| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Although human vaccine was eradicated in the world and the vaccination programs stopped, outbreaks of Vaccinia virus have been reported in several states and regions of Brazil with different types of vaccinia virus (VV) isolated and characterized.
Cantagalo virus, a vaccinia-type virus, was reported from cattle and milkers in the county of Cantagalo, Rio de Janeiro State, Brazil. The sequence of hemagglutinin protein gene (HA) was analyzed and the results demostrated a closer relation to VV-IOC, a vaccinal strain manufactured by Instituto Oswaldo Cruz in Brazil and used to eradicate smallpox from the region. Several polymorphisms most likely developed over the years suggesting virus adaptation into nature and a long persistence of VV in Brazil.1
Subsequently, a new Vaccinia virus was identified and isolated from different regions as Cotia virus, previously named SPAnv, and was isolated from sentinel mice in the border of the Amazon rain forest2; Araçatuba virus, reported in 2003 and isolated from cattle3; Belo Horizonte virus, isolated in an animal facility4; and Passatempo virus reported in 2005 in small properties in the town of Passa-Tempo, Minas Gerais State in Brazil. Lesions on the hands of milkers, headache, lymphadenopathy, and fever were observed.5 Vaccinia-like virus isolation, similar to Cantagalo virus, was described in 2004, from lesions of 74 patients collected during 2001–2003 originating from dairymaids, milkmen, and some farmers and their relatives from mainly the São Paulo State, Minas Gerais State, and Goiás State.6 In the same year, an outbreak of an exantemal disease in humans and cattle, caused by a Vaccinia virus, was reported in Zona da Mata, Minas Gerais State in Brazil. The disease was observed in 72 farms in 20 different counties in this region and in 87.5% of them human cases were observed. Only 24.6% of the persons affected were less than 25 years of age and consequently not vaccinated against variola. The authors reported the disease in house relatives suggesting horizontal transmission between humans.7
Recently, researchers studied the origin and diversity of Vaccinia virus isolated in Brazil and evaluated the possibility that those could represent an escaped vaccinal strain. The authors reinforce the persistence of VV in Brazil and other parts of the world despite the smallpox eradication campaign, and the concept that the vaccinal strain could no longer exist in nature and the need for epidemiologic studies.8
In this work, we report another outbreak of Vaccinia virus with lesions in cattle and humans in the southwest region of São Paulo State in Brazil. The disease was observed in four small dairy farms with manual milking. Lesions in cattle were observed on teats and udder (Figure 1
) and characterized by vesicules and ulcers. These lesions were sensitive to touch and presented increased local temperature. One lactating calve, 6 months of age, presented vesicules on muzzles and oral mucosae (Figure 2). The percentage of infected animals was 23%, 100%, 30%, and 44%, respectively. A fast dissemination was observed into the farms, with the incubation period of 1 to 2 days and the evolution period, for the animals submitted to symptomatic topical treatment, 15 days. The animals not submitted for treatment maintained the lesions for a long time. In one farm, the disease was detected after the evolution period and scars were observed in the teats.
|
|
Milkers presented lesions on the hands (Figure 3) and one child, 11 years of age, presented lesions in the mouth and nose. The age of the milkers ranged from 22 to 63 years of age. Three milkers were previously vaccinated against smallpox (47–63 years of age), and two milkers were not previously vaccinated (22 and 27 years of age, respectively). Systemic clinical signs were reported by all but one milker. They reported lymphadenopathy, headache, and fever that varied in severity and persisted for ~2–5 days. Lesions in the ocular region were observed in one milker 22 years of age, which correlated with several lesions on the hands (Figure 4). The affected persons sought medical help and symptomatic treatment was prescribed. Fragments of crusts from lesions and swabs of vesicular fluids were collected and sent to virus isolation and identification. The two affected milkers, not previously vaccinated against smallpox, were submitted to blood collection for antibody detection against Vaccinia virus.
|
|
Araçatuba virus, a Vaccinia virus previously isolated and classified, was used as antigen for the sera neutralization test and as a positive control for the PCR reaction.3
The test was performed in baby hamster kidney (BHK) cells according to Neeman and others.9 The initial and final sera dilution was 1:10 to 1:2560. Positivity was considered for titers superior to ten.
Virus isolation was prepared in monolayers of 2 x 105 Vero-CCL 81 cells, and MEM was used. The presence of characteristic cytopatic effect was considered positive for virus isolation and confirmed by PCR assay.
In PCR the DNA extraction was performed according Chomkzynski and others.10 Primers for the HA conserved regions of the Orthopoxvirus (EACP1, 5'-ATG ACA CGA TTG CCA ATA C-3'; EACP2, 5'-CTA GAC TTT GTT TTC TG-3') and B2L for Parapoxvirus (PP1, 5'-GTC GTC CAC GAT GAG CAG CT-3'; PP4, 5'-TAC GTG GGA AGC GCC TCG CT-3'; PP3, 5'-GCG AGT CCG AGA AGA ATA CG-3')11,12 were used. A Vaccinia virus strain Araçatuba was used as a positive control for the Orthopox-virus and the stirpe Orf virus as a positive control for Parapoxvirus amplification. Negative control was constituted by BHK cells.
