Prevalence of Eimeria spp . in calves from dairy farms in northern Paraná state , Brazil

Bovine coccidiosis is a disease of major importance in cattle herds across the world. The disorder mainly affects young calves, and E. bovis and E. zuernii are considered the most pathogenic species of the genus, however, E. alabamensis have been described in grazing calves. In this study, the prevalence of Eimeria spp. was evaluated in calves on dairy farms in the northern region of the state of Paraná, Brazil. Four hundred calves on 44 dairy farms were tested for the presence of coccidian oocysts. The positives were re-examined and the oocysts were morphometrically analyzed for species identification. All the farms were contaminated and 205 animals (51.25%) presented Eimeria spp. oocysts. Among these, 146 animals (71.22%) were co-infected by two or more species of coccidia. Ten species of Eimeria were identified: E. bovis (in 30.25% of the positive samples), E. alabamensis (26.75%), E. zuernii (22.00%), E. ellipsoidalis (18.50%), E. auburnensis (13.75%), E. canadensis (8.00%), E. cylindrica (7.25%), E. subspherica (5.00%), E. bukidnonensis (3.00%) and E. brasiliensis (0.75%). This study demonstrates the high prevalence of Eimeria spp. in the northern region of Paraná, Brazil, and detection for the first time in our region the pathogenic species E. alabamensis.


Introduction
Eimeriosis is caused by protozoa of the phylum Apicomplexa, family Eimeriidae and genus Eimeria (BRUHN et al., 2011).This disease is one of the most common parasitoses in cattle around the world, and it is especially important in animals less than one year old (DAUGSCHIES et al., 2004, BRUHN et al., 2011).
During its life cycle, the parasite destroys the host's enterocytes, causing loss of blood, water, albumin and electrolytes to the intestinal lumen.These effects may lead to diarrhea, dehydration, prostration and eventually death, depending on the period of exposure and infective dose (JONSSON et al., 2011;ALMEIDA et al., 2011).
Among a fourteen known pathogenic species, E. bovis and E. zuernii are considered to be the most important, due to the severity of clinical cases caused in younger animals (JONSSON et al., 2011;BANGOURA et al., 2011;FLORIÃO et al., 2016).However, some authors have emphasized the importance of E. alabamensis as the causative agent of coccidiosis in animals feeding on pastures (SVENSSON et al., 1993(SVENSSON et al., , 1994;;MARSHALL et al., 1998;SVENSSON, 2000).
This disease is responsible for major economic losses.An impact of US$ 400 million on the American market has been estimated, due to clinical cases alone, while over US$ 3.8 million are lost through treatments for bovine coccidiosis in Canada (MATJILA & PENZHORN, 2002;REHMAN et al., 2011).
The rate of occurrence of Eimeria spp. is highly variable, with prevalence rates ranging from 10 to 100% among animals tested in Europe and different regions in Brazil (LENTZE et al., 1999, STEWART et al., 2008, ALMEIDA et al., 2011, BRUHN et al., 2011, 2012;KOUTNY et al., 2012).There are just two works concerning about eimeriosis in cattle from south Brazil, both were conducted in Paraná state (GUIMARÃES et al., 1995;HILLESHEIM & FREITAS, 2016), the first one observed 16.7% of Eimeria spp.positivity at northern (GUIMARÃES et al., 1995), and the second one 48.2% of Eimeria spp.positivity at southwestern (HILLESHEIM & FREITAS, 2016).Thus, the objectives of the present study were to determine the prevalence of Eimeria spp.among dairy calves, in herds in the northern region of the state of Paraná, Brazil.This information is relevant because it forms a preliminary stage in devising control schemes against these parasites in dairy herds.

Samples
In the present work, a cross-sectional epidemiological study was performed, and four hundred male and female calves, holstein and jersey breed, aged between 20 days and 12 months, on 44 different farms in the northern region of the state of Paraná, Brazil, were sampled.The samples were collected from February 2012 to October 2013.The animals were selected randomly and around 10 animals were included per farm.Feces were collected directly from the rectal ampulla of each animal.All procedures performed in the present study were approved by the Animal Ethics Committee of the State University of Londrina (CEEA/UEL N. 121/2013).The sample size (384) was calculated assuming a prevalence of 50% with a confidence level of 95% and efficacy of 5% by Epi Info 3.5.4.The number of farms was chosen according to convenience, but it fulfilled the minimum sample size that had previously been calculated.

Study area
The climate of northern region of the state of Paraná is characterized as mesothermal with hot summers, infrequent hoar-frosts, and rains with tendency of concentration in the summer months.It presents the following annual averages: temperature of warmer months exceeding 22°C, and of colder months below 18°C; rainfall between 1,300 and 1,700 mm; and a relative humidity of 75%, without water deficiency (IPARDES, 2004).A map with all cities were samples were collected is showed in Figure 1.

Coprological examination and morphological identification
The samples were examined for the presence and number of oocysts per gram of feces (OOPG) using saturated NaCl solution and MacMaster chambers (modified from Gordon & Whitlock, as described in UENO & GONÇALVES, 1998).Positive samples were re-submitted to the flotation technique and oocysts were collected on coverslips, which were observed under an optical microscope for morphological evaluation (modified from Willis & Molay, as described in UENO & GONÇALVES, 1998).Fifty oocysts per sample were analyzed for size, presence of micropyle and wall conformation (REHMAN et al., 2011).The examination was carried out using a B1 microscope (Motic, China), under 400x magnification, with a TK-C138 camera (JVC, Japan) attached to the microscope for documenting Eimeria spp.found in the samples (CORNELISSEN et al., 1995;REHMAN et al., 2011;BANGOURA et al., 2011).Eimeria spp.unsporulated oocysts were identified in accordance with parameters described by Levine (1961), andEckert et al. (1995).

