Acta Scientiae Veterinariae, 2016. 44: 1411. RESEARCH ARTICLE ISSN 1679-9216 Pub. 1411 Spatial and Temporal Description of Laboratory Diagnosis of Bovine Rabies in the State of Rio Grande do Sul, Brazil Carla Rosane Rodenbusch1, José Carlos Ferreira1, Julio César Almeida da Rosa1, Victória Furtado Migliavacca2, Angélica Cavalheiro Bertagnolli2, Michelle Elias Peres1 & Laura Lopes de Almeida1 ABSTRACT Background: Rabies remains one of the most important zoonosis worldwide and in Brazil. In the Brazilian state of Rio Grande do Sul (RS) the disease is one of the main causes of death in bovine herds. The confirmation of suspected rabies cases upon clinical examination depends on specific laboratory diagnosis. The Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF) is the reference laboratory in RS to diagnose rabies. The objective of the present study was to present a spatial-temporal description of diagnostic procedures of rabies in cattle in the years 2011 to 2015 in RS. Materials, Methods & Results: The results of diagnosis procedures of rabies in cattle carried out in the Laboratory of Virology, IPVDF, between January 2011 and December 2015 were listed. Direct immunofluorescence (DIF) and biological assays for confirming rabies cases were used. Cattle population density per area of a municipality, as well as date, species involved, location of suspected case, and diagnosis were obtained from exams and from official rabies record, and input to a spreadsheet file. Spatial distribution maps of cattle rabies confirmed in laboratory and cattle population density per municipality were constructed using the software TerraView 4.2.2 (INPE). In the study period, 998 samples of brains of cattle presenting neurological symptoms compatible with rabies, of which 582 (58%) were confirmed in the laboratory. The frequency of positive cases on a yearly basis varied between 41 and 65%, with statistically lower number of positives in 2011 (P > 0.05). The annual incidence of rabies for a population of 10,000,000 bovines was 33, 88, 112, 108, and 79, respectively, for the years 2011 to 2015, in that order. A positive correlation was observed between the number of samples analyzed and the incidence of positive cases in the study period, and statistically significant difference in incidence between years (P > 0.05). Samples were from 175 municipalities, of which 114 (65%) had at least one confirmed rabies case and 61 did not have any. The five municipalities that sent most samples for analysis were Viamão (n = 66), Montenegro (n = 40), Camaquã (n = 39), Gravataí (n = 38), and São Lourenço do Sul (n = 37). The spatial distribution of rabies in cattle in a year did not follow a defined pattern, though most cases confirmed in the laboratory occurred in the greater Porto Alegre region and in southeast RS. Discussion: High incidence of rabies was observed in 2012 and 2013, and in the two following years the number of cases confirmed in laboratory remained high, compared with 2011. The high incidence of rabies cases confirmed in laboratory correlated with the number of samples analyzed. The fall in vaccine coverage during the study period may also have prompted the increase in suspected cases and in the number of samples examined in the laboratory. A large number of the municipalities presenting rabies cases in cattle was in the greater Porto Alegre region and in southeast RS, which are areas of high and intermediate risk for the disease, according to a previous epidemiological modeling study. The spatial distribution of positive cases was not associated with the cattle population density. The large number of rabies in cattle in RS underscores the importance of the disease and the high risk of exposure, both to humans and animals. The results presented herein may help define control and prevention actions against rabies in RS. Keywords: bovines; epidemiology, laboratory diagnosis, Rio Grande do Sul, rabies. Received: 15 May 2016 Accepted: 12 October 2016 Published: 11 November 2016 1Laboratório de Virologia e 2Laboratório de Histopatologia, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Fundação Estadual de Pesquisa Agropecuária (FEPAGRO), Eldorado do Sul, RS, Brazil. CORRESPONDENCE: L.L. Almeida [[email protected] - Tel.: +55 (51) 3481- 3711]. Laboratório de Virologia, Instituto de Pesquisas Veterinárias Desidério Finamor - IPVDF. Estrada do Conde n. 6.000. CEP 92990-000 Eldorado do Sul, RS, Brazil. 1 C.R. Rodenbusch, J.C. Ferreira, J.C.A. Rosa, et al . 2016. Spatial and Temporal Description of Laboratory Diagnosis of Bovine Rabies in the State of Rio Grande do Sul, Brazil. Acta Scientiae Veterinariae. 