Rev. Inst. Med. Trop. Sao Paulo
56(3):191-195, May-June, 2014
doi: 10.1590/S0036-46652014000300002
PRESENCE OF RESPIRATORY VIRUSES IN EQUINES IN BRAZIL
Dalva Assunção Portari MANCINI(1), Aparecida Santo Pietro PEREIRA(1), Rita Maria Zucatelli MENDONÇA(1), Adelia Hiroko Nagamori KAWAMOTO(1),
Rosely Cabette Barbosa ALVES(1), José Ricardo PINTO(1), Enio MORI(2), Leonardo José RICHTZENHAIN(2) & Jorge MANCINI-FILHO(3)
SUMMARY
Equines are susceptible to respiratory viruses such as influenza and parainfluenza. Respiratory diseases have adversely impacted
economies all over the world. This study was intended to determine the presence of influenza and parainfluenza viruses in unvaccinated
horses from some regions of the state of São Paulo, Brazil. Blood serum collected from 72 equines of different towns in this state was
tested by hemagglutination inhibition test to detect antibodies for both viruses using the corresponding antigens. About 98.6% (71) and
97.2% (70) of the equines responded with antibody protective titers (≥ 80 HIU/25µL) H7N7 and H3N8 subtypes of influenza A viruses,
respectively. All horses (72) also responded with protective titers (≥ 80) HIU/25µL against the parainfluenza virus. The difference
between mean antibody titers to H7N7 and H3N8 subtypes of influenza A viruses was not statistically significant (p > 0.05). The mean
titers for influenza and parainfluenza viruses, on the other hand, showed a statistically significant difference (p < 0.001). These results
indicate a better antibody response from equines to parainfluenza 3 virus than to the equine influenza viruses. No statistically significant
differences in the responses against H7N7 and H3N8 subtypes of influenza A and parainfluenza 3 viruses were observed according to
the gender (female, male) or the age (≤ 2 to 20 years-old) groups. This study provides evidence of the concomitant presence of two
subtypes of the equine influenza A (H7N7 and H3N8) viruses and the parainfluenza 3 virus in equines in Brazil. Thus, it is advisable
to vaccinate equines against these respiratory viruses.
KEYWORDS: Parainfluenza virus; Influenza virus; Serology; Hemagglutination inhibition test; Equines.
INTRODUCTION
Influenza is a contagious disease caused by negative eight-stranded
RNA viruses of the Orthomyxoviridae family. There are three major types:
A, B and C. Influenza A viruses are further classified into subtypes - 16
HA and 9 NA - according to the nature of the viral envelope glycoprotein:
hemagglutinin (H) or neuraminidase (N). Influenza A viruses stand
out because they are able to infect both humans and animals, which
characterizes influenza as a zoonotic disease. Also, their H and N antigens
undergo considerable variations, unlike the same proteins in types B and
C, which display minor alterations. Outbreaks of influenza type A occur
in outbreaks in late fall and all over the winter season. Pandemics caused
by influenza type A viruses frequently strike human populations1,12,19.
They target different cell types across species - strains of human (H1
and H3) and equine (H3 and H7) viruses, for example, attach to receptors
on tracheal epithelial cells, whereas avian influenza viruses attach to
cells in the intestine23,25.
Equine influenza A viruses (EIV) of subtype H3N8 have circulated
among horses in Brazil since its isolation by Mc QUEEN et al.10,
whereas subtype H7N7 viruses, isolated in 19762,18, are apparently
extinct or circulate at low levels in equine populations24, which is
consistent with recent observations by HEINEMANN et al.8, who
reported serologic evidence of the H7N7 subtype of equine influenza A
virus circulation in the north of Brazil. PENA et al.16 noted the presence
of the equine H3N8 influenza A virus in 35.9% of 1,592 unvaccinated
equines from farms situated in the south of the state of Pará-Brazil.
DIEL et al.5 have reported the prevalence of antibodies against equine
influenza (EIV) viruses among equines in Rio Grande do Sul (RS),
Brazil: 986 sera samples from 1,506 equines (65.4%) revealed positivity
for them. Recently, TONG et al.21 have isolated a new influenza A virus
from little yellow-shouldered bats captured in two different locations
in Guatemala and named it as H17. This virus lineage differs in genetic
constitution from all other known influenza A viruses, but it was not
recognized as a new H subtype until now.
The acute respiratory disease caused by the equine influenza virus
(EIV) features high morbidity and low mortality. The symptoms are:
high temperature (± 38 °C), anorexia, coughing, dyspnea, nasal discharge
and, depression. In some cases, it can escalate into a bacterial secondary
infection (e.g. pneumonia), which may result in the death of the infected
horses22. The disease also implies economic losses including turf race
cancellations, reduced athletic performance in horses and treatment costs.
