Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp. ) I: gross anatomical and histopathological findings in organs at necropsy

BACKGROUND Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Infección experimental en ovinos con el virus de leucosis bovina: dosis mínima de células BLV-FLK y virus libre de células, y actividad neutralizante de anticuerpos naturales / Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies

Abstract Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Resumen El virus de la leucosis bovina (bovine leukemia virus [BLV]) es un importante agente patógeno del ganado que causa importantes pérdidas económicas en todo el mundo, especialmente en los rodeos lecheros. El uso de modelos animales proporciona información valiosa sobre la patogénesis de las infecciones virales. Se realizaron infecciones experimentales en ovejas usando sangre de bovinos infectados con BLV, clones moleculares de BLV infecciosos o células derivadas de tumores. La línea celular Fetal Lamb Kidney, persistentemente infectada con el BLV (FLK-BLV), es uno de los cultivos a largo plazo más utilizados para la producción permanente de virus. Las células FLK-BLV o las partículas virales obtenidas del sobrenadante del cultivo libre de células podrían usarse como fuente de provirus o de virus para infectar experimentalmente ovejas. En este trabajo, nuestro objetivo fue determinar la cantidad mínima de células FLK-BLV o de sobrenadante libre de células que contiene BLV necesaria para producir infección en ovejas. También evaluamos la cantidad de anticuerpos bovinos anti-BLV necesaria para neutralizar la infección. Observamos que las dos ovejas inoculadas experimentalmente con 5000 células FLK-BLV se infectaron, y que una de las dos ovejas que recibieron 500 células FLK-BLV se infectó. Ninguno de los animales inoculados con 50 células FLK-BLV mostró evidencia de infección. El sobrenadante FLK-BLV libre de células demostró ser infectivo en ovejas hasta la dilución 1:1000. Los anticuerpos BLV específicos mostraron actividad neutralizante, ya que ninguna de las ovejas se infectó. Por el contrario, los animales que recibieron un suero BLV negativo mostraron signos de infección por BLV. Estos resultados contribuyen a la optimización de un bioensayo en ovejas útil para caracterizar la infección por BLV.

Experimental infection by Anaplasma marginale in buffaloes and cattle: clinical, hematological, molecular and pathological aspects / Infecção experimental de Anaplasma marginale em búfalos e bovinos: aspectos clínicos, hematológicos, moleculares e patológicos

The study aimed to evaluate and compare the clinical, laboratory and pathological aspects of buffalo and bovine experimentally infected with AmRio 2 strain of Anaplasma marginale. Four Murrah buffaloes and four crossbred cattle were used in the experiment, which two animals of each species were splenectomized. Strain AmRio 2 of A. marginale was inoculated in all experimental animals. Clinical exams, Packed Cell Volume (PCV), blood counts, blood smears, rickettsemia, necropsy and histopathology were performed in all cases. Semi-Nested-PCR (snPCR) for the msp5 and snPCR for the msp1α target gene for identification of A. marginale in blood samples from animals was done. From positive samples for msp1α snPCR, samples were analyzed for the amino acid sequences of this gene. Two splenectomized cattle presented apathy, pale mucous membranes, jaundice, hyperthermia, and severe anemia. The remaining experimental animals did not show clinical signs. The rickettsemia in all animals was less than 1%. The mean PCV of the splenectomized cattle was below 20% at two-time points after infection. On the blood count, the main changes were observed in splenectomized calves and were characterized by a decrease in red blood cells, hemoglobin, PCV and platelets (p <0.05). All animals presented leukocyte elevation by increased lymphocytes, however, with no significant difference. The average prepatent period was two days in all the animals. The average incubation period in cattle that became ill was 25.5 days, and death occurred, on average, 63 days after inoculation of the strain. The necropsy findings were characterized by pale carcass, ascites, enlarged liver, distended gallbladder, and thick bile. Histopathological findings included infiltration of macrophages and lymphocytes in various organs, hepatic sinusoidal dilatation, and necrosis of the large intestine. In snPCR for the msp5 gene, 100% of the animals were positive in at least one evaluation. And in the snPCR for the infection of the msp1α target gene was also found in all animals in at least one sample evaluated. However, sequencing revealed only five animals, including the bovine which died, with a similarity of the amino acid sequences with AmRio 2 strain of A. marginale. It is concluded that the splenectomized cattle died due to anaplasmosis caused by the inoculated strain and the buffalo were more resistant compared to cattle. Buffaloes can be an alternative to cattle rearing in areas with a high occurrence of clinical cases of anaplasmosis.(AU)

