Seroprevalence of Flavivirus in horses in Chaco , Argentina . Circulation during 2013-2014 Soroprevalência de Flavivirus em equinos no Chaco , Argentina Circulação durante 2013-2014

Griselda Inés Oria1,2, Lorena Ivana Spinsanti3, Verónica Laura Pirota4, Florencia Martinez5, Ornela Sofía Stechina6, Eduardo Etchepare7,8, Marta Silvia Contigiani9 & Marina Stein10,11 1,2Lic. in Zoology, Lic. Área Entomología, Instituto de Medicina Regional – IMR, Resistencia, Chaco, Argentina, and Facultad de Medicina, Universidad Nacional del Nordeste – UNNE, Corrientes, Argentina. 3Biology, Dra. Laboratorio de Arbovirus y Arenavirus, Instituto de Virología Dr. J. M. Vanella, Facultad de Ciencias Médicas FCM, Universidad Nacional de Córdoba – UNC, Córdoba, Argentina. 4Veterinary, Médico veterinário. Dirección de Fauna, Ministerio de la Producción, Resistencia, Chaco, Argentina. 5Biochemistry, Virology Specialist. Laboratorio de Arbovirus y Arenavirus, Instituto de Virología Dr. J. M. Vanella, Universidad Nacional de Córdoba – UNC, Córdoba, Argentina. 6Biology, Lic. Área Entomología, Instituto de Medicina Regional – IMR, Universidad Nacional del Nordeste – UNNE, Resistencia, Chaco, Argentina, Consejo Nacional de Investigaciones Científicas y Técnicas – CONICET, CCT Nordeste, Corrientes, Argentina. 7,8Biology, Dr. Laboratorio de Herpetología, Facultad de Ciencias Exactas y Naturales y Agrimensura – FACENA, Universidad Nacional del Nordeste – UNNE. Consejo Nacional de Investigaciones Científicas y Técnicas – CONICET, CCT Nordeste, Corrientes, Argentina. 9Biochemistry, Dra. Laboratorio de Arbovirus y Arenavirus, Instituto de Virología Dr. J. M. Vanella, FCM, UNC, Córdoba, Argentina. 10,11Biology, Dra. Área Entomología, Instituto de Medicina Regional – IMR, Universidad Nacional del Nordeste UNNE, Resistencia, Chaco, Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas – CONICET, CCT Nordeste, Corrientes, Argentina.


2/10
Seroprevalence of Flavivirus in horses in Chaco, Argentina. Circulation during 2013-2014 introduction Humans, horses and other vertebrates are considered the final hosts of different arboviruses transmitted by mosquitoes.Among them, Saint Louis Encephalitis virus (SLEV) and West Nile virus (WNV) both belonging to the Japanese encephalitis antigenic complex, genus Flavivirus (Flaviviridae) (King et al., 2012), caused epidemics and/or epizootics in Argentina and in the Americas.SLEV and WNV are mosquito-borne viral infections transmitted in natural cycles between birds and mosquitoes, particularly Culex spp.(Morales et al., 2017).
WNV originated in Africa, and was detected for the first time in America in 1999, causing human epidemics and massive bird and equine death toll in the USA (Petersen & Roehrig, 2001).Since the introduction of this virus, encephalitis outbreaks in humans, birds and equines have been detected from Canada to South America (Nash et al., 2001;Komar et al., 2003).
In Argentina, it was first isolated from diseased and dead horses in the province of Buenos Aires in 2006 (Morales et al., 2006); however, seroprevalence studies in birds from different provinces, including Chaco, confirmed its activity since 2004 (Díaz et al., 2008).Sporadic human cases have been reported in provinces of the northeast and central areas of the country (Chaco, Entre Ríos, Formosa, Santa Fe, and Córdoba provinces) but the impact on animal and human public health was considerably lower than in the northern hemisphere (Morales et al., 2017).
SLEV was first isolated in Missouri (United States of America) in 1933 and currently is widely distributed across the continent (Lumsden & Jellison, 1958).In Argentina, the distribution of SLEV is very broad; serological evidence (in some cases, up to 50%) for transmission has been found practically wherever it has been sought.The virus is endemic in subtropical provinces and in a number of temperate ones (Spinsanti et al., 2000).Also, in Cordoba province (temperate province in central Argentina), a human encephalitis outbreak caused by SLEV was reported in 2005 (Spinsanti et al., 2008).
Although the circulation of this viral infection has been reported in Argentina, the impact on horses health is unknown.
These Flavivirus have been largely ignored or neglected, because they have not a strong impact on the people who live in endemic areas.Due to the veterinary and medical importance of these infections, the aim of this work was to provide information on the activity of SLEV and WNV in three localities of the province of Chaco, through detection of antibodies in horses, based on background of previous researches in humans, birds and horses in the region (Sabattini et al., 1998;Díaz et al., 2008;Tauro et al., 2012); and provide useful information, contributing in surveillance and control programs for these diseases.
This study was part of a larger initiative to define Ecoepidemiology of arboviruses in Chaco province, conducted by the Department of Entomology of Instituto de Medicina Regional, Universidad Nacional del Nordeste.