(1) Virology Laboratory, Division of Scientific Development, Butantan Institute, São Paulo, SP, Brazil.
(2) Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine and Zootechnics, University of São Paulo, São Paulo, SP, Brazil.
(3) Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP. Brazil.
Correspondence to: Dalva Assunção Portari Mancini, Laboratório de Virologia, Divisão de Desenvolvimento Científico, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP,
Brasil. Phone: +55.11.2627-9833. E-mail: dapmancini@butantan.gov.br
MANCINI, D.A.P.; PEREIRA, A.S.P.; MENDONÇA, R.M.Z.; KAWAMOTO, A.H.N.; ALVES, R.C.B.; PINTO, J.R.; MORI, E.; RICHTZENHAIN, L.J. & MANCINI FILHO, J. - Presence
of respiratory viruses in equines in Brazil. Rev. Inst. Med. Trop. Sao Paulo, 56(3): 191-5, 2014.
Human parainfluenza viruses belong to the family Paramyxoviridae
(Paramyxovirus genus), and are classified into four species: 1, 3
(Respirovirus genus), 2, 4a and 4b (Rubulavirus genus). Human
parainfluenza 3 virus is a negative stranded RNA virus of the Respirovirus
genus7.
cities in the state of São Paulo, Brazil, were selected for blood serum
analysis (Fig. 1).
They thrive worldwide, in temperate and tropical climates, all year
round, through all seasons however, outbreaks of parainfluenza occur in
late fall and early winter. Parainfluenza viruses present HN glycoproteins
inserted in their envelopes, exhibiting hemagglutin (H) and neuraminidase
(N) features. In addition to infecting both humans and animals, influenza
and parainfluenza viral infections share most of their symptoms. Some
of the respiratory tract complications associated with the parainfluenza
virus infection are: legionellosis, viral pneumonitis, pneumococcal
pneumonia, melioidosis11.
In the latter half of 1994, a new respiratory virus, a member of the
Parainfluenza family, emerged in Australia and appears to be the cause
of an infectious disease of zoonotic nature, which has been responsible
for the deaths of humans and horses, two humans and 16 horses had
died before 199815. The authors suggested that the horses may have
been infected from contact with infected birds and bats. The autopsy of
these infected organisms revealed congested and hemorrhagic lungs, the
microscopic lung examination revealing alveolitis with giant cells, and
some syncytial formation. Sequenced analysis showed that PCR products
from this virus isolated from the horses and one patient were identical.
The phylogenetic analysis of the matrix protein segments indicated that
this virus was related to other Paramyxoviridae. Fatal encephalitis has
been reported in equines infected by a novel paramyxovirus14.
PHILBY et al.17 proposed menangle virus as a common name for
an apparently new virus in the Paramyxoviridae family, isolated from
stillborn piglets, in Australia. Sera from humans exposed to infected pigs
and fruit bats also revealed seropositivity to these viruses. Additionally,
CHANT et al.3 presented strong evidence of human infection by the same
virus, transmitted to humans by infected pigs, but they were unable to
explain the transmission mode.
FIELD et al. 6 have reviewed a recently-identified zoonotic
paramyxovirus, the Hendra virus which infects equines and then humans.
They suggest minimizing the human contact with its natural host a species
of fruit bats (Pteropus genus). Similarly, TAYLOR et al.20 reported cases of
human infection with Hendra virus through contact with equines infected
by fruit bats (natural reservoir). Two patients were followed up after two
and six years respectively and no evidence of virus shedding was found.
OBJECTIVE
This work was fueled by concerns about the circulation of influenza
and parainfluenza viruses among equines. Therefore, it aimed to
investigate antibody responses to both viruses in equine serum. This
study’s objective was to shed new light on the circulation of the influenza
and parainfluenza viruses in animals in Brazil.
MATERIAL AND METHODS
Animals: Seventy-two equines from stud farms situated in
Pirassununga (39), Águas de Lindóia (25) and Mairiporã (08), three
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Fig. 1- Location of Pirassununga, Águas de Lindóia and Mairiporã cities in the state of São
Paulo, Brazil.
The age, gender and breed of the equines from each city are described
as follows:
1) Pirassununga city - 19 equines between five and 10 years old, 14
between 11 and 18 years old and six of an unknown age. Eleven were
males and 28 females. As for the breed, 12 were Brazilian Turf, four
were Pure Blood Arabian, six were Appaloosa, two were Pêga, five
were Breton, one was Brazilian, one Schemule, one was Lusitano.
Six of them were of an undefined breed.
2) Águas de Lindóia city - 18 equines between ≤ 2 and 10 years old,
seven between 11 and 20 years old. Seventeen equines were males and
eight females. Twelve animals were Mangalarga Paulista, nine were
Mangalarga Mineiro; one was Appaloosa and one was Carapolina.