O estudo teve como objetivo avaliar e comparar os aspectos clínicos, laboratoriais e patológicos de búfalos e bovinos infectados experimentalmente com estirpe AmRio 2 de Anaplasma marginale. Para isso, foram utilizados quatro bubalinos Murrah e quatro bovinos mestiços, sendo dois animais de cada espécie, esplenectomizados. Estirpe AmRio 2 de A. marginale foi inoculada em todos os animais. Foram realizados exames clínicos, hematócrito, hemograma, esfregaço sanguíneo com avaliação de riquetsemia, necropsia e histopatologia, além de, Semi-Nested-PCR (snPCR) para o gene alvo msp5 e snPCR para o gene alvo msp1α para identificação de A. marginale nas amostras de sangue dos ruminantes. A partir das amostras positivas na snPCR msp1α, foram selecionadas amostras para análise das sequências de aminoácidos deste gene. Dois bovinos esplenectomizados apresentaram apatia, mucosas pálidas, icterícia, hipertermia e anemia severa. O restante dos animais não apresentou sintomatologia clínica. A riquetsemia em todos os animais foi menor que 1%. A média do hematócrito dos bovinos esplenectomizados esteve abaixo de 20% em dois momentos após infecção. Ao hemograma, as principais alterações observadas foram nos bovinos esplenectomizados e caracterizaram-se por redução de hemácias, hemoglobina, hematócrito e plaquetas (p<0,05). Todos os animais apresentaram elevação de leucócitos por aumento de linfócitos, porém, sem diferença significativa. O período pré-patente médio foi de dois dias em todos os animais. O período de incubação médio nos bovinos que adoeceram foi de 25,5 dias e estes morreram em média 63 dias após inoculação da estirpe. Os achados de necropsia caracterizaram-se por carcaça pálida, ascite, aumento de volume do fígado, vesícula biliar distendida e bile espessa. À histopatologia, verificou-se infiltração de macrófagos e linfócitos em diversos órgãos, dilatação dos sinusoides hepáticos e necrose do intestino grosso. A snPCR para o gene msp5, revelou 100% dos animais positivos em pelo menos um momento de avaliação. E na snPCR para o gene alvo msp1α também verificou-se infecção em todos os animais em pelo menos uma amostra avaliada. Entretanto, o sequenciamento revelou apenas cinco animais, incluindo os bovinos que morreram, com similaridade das sequências de aminoácidos com estirpe AmRio 2 de A. marginale. Conclui-se que os bovinos esplenectomizados morreram em virtude de anaplasmose provocada pela estirpe inoculada e os bubalinos foram mais resistentes em comparação aos bovinos. Finalmente, os búfalos podem ser uma alternativa à criação de bovinos em áreas com alta ocorrência de casos clínicos de anaplasmose.(AU)

Achados microbiológicos, moleculares e histopatológicos em pequenos ruminantes experimentalmente infectados com Actinobacillus seminis / Microbiological, molecular and histopathological findings in small ruminants experimentally infected with Actinobacillus seminis

O objetivo deste trabalho foi avaliar a patogenicidade, em ovinos, de uma cepa de Actinobacillus seminis isolada de caprino no Brasil. Foram utilizadas amostras de sêmen, punção e fragmentos de epidídimo, ducto deferente, testículos e glândulas seminíferas de dois caprinos (animais 1 e 2) e dois ovinos (animais 3 e 4), e foram realizados exame histopatológico, cultivo microbiológico e diagnóstico molecular. O inóculo foi preparado com solução salina na diluição de 10-2 correspondendo ao padrão 1,0 da escala de McFarland, com colônias previamente cultivadas de A. seminis e administrado no volume de 2 mL pelas vias intra-prepucial (animais 1 e 3) e na cauda do epidídimo (animais 2 e 4). Na avaliação clínica observou-se aumento unilateral de consistência firme após 30 dias no epidídimo e testículo do animal 4 que continuou até o dia da eutanásia, bem como o animal 1 apresentou discreto aumento unilateral dos testículos. As lesões macroscópicas e microscópicas observadas nos animais 3 e 4 foram compatíveis com aquelas causadas pela infecção por A. seminis. A. seminis foi isolado de material de punção e sêmen de um ovino (animal 4). Conclui-se que o modelo de infecção experimental utilizando caprinos e ovinos comprovou a patogenicidade da amostra de A. seminis, isolada de um caprino no semiárido brasileiro e reproduzida em um ovino, comprovando a predileção do agente pelo epidídimo, com quadro clinico, achados histopatológicos, isolamento bacteriano e diagnóstico molecular positivo.(AU)

