versão On-line ISSN 0717-7712
Parasitol. latinoam. v.63 n.1-2-3-4 Santiago dez. 2008
Parasitol Latinoam 63: 46 -50, 2008 FLAP
Frequency of seropositive equines for Theileria equines in the Southern Rio Grande do Sul State, Brazil
LEANDRO QUINTANA NIZOLI*, MARCELO MENDES GÖTZE*, SAMUEL RODRIGUES FÉLIX*. SERGIO SILVA DA SILVA** and CARLOS EDUARDO WAYNE NOGUEIRA***
* Centro de Biotecnología, Universidade Federal de Pelotas (UFPel).
** Departamento de Veterinária Preventiva, Faculdade de Veterinária, UFPel.
*** Departamento de Clínicas Veterinárias, Faculdade de Veterinária, UFPel.
Foi realizado um levantamento sobre theileriose equina em uma propriedade da região sul do estado do Rio Grande do Sul (RS). Amostras de sangue foram coletadas de 108 éguas de um haras localizado no municipio de Bagé, latitude 31°30' Sul e longitude 54Ú10' Oeste. A sorologia foi realizada com o uso da imunofluorescência indireta (IFAT) e utilizou-se a técnica de Nested Reacão em Cadeia Polimerase (nPCR). Do total de amostras examinadas, 22% (24) foram positivas por IFAT e 15% (16) por nPCR). Na análise por raca, 15.05% (14) dos animáis Puro Sangue Inglês (PSI) foram soro-positivos, e dos animáis da raça Crioula (RC), 55% (11) foram positivos por IFAT.
A study on equine theileriosis was carried out in the southern region of the Brazilian state of Rio Grande do Sul (RS). Blood samples were collected from 113 mares from an equine breeding farm located in the city ofBagé, latitude 31 "30'S and a longitude of 54 U10' W. The serological testing was carried out with the use of indirect fluorescence test (IFAT) and compared with Nested Polimerase Chain Reaction (nPCR). Among the sera collected from 118 horses, 25 were found positive to Theileria equi by the IFAT, while by nPCR 17 positive for T. equi was observed, corresponding to a frequency of22.1%> and 15.0%, respectively. The racial analysis showed 15.05% (14) thoroughbred and 55% (11) Crioulo breed horses to be positive by IFAT.
Key words: Theileria equi, IFAT, prevalence.
Equine piroplasmosis, caused by Theileria equi and Babesia caballi, is considered to be the most important tick-borne disease of horse in tropical and subtropical areas1. Clinical manifestation of the disease is variable including fever, anemia, icterus, lethargy, and in some cases death2.
In endemic countries like Brazil, the control of equine piroplasmosis is important to keep international markets open to the horse industry3. For this reason many countries prohibit the importation of horses because of the high prevalence of asymptomatic carrier animals in the region. The southern area of the Rio Grande do Sul State thoroughbred racing industry is particularly badly affected by equine piroplasmosis, acute infections resulting in missed training sessions and races and hence serious loss of income to owners and trainers. Income is also lost to owners through abortion in stud mares which are T. equi carriers.
It has also been shown that strenuous exercise, such as that experienced with horse-racing, can cause sub-clinical infections to become acute4,5.
Carrier animals are be responsible for the maintenance of the infection6. Parasites are present, in these animals, in very low numbers in the blood and they may not be detected in Giemsa-stained blood smears7.
Equine piroplasmosis can be made by microscope examination of Giemsa-satained blood smears or by specific polymerase chain reaction7, but complement fixation test is recommended for the detection of carrier animals8. Using the conventional thin blood smear examination, indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) babesiosis has been documented in Brazil913.
Dermacentor (Anocentor) nitens ticks are the vectors for B. caballi14 and, in Brazil, it is known that Rhipicephalus (Boophilus) microplus can transmit T. equi15'17.
The state of Rio Grande do Sul has an area of 281.749 Km2 and the population of horses is estimated to be 468. 447 head, equivalent to 8.15% of the national herd. The region has a subtropical weather, precipitation between 400 and 1200 mm per year. The climate is characterized by a distinct dry season (winter) from June to September and a wet season (summer) from December to March18.
In the present study, we performed an epidemiological study on equine piroplasmosis in one of the most important area for the international horse industry in Rio Grande do Sul State.
For the purpose of this study, blood samples from 113 mares were collected from an equine breeding farm located in the city of Bagé, Rio Grande do Sul State, Brazil, latitude 31°30' S and a longitude of 54Ú10'W.
From all mares 93 were Thoroughbred racehorses raised in stables and without limited access to the pasture and without contact with ticks and 20 of the Crioulo horse (male and female) breed with access to the pasture and raised with cattle infected with R. microplus.
The blood samples were collected from the jugular vein and placed into two tubes using a vacutainer blood collection set. One tube contained ethylenediaminetetraacetic acid (EDTA, at a concentration of 1 mg.ml1) was centrifuged (500 g for 5 min) and the supernatant and the buffy coat were removed. Blood samples were immediately used for DNA isolation. The second tube contained no anticoagulant, and the blood was left at room temperature to coagulate. Then the sera was collected and kept at -20°C until used.
