from two control animals, were tested by immunohistochemistry and in situ hybridization. Immunostaining and positive hybridization were observed in areas with lesions from infected animal samples, inside the cytoplasm of macrophages, epithelioid and giant cells. Immunostaining was intense in three samples and weak in one, while hybridization was weak in all cases. In situ hybridization was positive in negative areas of tissues analyzed by immunohistochemistry, which could be related to spheroplast detection as it was previously described for this method. Control samples resulted negative by these two methods. Both techniques were able to detect Map in formalin fixed and paraffin embedded tissues, however immunohistochemistry produced higher intensity staining and was easier to perform. Therefore, we believe that immunohistochemistry and in situ hybridization to be useful for the post-mortem diagnosis and research of Paratuberculosis.

Detection of reticuloendotheliosis virus by immunohistochemistry and in situ hybridization in experimentally infected chicken embryo fibroblasts

Reticuloendotheliosis virus (REV) infection can result in immunosuppression, runting syndrome, high mortality, acute reticular cell neoplasia, or T- and/or B-cell lymphoma, in a variety of domestic and wild birds. Histopathological changes of reticuloendotheliosis (RE) are not sufficient to differentiate it from Avian Leukosis (AL) and Marek's disease (MD). Currently there available diagnostic methods for detection of active REV infection. In order to develop immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of REV active infections, experimentally inoculated formalin-fixed and paraffin embedded DF-1 chicken embryo fibroblasts were used as an infection model. IHC and ISH assays proved to be efficient for the detection of several REV strains, and to differentiate those strains from representative strains of the avian leukosis/sarcoma group of retroviruses (ALSV).

Detection of reticuloendotheliosis virus by immunohistochemistry and in situ hybridization in experimentally infected Japanese quail embryos and archived formalin-fixed and paraffin-embedded tumours.

Reticuloendotheliosis virus (REV) infection can result in immunosuppression, a runting syndrome, high mortality, acute reticulum cell neoplasia, or T-cell and/or B-cell lymphomas, in a variety of domestic and wild birds. Histopathological changes in REV infection are not sufficient to differentiate it from avian lymphoid leukosis and Marek's disease, and currently there are no available in situ diagnostic methods for detection of active REV presence in pathologic specimens. To develop immunohistochemistry and in situ hybridization assays for detection of REV active infections, experimentally inoculated Japanese quail embryos, and archived formalin-fixed paraffin-embedded tissues from natural and experimental reticuloendotheliosis cases in chickens and turkeys, were examined. The in situ hybridization and immunohistochemistry assays proved to be efficient for the detection of several REV strains in Japanese quail embryos during active infection, whereas these assays were much less sensitive when applied to archived tissue samples from chronically infected birds with lymphoid tumours. The diagnostic assays developed in this study have potential as diagnostic tools for detection of active REV infections.

Immunohistochemical detection of receptors for oestrogen and progesterone in endometrial glands and stroma during the oestrous cycle in Nelore (Bos taurus indicus) cows.

The aim of the present study was to monitor endometrial distribution and concentrations of oestrogen receptors alpha (ER alpha) and progesterone receptors (PR) by immunohistochemistry in Nelore cows (Bos taurus indicus) during the oestrous cycle. Blood samples were collected for progesterone measurement and endometrial samples were taken from the uterine horn contra lateral to the corpus luteum in 16 cows at days 0 (ovulation), 5, 9, 13 and 19 of the oestrous cycle. Immunostaining evaluation for ER alpha and PR in the glandular epithelium and uterine stroma was performed by two methods: positive nuclei counting and staining intensity of the nuclei. Specific positive staining reactions for both receptors were limited to cell nuclei and they were not identified in the cytoplasm. The proportion of ER alpha positive nuclei had a temporal variation throughout the oestrous cycle in both cell types evaluated and was higher in uterine stroma than the glandular epithelium (p < 0.05). The greatest proportion of ER alpha stained nuclei was observed at oestrus and during the initial and mid luteal phase (days 5, 9 and 13) (p < 0.05) in the glandular epithelium and at days 0, 5 and 9 in the uterine stroma (p < 0.01). The proportion of PR positive nuclei remained constant throughout the entire oestrous cycle for both cell types evaluated (p > 0.05). A higher proportion of PR positive nuclei was measured in the uterine stroma compared with the glandular epithelium (p < 0.05). Intensity of staining for ER alpha and PR varied throughout the oestrous cycle (p < 0.01). There was a higher staining intensity at days 0 and 5 in the stroma for ER alpha (p < 0.01) and PR (p < 0.01) and in the glandular epithelium at days 0, 5, 9 and 13 for ER alpha (p < 0.01) and at days 0, 5 and 9 for PR (p < 0.01) when compared with the other evaluated days. These data demonstrate that ER alpha and PR expression varied throughout the oestrous cycle in Nelore cows, in general with highest concentrations at oestrus and the lowest during the luteal phase. This is similar to patterns observed in Bos taurus taurus.