The aim of this study was to evaluate, in sheep, the pathogenicity of an Actinobacillus seminis strain isolated from a goat in Brazil. Samples of semen, puncture and fragments of epididymis, deferent duct, testicles and seminal vesicles from two goats (animals 1 and 2) and two sheep (animals 3 and 4) were used, and histopathological, microbiological culture and molecular diagnoses were performed. The inoculum was prepared with saline solution at 10-2 dilution corresponding to 1.0 McFarland standard, with A. seminis colonies previously cultured and administered on 2mL volume by intra-preputial (animals 1 and 3) and epididymis tail (animals 2 and 4) routes. At clinical evaluation it were found unilateral swelling of firm consistency after 30 days in epididymis and testicle from animal 4 that continued until the day of euthanasia, as well as animal 1 shown discrete unilateral swelling of testicles. Gross and microscopic lesions in animals 3 and 4 were compatibles with that caused by A. seminis infection. A. seminis was isolated from material of puncture and semen of one sheep (animal 4). It is concluded that the experimental infection model using goats and sheep has proved the pathogenicity of the A. seminis strain isolated from a goat in the Brazilian semiarid and reproduced in a sheep, which confirm the prediletion of the agent for epididymis, with clinical signs, histopathological findings, bacterial isolation and positive molecular diagnosis.(AU)

Resposta imunológica a antígenos de Hysterothylacium deardorffoverstreetorum de peixes teleósteos / Imunne response against Hysterothylacium deardorffoverestreetorum from teleost fish

Anisaquidose é uma doença provocada por parasitos da família Anisakidae e se caracteriza por manifestações gastrointestinais e alérgicas. O Anisakis simplex é o parasito mais patogênico ao homem e altamente alergênico. Porém, outros anisaquídeos também são danosos aos humanos, mas é desconhecida a imunogenicidade dessas larvas. O objetivo deste trabalho foi avaliar o potencial imunogênico do parasito Hysterothylacium deardorffoverestreetorum (HD) em modelo murino. Camundongos da linhagem BALB/c foram divididos em três grupos experimentais e receberam as preparações antigênicas obtidas de larvas de HD: extrato bruto de larvas (EBH), extrato secretado/ excretado de larvas (ESH) e extrato bruto de larvas após excreção/secreção (EEH). Amostras séricas foram obtidas em diferentes dias após imunização para determinação dos níveis de anticorpos específicos pelo ensaio imunoenzimático (ELISA). Os resultados demonstram aumento na produção de imunoglobulina (Ig) G após a segunda imunização, com aumento progressivo após a terceira imunização. Já em relação à IgE, a reatividade foi mais tardia, demonstrando aumento progressivo após a terceira imunização. Foi avaliada a imunidade celular por meio da intradermorreação, como resultado estatisticamente significativo em relação ao controle utilizado. Este experimento é a primeira descrição da potencialidade patogênica desse parasito em mamíferos e representa um avanço no diagnóstico da anisaquidose humana.(AU)

Anisaquidosis is a disease caused by parasites of Anisakidae family and is characterized by gastrointestinal and allergic reactions. The Anisakis simplex is a more pathogenic Anisakidae to humans and is highly allergenic. However, other species of this family also have characteristics that are harmful to humans, but little is known about the immunogenicity this parasites. The objective of this study was to experimentally assess the immunogenic potential of the parasite Hysterothylacium deardorffoverestreetorum (H.D) in mice. Mice of inbred BALB/c strain were divided into three groups and received three immunizations of the following antigenic preparations obtained from L3 larvae H.D: Crude larval extract of H.D (CEH) Extract secreted / excreted larvae H.D. (ESH) and crude extract of larvae after excretion / secretion (EEH). Serum samples were obtained on different days after immunization to determine the levels of circulating specific antibodies by enzyme-linked immunosorbent assay (ELISA). The results show increased production of immunoglobulin (Ig) G after the second immunization with a gradual increase after the third immunization. Regarding IgE reactivity, this occurred later, demonstrating a progressive increase only after the third immunization. Cellular immunity was evaluated by intradermal, and showed statistically significant result compared to the control used. This experiment is the first description of the pathogenic potential of this parasite in mammals and represents a breakthrough in the diagnosis of human Anisakidosis.(AU)

Clinical and virological characteristics of calves experimentally infected with a Brazilian isolate of bovine viral diarrhea virus type 1a / Aspectos clínicos e virológicos da infecção experimental de bezerros com uma amostra brasileira do vírus da diarreia viral bovina 1a

ABSTRACT: To study the pathogenicity of the Brazilian bovine viral diarrhea virus (BVDV) type 1a 241.10 isolate, four calves were intranasally inoculated with a viral suspension containing 107.2 TCID50 mL-1. One calf was left uninoculated and kept in contact with the other calves to investigate viral transmissibility. After inoculation, the animals were monitored daily for clinical signs of infection. The presence of the virus in the blood and nasal secretions was confirmed by virus isolation in cell culture. White blood cells were quantified prior to and every 3 days after infection, and the presence of antibodies was checked every 7 days, starting at day 0 until day 42 post-inoculation (pi). After infection, nasal and ocular serous secretions were observed between days 1 and 5 pi, along with a mild cough from days 2 to 4 pi; however, no severe clinical signs were present. Body temperature was slightly elevated between days 4 and 6 pi. The control calf did not develop any of the signs observed in the infected animals. Cell culture-mediated virus isolation confirmed viremia between days 4 and 8 pi and the presence of the virus in the nasal secretions between days 1 and 10 pi. All infected animals showed a decrease in white blood cell count. Antibodies could be detected from day 14 pi, and these levels remained high until day 35 pi. The control calf had no viremia, viral presence in nasal secretions, or positive serology, indicating the absence of viral transmission. Thus, isolate BVDV 1a 241.10 has low pathogenicity and transmissibility but retains immunosuppressive capacity.

