FREQUENCY OF SPECIES OF THE GENUS Eimeria IN NATURALLY INFECTED CATTLE AND SHEEP OF RIO GRANDE DO SUL STATE, BRAZIL.

FREQUENCY OF SPECIES OF THE GENUS Eimeria IN NATURALLY INFECTED CATTLE AND SHEEP OF RIO GRANDE DO SUL STATE, BRAZIL. DALLA ROSA, Luciana 1 ; OLIVEIRA, Dionatan 2 ; COPETTI, Maíra M. 3 ; OLIVEIRA, Camila B. 4 ; GRESSLER, Lucas T. 5 ; MONTEIRO, Silvia G. 6 Resumo: A coccidiose é uma doença economicamente importante na pecuária, porque o parasito invade e destrói o epitélio intestinal dos seus hospedeiros, causando manifestações clínicas que variam de assintomática a diarreia grave com presença de sangue e tecido do revestimento intestinal e também perda de apetite, perda de peso ou redução no ganho de peso, resultando em efeitos potencialmente duradouros, principalmente nos animais mais jovens. O objetivo deste estudo observacional foi determinar a frequência de Eimeria spp. localizada no Rio Grande do Sul, Brasil. As amostras foram obtidas a partir de 6 diferentes cidades. As amostras foram coletadas via retal. As 122 amostras de fezes analisadas pela técnica de centrifugação-flutuação foram positivas para, pelo menos, uma espécie de Eimeria. Observou-se a presença de três espécies patogênicas em bovinos infectados: Eimeria bovis (34,1%), Eimeria zuernii (1,1%) e Eimeria alabamensis (3,3%). Em ovinos, foi identificada apenas uma espécie patogênica: Eimeria ovinoidalis (38,7%). Conclui-se que há infecção por coccídeos do gênero Eimeria no Rio Grande do Sul e devem ser tomadas medidas profiláticas para minimizar os efeitos causados por este parasita. Abstract: Coccidiosis is an economically important disease in livestock, because the parasite invades and destroys the intestinal epithelial of their host, causing clinical manifestations that vary from asymptomatic to severe diarrhea with blood and gut lining tissue in the feces and also loss of appetite, weight loss or reduced weight gain, resulting in potentially long lasting effects, mainly in younger animals. The aim of this cross-sectional observational study was to determine the frequency Eimeria spp. located in Rio Grande do Sul, Brazil. Samples were obtained from 6 different cities. The samples were obtained rectally. The 122 feces samples analyzed by the Centrifugal-flotation technique were positive for, at least, one Eimeria species. It was observed the presence of three species pathogenic in single infection by cattle: Eimeria bovis (34.1%), Eimeria zuernii (1.1%) and Eimeria alabamensis (3.3%). In sheep, pathogenic species were identified in single infection: Eimeria ovinoidalis (38.7%). It is concluded that there is infection by coccidia of the genus Eimeria in Rio Grande do Sul and prophylactic measures should be taken to minimize the effects caused by this parasite. Palavras- Chave: Coccidiose. Oocistos. Patogênica. Keywords: Coccidiosis. Oocysts. Pathogenic. 1 Professora do Curso de Medicina Veterinária UNICRUZ. E-mail:ldrosa@unicruz.edu.br 2 Médico Veterinário. E-mail: dionatan_to@hotmail.com 3 Médica Veterinária. E-mail: mairamc22@gmail.com 4 Pós-doutoranda em Parasitologia Veterinária UFSM. E-mail: camilabelmontevet@yahoo.com.br 5 Doutorando em Medicina Veterinária UFSM. E-mail: lucastrevi@hotmail.com 6 Professora do Curso de Medicina Veterinária UFSM. E-mail: sgmonteiro@uol.com.br 256