For the nest PCR, DNA from 250ul of each blood sample was prepared using the Purogene kit (Gentra Systems, Minneapolis, MN, USA) according to the manufactures protocol. The final pellet was dissolved in 30ul of water and reaction was performed as described by Nicolaiewsky19.
Blood from un-infected horse DNA was obtained from Veterinary College of Federal University of Pelotas, Rio Grande do Sul State, Brazil and used as a negative control. For examination of blood parasites, blood films were prepared at the time of sampling and stained with Giemsa stain. Both thin and thick blood smears were stained with Giemsa Stain21 and observed with Bright-field light microscopy at lOOOx for the presence of the T. equi and B. caballi. No apparent clinical signs were observed on all the sampled horses by macroscopic examination.
The slides were prepared with infected horses erythrocytes in which are visible as compact inclusion by IFAT staining. IFAT was performed according to the Cunha10, using Anti-Horse IgG conjugate with FITC produced in rabbit. The positive control was used a reference sample previously identified10. Serum samples were diluted at 1:80 dilution in phosphate buffered saline (PBS), pH 7.4 and applied at 10 il/well and were graded either as negative (-) or positive (+ to ++++). The size, appearance and density of staining were compared with the positive control where a positive reaction was seen as apple green fluorescent inclusion within the infected erythrocytes, while a negative reaction gave a uniformly red counter stain or uniform greenish staining (Nikon UV microscope, 490 nm excitation/520 nm emission). The differences between different parameters were evaluated by using the chi-square test (%2).
RESULTS AND DISCUSSION
Among the sera collected from 118 horses, 25 were found positive to T. equi by the IFAT, while by nPCR 17 positive for T. equi was observed, corresponding to aprevalence of 22.1% and 15.0%, respectively (Table 1). The distribution of the positive results for groups and the average prevalence of the infection for T. equi by IFAT are showed in Table 2. None of the horses showed signs of clinical piroplasmosis and B. caballi were not detected by examination of Giemsa-stained blood smears neither by nPCR. The agreement between the tests was of 87.6% and observed itself high association between the techniques (x2 = 42.13; p < 0.001). Using the nPCR we observed that the sensibility and the specificity of the EMA-1 gene of T. equi were the same found by Nicolaiewsky19.
In 3 samples the results of the nPCR were positive and the results of IFAT negative. This suggests that being a recent infection might not have the time necessary for an antibody response to the infection. Weiland22 observed that horses artificially infected with T. equi and T. caballi delayed of 3 to 20 days to produce antibodies, which might corroborate with our observation. In other hand, 11 animals were observed positive results for IFAT and negative for nPCR, probably these horse are not anymore in the acute phase of the infection or the sample are not well manipulated.
Transmission of piroplasmosis is usually influenced by the dynamics of vector populations, and these are directly influenced by climatic conditions24. In Rio Grande do Sul State the tick R. microplus is widespread and probably serve as agents of transmission for this hemoparasite15.
The percentage of seropositive animals in the studied population was relatively high but below than the seroplevalences to T. equi found in other studies in different states of Brazil. Authors have demonstrated occurrences varying from 49.2% to 100% in the southern and south-eastern states with different epidemiologic conditions but with high tick infections9"13,25.
The effects of the variation of the prevalence has been observed with different categories and breeding systems in different regions of Brazil25,26. It is evident in this study, whereas Crioulo breed horses were grazed with cattle in the same pasture had higher titers than the thoroughbred horses.
The management of the horses appears to be an important factor for the prevalence of T. equi infections. Among horses raised with access to pasture there was a significant difference in the seropositive percentage of reactors (15.05%) compared with horses without access to pasture (55%).
Our data are corresponding with another study that found 17.5% of the horses infected in professional stud farms, whereas the horses were grazed without contact with cattle16.
Among the positive animals the antibodies titers ranged from 1:80 to 1:1280, with 64% of the titers between 1:320 and 1:640, being these two titers more frequent. A similar serologic distribution was observed with horses breeding with access to pasture26.
The prevalence and the titers average of the Crioulo breed were higher than for animals without access to the pasture (thoroughbred) (Table 2 and Table 3). These results are explained for the breeding with herds of other species, in native pasture, where have a higher contact with bovine ticks.
These data are in agreement with described by other studies, whereas the authors found high prevalence in horses utilized for to work with cattle and breeding in pasture considered infected with ticks27,16.
Previous studies have shown that exist correlation between the occurrence of Rhipicephalus microplus and the prevalence of theileriosis27,14,28.
The prevalence of reagents' horses to T. equi in this study demonstrated that the region is an endemic area of equine theileriosis. In addition, the animals with contact to bovine herds are more exposed and prone to T. equi infection than stable animals.
Our results confirm the importance of the control of T. equi and demonstrate that the horse population of Southern Rio Grande do Sul State is exposed to T. equi infection and suggest that equine piroplasmosis is widespread in the area. Therefore, we anticipate that future research will maximize the effectiveness of the present.
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Corresponding author at: Dr. Leandro Quintana Nizoli
Centro de Biotecnología, Universidade Federal de Pelotas.
CP 354, 96010-900 Pelotas, Brasil. Tel: +55 53 32757350; fax: +55 53 32757555. E-mail: firstname.lastname@example.org