Comparative study of lymphocytes from individuals that were vaccinated and unvaccinated against the pandemic 2009-2011 H1N1 influenza virus in Southern Brazil

ABSTRACTINTRODUCTION:While no single factor is sufficient to guarantee the success of influenza vaccine programs, knowledge of the levels of immunity in local populations is critical. Here, we analyzed influenza immunity in a population from Southern Brazil, a region with weather conditions that are distinct from those in the rest of country, where influenza infections are endemic, and where greater than 50% of the population is vaccinated annually.METHODS:Peripheral blood mononuclear cells were isolated from 40 individuals. Of these, 20 had received the H1N1 vaccine, while the remaining 20 were unvaccinated against the disease. Cells were stimulated in vitro with the trivalent post-pandemic influenza vaccine or with conserved major histocompatibility complex I (MHC I) peptides derived from hemagglutinin and neuraminidase. Cell viability was then analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)]-based colorimetric assay (MTT), and culture supernatants were assayed for helper T type 1 (Th1) and Th2-specific cytokine levels.RESULTS:Peripheral blood lymphocytes from vaccinated, but not unvaccinated, individuals exhibited significant proliferation in vitro in the presence of a cognate influenza antigen. After culturing with vaccine antigens, cells from vaccinated individuals produced similar levels of interleukin (IL)-10 and interferon (IFN)-γ, while those from unvaccinated individuals produced higher levels of IFN-γ than of IL-10.CONCLUSIONS:Our data indicate that peripheral blood lymphocytes from vaccinated individuals are stimulated upon encountering a cognate antigen, but did not support the hypothesis that cross-reactive responses related to previous infections can ameliorate the immune response. Moreover, monitoring IL-10 production in vaccinated individuals could comprise a valuable tool for predicting disease evolution.

Bioaccumulation of animal adenoviruses in the pink shrimp

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

Seasonal variation on the presence of adenoviruses in stools from non-diarrheic patients

Human adenoviruses (HAdV), members of the Adenoviridae family, are excreted through the fecal route and may be present in the feces of humans consuming contaminated food or water. The presence of HAdV from different serotypes in the feces of healthy individuals was already reported using conventional polymerase chain reaction; however, real-time PCR (qPCR) may reveal not only the rates of detection as well as demonstrate the viral loads excreted by healthy persons. Aiming to identify and characterize the presence of adenoviruses in stool samples, 147 fecal samples from patients with no records of diarrhea were analyzed (74 from winter season and 73 from summer) by Real-Time PCR (qPCR) assay and conventional PCR. HAdV genome was present in 43.8% (32/73) of stools samples collected during summer season and 21.6% (16/74) during winter. The rate of detection of genomic copies (gc) ranged from 4.04×10² to 6.72×10⁵gc/g of feces among the 147 samples analyzed, of which the ranged of genomic copies of DNA HAdV was major in summer. All samples were negative when tested for rotaviruses (RV) and noroviruses (NoV) by PCR conventional and qPCR respectively. HAdV is excreted constantly by infected individuals in the absence of clinical signs and the occurrence may vary seasonally.

Bioaccumulation of animal adenoviruses in the pink shrimp

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems..

Epidemiological status of felid herpesvirus type-1 and feline calicivirus infections in Brazil / Situação epidemiológica das infecções pelo herpesvírus felino tipo 1 e calicivírus no Brasil

Feline calicivirus (FCV) and felid herpesvirus type-1 (FeHV-1) are the main infectious agents of domestic and wild felines worldwide. The FCV and FeHV-1 viruses were isolated in Brazil in 1988 and 2012, respectively. Serology surveys were performed among domestic feline in the State of Rio Grande do Sul and among wild felines in central Brazilian States. Felines with acute or chronic infections may become carriers for both viruses and, viral transmission occurs mainly by ocular and nasal secretions. In addition, FCV may be transmitted by oropharyngeal secretion and fomites. The clinical signs commonly observed in cats are fever, sneezing, coughing and nasal and ocular discharge; however, oral lesions are restricted to FCV infection. A systemic syndrome showing hemorrhagic lesions, alopecia, facial edema and jaundice has been associated with FCV. Attenuated as well as inactivated vaccines against FCV and FeHV-1 were developed in the middle 1970s, and they are effective at reducing the presentation/development of the diseases, but they are not capable of eliminating the persistence of FCV and FeHV-1. This article presents a brief review of the main aspects of the FCV and FeHV-1 infections, with an emphasis in the current situation on the domestic feline population from Brazil.