RESUMO: Com o objetivo de estudar a patogenicidade de uma amostra brasileira do BVDV-1a (241.10), quatro bovinos foram inoculados pela via intranasal com uma suspensão viral contendo 107,2 TCID50 mL-1. Um bezerro foi mantido em contato com o grupo infectado para avaliar a transmissibilidade. Após a inoculação, os animais foram monitorados diariamente para observação dos sinais clínicos. A presença de vírus no sangue e na secreção nasal foi confirmada pelo isolamento viral em cultivo celular. A contagem total de leucócitos no sangue foi realizada com intervalos de três dias pré- e pós-infecção e a detecção de anticorpos a cada sete dias, iniciando-se no dia 0 até o dia 42 pós-inoculação (pi). Após a inoculação, não foram observados sinais clínicos evidentes, apenas secreção nasal e ocular serosa entre os dias 1 e 5 pi e tosse discreta entre os dias 2 e 4 pi. A temperatura corporal teve leve aumento entre os dias 4 e 6 pi. O animal controle não desenvolveu os sinais observados no grupo infectado. O isolamento viral indicou presença de viremia entre os dias 4 a 8 pi e excreção viral na secreção nasal entre os dias 1 e 10 pi. Os animais inoculados apresentaram redução na contagem total de leucócitos no sangue. A detecção de anticorpos iniciou-se no dia 14 pi e os níveis mantiveram-se elevados até o dia 35 pi. No animal controle, não foi observada viremia, presença de vírus na secreção nasal e sorologia positiva, demonstrando ausência da transmissão. Assim sendo, conclui-se que a amostra de BVDV 1a 241.10 possui baixa patogenicidade, mantém a capacidade imunossupressora e tem baixa transmissibilidade.

Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum using murine model and artificial feeder

Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites.

Experimental infection of Rio Mamore hantavirus in Sigmodontinae rodents

This study shows an experimental spillover infection of Sigmodontinae rodents with Rio Mamore hantavirus (RIOMV). Necromys lasiurus and Akodon sp were infected with 103 RNA copies of RIOMV by intraperitoneal administration. The viral genome was detected in heart, lung, and kidney tissues 18 days after infection (ai), and viral excretion in urine and faeces began at four and six ai, respectively. These results reveal that urine and faeces of infected rodents contain the virus for at least 18 days. It is possible that inhaled aerosols of these excreta could transmit hantavirus to humans and other animals.

Experimental infection of horses with Vaccinia virus / Infecção experimental de equinos com o vírus Vaccínia

ABSTRACT: Vaccinia virus (VACV) is the etiologic agent of bovine vaccinia, an emerging zoonotic disease with potential health issues for dairy herds and humans. VACV may occasionally infect other species, including horses. In this sense, an outbreak of VACV disease in horses was described in Pelotas, RS, in 2008, where a co-infection with two VACV strains (named Pelotas Virus 1 [P1V] and Pelotas Virus 2 [P2V]) was detected. Considering the rare occurrence of VACV infection in horses, the objective of this study was to investigate the susceptibility and pathogenesis of VACV infection in this species. Six adult horses were inoculated with VACV P1V or P2V (106.3TCID50/ml) through scarification of the nasolabial surface and monitored for virological and clinical aspects during 28 days. Four inoculated horses (4/6) developed mild lesions in the site of inoculation. Ulcers and scabs restricted to inoculated areas were observed between days 2 and 8 post-inoculation (pi). Microscopically there were acanthosis, ballooning degeneration of the stratum spinosum, necrosis and loss of the epidermis. Infiltration of neutrophils, macrophages and lymphocytes were observed in the dermis. Intracytoplasmic eosinophilic inclusions were infrequently observed in degenerate keratinocytes from adjacent necrotic areas. Virus shedding was detected between days 4 and 8 pi by PCR and virus isolation (infectious virus) from the lesions of one horse inoculated with P2V. No neutralizing antibodies were detected in inoculated animals at day 28 pi. In summary, inoculation of horses with VACV P1V and P2V isolates resulted in a low level of replication and at low frequency, with mild cutaneous lesions, when compared with the course of infection of other susceptible species to VACV. Therefore, horses possibly have a low potential for viral maintenance and transmission to other species, albeit being susceptible to VACV infection.