Two of them were of an undefined breed.
3) Mairiporã city - five equines between five and nine years old, three
over 10 years old. Seven were males and one was female. As for the
breed, three were Arabian, one was Pure Blood English, one was
Paint-equine, one was Appaloosa, one was Anglo-Arabian and one
was Quarter Horse.
Serum: Blood serum samples were taken, heat-inactivated
(56 °C/30 min) and then treated with Kaolin (20%) and erythrocytes (50%)
according to MANCINI et al.9, DESHPANDE et al.4 and OIEN et al.13.
Serology: The hemagglutination inhibition (HI) test was performed
according to MANCINI et al.9. Four HA units in 25 µL of viruses were
used. Titers were expressed as hemagglutination inhibition units per 25
µL of sera (HIU/25 µL).
Antigens: Antigens of the influenza viruses types A/Eq1/SP/56 (A
Eq1 H7N7) and A/Eq2/1/SP/85 (A Eq2 H3N8) and Parainfluenza 3 virus
identified and provided by Adolfo Lutz Institute São Paulo, Brazil were
used. These virus samples were grown in MDCK (Madin Darby canine
kidney) cell cultures according to MANCINI et al.9.
MANCINI, D.A.P.; PEREIRA, A.S.P.; MENDONÇA, R.M.Z.; KAWAMOTO, A.H.N.; ALVES, R.C.B.; PINTO, J.R.; MORI, E.; RICHTZENHAIN, L.J. & MANCINI FILHO, J. - Presence
of respiratory viruses in equines in Brazil. Rev. Inst. Med. Trop. Sao Paulo, 56(3): 191-5, 2014.
Table 1
Antibody titers for influenza and parainfluenza viruses in equine sera
Virus
Samples
HIU/25µL
≤40
80
160
320
640
1280
2560
Total
PI
72
-(0)
2.80 (02)
16.66(12)
18.05(13)
30.55(22)
25.0(18)
6.94(05)
100(72)
H7N7
72
-(0)
20.83(15)
30.55(22)
35.72(25)
11.11(08)
1.38(01)
-(0)
98.6(71)
H3N8
72
-(0)
2.77 (02)
29.16(21)
44.44(32)
18.05(13)
2.77(02)
-(0)
97.22(70)
(HIU = Hemagglutination Inhibition Units).
RESULTS
The serology results obtained through the hemagglutinating inhibition
(HI) test showed that 100, 98.6 and 97.22% of the equines responded
with mean titers of ≥ 80 HIU/25µL to parainfluenza 3 and H7N7 and H3N8
subtypes of influenza A viruses, respectively (Table 1).
Figure 2 depicts the antibody responses to parainfluenza 3 virus in
sera, in equines from Pirassununga and Águas de Lindóia and reveals
a higher prevalence of the virus in equines from Lindóia city (HI =
1,324.48) than in the ones from Pirassununga city and from either of
the age groups. The difference among mean titers was statistically
significant (p = 0.0001). Mean HIU/25µL values for under nine-year-old
and over 10-year-old equines, 917.89 and 738.06, respectively, showed
no statistically significant difference (p > 0.05).
Fig. 3 - Antibody response to influenza A (H7N7) and A (H3N8) viruses in equines from
Pirassununga and Águas de Lindóia cities and in two age groups. The results were expressed
as means ± S.E.M.
type 3 viruses, in equines from Pirassununga, Águas de Lindóia and
Mairiporã, three cities of the state of São Paulo State, Brazil. The distance
between these cities is around ≥ 81.25 miles therefore, contact between
the animals seems very unlikely.
Given that these equines had not been vaccinated against equine
influenza or parainfluenza viruses, their antibody responses indicated a
viral infection. They may have been infected from contact with infected
birds, as previously suggested by PATERSON et al.15 (Table I).
Fig. 2- Antibody response to parainfluenza 3 virus in equines from Pirassununga and Águas
de Lindóia cities and in two age groups. The results were expressed as means ± S.E.M.
Figure 3 depicts the mean titers of influenza A viruses (H7N7
and H3N8) for equines over ten years old (HIU/25µL of 266.35 and
381.93, respectively). Horses under nine years old showed mean titers
(HIU/25µL) of 324.67 for influenza A(H7N7) and 353.68 for influenza
A(H3N8). No statistically significant difference was observed.
Overall antibody responses to influenza A (H7N7) and A (H3N8)
viruses and parainfluenza 3 virus were different. No statistically
significant difference was observed between both influenza virus
subtypes, however the mean titers for influenza and parainfluenza viruses
were statistically different (p < 0.01) (Table 1).