The PCR reaction for Orthopoxvirus using 5 µL of extracted DNA, 1.5 µL of 50 mM of MgCl2 and 0.5 mM of each primer in a 1x PCR Mix (Invitrogen, CA) was performed with 95°/5 minutes, 40 cycles of 94°/1 minute, 48°/1 minute, 72°/1 minute, and followed for one extention cycle of 72°/5 minutes. This protocol amplifies 846 bp.
The hemi-nested (hn) PCR reaction for Parapoxvirus using 5µL of extracted DNA in the first amplification (594 bp) and 2 µL of the amplificated product in the second amplification (235 bp) was performed in 1x PCR Mix containing 1.5 µM/50 mM MgCL2, and 0.5 µM of each primer in both amplifications. The conditions for the reactions were 94°/5 minutes followed by 30 cycles of 94°/45 seconds, 61°/45 seconds, 72°/45 seconds, and a final extension of 72°/5 minutes.
In the restriction fragment length polymorphism (RFLP) technique, after the amplification the amplicons were digested with the Hae III (Amershan, CA) at 37°C for 3 hours in a 30 µL mix reaction constituted by 2.25 µL buffer reaction, 0.75 µL HaeIII, 15 µL DNA, and 4 µL ultrapure water according to the laboratory producer protocol; originating a 748 bp product.
For electron microscopy diagnosis, fragments of skin lesions were processed by negative contrast methodology according to the authors.13–15
Virus isolation in cell culture was characterized by cytophatic effects (Figure 5). Positive materials were obtained from crusts and confirmed by electron microscopy (Figure 6) with a large number of particules measuring 250 nm x 300 nm; similar to poxvirus and characteristics of Orthopoxvirus, and tubular irregular disposition on external membranes were visualized. Positivity for PCR (Figure 7) and RFLP (Figure 8) was detected in all crusts materials, and characterizing the isolation as Vaccinia virus strain. No positive results were observed from swabs of vesicular fluids (Figure 7). The serum neutralization assay performed with sera from two affected milkers presented titers lower than 10 IU/mL and results were considered negative.
|
|
|
|
Lesions on the hands of affected people were localized on points of contact with the lesions on the teats and udder of cows during the milking as reported by several researchers.3–6 Lesions on ocular regions are probably a consequent of the lesions from the hands demonstrating the possibility of viral transmission to himself and close contacts. It was not possible to detect the origin of contact to the children that presented lesions on the nose and mouth. The possibility of infection during the milking was considered, although the absence of lesions on the hands was not confirmed.
Significative antibody titers were not detected in the milkers, which can be explained by the absence of smallpox vaccination once people previously vaccinated maintain serologic titers for many years after vaccination, and the acute phase of the disease without detectable antibody titers.16 The severity of lesions from previously vaccinated and not vaccinated milkers were similar except for the presence of lesions on the ocular area in one young milker and in the mouth and nose of the children, both not previously vaccinated against smallpox. Although less severe lesions in previously vaccinated persons were reported, it was not observed in these cases.17
Although several reports of Vaccinia virus outbreaks have been done in Brazil, it was not yet reported in this region (Figure 9). This report reinforces the viral circulation in our country as reported.8 The disease in previously vaccinated and in not vaccinated persons against smallpox reinforces the absence of immunity, the risk to the human health, and the needs for more epidemiologic and immunologic studies.
|
Received March 20, 2008. Accepted for publication July 5, 2008.
* Address correspondence to Jane Megid, Universidade Estadual Paulista "Julio de Mesquita Filho," Faculdade de Medicina Veterinária e Saúde Pública, Departamento de Higiene Veterinária e Saúde Pública, Distrito de Rubião Junior, sem número, CEP 18618-000 Botucatu, São Paulo, Brazil. E-mail: jane{at}fmvz.unesp.br 
Authors’ addresses: Jane Megid, Camila Michele Appolinário, and Hélio Langoni, Universidade Estadual Paulista "Julio de Mesquita Filho," Faculdade de Medicina Veterinária e Zootecnia, Departamento de Higiene Veterinária e Saúde Pública, Distrito de Rubião Junior, sem número, CEP 18618-000 Botucatu, São Paulo, Brazil, Tel/Fax: 55-14-3815-2343, 55-14-3811-6270, E-mails: jane{at}fmvz.unesp.br, camila_app{at}yahoo.com.br, and hlangoni{at}fmvz.unesp.br. Edviges Maristela Pituco and Liria Hiromi Okuda, Instituto Biológico de São Paulo, Laboratório de Viroses de Bovídeos, Av. Conselheiro Rodrigues Alves, 1.252, CEP 04014-000 São Paulo, São Paulo, Brazil, Tel/Fax: 55-11-5087-1701, E-mails: pituco{at}biologico.sp.gov.br and liriaho{at}yahoo.com.br.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
