Juliana Felipetto Cargnelutti

Lipid peroxidation associated with anemia in rats experimentally infected with Trypanosoma evansi

This study aimed to assess the plasma lipid peroxidation and the susceptibility of erythrocytes to in vitro peroxidation as indicators of oxidative damage in erythrocytes and their roles in the pathogenesis of anemia during the early acute phase of Trypanosoma evansi infection in rats. Fifty male Wistar rats were randomly distributed into seven groups: three trypanosome-infected groups (T(2), T(4) and T(6); n=10 animals per group) and four uninfected controls (C(0), C(2), C(4) and C(6); n=5 animals per group). Animals from trypanosome-infected groups were inoculated intraperitoneally with 10(6) trypanosomes. Blood samples were collected by cardiac puncture before infection (day 0; group C(0)) or on the 2nd (C(2) and T(2)), 4th (C(4) and T(4)) and 6th (C(6) and T(6)) day post-infection (dpi). Samples were analyzed for red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), plasma malondialdehyde (MDA) and in vitro peroxidation of erythrocytes. The mean values of the hematological indices gradually decreased in the infected rats compared with the control. MDA was significantly increased (P<0.001) on the 6th dpi in infected versus control animals and was negatively correlated with PCV (P<0.001; R(2)=0.372). The values for erythrocyte in vitro peroxidation were higher for groups T(4) and T(6) than for the control rats (P<0.01). A positive correlation between erythrocyte peroxidation and MDA (P<0.001; R(2)=0.414) was observed. The results of this study indicate that T. evansi infection in rats is associated with oxidative stress, indicated by lipid peroxidation and oxidative damage in erythrocyte membranes, as demonstrated by in vitro peroxidation. This may be one of the causes of anemia in acute trypanosomosis.

Resposta eritropoética de ratos em diferentes graus de parasitemia por Trypanosoma evansi

O Trypanosoma evansi e um protozoario hemoflagelado que causa, em varias especies, uma doenca caracterizada por altos niveis de parasitemia, com rapido desenvolvimento de anemia. Este trabalho teve como objetivo investigar a relacao entre o grau de parasitemia e a alteracao na eritropoese de ratos (Rattus norvegicus) da linhagem Wistar infectados experimentalmente com T. evansi. Foram utilizados 42 ratos, dos quais 36 foram inoculados pela via intraperitoneal com 0,2ml de sangue, contendo 2,5 x 104 parasitas. Seis ratos nao-inoculados foram utilizados como controles. Apos inoculacao, a parasitemia foi avaliada a cada 12h. Os grupos para analise foram estipulados de acordo com a media de tripanossomas em 10 campos homogeneos focados aleatoriamente, sendo: A, controle; B, animais que apresentaram um grau de parasitemia entre 1-10 tripanossomas/campo; C, ratos com 11-20 tripanossomas/campo; D, ratos com 21-30 tripanossomas/campo; E, ratos com 31-40 tripanossomas/campo; F, 41-50 tripanossomas/campo; e G, ratos com mais de 51 tripanossomas/campo. Quando os animais apresentaram o numero de protozoarios equivalente ao grupo, foram coletadas amostras de sangue para realizacao de hemograma e dosagem de ferro, e foi realizada citologia de medula ossea para avaliacao da relacao mieloide:eritroide. A analise estatistica mostrou reducao significativa das hemacias e do hematocrito a partir de 31 tripanossomas/campo (grupos E, F e G; P<0,005) e a reducao de hemoglobina ocorreu a partir de 41 tripanossomas/campo (grupos F e G; P<0,005). A relacao mieloide:eritroide foi reduzida de 0,7 para 0,6 a partir de 41 tripanossomas/campo (grupos F e G; P<0,005). Nao foram detectadas variacoes na concentracao de ferro. Os dados obtidos demonstraram que ratos com parasitemia acima de 31 tripanossomas por campo desenvolvem uma anemia aguda, com um aumento compensatorio na atividade hematopoetica.

