Serologic and Molecular Evidence of Vaccinia Virus Circulation among Small Mammals from Different Biomes, Brazil.

Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain.

Detection of Vaccinia Virus in Dairy Cattle Serum Samples from 2009, Uruguay.

We detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in Uruguay. Two samples were PCR-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in Brazil. Autochthonous circulation of vaccinia virus in Uruguay and other South American countries cannot be ruled out.

Educational Approach to Prevent the Burden of Vaccinia Virus Infections in a Bovine Vaccinia Endemic Area in Brazil.

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonotic disease characterized by exanthematous lesions on the teats of dairy cows and the hands of milkers, and is an important public health issue in Brazil and South America. BV also results in economic losses to the dairy industry, being a burden to the regions involved in milk production. In the past 20 years, much effort has been made to increase the knowledge regarding BV epidemiology, etiologic agents, and interactions with the hosts and the environment. In the present study, we evaluated milking practices that could be associated with VACV infections in an endemic area in Brazil and proposed an educational tool to help prevent VACV infections. In our survey, 124 individuals (51.7%) from a total of 240 had previously heard of BV, 94 of which knew about it through BV outbreaks. Although most individuals involved in dairy activities (n = 85/91) reported having good hygiene practices, only 29.7% used adequate disinfecting products to clean their hands and 39.5% disinfected cows' teats before and after milking. Furthermore, 46.7% of individuals reported having contact with other farm and domestic animals besides dairy cattle. We also evaluated the presence of IgG and IgM antibodies in the surveyed population. Overall, 6.1% of likely unvaccinated individuals were positive for anti-Orthopoxvirus IgG antibodies, and 1.7% of all individuals were positive for IgM antibodies. Based on our findings, we proposed educational materials which target individuals with permanent residence in rural areas (mainly farmers and milkers), providing an overview and basic information about preventive measures against VACV infections that could enhance BV control and prevention efforts, especially for vulnerable populations located in endemic areas.

Twenty Years after Bovine Vaccinia in Brazil: Where We Are and Where Are We Going?

Orthopoxvirus (OPV) infections have been present in human life for hundreds of years. It is known that Variola virus (VARV) killed over 300 million people in the past; however, it had an end thanks to the physician Edward Jenner (who developed the first vaccine in history) and also thanks to a massive vaccination program in the 20th century all over the world. Although the first vaccine was created using the Cowpox virus (CPXV), it turned out later that the Vaccinia virus was the one used during the vaccination program. VACV is the etiological agent of bovine vaccinia (BV), a zoonotic disease that has emerged in Brazil and South America in the last 20 years. BV has a great impact on local dairy economies and is also a burden to public health. In this review, we described the main events related to VACV and BV emergence in Brazil and South America, the increase of related scientific studies, and the issues that science, human and animal medicine are going to face if we do not be on guard to this virus and its disease.

Serological Evidence of Orthopoxvirus Circulation Among Equids, Southeast Brazil.

Since 1999 Vaccinia virus (VACV) outbreaks involving bovines and humans have been reported in Brazil; this zoonosis is known as Bovine Vaccinia (BV) and is mainly an occupational disease of milkers. It was only in 2008 (and then again in 2011 and 2014) however, that VACV was found causing natural infections in Brazilian equids. These reports involved only equids, no infected humans or bovines were identified, and the sources of infections remain unknown up to date. The peculiarities of Equine Vaccinia outbreaks (e.g., absence of human infection), the frequently shared environments, and fomites by equids and bovines in Brazilian farms and the remaining gaps in BV epidemiology incited a question over OPV serological status of equids in Brazil. For this report, sera from 621 equids - representing different species, ages, sexes and locations of origin within Minas Gerais State, southeast Brazil - were examined for the presence of anti-Orthopoxvirus (OPV) antibodies. Only 74 of these were sampled during an Equine Vaccinia outbreak, meaning some of these specific animals presented typical lesions of OPV infections. The majority of sera, however, were sampled from animals without typical signs of OPV infection and during the absence of reported Bovine or Equine Vaccinia outbreaks. Results suggest the circulation of VACV among equids of southeast Brazil even prior to the time of the first VACV outbreak in 2008. There is a correlation of OPVs outbreaks among bovines and equids although many gaps remain to our understanding of its nature. The data obtained may even be carefully associated to recent discussion over OPVs history. Moreover, data is available to improve the knowledge and instigate new researches regarding OPVs circulation in Brazil and worldwide.

