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On-line version ISSN 1678-9946
Rev. Inst. Med. trop. S. Paulo vol.54 no.6 São Paulo Nov./Dec. 2012
Incidência do vírus influenza em cães adultos criados em áreas rural e urbana do estado de São Paulo, Brasil
Dalva Assunção Portari ManciniI; Rita Maria Zucatelli MendonçaI; Aparecida Santo Pietro PereiraI; Adélia Hiroko Nagamori KawamotoI; Camila Infantosi VannucchiII; José Ricardo PintoI; Enio MoriIII; Jorge Mancini FilhoIV
ILaboratório de Virologia, Divisão de Desenvolvimento Científico, Instituto Butantan. São Paulo, SP, Brazil
IIClínica Médica Veterinária, Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, USP. São Paulo, SP, Brazil
IIIDepartamento de Medicina Veterinária Preventiva e Saúde Animal Laboratório de Biologia Molecular Aplicada e Sorologia, Faculdade de Medicina Veterinária e Zootecnia, USP. São Paulo, SP, Brazil
IVLaboratório de Lípides, Departamento de Bioquímica do Alimento, Faculdade de Farmácia, USP. São Paulo, SP, Brazil
In 1970, searching for the interspecies transmission of influenza viruses led to the first study on influenza viruses in domestic animals. Birds and mammals, including human beings, are their natural hosts; however, other animals may also play a role in the virus epidemiology. The objective was to investigate the incidence of influenza viruses in adult dogs raised in rural (9, 19.56%) and urban (37, 80.43%) areas in the state of São Paulo, Brazil. Dog serum samples were examined for antibodies to influenza viruses by the hemagglutination inhibition (HI) test using the corresponding antigens from the circulating viruses in Brazil. Dogs from rural areas presented antibodies to influenza A H3N2, and influenza A H7N7 and H3N8. In rural areas, dog sera displayed mean titers as 94.37, 227.88, 168.14, 189.62 HIU/25 µL for subtypes H1N1, H3N2, H7N7, H3N8, respectively. About 84% and 92% of dogs from urban areas exhibited antibodies to human influenza A H1N1 and H3N2, respectively, with statistical difference at p < 0.05 between the mean titers of antibodies to H1N1 and H3N2. About 92% and 100% were positive for H7N7 and H3N8, respectively. In dogs from urban areas, the mean titers of antibodies against influenza A H1N1, H3N2, H7N7 and H3N8, were 213.96, 179.42, 231.76, 231.35 HIU/25 µL respectively. The difference among them was not statistically significant at p > 0.05. In conclusion, these dogs were positive for both human and equine influenza viruses. The present study suggests the first evidence that influenza viruses circulate among dogs in Brazil.
Keywords: Influenza A; Dogs; Serology; Epidemiology.
A transmissão interespecífica do vírus influenza é relatada em estudo sobre influenza com animais domésticos desde 1970. Pássaros e mamíferos, incluindo o homem, são seus hospedeiros naturais, porém outros animais podem participar da sua epidemiologia. Foi investigada a incidência do vírus influenza em cães adultos criados em zonas rural (9, 19,56%) e urbana (37, 80,43%), do Estado de São Paulo. Os soros dos cães foram examinados pelo teste de inibição da hemaglutinação (IH), usando antígeno dos vírus influenza circulantes no Brasil. Nos cães rurais foram detectados títulos médios de 94,37, 227,88, 168,14 e 189,62 UIH/25 mL (unidades inibidoras de hemaglutinação/25 mL) para os subtipos H1N1, H3N2, H7N7, H3N8 de vírus influenza A, respectivamente, com diferenças estatisticamente significativas (p<0,05) entre as médias de títulos de anticorpos contra H1N1 e H3N2. Cerca de 84% e 92% dos cães urbanos responderam aos vírus influenza A humano H1N1 e H3N2, respectivamente e destes 92% e 100% foram positivos para os vírus eqüinos H7N7 e H3N8, respectivamente. Para esses cães as médias de títulos de anticorpos para os vírus influenza A H1N1, H3N2, H7N7 e H3N8 foram 213,96, 179,42, 231,76 e 231,35UIH/25 mL, respectivamente. As diferenças entre as médias não foram estatisticamente significativas (p>0,05). Conclui-se que os cães apresentaram positividade para ambos vírus influenza humano e equino. O presente estudo sugere, pela primeira vez, evidências de que há circulação do vírus influenza em cães, no Brasil.