Coinfection by Vaccinia virus and an Orf virus–like parapoxvirus in an outbreak of vesicular disease in dairy cows in midwestern Brazil

The current report describes an outbreak of vesicular disease affecting dairy cows in midwestern Brazil in which a coinfection with 2 poxviruses—Vaccinia virus (VACV) and a parapoxvirus—was demonstrated. Milking cows presented vesicles, painful reddish or whitish papules, and scabby proliferative lesions in the teats and udder, in a clinical course of approximately 10–21 days. Histologically, multifocal areas of moderate to severe acanthosis, spongiosis, hypergranulosis, and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers were observed in the epidermis. Rounded eosinophilic inclusion bodies were observed in the cytoplasm of epithelial cells of areas with acanthosis or necrosis. Moderate inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, and macrophages were observed in some dermal areas. Two people milking the affected cows developed lesions on the hands, painful papules which progressed to ulcerative and scabby lesions in 4–7 days. Electron microscopy of scabs from 1 cow revealed the concomitant presence of orthopoxvirus and parapoxvirus particles. Scabs from 2 cows were positive by polymerase chain reaction for the parapoxvirus B2L gene; 1 of the scabs was also positive for the VACV vgf gene. Nucleotide sequencing of the B2L amplicon revealed a similarity of 96–99% with Orf virus (ORFV) and lower identity with Pseudocowpox virus (92–95%) and Bovine papular stomatitis virus (85–86%). Nucleotide sequencing of a region of parapoxvirus DNA polymerase gene revealed a high similarity (98–100%) with ORFV sequences. Thus, an unusual coinfection with VACV and a parapoxvirus, likely ORFV, was demonstrated in the outbreak.

Bovine papular stomatitis affecting dairy cows and milkers in midwestern Brazil

Bovine papular stomatitis virus (BPSV) is a parapoxvirus associated with papular and erosive lesions on the muzzle, lips, and oral mucosa of cattle. Teats of milking cows are occasionally affected, and the infection is frequently transmitted to human beings. The present report describes an outbreak of BPSV infection affecting cows in midwestern Brazil, with human involvement. The disease was observed in neighboring small hand-milking farms, affecting 20 milking cows. The signs included painful reddish papules, ulcers, and scabby proliferative lesions on the teats, with a clinical course of 7–12 days. Affected cows presented severe local pain, not allowing the completion of milking. Histologically, acanthosis, spongiosis, and parakeratotic hyperkeratosis with adjacent focally extensive ulcers and multifocal inflammatory infiltrate were observed in the epidermis. Eosinophilic inclusion bodies were noted in the cytoplasm of epithelial cells. Personnel milking the affected cows developed lesions on the hands, painful papules that progressed to ulcerative and scabby lesions in 4–7 days. A polymerase chain reaction using a set of pan-parapoxvirus primers for the B2L gene performed on DNA extracted from scabs amplified a 590-bp product, which when sequenced, revealed similarities of 99%, 85%, and 84% with BPSV, Pseudocowpox virus, and Orf virus, respectively. A phylogenetic tree based on the B2L sequence was constructed, showing that the virus clustered with BPSV isolates. Although clinical cases compatible with BSPV infection have been frequently described in Brazil, the present report identifies the agent associated with cattle and human disease in the country.

An outbreak of pseudocowpox in fattening calves in southern Brazil

Pseudocowpox virus is a parapoxvirus frequently associated with papulovesicular and scabby lesions on the teats and udders of milking cows and is often transmitted to human beings. An unusual outbreak of skin disease in fattening calves in southern Brazil is described. Fourteen of 17 male cattle (82%), aged 6–48 months, feeding on grass pastures were affected. Animals developed papules, vesicles, and scabby proliferative lesions on the muzzle in a clinical course of approximately 10–15 days. The scabby lesions often presented with exudation and bleeding. Histological examination of mucocutaneous tissue in detached scabs revealed acanthosis with thickening of the corneal layer and premature keratinization (parakeratotic hyperkeratosis). The dermis had multifocal lymphoplasmacytic infiltrates. Electron microscopic examination of scab specimens revealed typical parapoxvirus particles: oval shaped (260 nm × 160 nm), enveloped, and covered with a helical layer. Polymerase chain reaction using a set of pan-parapoxvirus primers for the B2L gene amplified a 590-bp product out of DNA extracted from scabs. Nucleotide sequencing of the amplicons revealed a nucleotide homology of 97% with Pseudocowpox virus and lower homology with other parapoxviruses: Bovine papular stomatitis virus (84%) and Orf virus (94%). A phylogenetic tree based on the B2L sequence was constructed, showing that the virus clustered with Pseudocowpox virus isolates.

