Enio Mori

Reference Values on Serum Biochemical Parameters of Brazilian Donkey (Equus asinus) Breed

Abstract Summary Seventeen serum biochemical variables were determined in 40 donkeys of the Brazilian breed (34 females and 6 males) aged from 3 to 19 years. Mean ± standard deviation (SD) (minimum–maximum) values, obtained by automated analysis, were as follows: glucose, 58.35 ± 10.40 (44.00–90.00) mg/dL; cholesterol, 88.41 ± 9.86 (73.58–124.26) mg/dL; serum protein, 6.82 ± 0.40 (6.00–7.52) g/dL; albumin, 3.13 ± 0.21 (2.65–3.69) g/dL; creatinine, 1.80 ± 0.14 (1.51–2.19) mg/dL; urea, 24.25 ± 5.37 (14.12–34.39) mg/dL; lactate, 20.10 ± 4.58 (12.99–33.47) mg/dL; aspartate aminotransferase (AST), 295.81 ± 62.79 (173.71–466.07) IU/L; creatine kinase (CK), 158.00 ± 76.94 (51.69–440.33) IU/L; γ-glutamil-transferase (GGT), 45.82 ± 13.34 (26.17–86.38) IU/L; lactate dehydrogenase (LDH), 576.02 ± 156.32 (213.53–1162.81) IU/L; alkaline phosphatase (AP), 345.36 ± 65.90 (227.25–490.16) IU/L; calcium (Ca), 8.54 ± 0.18 (8.19–8.90) mg/dL; phosphorus (P), 2.76 ± 0.38 (1.99–3.97) mg/dL; chloride (Cl), 106.05 ± 3.20 (99.00–112.00) mEq/L; sodium (Na), 121.50 ± 4.14 (116.00–132.00) mEq/L; and potassium (K), 3.70 ± 0.42 (2.80–4.40) mEq/L. Comparisons of biochemical ranges obtained for the Brazilian donkey breed with reference ranges for other donkey breeds suggested that most values were similar. Biochemical values determined in the present study serve as reference ranges for donkey populations and can be used for health control and diagnosis of diseases.

Equid herpesvirus type-1 exhibits neurotropism and neurovirulence in a mouse model.

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses.

Citologia dos lavados traqueobrônquico (LTB) e broncoalveolar (LBA) de bezerros holandeses sadios durante o primeiro mês de vida

The neonatal calf is a critical moment for adaptation of the newborn to extra uterine life. The respiratory tract is functionally very demanded and often affected by disease, resulting in direct loss of their function and causing serious economic losses in livestock. The basic point to reduce these losses is appropriate clinical evaluation of neonates; but the diagnosis based solely in physical examination is very difficult to establish. The use of complementary analysis such cytology of the respiratory tract becomes an important diagnostic tool; however their findings must be standardized in the face of different techniques employed. This research studied the dynamics of the cellularity of the bronchoalveolar and tracheobronchial region obtained through lung lavage harvested by nasotracheal catheterization technique and tracheocenthesis respectively, during the first month of life of healthy calves. The tracheobronchial cytology was influenced by the time, showing decreased number of alveolar macrophages and greater number of neutrophils, possibly increased by local irritation caused by the technique, which was repeated sequentially, and/or through greater stimulation of inhaled microorganisms deposited in this region. In the bronchoalveolar region no variation in the cellular constituents in function of time was found. The results allowed the conclusion the cell population of the tracheobronchial region has changed over the week-old calves, possibly due to the technique used and/or to the normal region physiology, represented by higher magnitudes of neutrophils. Otherwise, the cells of the broncholaveolar region showed a stable behavior during the first month of life of newborn calves, presenting numerical predominance of alveolar macrophages.

Influenza viruses in adult dogs raised in rural and urban areas in the state of São Paulo, Brazil

In 1970, searching for the interspecies transmission of influenza viruses led to the first study on influenza viruses in domestic animals. Birds and mammals, including human beings, are their natural hosts; however, other animals may also play a role in the virus epidemiology. The objective was to investigate the incidence of influenza viruses in adult dogs raised in rural (9, 19.56%) and urban (37, 80.43%) areas in the state of São Paulo, Brazil. Dog serum samples were examined for antibodies to influenza viruses by the hemagglutination inhibition (HI) test using the corresponding antigens from the circulating viruses in Brazil. Dogs from rural areas presented antibodies to influenza A H3N2, and influenza A H7N7 and H3N8. In rural areas, dog sera displayed mean titers as 94.37, 227.88, 168.14, 189.62 HIU/25 µL for subtypes H1N1, H3N2, H7N7, H3N8, respectively. About 84% and 92% of dogs from urban areas exhibited antibodies to human influenza A H1N1 and H3N2, respectively, with statistical difference at p < 0.05 between the mean titers of antibodies to H1N1 and H3N2. About 92% and 100% were positive for H7N7 and H3N8, respectively. In dogs from urban areas, the mean titers of antibodies against influenza A H1N1, H3N2, H7N7 and H3N8, were 213.96, 179.42, 231.76, 231.35 HIU/25 µL respectively. The difference among them was not statistically significant at p > 0.05. In conclusion, these dogs were positive for both human and equine influenza viruses. The present study suggests the first evidence that influenza viruses circulate among dogs in Brazil.