RESUMO: O vírus Vaccinia (VACV) é o agente etiológico da vaccínia bovina, uma doença zoonótica re-emergente e de importância sanitária e econômica para rebanhos leiteiros. O VACV também pode ocasionalmente infectar outras espécies, incluindo equinos. Nesse sentido, um surto de VACV em equinos foi descrito em Pelotas, RS, em 2008, no qual uma coinfecção com dois isolados de VACV (denominados Pelotas 1 [P1V] e Pelotas 2 [P2V]) foi detectada. Considerando a rara ocorrência da infecção pelo VACV em equinos, o objetivo deste estudo foi investigar a susceptibilidade e a patogenia experimental da infecção pelo VACV nessa espécie. Para isso, seis equinos adultos foram inoculados com P1V ou P2V (106,3DICC50/ml) após escarificação na superfície nasolabial e monitorados para os aspectos virológicos, clínicos e patológicos por 28 dias. Quatro animais inoculados (4/6) desenvolveram lesões macroscópicas leves nos locais da inoculação, consistentes com aquelas causadas por VACV, caracterizadas por úlceras e crostas focais restritas à área inoculada, entre os dias 2 e 8 pós-inoculação (pi). Microscopicamente, havia acantose, degeneração hidrópica do estrato espinhoso e necrose da epiderme, com bactérias intralesionais. Infiltração moderada de neutrófilos, macrófagos e linfócitos foi observada na derme superficial. Inclusões intracitoplasmáticas eosinofílicas foram infrequentemente observadas nos queratinócitos degenerados adjacentes às áreas necróticas. Excreção viral foi detectada por PCR e isolamento viral das lesões de um equino inoculado com o P2V, entre os dias 4 e 8pi. Não foi detectada produção de anticorpos específicos para o VACV em nenhum animal inoculado. Em resumo, a inoculação de cavalos com os isolados P1V e P2V resultou em um baixo nível de replicação viral e em baixa frequência, resultando em lesões cutâneas mais discretas em comparação com outras espécies susceptíveis ao VACV. Apesar dos equinos serem susceptíveis à infecção pelo VACV, possivelmente apresentam baixo potencial de manutenção e de transmissão do vírus para outras espécies.

Experimental infection with Brazilian Newcastle disease virus strain in pigeons and chickens

Abstract This study was designed with the goal of adding as much information as possible about the role of pigeons (Columba livia) and chickens (Gallus gallus) in Newcastle disease virus epidemiology. These species were submitted to direct experimental infection with Newcastle disease virus to evaluate interspecies transmission and virus-host relationships. The results obtained in four experimental models were analyzed by hemagglutination inhibition and reverse transcriptase polymerase chain reaction for detection of virus shedding. These techniques revealed that both avian species, when previously immunized with a low pathogenic Newcastle disease virus strain (LaSota), developed high antibody titers that significantly reduced virus shedding after infection with a highly pathogenic Newcastle disease virus strain (São Joao do Meriti) and that, in chickens, prevent clinical signs. Infected pigeons shed the pathogenic strain, which was not detected in sentinel chickens or control birds. When the presence of Newcastle disease virus was analyzed in tissue samples by RT-PCR, in both species, the virus was most frequently found in the spleen. The vaccination regimen can prevent clinical disease in chickens and reduce viral shedding by chickens or pigeons. Biosecurity measures associated with vaccination programs are crucial to maintain a virulent Newcastle disease virus-free status in industrial poultry in Brazil.

Experimental infection and pathology of clade 2.2 H5N1 virus in gulls

During 2006, H5N1 HPAI caused an epizootic in wild birds, resulting in a die-off of Laridae in the Novosibirsk region at Chany Lake. In the present study, we infected common gulls (Larus canus) with a high dose of the H5N1 HPAI virus isolated from a common gull to determine if severe disease could be induced over the 28 day experimental period. Moderate clinical signs including diarrhea, conjunctivitis, respiratory distress and neurological signs were observed in virus-inoculated birds, and 50% died. The most common microscopic lesions observed were necrosis of the pancreas, mild encephalitis, mild myocarditis, liver parenchymal hemorrhages, lymphocytic hepatitis, parabronchi lumen hemorrhages and interstitial pneumonia. High viral titers were shed from the oropharyngeal route and virus was still detected in one bird at 25 days after infection. In the cloaca, the virus was detected sporadically in lower titers. The virus was transmitted to direct contact gulls. Thus, infected gulls can pose a significant risk of H5N1 HPAIV transmission to other wild migratory waterfowl and pose a risk to more susceptible poultry species. These findings have important implications regarding the mode of transmission and potential risks of H5N1 HPAI spread by gulls.

Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice / Expressão de citocinas e detecção molecular de Mycobacterium avium subspecies paratuberculosis em órgãos de camundongos infectados experimentalmente

Mycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Mycobacterium avium subespécie paratuberculosis (MAP) pode infectar ruminantes e permanecer subclínica por longos períodos nos rebanhos. A identificação de órgãos mais susceptíveis à infecção e a avaliação da expressão das citocinas no local da infecção são importantes para compreender a patogênese de MAP. Neste estudo foi avaliada a probabilidade de detecção de DNA de MAP e a expressão de citocinas em órgãos de camundongos C57BL/6 infectados por via intraperitoneal durante 120 dias. Dentre os órgãos avaliados, o baço (85%), cólon (75%) e fígado (60%) tiveram as maiores frequências de positividade. Quando comparadas essas frequências entre os órgãos, verificou-se que o baço teve 1,54 vezes mais probabilidade de ser positivo em relação ao íleo, e 2,0 vezes mais probabilidade em relação às placas de Peyer. Além disso, aos 60 dias pós infecção, o baço e o fígado foram responsáveis pela maior expressão de IFN-γ e o íleo pela TNF-α e IL-4. Os resultados indicam que o baço é o melhor órgão para avaliar uma infecção experimental por MAP, principalmente nos períodos iniciais da infecção. Além disso, demonstrou que o baço, fígado e íleo têm importância direta na resposta inflamatória de modelos experimentais.

Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

Objective: To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods: A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions: The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

OBJECTIVE@#To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models.@*METHODS@#A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates.@*RESULTS@#Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons.@*CONCLUSIONS@#The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

Brazilian Angiostrongylus cantonensis haplotypes, ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.

Metabolic alterations in broiler chickens experimentally infected with sporulated oocysts of Eimeria maxima / Alterações metabólicas em frangos de corte infectados experimentalmente com oocistos esporulados de Eimeria maxima

Metabolic and morphometric alterations of the duodenal villi caused by parasitism of chickens by Eimeria maxima were evaluated, using 100 male Cobb birds, randomly distributed into two groups (control and infected). The infected group was inoculated with 0.5 ml of a solution containing 5×103 sporulated oocysts of Eimeria maxima. Ten birds per sample were sacrificed on the 6th, 11th, 22nd and 41st days post-infection (dpi). In order to evaluate the alterations, samples of duodenum, jejunum and ileum fragments were collected after necropsy for histological analysis. Villus biometry was determined by means of a slide graduated in microns that was attached to a binocular microscope. To evaluate the biochemical data, 5 ml of blood were sampled from the birds before sacrifice. The statistical analyses were performed using the GraphPad 5 statistical software for Windows. Tukey's multiple comparison test (p <0.05) was performed for the different dpi's and the unpaired t test for the difference between the groups. Infection by E. maxima causes both qualitative and quantitative alterations to the structure of the intestinal villi, thereby interfering with the absorption of nutrients such as calcium, phosphorus, magnesium, protein and lipids, with consequent reductions in the birds' weights.

Foram avaliadas alterações metabólicas e morfométricas das vilosidades intestinais causadas pelo parasitismo de frangos por Eimeria maxima, sendo utilizadas 100 aves da linhagem Coob, machos, distribuídos aleatoriamente em dois grupos experimentais: grupo controle, inoculado com 0,5 ml de água destilada; grupo infectado, inoculado com 0,5ml de solução contendo 5×103 oocistos esporulados de Eimeria maxima. Foram sacrificadas 10 aves por coleta no 0, 6, 11, 22 e 41 dias pós-infecção. Para avaliar as alterações foram retiradas, após necropsia, amostras de fragmentos do duodeno, jejuno e íleo para análise histológica. A determinação da biometria de vilosidades foi realizada por meio de lâmina milimetrada acoplada a um microscópio binocular. Para avaliação dos dados bioquímicos foram coletados 5 ml de sangue das aves antes da eutanásia. As análises estatísticas foram realizadas, utilizando-se o programa estatístico Graphpad Prism. 5 – Windows e realizado o teste de comparações múltiplas de Tukey (p <0,05) para os diferentes dpi's e o Teste T não Pareado para diferença entre os grupos. A infecção por E. maxima provoca alterações qualitativas e quantitativas das vilosidades intestinais, interferindo na absorção de nutrientes, como cálcio, fósforo, magnésio, proteínas e lipídios, com consequente redução no peso das aves.

El factor de crecimiento del endotelio vascular hepático en la infección murina por Schistosoma mansoni / The endothelial growth factor vascular infection in murine liver by Schistosoma mansoni

Los antígenos secretados por los huevos de S. mansoni inducen la proliferación de células endoteliales in vivo, así como la producción del Factor de Crecimiento del Endotelio Vascular (VEGF), sugiriendo que la patogénesis en la esquistosomiasis se relaciona con eventos angiogénicos. Se evaluó la expresión del VEGF, como una medida de angiogénesis estimulada por los huevos y los gusanos de S. mansoni, en la infección Bisexual (BIS) y los gusanos adulto (infección UNI) en el hígado de ratones Balb/c, antes y después del tratamiento con PZQ. Los resultados indican que tanto la infección UNI como BIS son capaces de estimular la producción de VEGF en tejido hepático, lo que explica la vascularizacón anómala durante este cuadro infeccioso. Este proceso se acompaña con la presencia de un elevado número de infiltrados leucocitarios en los sitios donde se observa lesión tisular; la producción de VEFG remite tras 48 de tratamiento con PZQ. Estos resultados indican que la producción anómala de VEGF junto con la intensa respuesta pro-inflamatoria asociada no solo a la actividad de VEGF sino también a los infiltrados leucocitarios observados en el tejido hepático, causada tanto por los huevos secretados como por las formas adultas de S. mansoni son los mecanismos que subyacen a las lesiones granulomatosas observadas durante el curso de la esquistosomiasis, pudiendo al menos revertirse el incremento en la vascularización mediante el uso de PZQ.