Calicivírus felino (feline calicivirus - FCV) e herpesvírus felino tipo - 1 (felid herpesvirus type 1 - FeHV-1) são os principais agentes envolvidos descritos mundialmente infectando felinos domésticos e selvagens. No Brasil, o FCV e o FeHV-1 foram isolados e caracterizados em 1988 e 2012, respectivamente. Estudos sorológicos em felinos domésticos foram realizados no estado do Rio Grande do Sul e em felinos selvagens em alguns estados da região central do país. Felinos com infecção aguda e/ou infecção crônica podem tornar-se portadores, para ambos os vírus, e a transmissão ocorre principalmente por secreções oculares, nasais. Além disso, o FCV pode também ser transmitido por secreções orofaringeanas e fômites. Os sinais clínicos comumente observados em felinos afetados são: febre, espirros, tosse, descarga nasal e ocular; lesões orais se restringem à infecção pelo FCV; além disso, foi descrita uma síndrome sistêmica que apresenta um quadro hemorrágico, alopecia, edema de face e icterícia. Vacinas vivas e inativadas que amenizam o quadro clínico, mas não previnem infecções persistentes pelos vírus, foram desenvolvidas nos anos 70. Este artigo apresenta uma breve revisão dos principais aspectos da infecção pelo FCV e FeHV-1, com ênfase na atual situação na população de felinos domésticos do Brasil.

Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil

Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and co-infection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary.

Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada / Fetal protection against bovine viral diarrhea virus (BVDV) in pregnant cows previously immunized with an experimental attenuated vaccine

Esse artigo relata a avaliação da resposta sorológica e proteção fetal conferida por uma vacina experimental contendo duas amostras atenuadas do vírus da diarréia viral bovina tipos 1 (BVDV-1) e 2 (BVDV-2). Vacas foram imunizadas com a vacina experimental (n=19) e juntamente com controles não-vacinadas (n=18) foram colocadas em cobertura e desafiadas, entre os dias 60 e 90 de gestação, pela inoculação intranasal de quatro amostras heterólogas de BVDV-1 e BVDV-2. A resposta sorológica foi avaliada por testes de soro-neutralização realizados a diferentes intervalos após a vacinação (dias 34, 78 e 138 pós-vacinação [pv]). A proteção fetal foi monitorada por exames ultra-sonográficos e clínicos realizados durante o restante da gestação; e pela pesquisa de vírus e anticorpos no sangue pré-colostral coletado dos fetos abortados e/ou dos bezerros recém nascidos. No dia do desafio (dia 138 pv), todas as vacas vacinadas apresentavam anticorpos neutralizantes em títulos altos contra o BVDV-1 (1.280- >10.240) e, com exceção de uma vaca (título 20), todas apresentavam títulos médios a altos contra o BVDV-2 (80-1.280). O monitoramento da gestação revelou que, dentre as 18 vacas não-vacinadas, apenas três (16,6 por cento) pariram bezerros saudáveis e livres de vírus. As 15 restantes (83,3 por cento) apresentaram indicativos de infecção fetal e/ou falhas reprodutivas. Sete dessas vacas (38,8 por cento) pariram bezerros positivos para o vírus, sendo que cinco eram saudáveis e sobreviveram (27,7 por cento); e dois apresentavam sinais de prematuridade ou fraqueza e morreram três e 15 dias após o nascimento, respectivamente. As oito vacas controle restantes (44,4 por cento) abortaram entre o dia 30 pós-desafio e às proximidades do parto, ou deram à luz bezerros prematuros, inviáveis ou natimortos. Por outro lado, 17 de 19 (89,4 por cento) vacas vacinadas deram à luz bezerros saudáveis e livres de vírus. Uma vaca vacinada abortou 130 dias pós-desafio, mas...(AU)

This paper reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of bovine viral diarrhea virus (BVDV-1 and BVDV-2). Cows (n=19) were vaccinated twice, with a 34 days-interval, with the experimental vaccine and together with non-vaccinated controls (n=18), were mated and challenged between days 60 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1,280->10,240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1,280). At the end of the monitoring, only three out of 18 control cows (16.6 percent) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3 percent) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8 percent) delivered virus-positive calves; five were healthy and survived (27.7 percent); two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4 percent) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4 percent) vaccinated cows delivery virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet this fetus could not be examined for the presence of virus. Another cow delivered a virus-positive calf (5.2 percent). In summary...(AU)

Aspectos virológicos e clínico-patológicos da infecção genital aguda e latente pelo herpesvírus bovino tipo 1.2 em bezerras infectadas experimentalmente / Virological and clinico-pathological features of acute vulvovaginitis and latent infection by bovine herpesvirus 1.2 in heifers experimentally infected

