A novel in situ Polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimization of an amperometric biosensor for the detection of hepatitis C virus using fractional factorial designs

Fractional factorial design and factorial with center point design were applied to the development of an amperometric biosensor for the detection of the hepatitis C virus. Biomolecules were immobilized by adsorption on graphite electrodes modified with siloxane-poly(propyleneoxide) hybrid matrix prepared using the sol-gel method. Several parameters were optimized, such as the streptavidin concentration at 0.01 mg mL(-1) and 1.0% bovine serum albumin, the incubation time of the electrodes in the complementary DNA solution for 30 minutes and a 1: 1500 dilution of the avidin-peroxidase conjugate, among others. The application of chemometric studies has been efficient, since the best conditions have been established with a restricted number of experiments, indicating the influence of different factors on the system.

Isolation of bovine coronavirus (BCoV) in monolayers of HmLu-1 cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bull semen varibles after experimental exposure with Bovine Herpesvirus type 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of neuron-like cells derived from bovine Wharton's jelly to bovine herpesvirus type 5 infections

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Astrocyte intermediate filaments are important markers in olfactory bulb of bovine herpesvirus type 5 natural infections

Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear.

Poult intestinal organ culture for propagation of Turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.

Importância da criptosporidiose como zoonose

The protozoa of the genus Cryptosporidium sp. are parasites that complete their life cycle on the surface of epithelial cells of the respiratory, gastrointestinal and urinary tracts of mammals, birds, reptiles and fish. Affected animals generally are asymptomatic but gastrointestinal disorders sometimes can be present. Criptoporidiosis is considered a zoonosis. Infected animals, especially cattle, are a source of infection for the environment and humans, because they eliminate large numbers of oocysts in their feces. The symptoms when present is characterized by watery diarrhea, dehydration, abdominal pain, weight loss and death, especially in immunosuppressed individuals, and especially in patients with human immunodeficiency virus (HIV) and children. Therapeutic methods effective to eliminate this agent in animals and humans has not been developed but it is necessary to applied support treatment. Criptosporidiosis is considered by the World Health Organization as an emerging disease. Basic sanitation, use of appropriate methods for the inactivation of oocysts and security of personal hygiene are recommended as Prophylactic methods to minimize the spread of Cryptosporidium. This literature review was aimed at describing the importance of cryptosporidiosis in public health.

Serological study of vaccinia virus reservoirs in areas with and without official reports of outbreaks in cattle and humans in São Paulo, Brazil

Vaccinia virus (VACV), the etiological agent of an exanthematic disease, has been associated with several bovine outbreaks in Brazil since the end of the global vaccination campaign against smallpox. It was previously believed that the vaccine virus used for the WHO global campaign had adapted to an unknown wild reservoir and was sporadically re-emerging in outbreaks in cattle and milkers. At present, it is known that Brazilian VACV is phylogenetically different from the vaccinia virus vaccinal strain, but its origin remains unknown. This study assessed the seroprevalence of orthopoxviruses in domestic and wild animals and farmers from 47 farms in three cities in the southwest region of the state of São Paulo with or without official reports of outbreaks in cattle or humans. Our data indicate a low seroprevalence of antibodies in wild animals and raise interesting questions about the real potential of wild rodents and marsupials as VACV reservoirs, suggesting other routes through which VACV can be spread. © 2013 The Author(s).

Epidemiology of human immunodeficiency virus-visceral leishmaniasis-co-infection

In Brazil, the rates of mother-to-child-transmission (MTCT) of human immunodeficiency virus (HIV) decreased from 20% to 1-2% in some regions. However, the country contains 90% of individuals infected with visceral leishmaniasis (VL) in Latin America, and the west region of São Paulo state faces an alarming expansion of the disease. We describe the epidemiological aspects of the expanding infection of VL and a case report of an HIV-VL-co-infected child from the west region of São Paulo state. The patient was an AIDS-C3 with low levels of CD4, high viral load, severe diarrhea, oral and perineal candidiasis, severe thrombocytopenia, and protein-caloric malnourishment. She evolved with sepsis, renal and cardiac failure. An rK rapid diagnosis test, indirect fluorescent antibody test (IFAT), and bone marrow aspirate were performed for VL. Her symptoms improved significantly after liposomal amphotericin B administration. From the 45 municipalities that compose the Regional Health Department of Presidente Prudente, Lutzomyia longipalpis vectors were found in 58% of them. VL infected dogs were found in 33% of those municipalities, infected dogs and humans were found in 29%, 20% are starting and 33% of the municipalities are preparing VL investigation. It is likely, in this patient, that VL advanced the clinical progression of the HIV disease and the development of AIDS severity. Supported by favorable conditions, the region becomes a new frontier of VL in Brazil. © 2013.

Bovine herpesvirus type 5 infection regulates Bax/BCL-2 ratio

Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny. © FUNPEC-RP.