Study area
The Province of Chaco is located (28° and 25°39'21" S; 61°42' 40'' and 62°20'30' W) in the Northeastern of Argentina (Figure 1).The climate is subtropical with a dry season (May to November).In summer (December-February) the mean annual temperature is 27 °C, with an absolute maximum that can exceed 43 °C; the winter (June-August) is moderate, the mean monthly temperature from the coldest month is approximately 12 °C with some frost (5 to 10 days per year).The daily thermal oscillation is significant and the differences of 10 °C are frequent (Karlin, 2012).The mean rainfall between November and April is 1280 mm and between June and September is 650 mm (Bruniard, 1978).

Studied localities
1) Resistencia (RS) is the capital city of Chaco province (27° 28' S, 58° 59' W) (Figure 1) belonging to San Fernando Department with 100% urban population, estimated in 290,723 inhabitants (Instituto Nacional de Estadística y Censo, 2011).It is located within the flooded valley of Negro and Parana Rivers which makes it especially vulnerable to rain and river flooding.
2) General José de San Martín (GSM) city (26° 32' 11'' S, 59° 20' 29'' W) is distant 90 km from the capital city.It has 28,124 inhabitants belonging to the Libertador General San Martín Department, which is located in the central-eastern region of the province (Figure 1).It is an important region where the people participate in agricultural activities and livestock (Instituto Nacional de Estadística y Censo, 2011).This city is crossed by several provincial routes that connect it with other localities, being the place of passage of transport activities of different commercial products.The people had access to piped water.
3) Pampa del Indio (PI) city (26° 2'W''S, 59° 55'W) is distant 235 km from the capital city of the Chaco province.This city is located to the northeastern of the province and it has a population of 9,204 inhabitants (Instituto Nacional de Estadística y Censo, 2011) between creoles and aborigines of the Qom ethnic group.It is the second most populated city of the Libertador General San Martín Department and the 5th most populated city of the province but it has the lower urban population compared with the provincial population mean.In the urban area, the main commercial and political activities are developed.In the rural area, different economic activities are developed such as: livestock, agriculture, sawmills brickworks and coal production.The aborigine population also carries out traditional ceramics, sculpture, basketry, textiles as second source of money income.The rural population is distributed in 25 spots around the urban area of PI.Provincial Route N° 3 connects this locality with GSM city (Figure 1).In the urban area, most of the people have access to piped water, but those living in the rural area collect rainwater for daily use (Chaco, 2010).

Samples studied and analyzed
The blood samples from 68 asymptomatic horses belonging to three locations of Chaco province were taken sequentially.They were collected from the jugular vein and sent to the laboratory, refrigerated and kept at 4°C until processing.None had a history of previous vaccination.Samples were obtained in May, September and December 2013 and May 2014.Twenty one male horses, with an age range of 6-28 years belonging to the police of the Chaco province were studied in

4/10
Seroprevalence of Flavivirus in horses in Chaco, Argentina. Circulation during 2013-2014 RS; from GSM, 27 horses (including only 6 females) with an age range of 2-27 years were studied and finally, in PI 20 horses aged between 5 and 22 were studied.

Plaque reduction neutralization test (PRNT)
Neutralizing antibodies (NTAb) detection was performed using a PRNT with Vero cells (African green monkey kidney cells) (Earley et al., 1967).The samples were initially tested in a 1/10 dilution and those that neutralized at least 80% of the inoculated viral plaques forming units were considered positive.SLEV strain CbaAr4005 isolated from Cx. quinquefasciatus mosquitoes from Córdoba (Diaz, 2006) and WNV E/7229/06 isolated from sick equines in Buenos Aires were used (Morales et al., 2006).Viral suspensions were prepared using the brains of infected lactating mice and 10% (weight/volume) minimal essential medium with Earle salts supplemented with L-glutamine, non-essential amino acids, 10% fetal bovine serum and 1% antibiotics.The suspensions were centrifuged at 10,000 rpm for 30 min using a refrigerated centrifuge.
Variance analysis (ANOVA) was used to assess the degree of statistical difference, in the presence of antibodies by age classes.
The project that gave rise to this manuscript was approved by the ethics committee (protocol 2012) of the Institute of Regional Medicine (UNNE), 2012.