Influenza is an acute and highly contagious viral disease that afflicts the respiratory tract and is transmitted through contact with nasopharyngeal secretions. It is known to be caused by RNA viruses of the Orthomyxoviridae family. In their lipoprotein envelope are two glycoproteins, hemagglutinin (H) and /neuraminidase (N), whose structures differ among virus strains. Sixteen hemagglutinin (H1 - H16) and nine neuraminidase (N1 - N9) subtypes have been identified in influenza A viruses. Influenza A viruses can infect both humans and animals, but influenza B viruses only infect humans1,17.
Hemagglutinin (H) and neuraminidase (N) undergo considerable antigenic variation in influenza A and B viruses, whereas these same proteins display minor alterations in types B and C. Such changes lead to the emergence of new strains. Influenza incidence peaks during winter and can be very recurrent. The main symptoms are: high temperature (± 38 °C), sneezing and coughing, which can evolve into pneumonia in both humans and animals. Influenza types A and B are responsible for seasonal flu epidemics. Type C also causes the disease; however, the symptoms are milder10.
Influenza virus receptors on host cells differ among mammalian species, viruses bind to receptors on tracheal epithelial cells, while duck viruses attach to the cells of the intestine. Avian (H1, H3, H9, H11) and equine (H3 and H7) strains have receptor-binding specificity for acetylneuraminic acid with an alpha 2,3-N-linkage to galactose (a‑2,3 NeuAcGal). Human strains (H1 and H3), on the other hand, have specificity for the alpha 2,6-linkage. Pigs present both receptors, which makes them susceptible to human and avian influenza viruses. They are considered to be intermediate hosts for the adaptation of avian influenza viruses to humans and "mixing vessels" for novel combinations between avian and human influenza viruses, which can result in the emergence of new pandemic strains. The resulting new viruses may then be able to infect horses and humans13. The proteolytic cleavage of the viral receptor proteins is also a very important activation mechanism in the pathogenesis of influenza viruses18,20.
The host range of influenza viruses has been widely studied in order to characterize the role of some animals in the transmission chain - once considered unlikely to infect others. The susceptibility of domestic animals to an influenza type A virus was first reported in 197212. Recently, influenza viruses were isolated from dogs with respiratory infections. Among the isolated viruses, a specific strain, named Influenza A/canine/Florida/43/2004, was observed to be very closely related to the equine influenza A subtype H3N8. This monophyletic origin of US canine influenza viruses of subtype H3N8, which have circulated among dogs since the early 1990s, very likely indicates a single trans-species transmission event directly from horse to dog, possibly facilitated by direct contact in a racing environment3,15.
Dog-to-dog contact transmission of avian-origin influenza A viruses subtypes H3N2 and H3N8 has been observed in a recent experimental study of Beagle dogs. The dogs showed clinical signs after 14 days post-inoculation, confirming the interspecies transmission of the influenza virus16. Domestic animals (cats and dogs) may also enable the adaptation of avian influenza viruses to mammalians, especially carnivores that feed on infected birds5.
Recently, HEINEMANN et al.6 reported serologic evidence of the equine influenza virus A subtype H7N7 circulation in some regions of north Brazil.
Reptilian and crocodilian species, as well as domestic animals (cats and dogs), can be potential hosts of influenza viruses. Recently, influenza type A viruses were detected in crocodilians from two locations in Florida (USA)2.
In one of our previous studies, poisonous snakes (heterothermic animals) showed seropositivity for human influenza A viruses subtypes H1N1 and H3N29.
To date, no studies on influenza viruses in dogs have been carried out in Brazil.
This work was fueled by concerns about the dog-to-human contact transmission of influenza viruses. Therefore this work aimed to study the role of adult dogs from rural and urban areas in the state of São Paulo, Brazil, in the epidemiology of the influenza virus infections. Considering the originality of the present study, the hope is that it could contribute towards the research and the knowledge of this study field.
MATERIALS AND METHODS
Animals: Forty-six healthy dogs, assisted at the clinic of the Faculty of Veterinary Medicine at the University of São Paulo - Brazil, were grouped according to whether they were raised in nine rural or 37 urban areas in the state of São Paulo. The protocol for animal studies, CEEA number 2230/2011, was approved by the Animal Experimentation Ethics Committee of the School of Veterinary and Animal Sciences of the University of São Paulo.
Serum: Blood serum samples were taken, inactivated by heat treatment at 56 °C for 30 min and then treated with Kaolin (20%) and erythrocytes (50%) according to MANCINI et al.8, DESHPANDE et al.4 and OIEN et al.11.