Partial sequence analysis of B2L gene of Brazilian orf viruses from sheep and goats

We herein describe the partial nucleotide sequencing and phylogenetic analysis of the B2L gene of seventeen Brazilian orf viruses (ORFV). Seventeen viruses were recovered from outbreaks of contagious ecthyma in sheep and goats in four states in Southern and Northeast country, and three from commercial vaccines. Most analyzed viruses were associated with outbreaks of classical contagious ecthyma, with lip, nostrils and labial commissure involvement, yet udder/teat, feet, vulvar and disseminated lesions were also reported in some cases. Nucleotide sequence analysis revealed a high degree of B2L similarity among sheep sequences (>99%) regardless the geographic origin, and a remarkable high identity for the two goat isolates (>99.8%), with similarity dropping to below 99% when comparing viruses from the two species. A phylogenetic tree grouped most sheep and goat viruses on different branches. In addition, sequence alignment allowed the identification of up to six scattered nucleotide changes that were predominant and more consistent in goat isolates, including a number of sequences from other continents. Thus, in spite of the high nucleotide similarity, different degrees of similarity and discrete nucleotide changes in the B2L gene may help in grouping ORFV viruses according to host species.

Antiviral Action of Diphenyl Diselenide on Herpes Simplex Virus 2 Infection in Female BALB/c Mice.

Diphenyl diselenide, (PhSe)2, is an organoselenium compound with pharmacological actions mostly related to antioxidant and anti‐inflammatory properties. The study investigated its antiviral and virucidal actions against herpes simplex virus 2 (HSV‐2) infection in vitro and in a vaginal infection model in mice. The plaque reduction assay indicated that (PhSe)2 showed virucidal and antiviral actions reducing infectivity in 70.8% and 47%, respectively. The antiviral action of (PhSe)2 against HSV‐2 vaginal infection was performed by infecting mice (105 PFU/ml−1) at day 6. The treatment with (PhSe)2 (5 mg/kg/day, intragastric [i.g.]) followed 5 days before and for more 5 days after infection. The extravaginal lesion score was evaluated from days 6 to 10. At day 11, animals were killed, and histological evaluation, determination of viral load, and TNF‐α and IFN‐γ levels were performed in supernatants of homogenized vaginal tissue. The levels of reactive species (RS), protein carbonyl, non‐protein thiols (NPSH), nitrate/nitrite (NOx), and malondialdehyde (MDA), and the activities of myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. (PhSe)2 reduced the histological damage, extravaginal lesion scores, the viral load of vaginal tissue, and the activity of MPO, but increased the levels of TNF‐α, IFN‐γ. (PhSe)2 attenuated the increase of RS, MDA, NOx levels and the activity of GR caused by infection. (PhSe)2 also attenuated the reduction of NPSH content and the inhibition of CAT, SOD, and GPx activities. The antiviral action of (PhSe)2 against HSV‐2 infection was related to its immunomodulatory, antioxidant, and anti‐inflammatory properties. J. Cell. Biochem. 117: 1638–1648, 2016.

Virological and clinico-pathological features of orf virus infection in experimentally infected rabbits and mice.

Many aspects of the biology of orf virus (ORFV) infection remain poorly understood and attempts to establish animal models have yielded conflicting and non-reproducible results. We herein describe the characterization of ORFV infection and disease in rabbits and mice. A protocol of intradermal inoculation was employed to inoculate 10(8.5)TCID₅₀/mL of ORFV strain IA-82 in the skin of ears, of the back and labial commissures. All inoculated rabbits presented a clinical course characterized by erythema, macules, papules/vesicles or pustules that eventually dried originating scabs. Local signs started around days 3 and 4 post-inoculation (pi) and lasted 3-10 days. Virus was recovered from lesions between days 2 and 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit milder clinical course occurred in 5/10 inoculated mice; virus was recovered from lesions from three animals. Inoculated lambs - used as controls - developed severe lesions of contagious ecthyma. VN tests performed at day 28pi failed to detect neutralizing antibodies in all inoculated animals. In contrast, convalescent rabbit sera were positive by ELISA at dilutions from 100 to 400. These results show that rabbits are susceptible to ORFV infection and thus may be used to study selected aspects of ORFV biology.

Estimulação elétrica neuromuscular de média freqüência (russa) em cães com atrofia muscular induzida