results
Seropositive animals were identified in the three surveyed locations of the Chaco province.Table 1 shows the results of the serology.Most (97.5%) of the horses studied were seropositive for SLEV, WNV or both.Seventeen (25%) of the horses presented monotypic reactions to SLEV, and 6 (8.82%) presented monotypic reactions to WNV.
The highest prevalence for SLEV (38.09%), and WNV (14.28%) of all samples analyzed, considering only monotypic infections was detected in RS.
In PI was observed the greatest frequency of secondary infections (85%) (Table 1).
Table 2 shows all positive samples and the horses that demonstrate recent virus circulation.A total of 25 seronegative horses in May 2013 became seropositive in later periods.Fifty two percent of recent infections were caused by SLEV, 36% due to WNV and 12% of horses were infected with both viruses.
The period of greatest transmission was between June 2013 and September 2013 (N=13) and occurred mainly in GSM (Table 2).
For the three localities studied no significant differences between age classes and the prevalence of Flavivirus were found (F=0.52;p>0.59).

Discussion
Several arboviruses have been emerged and reemerged in the New World in the past decades.WNV was reported in North America since 1999 and it rapidly extended causing human and equine encephalitis outbreaks every year and the decline of some wild bird populations (Roehrig, 2013).Today it is active in most countries of the Americas.The biological transmission cycle of WNV is maintained by Culex spp.mosquitoes, which act as vectors and Passeriformes birds as vertebrate hosts (Komar et al., 2003).Díaz et al. (2008) found antibodies against WNV in birds from three provinces in Argentina including the province of Chaco, and suggested that the species of the Furnariidae family are competent hosts of the virus and could play an important role in the transmission of this virus in Argentina.Although, to date, the species of birds and mosquitoes involved in the transmission cycle of WNV in Argentina are still unknown.This is the first report of seroprevalence of WNV in equines, in Chaco province.Although equines are important WNV vertebrate hosts, only a limited number of dead or diseased horses have been diagnosed in America (Díaz et al., 2013).These authors suggest that prior exposure to others Flavivirus could modulate the disease in subsequent exposures to WNV, which could be a possible explanation that none of the horses infected with this virus in the present study have developed a clinical response.
The behavior of this virus differs from that observed in the USA, mainly due to the absence of epizootics in horses and wild birds and epidemics in human populations, similar to that happens in other South American countries.The causes of these differences are unknown.

conclusion
In conclusion, the detection of antibodies against the two viruses studied, especially revealed in the second and / or third samples in horses in three localities of the present research, show active transmission of them in the province.The high prevalence of positive equines in the three studied localities allows us to suggest that the province of Chaco it is also favorable place for the presence of vectors and hosts for these viral agents with wide distribution of them in the study area.
SLEV is known to cause subclinical infections in horses nevertheless; the first isolation of SLEV from a horse that died due to a neurological disease in Brazil is a significant event.This is the first observation that SLEV can cause disease in horses, which indicates that some aspects of its viral cycle and its ability to cause disease need further studies (Díaz et al., 2013).
This should be considered with attention by the health authorities not only because the possibility of epidemic outbreaks but also because it complicates diagnosis in persons repeatedly exposed to multiple flaviviruses in the study area, including dengue viruses.On the other hand, it is also necessary to deepen the studies on these infectious agents, mosquito vectors and vertebrate hosts involved in the cycles of transmission in the province of Chaco.

Figure 1 .
Figure 1.Map of Argentina.Chaco province and location of sampling sites.

table 1 .
Prevalence of neutralizing antibodies against SLEV and WNV, in blood samples horses, from Chaco province, Argentina, collected between May 2013 and May 2014.Seroprevalence of Flavivirus in horses inChaco, Argentina.Circulation during 2013-2014

table 2 .
Positive samples for antibody detection against SLEV and WNV collected between May 2013 and May 2014, from horses in Chaco province.Samples with gray shading showed recent virus circulation.Seroprevalence of Flavivirus in horses inChaco, Argentina.Circulation during 2013-2014

table 2 .
Continued...These results suggest the need for additional studies on viral circulation in mosquitoes and hosts.