Serology: The hemagglutination inhibition (HI) test was performed according to MANCINI et al.8. 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 as A/SP/2/95 = A/Beijing/353/89 (H3N2), A/SP/1/91= A/Singapore/6/86 (H1N1), A/Eq1/SP/56 (A Eq1 H7N7) and A/Eq2/1/SP/85 (A Eq2 H3N8) were used. Influenza virus samples were grown in MDCK (Madin Darby canine kidney) cell cultures according to MANCINI et al.8.
The serological analysis was obtained through the hemagglutination inhibition (HI) test which showed that 15.27, 6.52 and 6.52% of the dogs exhibited low antibody titers (≤ 40 HIU/25 µL) to influenza A viruses subtypes H1N1, H3N2 and H7N7, suggesting no protective response against these subtypes. The other animals tested presented higher antibody titers, ranging from 40 to ≥ 320 HIU/25 µL (Table 1).
Many serologic methods such as the neutralization, Elisa, could be used to search the antibodies to influenza virus. But the hemagglutination inhibition test is usually the choice for investigating the influenza virus13,14.
As seen in Figure 1, the average number of protective antibody titers (≥ 40 HIU/25 µL) to human H1N1 and H3N2 and equine influenza H7N7, H3N8, in rural areas were 94.37, 227.88, 168.14, 189.62 HIU/25 µL, respectively. In this case, the difference (p < 0.05) among mean titers for human influenza A subtypes H1N1 and H3N2 was statistically significant.
Figure 2 shows the average of protective antibody titers (≥ 40HIU/25 µL) against human and equine influenza viruses in dogs from urban areas. Mean titers of 213.96, 179.42, 231.76 and 231.35 HIU/25 µL were observed for human influenza A subtypes H1N1 and H3N2 and equine influenza A subtypes H7N7 and H3N8 viruses, respectively. No statistically significant difference among mean titers was observed (p > 0.05). When dogs from urban and rural areas were compared for the presence of protective antibody titers (≥ 40 HIU/25 µL) against human and equine influenza A subtypes H1N1, H3N2, H7N7 and H3N8, no statistically significant difference (p > 0.05) was observed (Fig 3).
Nine (19.56%) and 37 (80.43%) dogs from rural and urban areas, respectively, exhibited high mean titers of antibodies (≥ 40 HIU/25 µL) against influenza A viruses subtypes H1N1, H3N2, H7N7 and H3N8, suggesting that these dogs had been in contact with human and equine influenza viruses. Our results are in agreement with a recent serological study performed via the hemagglutination inhibition test on pet dogs participating in a flyball tournament in Pennsylvania (PA, USA), which showed positive results for antibodies against an influenza A virus subtype H3N814.
Healthy dogs, kept in close contact with equine influenza virus (EIV)-infected horses, were reported as developing antibodies against the virus, which suggests that interspecies transmission of EIV to dogs could have occurred as result of contact between these animal species19.
Similar observations were also reported for dogs in Japan. Their serum samples responded positively to human and equine influenza A viruses subtype H3 in the hemagglutination inhibition test (HI)13.
A careful analysis of acute and convalescent dog serum samples, history and clinical signs are important in diagnosing canine influenza. In a study on the etiology of the disease, its causative agent, a type A virus, was found to be closely related to the equine influenza A virus subtype H3N87.
Results from this study and the literature suggest evidence that dogs can harbor the influenza viruses and may play a role in the interspecies transmission and epidemiology of the virus. Furthermore, recent studies on the distribution of receptor molecules in the canine respiratory tract detected that SAa2,3Gal was the dominant receptor, although SAa2,6Gal could be detected in the trachea, bronchi and bronchioles. These reports are consistent with the hypothesis that the sero-positive dogs found in this study were infected with H3N2 influenza A virus originating from human sources13.
The high percentage (100%) of the protective antibodies to influenza virus detected in all evaluated dogs from rural and urban areas suggests that animals have had several contacts with different virus subtypes, during their lives. The subtype H7N7, considered the original avian strain revealed to be present in the dogs population. Also, HEINEMANN et al. (2009) reported that the influenza A, subtype H7N7, was present in equine populations raised in north from Brazil6.
It’s also possible to conclude that influenza viruses could be circulating among dogs in many parts of the world, including Brazil.
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico for their financial support(CNPq Project 471876/2009/2010).
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Dalva Assunção Portari Mancini, Laboratório de Virologia, Divisão de Desenvolvimento Científico, Instituto Butantan
Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brasil.
Tel.: 55 11 3726-7222 Ext. 2152.
Received: 12 December 2011
Accepted: 15 June 2012