Am. J. Trop. Med. Hyg., 79(5), 2008, pp. 647–651
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene
Short Report: Vaccinia Virus in Humans and Cattle in Southwest Region of
São Paulo State, Brazil
Jane Megid,* Camila Michele Appolinário, Hélio Langoni, Edviges Maristela Pituco, and Liria Hiromi Okuda
University of São Paulo State, School of Veterinary Medicine, Department of Veterinary Hygiene and Public Health, Botucatu,
Brazil; Instituto Biologico de São Paulo, Laboratorio de Viroses de Bovideos, São Paulo, Brazil
Abstract. A new outbreak of Vaccinia virus was observed in Southwest region of São Paulo State, Brazil. The disease
was observed in four small dairy farms with manual milking. Lesions were detected in cattle and in humans previously
vaccinated and not vaccinated against smallpox. Although several reports of Vaccinia virus outbreaks have been
occurring in Brazil, it was not yet reported in this region. This outbreak reinforces the viral circulation in our country.
The disease in persons previously vaccinated and not vaccinated against smallpox reinforces the absence of immunity,
the risk to the human health, and the need for more epidemiologic and immunologic studies.
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.
The disease was reported during the same period in three
properties and three months after the first report. In two of
the farms, the same person was responsible for the milking.
The properties were close, but no correlation was detected
regarding the other properties with a greater distance and
natural geographical barriers between them.
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.
Crusts and swabs were collected from the animals that presented lesions in initial and healing stages. Crusts and swabs
were maintained in Eagle’s minimal essential medium (MEM)
with penicillin (500 U/mL) and mycostatin (10 ␮g/mL) in re-
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
* 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 18618000 Botucatu, São Paulo, Brazil. E-mail: [email protected]
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MEGID AND OTHERS
FIGURE 1.
FIGURE 2.
Vaccina virus lesions in teats of lactating cows. This figure appears in color at www.ajtmh.org.
Vaccinia virus lesions on mouth and nose of 6 months of age lactating calve. This figure appears in color at www.ajtmh.org.
FIGURE 3.
FIGURE 4.
Vaccinia virus lesions on milkers hands. This figure appears in color at www.ajtmh.org.
Vaccinia virus (A) ocular and (B) hand lesion in milker. This figure appears in color at www.ajtmh.org.
VACCINIA VIRUS IN HUMANS AND CATTLE
FIGURE 5.
cells (400×).
Cytopathic effect of Orthopoxvirus isolation in Vero
FIGURE 6. Positive electron microscopy for Poxvirus. Poxvirus
particules in skin suspension, negatively contrasted with amonium
molibdate. Observe irregular tabular disposition on external membrane. Bar correspondent to 100 nm (Philips EM 208).
649
frigeration and sera was frozen until processing for virus isolation, characterization, and polymerase chain reaction (PCR).
Human sera were collected for serum neutralization assay.
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 × 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 Orthopoxvirus 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 ␮L of extracted DNA, 1.5 ␮L of 50 mM of MgCl2 and 0.5 mM of each
primer in a 1× 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
5 ␮L of extracted DNA in the first amplification (594 bp) and
2 ␮L of the amplificated product in the second amplification
(235 bp) was performed in 1× PCR Mix containing 1.5 ␮M/50
mM MgCL2, and 0.5 ␮M 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
FIGURE 7. Polymerase chain reaction (PCR) for Orthopoxvirus in crusts and fluid vesicular swabs. Lanes 2, 4, 6, 8, 10 positive crusts samples;
1, 3, 5, 7, 9 negative swabs from vesicular fluids correspondents to the crusts; br: negative control.10–12
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MEGID AND OTHERS
FIGURE 8. Restriction fragment length polymorphism (RFLP)
from Orthopoxvirus positive polymerase chain reaction (PCR).
Lanes 1, 3–6 orthopoxvirus positive samples digested by Hae III; lane
7 digested positive control; lane 8 positive control not digested.13–15
in a 30 ␮L mix reaction constituted by 2.25 ␮L buffer reaction, 0.75 ␮L HaeIII, 15 ␮L DNA, and 4 ␮L 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 × 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.
Several strains of Vaccinia virus have been isolated and
reported in Brazil, which are affecting cattle and persons in
various states.1–7 The affected properties were small farms
with no transit of animals and did not report any epidemiologic common aspect between them. This raises a question
related to wild animals as a natural reservoir of Vaccinia virus, although no specific wild animal was reported in these
farms. The disease was detected initially in one animal, without any specific characteristic or epidemiologic aspects, and
transmitted to the others during the milking. Persons were
infected by contact with sick animals and responsible for viral
dissemination to the other animals. The percentage of infection was directly correlated to the introduction of control
measures.
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.
FIGURE 9. Localization of this Vaccinia virus outbreak in map of Brazil showing previous cases reported and states and ecological biomes
outbreak of Vaccinia virus. Adapted from Lobato et al. (2007).7 This figure appears in color at www.ajtmh.org.
VACCINIA VIRUS IN HUMANS AND CATTLE
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.
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@fmvz
.unesp.br, [email protected], and [email protected].
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: [email protected]
.gov.br and [email protected].
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