The medium frequency neuromuscular electrical stimulation (NMES) (Russa) or Kotz is designed for recuperation of muscle mass in dogs with muscular atrophy in disuse. This study aims to utilize medium frequency NMES on the femoral quadriceps of dogs with induced muscular atrophy and evaluate the occurrence of gain in mass. Eight dogs in two groups denominated GI, or control, and GII, or treated were used. For the induction of muscular atrophy, the left femoral-tibial-patellar joint was immobilized for 30 days. NMES treatment began 48 hours after the removal of the immobilization device on dogs from group II and was carried out three times per week, with an interval of 48 hours between each session, during 60 days. The following parameters were measured: thigh perimeter, goniometry of the knee, creatine kinase (CK) enzymes and morphometry of the muscular fibers in transversal cuts of the vastus lateralis muscle, collected through a muscular biopsy. EENM was utilized on the femoral quadriceps at a frequency of 2500 Hz, with pulse duration of 50%, and the time on/off was at a proportion of 1:2. There was no significant difference between the thigh perimeter and the activity of enzyme CK between groups I and II. As for the goniometry a significant increase (P<0,05) was observed among 0 and 30 days after the immobilization in group II. As for the morphometry of the fibers of the vastus lateralis, a significant increase (P<0,05) was observed in the transversal area of the treated group on the 90th day when compared with that observed at the time of immobilization and among the groups, group II presented a greater transversal area (P<0.05) on the 90th day. The medium frequency NMES brings about a hypertrophy of the vastus lateralis muscle in dogs after induced muscular atrophy.A estimulacao eletrica neuromuscular (EENM) de media frequencia (Russa) ou de Kotz pode ser empregada para a recuperacao de massa muscular em animais apresentando atrofia muscular por desuso. Assim, o objetivo deste trabalho foi empregar a EENM de media frequencia no quadriceps femoral de caes com atrofia muscular induzida, avaliando-se a ocorrencia de ganho de massa. Foram utilizados oito caes em dois grupos denominados de GI ou controle e de GII ou tratado. Para a inducao da atrofia muscular, a articulacao femoro-tibio-patelar esquerda foi imobilizada por 30 dias. Apos 48 horas da remocao, foi realizada a EENM nos caes do grupo II, tres vezes por semana, com intervalo de 48 horas cada sessao, pelo periodo de 60 dias. Foram avaliadas a mensuracao da perimetria da coxa, da goniometria do joelho, as enzimas creatina-quinase (CK) e morfometria das fibras musculares em cortes transversais do musculo vasto lateral, colhido mediante a biopsia muscular. A EENM foi empregada no musculo quadriceps femoral numa frequencia de 2.500Hz, largura de pulso de 50% e relacao de tempo on/off de 1:2. Nao houve diferenca significativa quanto aos valores de perimetria da coxa e a atividade da enzima CK entre os grupos I e II. Na goniometria, houve diminuicao significativa (P<0,05) da amplitude articular apos a remocao do aparelho de fixacao externa somente nos animais do grupo II, em comparacao a com tempo zero. Quanto a morfometria das fibras do musculo vasto lateral, foram notados valores maiores de area das fibras no grupo Tratado, em relacao ao Controle (P<0,05), no dia 90, e, no grupo Tratado, entre os dias zero e 90. A EENM de media frequencia ocasiona hipertrofia do musculo vasto lateral em caes apos a atrofia muscular induzida.

Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goiás State, Brazil (2010-2012)

ABSTRACT.- Sant’Ana F.J.F., Leal A.A., Rabelo R.E., Vulcani V.A.S., Ferreira Junior J.A., Carg-nelutti J.F. & Flores E.F. 2013. Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goias State, Brazil (2010-2012) . Pesquisa Veterinaria Brasileira 33(7):860-866. Laboratorio de Patologia Veterinaria, Universidade de Brasilia, Brasilia, DF 70910-900, Brazil. E-mail: santanafjf@yahoo.comCases of vesicular and exanthematic disease by Vaccinia virus (VACV) have been reported in dairy herds of several Brazilian regions, occasionally also affecting humans. The present article describes eight outbreaks of vesicular disease caused by VACV in dairy herds of six counties of Goias state, Midwestern Brazil (2010-2012), involving a total of 122 cows, 12 calves and 11 people. Dairy cows (3 to 9 years old) were affected in all cases and calves (2 to 9 months old) were affected in five outbreaks, presenting oral lesions. The morbidity ranged between 8 and 100% in cows, and 1.5 to 31% in calves. In the cows, the clinical signs started with vesicles (2-7mm), painful and coalescent papules (3-8 mm), which resulted in ulcers (5-25mm) and scabs in teats, and, occasionally, in the muzzle. The clinical course lasted from 16 to 26 days. The histopathology of bovine skin samples revealed superficial perivascular in-flammatory infiltrate of lymphocytes, plasma cells, neutrophils, macrophages and multifocal areas of acanthosis, spongiosis, hipergranulosis and parakeratotic or orthokeratotic hyperke-ratosis with adjacent focally extensive ulcers. Eosinophilic inclusion bodies were noted in the cytoplasm of the keratinocytes. PCR to