44: 1411. INTRODUCTION mation volume (under 40%) was observed for the Rabies remains one of the most important dis- item ‘clinical signs’ and therefore this category of eases worldwide, accounting for approximately 60,000 data was excluded. The search also included cattle deaths a year [20]. In Brazil, besides the impact on hu- population data on a municipality basis, considering man health, rabies seriously affects livestock [14,18], the respective area in the calculation of cattle popu- and in the state of Rio Grande do Sul (RS) the disease lation density. The cattle population used was that has been considered the main cause of death of cattle officially surveyed in May of each year, during the [9]. More specifically, in cattle rabies normally takes annual foot-and-mouth disease vaccination campaign the clinical paralytic form. The main signs include in- (Marcelo Gocks, personal communication, 2016). coordination, flaccid paralysis of tail, weakness, pelvic The area of each municipality was retrieved from limb paralysis, recumbence and convulsions [6,7,14]. the website of the Instituto Brasileiro de Geografia Confirmation of clinical suspected cases de- e Estatística (IBGE). pends on a laboratory diagnosis. Fragments of brain Laboratory diagnosis of rabies tissues are submitted to direct immunofluorescence Suspected rabies cases in cattle were in- (DIF), whose results are complemented with a bio- vestigated by the Official Veterinary Service. Fresh logical assay [2,4,5]. As the reference center for the or frozen brain tissues were sent for analysis and a diagnosis of rabies in RS, historically most positive specific form to report neurological syndrome [12]. diagnoses of rabies made in the Instituto de Pesqui- In the laboratory, fragments of brain, cerebellum, and sas Veterinárias Desidério Finamor (IPVDF) were in medulla tissues were submitted to the direct immuno- cattle, representing 88.7% of all analyses carried out fluorescence antibody (DIF) test for rabies [4]. This from 1985 and 2007 [17]. In addition to the fact that assay was complemented with a biological test proto- cattle and humans often share the same environment col consisting of intracerebral inoculation of lactating and due to the complexity and epidemiology of rabies, mice [5,12]. The results were sent to the Department diagnosis of the disease is essential in the process to of Livestock Agriculture and Irrigation (SEAPI) and decide the actions towards prevention and control to the Health Secretary of RS. Any sample poorly stored be taken against the disease in the several species that in inappropriate preserving agent or in advanced decay are susceptible to it. So, the objective of the present status were excluded. study was to produce a spatial-temporal description of the laboratory diagnosis of cattle rabies in the State of Spatial distribution Rio Grande do Sul from 2011 to 2015. The spatial distribution maps depicting the laboratory diagnosis of rabies and cattle population MATERIALS AND METHODS density were constructed using the software TerraView 4.2.2 (INPE) [19] based on the geopolitical map of Data and inclusion criteria RS (IBGE). One municipality was considered to have This retrospective study was carried out based positive value when at least one rabies case was diag- on an examination of laboratory tests conducted to nosed in cattle and confirmed in the laboratory during diagnose rabies in cattle in the Virology Labora- the study period. A negative value was attributed to tory - IPVDF between January 2011 and December municipalities in which no positive diagnosis of rabies 2015. The information about the requests to diagnose was confirmed in the laboratory. cattle rabies received by the laboratory were retrieved from exam request forms and Rabies Record Book, Statistical analyses and included date, species involved, location of the Based on the notion that rabies is a fatal clinical suspicion, and result of laboratory diagnosis. disease [8], it was assumed that the whole herd was A spreadsheet containing all information collected free of rabies, except the cases that were diagnosed was created in specific software (Microsoft Excel™ positive. Therefore, incidence was calculated using 2010). Location data included the municipality, the number of positive cases divided by the bovine since actual geographic coordinates were available population recorded for the same year and adjusted in only 60% of the request forms. The poor infor- to 10,000,000 bovines. The effect of the number of 2 C.R. Rodenbusch, J.C. Ferreira, J.C.A. Rosa, et al . 2016. Spatial and Temporal Description of Laboratory Diagnosis of Bovine Rabies in the State of Rio Grande do Sul, Brazil. Acta Scientiae Veterinariae. 44: 1411. suspected cases submitted to diagnosis procedures The suspected cases analyzed were from 175 on the actual incidence of rabies was analyzed using municipalities, of which 114 (65%) had at least one the Pearson’s linear correlation coefficient, while an- positive case of rabies in the study period. Sixty-one nual frequencies of positive cases in the 5-year period municipalities did not record any rabies case (Table 2). considered was analyzed using the chi-square test with In the years 2013 and 2014, the number of municipali- 0.05 significance level. The analyses were carried out ties that investigated suspected rabies cases was higher in the software SAS 9.4 with PROC GLM and PROC than in the other years.