The antigens secreted by eggs of S. mansoni induce the proliferation of endothelial cells in vivo, as well as the production of Factor Vascular Endothelial Growth factor (VEGF), suggesting that the pathogenesis of schistosomiasis relations with angiogenic events. We evaluated the expression of VEGF, as a measure of angiogenesis stimulated by the eggs and worms S. mansoni in infection bisexual (BIS) and adult worms ( UNI infection ) in the liver of BALB / c mice before and after treatment with PZQ. These results indicate that abnormal production of VEGF with intense pro-inflammatory response not only associated with the activity of VEGF but also leukocyte infiltrates observed in the liver tissue caused by both secreted and eggs by adult forms S. mansoni are the mechanisms underlying granulomatous lesions observed during the course of schistosomiasis, and can be reversed at least the increased vascularization by using PZQ .

Infecção experimental por Mycoplasma gallisepticum e Escherichia coli em perus / Experimental infection with Mycoplasma gallisepticum and Escherichia coli in turkeys

Mycoplasma gallisepticum (MG) é responsável por provocar sinusite infecciosa em perus. A infecção por Mycoplasma spp. torna a ave susceptível a infecção por Escherichia coli. O objetivo deste estudo foi desenvolver em perus, um modelo experimental para a sinusite infecciosa. Utilizou-se 250 peru,s machos da linhagem Nicholas (Aviagen®) divididos em grupo não infectado (T1) e grupo desafiado (T2) que recebeu por via ocular, com um dia de idade, Mycoplasma gallisepticum cepa F e aos 21 dias de idade E. coli por via saco aéreo. Analisou-se a mortalidade, os sinais clínicos e lesões em sacos aéreos, fígado e coração. Concluiu-se que o delineamento experimental utilizado foi eficaz para simular a infecção natural por MG e E. coli, sendo que a vacina contra MG-F utilizada para poedeiras é patogênica para perus.

Mycoplasma gallisepticum (MG) causes infectious sinusitis in turkeys, and is commonly associated with Escherichia coli. The objective of this study was to develop in turkeys an experimental model for infectious sinusitis. Two hundred and fifty male turkeys of Nicholas breed (Aviagen®) were divided into negative control group and challenged, animals were housed until 42 days old. The birds were inoculated in the first day of age with the MG vaccine (F-VAX ® Schering Plough) and on day 21 with E. coli. We analyzed the mortality, clinical signs and lesions in air sacs, liver and heart. The results showed that the vaccine against Mycoplasma gallisepticum (MG-F) is pathogenic for turkeys and that the experiment was able to simulate natural infection with MG and E. coli.

Infection of sparrows (Passer domesticus) and different mice strains with Lawsonia intracellularis / Infecção de pardais (Passer domesticus) e diferentes linhagens de camundongos com Lawsonia intracellularis

The susceptibility of sparrows (Passer domesticus) and strains of mice (Swiss, BALB/c, C-57 and DB-A) to Lawsonia intracellularis infection was studied. Thirty-two sparrows were inoculated with pure culture of L. intracellularis and eleven received sham inoculum. Feces were collected on -1, 7, 14 and 21 days post infection (dpi) for detection of L. intracellularis by PCR. After 21 days, all sparrows were euthanized and the tissues processed for histology and immunohistochemistry (IHC). One hundred sixty mice of four different strains (n=40, per strain) were used. For each mouse strain, 16 animals received mucosa homogenate from a pig infected with L. intracellularis, 16 received pure culture of L. intracellularis and eight animals received sham inoculum. Two control and four inoculated mice from each group were euthanized on 7, 14, 21 and 28 dpi. Sections of intestine were collected for histologic analysis and IHC and pooled feces were collected for L. intracellularis PCR. None of the sparrows had any histologic lesions characteristic of proliferative enteropathy or antigen labeling by IHC. All sparrow fecal samples were negative by PCR. All mice strains studied had histopathological lesions typical of PE and IHC labeling consistent with L. intracellularis infection, especially those animals inoculated with pure culture. The most severe lesions were observed in DB-A and Swiss mice. Fecal shedding was detected in all mice strains, with peak at 14 dpi. We conclude that sparrows do not seem to be relevant in the epidemiology of L. intracellularis. The results showed variations in the lesions among the four mice strains used.