PapillomatosIs in cattle: in situ detection of bovine papillomavirus DNA sequences in reproductive tissues

Bovine papillomatosis is a common viral infection in Brazil that is caused by a bovine papillomavirus(BPV). Dissemination is by direct contact between infected animals, although the investigation of othermodes of transmission is a very important aspect in the management of this condition. BPV DNA sequenceshave been detected in many tissues by using the polymerase chain reaction. In this work, we used in situhybridization to detect BPV DNA sequences in bovine reproductive tissues and cells. The detection ofBPV in these tissues strongly suggests that these sequences could be an important alternative of viraltransmission that could contribute to the widespread incidence of bovine papillomatosis and its complexpathology. Alternatively, the viral sequences could result from cell apoptosis and may therefore not bedirectly involved in the infection.

Fecal samples as DNA source for the diagnosis of Necrotizing Hepatopancreatitis (NHP) in Penaeus vannamei broodstock.

Necrotizing Hepatopancreatitis (NHP) is a severe disease of cultivated penaeid shrimp caused by a pleomorphic, gram-negative, intracellular rickettsia-like bacterium. Current diagnostic methods for this disease are invasive, requiring dissection of the animal to perform histopathological analysis. In Colombia, NHP affects mainly broodstock, being a major cause of mortalities in maturation laboratories. In order to identify the presence of NHP without having to dissect the animal, we developed a PCR-based method using fecal samples as the DNA source. The DNA was extracted using a quick isolation protocol followed by amplification with primers specific for 16S ribosomal RNA gene sequences. To verify the sensitivity and specificity we analyzed samples from the same animal by PCR and in situ hybridization, and found 100% agreement. In addition, we amplified DNA extracted form paraffin blocks to confirm NHP diagnosis. PCR amplification from fecal samples and paraffin blocks yielded the expected 440 bp fragment. We conclude that PCR amplification from fecal samples is a valuable tool for the diagnosis of NHP in broodstock organisms, and that paraffin-fixed tissues can be used as a source of DNA for PCR amplification of NHP.

Periodic occurrence of epithelial viral necrosis outbreaks in Penaeus vannamei in Ecuador.

Epizootics of an infectious cuticular epithelial necrosis virus (ICENV) occurred in cultured Penaeus vannamei in Ecuadorian shrimp farms from 1994 to 1996. There were few reports of outbreaks during 1997, but in the second half of 1998 epizootics were again reported. Histopathological examination revealed extensive tissue changes and necrosis as described for infections by what others have called Taura syndrome virus (TSV). Infiltration of haemocytes in the cuticular epithelium was also one of the characteristics in the subacute and acute forms of this disease. Electron microscopy of affected tissues demonstrated the presence of a single type of virus particle in the cytoplasm of diseased shrimps from these outbreaks and it corresponded to the published descriptions for TSV. The epizootics of ICENV were periodic in occurrence, and data indicated that they might be related to the oceanographic and climatic variations reported in the eastern Pacific from 1994 to 1998. Using 3 mo rolling averages, a statistically significant negative correlation was found between prevalence of ICENV and temperature but not temperature change. By contrast, there was a statistically significant positive correlation between prevalence of ICENV and salinity change but not salinity value. Although these data do not establish causal relationships, they suggest that laboratory tests should be conducted to determine whether low temperature and upward changes in salinity can increase shrimp susceptibility to infection and mortality by ICENV.

Oral focal epithelial hyperplasia in a howler monkey (Alouatta fusca).

Oral focal epithelial hyperplasia is a rare and seldom reported disease in animals and humans induced by a papillomavirus. The present report is the first description of this disease in a Neotropical primate, a howler monkey (Alouatta fusca). The diagnosis was based on gross and microscopic findings. The generic papillomavirus antigen was identified by immunohistochemistry and was found not to be related to any human papillomavirus DNA tested by in situ hybridization. This virus is probably a specific papillomavirus of the howler monkey (HMPV).

Chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys.

The chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys was analyzed in eight species by in situ hybridization, using a probe isolated from C. porteousi. The hybridization patterns showed different numbers of chromosomes with positive pericentromeric regions and/or complete short arms. In some species, a positive signal was scarce (or not detectable, as in C. opimus), and was usually located in the pericentromeric areas (C. occultus and C. latro). In those species where the satellite was highly amplified, its chromosomal localization tended to encompass the entire length of the short arms. These patterns were compared with C-band distribution patterns in the same species. We discuss the putative evolutionary trend of this satellite DNA in the genus Ctenomys and suggest that it evolved from a strictly pericentromeric localization to comprising the whole short arms of some chromosomes.