INTRODUCTION Parasitic infections are one of the major constraints for dairy industry and livestock in tropical and subtropical countries (SINGH et al., 2012), including Brazil. Eimeriosis is a disease caused by coccid protozoa belonging to the phylum Apicomplexa, family Eimeriidae and genus Eimeria that infect specific hosts, such as cattle and sheep, by invading and destroying the intestinal epithelial cells through rapid multiplications (LEVINE, 1985). More commonly but not exclusively, occurs in younger animals (CORNELISSEN et al., 1995), causing clinical manifestations that vary from asymptomatic to severe diarrhea with blood and gut lining tissue in the feces and also loss of appetite, weight loss or reduced weight gain, resulting in potentially long lasting effects (SARTIN et al., 2000; MUNDT et al., 2005). Moreover, economic losses that they cause in relation to low herd productivity, delayed animal development, death and significant expenses on management and medication. Lassen and Østergaard (2012) compared group control with group infected with Eimeria and found annual losses to the gross margin of the herd due to Eimeria ranged 8 9% and the losses may amount to US$ 120 million when costs associated with disease (SILVA, et al. 2011). The infection of ruminant animals occurs by the ingestion of oocysts with food and water contaminated by feces. Under conditions of intensive rearing, with high population density, disease transmission takes place more readily and oocysts are highly available within the environment. Stressful situations such as weaning, transportation, starting of confinement, changes in the weather and debility caused by other pathogens may induce outbreaks of eimeriosis (SÁNCHEZ et al., 2008; REHMAN et al., 2010). Studies have shown that the intensity of Eimeria parasite species and gastrointestinal parasites in cattle varies between different regions of Brazil (REBOUÇAS et al., 1994; ALMEIDA et al., 2011). Pathogenicity varies with species and few are considered pathogenic enough to cause clinical manifestations of the disease (LIMA, 2004). Many species of Eimeria have been cited as causing production and economic losses around the world, and E. zuernii and E. bovis are considered to be the most pathogenic species from cattle and E. crandallis and E. ovinoidalis from sheep (CORNELISSEN et al., 1995; LIMA, 2004; TAUBERT et al., 2008). This indicates the importance of knowledge of the frequency of occurrence of each species in herds. The present study had the aim of investigating the frequencies of occurrence of species of the genus Eimeria in cattle and sheep of Rio Grande do Sul State, Brazil. 257

MATERIAL AND METHODS Feces samples from 104 calves and 18 sheeps of eight farms located in Rio Grande do Sul State in the municipalities of Bagé, Caçapava do Sul, Santiago, Santo Antônio das Missões, São Borja and Silveira Martins were collected. Individual fecal samples were collected directly from the rectal ampulla and were packed in plastic bags that were identified with the farm name and date of sampling. This fecal samples were stored under refrigerated conditions until were tested for Eimeria spp. oocysts in the laboratory. For individual quantification of oocysts (OPG) per gram of feces, the technique of Gordon and Whitlock modificated (1939) was used. After counting, the oocysts were separated using the centrifugal flotation technique in Sheather s sugar flotation solution (DUSZYNSKI; WILBER, 1997). Oocysts were examined at 40X and 100X magnification on optical microscope. Eimeria species were described based on morphological (size, shape, color, absence or presence of micropyle and micropylar cap, texture of the external wall, shape of sporocysts, absence or presence and characteristics of sporocyst residue, and Stieda body and morphometric characteristics (dimensions) of oocysts and sporulating oocysts as proposed by Taylor et al. (2009). After, the data were submitted to one-way analysis of variance followed by the Student s t test (P<0.05). Values were represented as mean ± standard deviation. RESULTS AND DISCUSSION In cattle the 104 samples, 102 (98.1%) had oocysts of Eimeria. Of these, 89.2% (91) were Eimeria considered pathogenic (E. bovis, E. zuernii and E. alabamensis). Single and multiple infections were found. 80,8% samples showed E. bovis being 30.1% samples with single infection, 17.6% multiple infection E. bovis and E. zuernii and 39% multiple infection E. bovis and E. alabamensis (Figure 1). São Borja and Santo Antônio das Missões cities (Figure 2) presented 100% samples with E. bovis. Regarding to E. zuernii 27.9% samples were positives, being that Santo Antônio das Missões City (Figure 2) had the bigger presence, 54.5% samples. These positive E. zuernii samples, only 1.1% were single infection (Figure 1). 258