Statistical analysis
The descriptive analysis were performed by the use of Excel software package.For prevalence, and confidence interval at 95% from apparent prevalence OpenEpi version 3.01 was used.

Results and Discussion
Eimeria spp.parasites were widespread and very common in the cattle herds included in this study.From the 400 samples analyzed, 205 (51.25%) were positive to Eimeria spp.. Within the positives, 146 (71.22%) were cross infected with multiple Eimeria spp.and 59 (28.78%) presented only one species of this parasite genus.The oocyst counts ranged from 50 to 207,800 OOPG and 10 different species were identified by morphological examination (Table 1).The identification of parasitic species, the prevalence of infection in animals and farms and morphometric measurements of analyzed specimens are presented in Table 1.
The mean of prevalence in the herds observed here was 56.8% (CI95% = 48.66-64.95).Investigations carried out in different regions of Brazil and around the world have found variable prevalence of Eimeria spp. in cattle in dairy and beef herds (CORNELISSEN et al., 1995;CHIBUNDA et al., 1997;REBOUÇAS et al., 1994;SÁNCHEZ et al., 2008;ALMEIDA et al., 2011;REHMAN et al., 2011;BANGOURA et al., 2011;DONG et al., 2012;ENEMARK et al., 2013).Almeida et al. (2011) found that the prevalence of Eimeria spp.contamination among young and adult cattle on dairy farms in the northeast of the state of Bahia, Brazil, was 33.33%.However, the prevalence of this parasite among calves younger than one year of age was One of the problems for the diagnosis of eimeriosis is the fact that it takes several days to sporulated oocysts for photomicrography what is time consuming.Here in the present study, we focused on epidemiological study, by using a faster approach instead time consuming one such as: sporulation and photomicrography.For this, we followed methodology described by Cornelissen et al. (1995), Rehman et al. (2011), Bangoura et al. (2011), and Stewart et al. (2008).
The high frequency of positive samples found in the animals studied here was probably due to the focus on young calves, which is the age group of cattle that is most susceptible to coccidiosis.In dairy herds in Holland, 46% of young calves and 43% of older calves presented infection by Eimeria spp., whereas only 16% of cows in the same herds were infected (CORNELISSEN et al. 1995).This suggests that younger animals have higher susceptibility to coccidia, given that clinical cases are almost exclusively seen among animals in this age range (STOCKDALE, 1981;CHIBUNDA et al., 1997;JONSSON et al., 2011;BANGOURA et al., 2011).
Eimeria spp. that are considered to be the most pathogenic to cattle are E. bovis and E. zuernii (TOMCZUK et al., 2015), which were two of the three most frequent species found in the present study, thus this could suggest that the farms studied here were at high risk of cases of clinical coccidiosis.Both of these species have previously been reported in other states of Brazil, such as São Paulo, Bahia and Minas Gerais (REBOUÇAS et al., 1994;ALMEIDA et al., 2011;BRUHN et al., 2011BRUHN et al., , 2012)).The prevalence of E. zuernii ranged from 6.83% to 22.6% in those studies, i.e. very close to the frequency of 22.00% that was found in the present study.The spatial distribution of E. bovis in Bahia, where 90% of the farms were contaminated (ALMEIDA et al., 2011), was similar to the distribution of E. bovis found in the present study (86.36%).
E. alabamensis has been reported as the causative agent of coccidiosis among grazing calves (SVENSSON et al., 1993(SVENSSON et al., , 1994;;MARSHALL et al., 1998;SVENSSON, 2000).All over Brazil, including the northern region of the state of Paraná, livestock are fed on pastures more often than in stalls.This may explain why the occurrence of this parasite species among the individuals analyzed was the second highest among all results so far (26.75%), and may also explain the broad distribution across the farms (90.91%).The high prevalence of E. alabamensis amongst the animals tested matched the results previously found in Austria, where 45.16% of the animals presented this species (KOUTNY et al., 2012).However, the infection levels found in the present study were much higher than those found elsewhere in Brazil, where the proportion of positive samples ranged from 0.4% to 4.3% among the animals tested and up to 25% of the farms were contaminated (REBOUÇAS et al., 1994;ALMEIDA et al., 2011;BRUHN et al., 2011BRUHN et al., , 2012;;KOUTNY et al., 2012;ENEMARK et al., 2013).
A similar study carried out two decades ago in the same region of the present work found that 16.69% of the animals were infected by Eimeria spp.(GUIMARÃES et al. 1995), what it is much lower than the occurrence of 51.25% found in the present study, however, Hillesheim & Freitas (2016) whose worked at southwestern of Paraná, observed 48.2% of samples positives for Eimeria spp., this was very similar with prevalence of the present study.Eimeria alabamensis was found in 26.75% of the samples of the present study, and 9.1% in the work of Hillesheim & Freitas (2016), but was not described by Guimarães et al. (1995).These differences between our study and Guimarães et al. (1995) work could be associate with the type of samples used, here feces samples were collected among animals without clinical signals, and the other one, most part of the animals (85.5%) had diarrhea.
The broad distribution of Eimeria spp., their increasing prevalence and detection for the first time in our region the pathogenic species E. alabamensis emphasize the importance of bovine coccidiosis in the northern region of the state of Paraná, Brazil.The epidemiology of coccidiosis, occurrences of clinical cases and evaluations on their economic impact on the respective farms need to be assessed in greater detail.Such studies may provide support for development of a control plan for bovine coccidiosis, which could be extrapolated to other Brazilian regions and states.

Figure 1 .
Figure 1.Map of Paraná state, Brazil showing municipalities where calf feces samples were collected in for Eimeria spp.occurrence, in the years 2012 and 2013.