An Update on the Known Host Range of the Brazilian Vaccinia Virus: An Outbreak in Buffalo Calves.

Even nearly forty years after the eradication of smallpox, members of the Poxviridae family continue to be the focus of an increasing number of studies. Among these studies, prominently stands vaccinia virus, an orthopoxvirus that is associated with bovine vaccinia outbreaks. Although more frequently associated with infections in cattle and humans, the host range of vaccinia virus is not restricted only to these hosts. There are several instances of molecular and serological evidence of circulation of vaccinia virus among wildlife species. In addition, viral isolation has confirmed a broad spectrum of vaccinia virus hosts. In this report, we provide a brief update on the host range of Brazilian vaccinia virus, and present a case description of an outbreak in domestic buffalo calves from Northeastern Brazil that corroborates previous serological and molecular studies. Furthermore, in the present study, vaccinia virus has been isolated for the first time in buffaloes, and referred to as vaccinia virus Pernambuco (VACV-PE). Phylogenetic reconstruction was based on A56R clustered VACV-PE with vaccinia virus isolates belonging to group 1 Brazilian vaccinia virus. Furthermore, the vaccinia virus genome was detected in the milk of a lactating cow, which thereby revealed a pathway for future studies on the possible impact of vaccinia virus on buffalo milk and milk products. Taken together, these results provide the first description of clinical disease caused by vaccinia virus in buffaloes in South America. They also raise new questions about the chain of transmission of this virus.

Bovine Vaccinia: Insights into the Disease in Cattle.

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions in the teats of dairy cows and the hands of milkers and is an important public health issue. Severe VACV-induced lesions in the teats and udder of cows and buffaloes could lead to mastitis and other secondary infections, thereby reducing productivity and resulting in economic losses to the dairy industry. In Brazil, BV re-emerged in the late 1990s and is now endemic in most of the Brazilian territory. In the last 15 years, much effort has been made to know more about this disease and its epidemiology, etiologic agents, and interactions with the host and the environment. In this review, we describe the known dynamics of VACV infection in cattle and the viral shedding routes, as well as the relevance of BV for animal and public health.

Vaccinia virus detection in dairy products made with milk from experimentally infected cows.

Vaccinia virus (VACV) is the agent of bovine vaccinia (BV), an emerging zoonosis that causes exanthematic lesions on the teats of dairy cows and on the hands of milkers. The virus has been detected in the milk of naturally infected cows. The objective of this study was to investigate and quantify VACV DNA as well as the presence of infectious virus particles in samples of cheese curd, cheese whey and pasteurized milk produced using milk from cows experimentally inoculated with VACV-GP2, a Brazilian isolate of VACV (VACV-BR). VACV DNA was detected in samples of cheese and pasteurized milk at different time points, even after the resolution of the typical lesions caused by VACV, which occurred after 22 days post-infection (dpi), on average. Moreover, it was possible to detect infectious viral particles in cheese samples on alternate days until 27 dpi. The presence of both VACV DNA and infectious viral particles in cheese samples throughout the clinical course of BV and even after the disappearance of the typical clinical signs of disease draws attention to the risk associated with consumption of the cheese. Furthermore, VACV-contaminated milk and cheese may represent an occupational risk to cheesemakers who often manipulate milk and cheese curd without wearing gloves.