DISCUSSION
Serum analysis confirmed the presence of influenza and parainfluenza
Antibody responses, to equine influenza and parainfluenza viruses
were not statistically different between the two age groups (equines
under nine and over ten years old). Overall, all equines showed greater
antibody response to parainfluenza 3 virus than to the equine influenza
A viruses (H7N7 and H3N8).
Equines from Águas de Lindóia displayed higher antibody levels
(HIU/25µL) against parainfluenza 3 virus than against equine influenza
viruses, indicating no cross-reactivity among viruses. As for the influenza
A viruses, (H7N7) subtype was more prevalent in the same city than
the (H3N8) subtype, which, in turn, occurred more predominantly in
Pirassununga.
The city of Mairiporã provided equines at the smallest sample size and
lowest breed diversity. For this reason, it was not used in the comparison
analysis of antibody responses.
Outbreaks of equine influenza in Brazil have been reported by
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MANCINI, D.A.P.; PEREIRA, A.S.P.; MENDONÇA, R.M.Z.; KAWAMOTO, A.H.N.; ALVES, R.C.B.; PINTO, J.R.; MORI, E.; RICHTZENHAIN, L.J. & MANCINI FILHO, J. - Presence
of respiratory viruses in equines in Brazil. Rev. Inst. Med. Trop. Sao Paulo, 56(3): 191-5, 2014.
McQUEEN et al.10, PIEGAS et al.18, CUNHA et al.2, PENA et al.16 and
HEINEMANN et al.8, who recently observed the prevalence of equine
influenza A(H7N7) virus in sera of horses from Uruará, State of Pará,
Brazil. DIEL et al. 20055 have also reported the prevalence of equine
influenza in animals in the south of Brazil.
The zoonotic characteristics of influenza viruses can be attributed to
their wide host range, from birds to mammals and even heterothermic
animals, as participants in virus circulation1,4,9. Fruit bats have also been
regarded as a reservoir for both influenza and parainfluenza viruses6,14,17,20.
Reports on the development of the parainfluenza 3 virus in equines
are scarce, either nationally or internationally. In one relevant study
from 1998, in Australia, PATERSON et al.15 reported the isolation of
a new respiratory virus from horses and humans. Its characterization
as a member of the Paramyxovirus genus was initially performed by
virological tests and later confirmed by sequence analysis of the PCR
products from the virus and the phylogenetic analysis of its matrix. The
disease has been responsible for the deaths of human and horses, as
observed in recent studies by TAYLOR et al.20 and O’SULLIVAN et al.14,
indicating its zoonotic nature. Equine influenza A(H7N7) and A(H3N8)
viruses are currently common causes of disease in equines and have
recently been reported to have infected cats and dogs1,4,25. Considering
that these viruses have adversely impacted economies all over the world,
it is advisable to vaccinate equines.
In conclusion, our serology results confirmed the wide distribution
of respiratory viruses, such as equine influenza and parainfluenza 3
virus, among equines in different cities of the state of São Paulo, Brazil.
Because the animals had not been vaccinated against these viruses
prior to our study, it is possible to conclude that the equines have been
infected by them.
The presence of parainfluenza 3 virus is also evidenced by the fact
that this virus is not equine-specific and that no cross-reactions were
observed among the viruses studied here.
Further detailed studies of parainfluenza 3 virus infection in equines
should be performed in order to develop preventive and protective
measures for equine populations in Brazil.
o vírus influenza A não foi estatisticamente significante (p > 0,05). As
médias de títulos dos vírus influenza e parainfluenza, por outro lado,
demonstraram diferença estatisticamente significante (p < 0,001). Esses
resultados indicam melhor resposta de anticorpos pelos equinos ao vírus
parainfluenza 3 do que ao vírus da influenza equina. Nenhuma diferença
estatística foi observada nas respostas contra os vírus da influenza equina
A (H7N7 e H3N8) e parainfluenza 3, com relação ao gênero (fêmeas e
machos) e grupo etário (≤ 2 até 20 anos) nos equinos avaliados. Este
estudo fornece evidência da presença concomitante dos dois subtipos
vírus influenza A (H7N7 e H3N8) e do parainfluenza 3 em cavalos no
Brasil. Portanto, é aconselhável a vacinação dos cavalos contra esses
vírus respiratórios.
ACKNOWLEDGMENTS
The authors acknowledge the financial support of Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq Project
471876/2009/2010) and Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP- 2011/03234-7).
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RESUMO
Presença de vírus respiratórios em equinos do Brasil
Os equinos são susceptíveis aos vírus respiratórios, como o vírus
influenza, e também tem sido citado o vírus parainfluenza. Doenças
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194
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Received: 24 October 2012
Accepted: 25 October 2013
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