Anti-human influenza protector antibody detected in horses as a zoonotic viruses

O objetivo deste estudo foi investigar em cavalos, a incidencia do virus influenza e seu ciclo de transmissao interespecies. Portanto, levantamento sorologico foi realizado em soro de cavalos, confrontados com ambas cepas, as especificas (equino) e nao especificas (humana) deste virus. Sangrias de cavalos realizadas nos anos de 1999 e de 2000, forneceram soros que, apos tratamento com Caolim (20%) e hemacias de galo(50%) para remocao dos anticorpos inespecificos, foram titulados contra ambas referidas cepas, atraves do teste de Inibicao da Hemaglutinacao (recomendado pela OMS). Os resultados, demonstraram que as respostas sorologicas dos cavalos apresentaram reacao cruzada entre as cepas especificas e as nao especificas. As porcentagens de titulos IH obtidos foram de 62,75% e de 60,65% para as cepas especificas A/Eq1 (H7N7) e A/Eq2 (H3N8), respectivamente. E as cepas nao especificas essas porcentagens foram de: 79,05% para A (H1N1), de 94,45% para A (H3N2) e de 77,75% ao tipo B. O mais relevante nestes dados comparativos com virus influenza, foi a alta porcentagem de resposta protetora a cepa nao especifica comparada aquela especifica, detectada nos soros equinos. Considerando o fato de que o tipo B, deste virus, ser restrito a especie humana, portanto a resposta de protecao nos cavalos sugere uma direta transmissao interspecies, como em viroses zoonoticas. Os autores relatam pela primeira vez este tipo de evento no Brasil.

Evaluation of alveolar macrophage function after experimental infection with equine herpesvirus-1 in horses

O papel dos macrofagos alveolares (MA) nos mecanismos de defesa pulmonar foi estudado em cavalos infectados pelo herpesvirus equino tipo 1 (EHV-1). Cinco cavalos adultos foram inoculados com 106,6 TCID50 do EHV-1, por instilacao intranasal. A citologia do lavado broncoalveolar (LBA) foi feita usando-se citocentrifugacao das amostras e confeccao de lâminas coradas por Rosenfeld. A concentracao celular foi ajustada para 2´106 celulas/ml, para mensuracao da atividade macrofagica - espraiamento, fagocitose de particulas de zymosan e liberacao de peroxido de hidrogenio (H2O2). Observou-se soroconversao no 14o dia pos-inoculacao (DPI) e isolamento viral positivo no segundo, terceiro e quinto DPI. Na citologia do LBA observou-se linfocitose no 16o DPI. Os testes de mensuracao da atividade macrofagica demonstraram aumento significativo nos indices de espraiamento no 23o e 30o DPI e diminuicao no indice de fagocitose no segundo DPI, retornando a valores semelhantes aos observados antes da inoculacao a partir do 23o DPI. Houve diminuicao na liberacao de H2O2 no segundo DPI, e no 16o DPI esses valores retornaram aos observados antes da inoculacao. O declinio da atividade fagocitica dos MA, na fase aguda da infeccao, indicou que essas celulas atuam de forma importante nos mecanismos de defesa pulmonar contra esse agente.

Avaliação da função de macrófagos alveolares em cavalos clinicamente sadios

Due to the importance of alveolar macrophages (AM) in pulmonary defense mechanisms, studies were performed in order to evaluate the activity of these cells. Bronchoalveolar lavages (BAL) were obtained from five healthy horses, and cytology was performed on glass slides after cytocentrifugation of the samples. Slides were stained by Rosenfeld. All BAL samples were centrifuged and cell concentration was adjusted to 2´106 cells/ml, for the measurement of AM activity (spreading, phagocytosis and hydrogen peroxide release tests). Differential counting of the BAL cells demonstrated that macrophages were the predominant type of cell (59.0± 6.9%). Measurement of AM activity presented the following results: spreading rate, 25.1± 19.7%, phagocytosis rate, 89.4± 6.2% and hydrogen peroxide release, 1.6± 0.3nmoles/2´105 cells (without PMA- phorbol 12-myristate 13-acetate) and 1.8± 0.4nmoles/2´105 cells (with PMA). Results presented a pattern for the activity of AM in healthy horses enabling the demonstration of an activation rate even without known previous eliciting factors. These results indicate that the tests of macrophage activity measurement are adequate for evaluation of phagocytic activity of AMs.

Genotipagem de polimorfismos no gene prnp em ovinos da raça Santa Inês no Estado de São Paulo

Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.

Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil.

BackgroundBats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil.FindingsThree hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus.ConclusionsThis study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.