Subclinical bovine vaccinia: An important risk factor in the epidemiology of this zoonosis in cattle.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that mainly affects lactating cows and dairy farm milkers. The epidemiological role(s) of other cattle categories such as dry cows, bulls, and heifers in BV remains unclear. This study was performed to investigate VACV in affected dairy cattle herds and perifocal farms during an outbreak in Brazil. Crusts from lesions of cows' teats were collected from all farms with BV outbreaks. Milk, feces, blood, and serum were collected from symptomatic and asymptomatic lactating cows. Blood and serum were also sampled from other cattle categories (calves, heifers, dry cows, and bulls). The samples were tested for VACV by PCR, and to confirm VACV viability, VACV-positive samples were inoculated in BSC-40 cells and stained using immunoperoxidase. Neutralizing antibodies were investigated using plaque reduction neutralization test. Viral DNA was detected in milk, blood, and feces samples of symptomatic and asymptomatic dairy cows and in blood samples from other cattle categories on farms with and without confirmed BV outbreak. In affected farms, viable virus was identified in feces and milk samples from lactating cows and in blood samples from asymptomatic dry cows. Viable VACV was also identified in feces from lactating cows and one bull's blood sample from perifocal farms. Neutralizing antibodies were detected in 81.6% of the herds affected by BV and in 53.8% of the herds on perifocal farms. The presented data indicate a potential source of viral dissemination, which contributes to the persistence and spread of VACV in the environment.

Bovine vaccinia: Inactivated Vaccinia virus vaccine induces protection in murine model.

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions on the teats of dairy cows and the milkers' hands. Since 1999, due to the occurrence of many BV outbreaks in dairy farms across all Brazilian regions, there is a need to improve the control and prevention measures of the disease. Vaccination is one of the major tools to prevent viral diseases, and it could be an alternative for BV prevention. The main objective of this study was the development of vaccine formulations against BV using the inactivated VACV strain GP2 as antigen combined with different adjuvants. Potency tests were performed in mice, which were vaccinated with two doses at a 21-day interval, and then challenged with the vaccine homologous virus. VACV strain GP2 inactivated by beta-propiolactone (BPL) in association with adjuvants was effective in inducing a humoral immune response against VACV, as measured by neutralizing antibody (NA) titers, and was variable depending on the adjuvant used in each vaccine formulation. The vaccine formulation containing aluminum hydroxide (AH) associated with saponin as adjuvant induced the production of high NA titers in all vaccinated mice, giving 100% protection in Balb/c murine model after challenge with homologous virus.

Dairy production practices and associated risks for bovine vaccinia exposure in cattle, Brazil.

A cross-sectional serosurvey was performed to identify environmental features or practices of dairy farms associated with risk for exposure to vaccinia-like viruses in dairy cattle in Brazil. Sera from 103 cows from 18 farms in Minas Gerais state were examined for Orthopoxvirus-neutralizing antibodies. A database of 243 binary or multiple-selection categorical variables regarding the physical features and surrounding ecology of each property was obtained. Thirteen of 46 presumptive predictor variables were found to be significantly associated with Orthopoxvirus serostatus by univariate logistic regression methods. Use of teat sanitizer and having felids on the property were independently associated with virus exposure by multivariable analysis. Rodents have long been suspected of serving as maintenance reservoirs for vaccinia-like viruses in Brazil. Therefore, domestic felids are not only effective predators of small rodent pests, but also their urine can serve as a deterrent to rodent habitation in buildings such as stables and barns. These results corroborate previous evidence of the high significance of rodents in the Vaccinia virus transmission cycle, and they also raise questions regarding the common use of teat sanitizers in dairy production areas.

Clinical, hematological and biochemical parameters of dairy cows experimentally infected with Vaccinia virus.

Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an important zoonosis that affects dairy cattle. There are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of VACV experimentally infected cows were evaluated. In the first part of the study, lactating cows were infected with VACV-GP2 strain. In the second part, animals previously infected with VACV-GP2 were divided into two treatment groups: Group 1, immunosuppressed cows; and Group 2, re-infected cows. In this study, BV could be experimentally reproduced, with similar lesions as observed in natural infections. Moreover, a short incubation period and local lymphadenopathy were also observed. VACV could be detected by PCR and isolated from scabs taken from teat lesions of all inoculated and re-inoculated animals. Lymphocytosis and neutrophilia were observed in all animals from the first part of the experiment, and lymphopenia and relative neutrophilia were observed in the immunosuppressed animals. Detection of viral DNA in oral mucosa lesions suggests that viral reactivation might occur in immunosuppressed animals. Moreover, clinical disease with teat lesions may occur in previously VACV-infected cows under the experimental conditions of the present study.

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.