A infecção genital de vacas pelo herpesvírus bovino tipo 1.2 (BoHV-1.2) pode resultar em vulvovaginite e infertilidade temporária. Após a infecção aguda, o BoHV-1 estabelece infecção latente, que pode cursar com episódios periódicos de reativação. O presente trabalho descreve os aspectos virológicos e clínico-patológicos da vulvovaginite aguda e infecção latente resultantes da inoculação de bezerras com uma amostra de BoHV-1.2 isolada de casos de balanopostite em touros. A inoculação do vírus em quatro bezerras pela via genital (10(8.1)TCID50/animal) resultou em replicação viral na mucosa genital e no desenvolvimento de vulvovaginite moderada a severa. Os animais inoculados excretaram o vírus nas secreções genitais até o dia 10 pós-inoculação (p.i.) com título máximo de 10(7.3)TCID50/mL. Foram observados congestão e edema da mucosa vulvovestibular, e formação de pequenas vesículas e pústulas. Durante a progressão clínica, as vesículas e pústulas aumentaram de tamanho e eventualmente se tornaram coalescentes e recobertas por um exsudato fino de coloração amarelada. Estes sinais foram observados a partir do dia 2 p.i. e aumentaram progressivamente de severidade até os dias 5-8 p.i. A administração de dexametasona no dia 55 p.i. resultou em excreção viral nas secreções genitais dos quatro animais por até 10 dias. A reativação da infecção latente foi acompanhada de recrudescência clínica, porém com sinais menos severos e com menor duração do que na infecção aguda. O DNA viral latente foi detectado por PCR, aos 36 dias pós-reativação (p.r.), nos seguintes tecidos: gânglio sacrais: pudendo (4/4); genitofemoral e retal caudal (3/4) e obturador (4/4) e em alguns linfonodos regionais. Estes resultados demonstram que o isolado SV-56/90 é virulento para fêmeas soronegativas, após inoculação genital, e pode ser utilizado em estudos de patogenia e de desafio vacinal.

Venereal infection of heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) may result in vulvovaginitis and transient infertility. The acute infection is followed by the establishment of latent infection which can be periodically reactivated. We herein describe the virology and clinico-pathological aspects of acute and recrudescent vulvovaginitis in heifers inoculated with a Brazilian BoHV-1.2 isolate recovered from an outbreak of balanoposthitis. Genital inoculation of isolate SV-56/90 (10(8.1)TCID50/animal) in four eight-months-old heifers resulted in efficient virus replication in the genital mucosa and the development of moderate to severe vulvovaginitis. The inoculated heifers shed virus in genital secretions in titers up to 10(7.3)TCID50/mL until day 10 pi and developed genital congestion, swelling, vesicles and pustules. The vesicles and pustules increased in size eventually coalesced and became covered with a yellowish exsudate. These signs appeared at day 2 pi, increased in severity up to days 5 - 8 pi and progressively subsided thereafter. Dexamethasone administration at day 55 pi resulted in virus shedding in vaginal secretions for up to 10 days. Virus reactivation in all animals was accompanied by clinical recrudescence of the disease, yet less severe than during acute infection. Examination of sacral ganglia and lymph nodes by PCR at day 36 post-reactivation revealed the presence of latent viral DNA in the pudendal (4/4), genito-femoral, sciatic and rectal caudal (3/4) and obturator nerve ganglia (1/4); in addition to several regional lymph nodes. These results demonstrate the virulence of isolate SV-56/90 for heifers and pave the way for its use in further pathogenesis studies and vaccine-challenge trials.

Atividade antibacteriana de extrato hidro-alcoólico de folhas de jambolão (Syzygium cumini (L. ) Skells) / Antibacterial activity of hydro-alcoholic extract leaves of jambolan (Syzygium cumini (L. ) Skells)

A atividade antimicrobiana de plantas medicinais tem sido pesquisada em diversas espécies, tanto no Brasil quanto em outros países. O objetivo deste trabalho foi o de avaliar a existência de efeito antibacteriano de extrato hidro-alcoólico a 10 por cento (m/v) de folhas de jambolão. Utilizaram-se 17 isolados bacterianos, Gram positivos e Gram negativos. A ação antibacteriana foi avaliada através da inoculação de placas de ágar Mueller Hinton, com um inóculo bacteriano de 1x10(6) ufc mL-1, onde se colocaram quatro discos de papel; o primeiro de antimicrobiano comercial e os demais embebidos em 25mL do extrato, de solução salina ou de etanol. As placas foram incubadas a 37°C por 24 horas, sendo posteriormente realizada a leitura do diâmetro dos halos de inibição. O extrato inibiu o crescimento de 100 por cento das bactérias testadas e os isolados Gram positivos apresentaram um halo médio de 19,5mm, enquanto o dos Gram negativos foi de 18,8mm. Não houve inibição significativa de crescimento nos tratamentos com salina e etanol. Conforme os resultados deste estudo, o extrato testado apresenta atividade antibacteriana frente às amostras testadas, sem diferença de sensibilidade entre microrganismos Gram positivos e Gram negativos.