A susceptibilidade de pardais (Passer domesticus) e linhagens de camundongos (Swiss, BALB / C, C-57 e DB-A) à infecção por L. intracellularis foi testada. Trinta e dois pardais foram inoculados com cultura pura de L. intracellularis e onze receberam placebo. As fezes foram coletadas nos dias -1, 7, 14 e 21 após a infecção (dpi) para a detecção de Lawsonia intracellularis por PCR. Após 21 dias, todos os pardais foram eutanasiados e os tecidos processados para a realização da histologia e imuno-histoquímica (IHQ). Cento e sessenta camundongos de quatro linhagens diferentes (n=40, por linhagem) foram utilizados. Para cada linhagem de camundongo, 16 receberam homogeneizado de mucosa preparado a partir de um suíno infectado com L. intracellularis, 16 receberam cultura pura de L. intracellularis e oito animais receberam placebo. Dois camundongos controle e quatro camundongos inoculados de cada grupo foram sacrificados aos 7, 14, 21 e 28 dpi. Seções de intestino foram coletadas para análise histológica e IHQ e amostras de fezes foram coletadas para a realização da PCR para detecção de L. Intracellularis. Nenhum dos pardais apresentou lesões histológicas características da enteropatia proliferativa ou marcação positiva por meio da IHQ. As amostras de fezes dos pardais foram negativas na PCR. Todas as linhagens de camundongos estudadas tinham lesões histopatológicas típicas de enterite proliferativa e IHQ positiva para a infecção por L. intracellularis, especialmente aqueles animais inoculados com a cultura pura. As lesões mais graves foram observadas em camundongos DB-A e Swiss. A eliminação fecal foi detectada em todas as linhagens de camundongos, com pico 14 dpi. Conclui-se que os pardais não são relevantes na disseminação da L. intracellularis. Os resultados mostraram variações nas lesões entre as quatro linhagens de camundongos utilizadas, indicando o potencial risco que os camundongos representam na transmissão de L. Intracellularis.

Parameters for evaluation of clinical trial in mice infected with Trypanosoma cruzi / Parâmetros para avaliação clínica de camundongos infectados pelo Trypanosoma cruzi em experimentação

The effectiveness of clinical parameters in the evaluation of Trypanosoma cruzi infection was analyzed in male Swiss mice at 8 weeks old Animals were divided into HG (healthy) and IG (1400 trypomastigotes, intraperitoneally, Y strain - Trypanosoma cruzi). Quantitative and qualitative parameters were evaluated in non-consecutive days in the period, from 7th-11th and 15th-18th days of infection. There were significant differences (P<0.05) between both groups in both periods regarding water consumption, abdominal circumference and weight. The second group presented differences in amount of excreta, body temperature, move-up and mortality. There was no difference (P>0.05) between the groups in food consumption, exploration of self-cleaning and skin staining. The fecal feature differed between the groups in the second period. The occurrence of isolation was not practical. Differences were observed in the hair between groups, although the parameter had been interfered by fights between animals. The consumption of water, feed, excreta production, characteristic of the faeces, body temperature, abdominal circumference, move up, weight and mortality parameters are easy to be measured and effective in clinical differentiation of healthy mice infected with T. cruzi, elected in protocols for clinical study with mice, which is the first work to gather information of qualitative and quantitative clinical parameters evaluated in these animals.

Analisou-se a eficiência de parâmetros clínicos na avaliação da infecção pelo Trypanosoma cruzi em camundongos suíços, machos de 8 semanas. Os grupos foram divididos em GS (sadios) e GI (1400 tripomastigotas, intraperitoneal, cepa Y - Trypanosoma cruzi). Avaliaram-se parâmetros quantitativos e qualitativos em dias não consecutivos nos períodos, 7º-11º e 15º-18º dias de infecção. Observaram-se diferenças (P<0.05) significativas entre os grupos, nos dois períodos: consumo de água, circunferência abdominal e peso; apenas no segundo período: quantidade de excretas, temperatura corporal, movimento-levantar e mortalidade. Não houve diferença (P>0.05) entre os grupos: consumo de ração, exploração de auto-limpeza e coloração da pele. As fezes diferiram entre os grupos no segundo período. A ocorrência de isolamento não se mostrou prática. Diferenças no pêlo foram observadas entre os grupos, embora o parâmetro sofra interferência de brigas entre os animais. O consumo de água, ração, produção de excretas, característica das fezes, temperatura corporal, circunferência abdominal, movimento-levantar, peso e mortalidade são parâmetros fáceis de serem medidos e eficientes na diferenciação da clínica de camundongos sadios e infectados pelo T. cruzi, eleitos para protocolos de estudos clínicos com camundongos, sendo este o primeiro trabalho a reunir informações de parâmetros clínicos qualitativos e quantitativos avaliados nesses animais.