The presence of E. alabamensis was significantly lower, with only 9.6% of positive samples., but of these, 3.3% were single infections (Figure 1). Santiago (Figure 2) was the city that had the bigger positive, 15.3% of samples coming from this region. Multiple infection by pathogenic and nonpathogenic Eimeria were found in 33.3% samples and only nonpathogenic Eimeria were found in 10.6% samples. Figura 1. Percentage of Eimeria observed in bovine fecal samples (n = 104) of the Rio Grande do Sul State, Brazil, analyzed during the period June to December 2012. 259

Figure 2. Map of the Rio Grande do Sul State, Brazil representing the cities where the bovine and ovine fecal samples were originated in the period June to December 2012. (1= Santo Antônio das Missões, 2=São Borja, 3= Santiago, 4 = Silveira Martins, 5 Caçapava do Sul, 6 = Bagé) According Lassen et al. (2009) E. zuernii and E. bovis, could be found together in 72% of the herds, and potentially able to cause disease if the right conditions manifest. On pastures E. alabamensis is known to cause problems in first grazing animals (SVENSSON, 2000). The reason for the different frequencies observed are due host factors such as immune status, race, age, physiological state; related with the parasite such as strain and specificity; related with the environmental such as humidity, temperature; and, finally, linked to the management system, which can favor the sporulation of oocysts of Eimeria spp. and its transmission (TEMBUE et al., 2009; PENZHORN et al., 1994). This is mainly because the coccidiosis affects animals of all ages, it is more prevalent in young animals and especially in newly weaned and confined animals. And these last are most susceptible to the disease and account for most clinical cases (FOREYT, 1990). Also because, the oocysts are very resistant structures that can remain viable in the environment for several months under favorable conditions (LIMA, 2004). In sheep, 100% of samples were positive, and 94.4% (17) of the samples were positive for Eimeria considered pathogenic (E. crandallis e E. ovinoidalis). According Silva et al. (2011) in a study of Rio Grande do Norte State, the most lambs had multiple infections while fecal samples containing oocysts from three or more different species (Figure 3). 260

Figure 3. Percentage of Eimeria observed in ovine faecal samples (n = 18) of the Rio Grande do Sul State, Brazil, analyzed during the period June to December 2012. Relative to E. crandallis 16.7% of samples were positive, and all in multiple infections with E. ovinoidalis and nonpatogenics Eimeria. Samples from Caçapava do Sul showed the largest percentage of this species, 60% of the samples. E. ovinoidalis was present in 88.9% of the samples analyzed, being 38.9% single infection. Multiple infections between pathogenic (E. crandallis and/or E. ovinoidalis) and nonpathogenic Eimeria were found of 55.6% samples. One sample (5.6%) was positive only to nonpathogenic Eimeria. In sheep, the oocyst elimination peak of E. ovinoidalis, which is one of the most pathogenic of sheep parasites (McDOUGALD, 1979; GREGORY et al., 1989), occurred at week 6 and coincided with that of E. crandallis. Infection of animals occurs after the ingestion of these oocysts along with water and/or food contaminated with fecal matter (SILVA et al., 2007). Long periods of constant low oocyst uptake are more likely to reflect the natural conditions of calves and the subsequent impact on growth rate. This is in contrast to experimental single-high-dose infections often resulting in clinical disease, recovery, and protective immunity (NIELSEN et al., 2003; LARSSON et al., 2006). Bruhn et al. (2012) showed that dairy farms in the south of Minas Gerais, the use of modern management practices, with the aim of increasing productivity, has not been accompanied by greater sanitary care for the herd. This situation has led to the existence of 261

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