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Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The aim of the present study was to evaluate the natural and experimental infection of B. equi
in nymphs and adult Boophilus microplus using optical microscopy and molecular analysis.
The experimental infection was observed in nymphs, males and females of B. microplus fed
on equines chronically infected with B. equi and in non fed larvae and eggs. For the
experiments two equines, of not defined breed, proven to be infected with B. equi were used.
The animals were infested with B. microplus larvae of generation F4 obtained from a
laboratory maintained population, known to be free of Babesia spp. infection. Daily
collections of ticks were realized using as starting point the ecdisys to nymph state. After the
collection the hemolymph was examined and the intestine and salivary glands were dissected,
smashed on glass slides for microscopy, methanol treated and stained with Giemsa. There
were dissected 860 specimens (432 nymphs, 280 females and 148 males). The dissected
salivary glands were divided into two parts. The first one was smashed on microscopy glass
slides, stained and examined by optical microscopy for morphologic analyses. From the
second part was realized DNA extraction and PCR with specific primers for the 18S rRNA.
On optical microscopy of nymph hemolymph was possible to be observed the presence of
kinetes with claviform aspect characteristic for the genus Babesia. In the salivary glands of B.
microplus nymphs the morphology and the sequence of developmental stages of B. equi were
observed initiating on day 4 after ecdysis, being possible to see in acinary cells the formation
of sporoblasts and ovoid sporozoites measuring 1.5 μm of diameter and elongated sporozoites
with 3.8 to 5.2μm of length and 0.8 to 1.5 μm of width. The PCR confirmed the presence of
B. equi in DNA samples extracted from salivary glands of nymphs, male and female ticks as
well as in larvae and eggs. To evaluate the natural infection were collected nymphs and adult
B. microplus from two equines naturally infested by these ticks and naturally infected with B.
equi. These horses were originated from Seropedica city in the state of Rio de Janeiro. There
were dissected 324 specimens (145 nymphs, 138 females and 41 males). The proceedings
with the salivary glands were identical to the previously described for the experimental
infection. Of the salivary glands submitted to PCR, 70% showed to be infected with B. equi.
Microscopical analysis of the salivary glands revealed the presence of sporoblast stages and
the development of elongated sporozoites in acinary glands. The morphologic, morphometric
and molecular analysis confirmed the experimental and natural infection of the salivary
glands of nymphs and adult B. microplus with B. equi. The results of the present study show
the ability of B. equi to develop in this tick species. The detection of B. equi DNA in eggs and
larvae also suggests the possibility of transovarian transmission in B. microplus. The results
allow to consider the tick B. microplus as a potential biologic vector of B. equi in horses from
the studied region. / Este trabalho teve como objetivo avaliar a infec??o natural e experimental de Babesia equi em
ninfas e adultos de Boophilus microplus por meio de microscopia ?ptica e an?lise molecular.
A infec??o experimental foi observada a partir de ninfas, machos, f?meas, ovos e larvas n?o
alimentadas de B. microplus alimentados em equinos com infec??o cr?nica por B. equi. Para a
realiza??o do experimento foram utilizados dois eq?inos, mesti?os, com infec??o por B. equi.
Os animais foram infestados com larvas de B. microplus de gera??o F4 obtidas de col?nia
mantida em laborat?rio e livres de infec??o por Babesia spp. A partir da ecdise para ninfa
foram realizadas coletas di?rias. Ap?s a coleta foram realizados os exames de hemolinfa e
extra??o do intestino e das gl?ndulas salivares os quais foram macerados em l?minas de vidro
para microscopia, fixados em metanol e corados com Giemsa. As gl?ndulas salivares
dissecadas foram divididas em duas partes. A primeira parte foi macerada em l?minas de
vidro para microscopia, corada com corante Giemsa e observada em microsc?pio ?tico para
an?lise morfol?gica. A segunda parte foi realizada a extra??o de DNA, sendo submetida a
PCR com primers especif?cos para o gene 18S rRNA para B. equi. Foram dissecados 860
esp?cimes (ninfas= 432; f?meas= 280 e machos= 148). Na microscopia ?ptica foi poss?vel
observar nas hemolinfas das ninfas a presen?a de cinetos com aspecto claviforme t?picos do
g?nero Babesia. Nas gl?ndulas salivares, a morfologia e a seq??ncia dos est?gios de
desenvolvimento de B. equi das ninfa s de B. microplus, foram observadas a partir do 4? dia
ap?s ecdise, sendo poss?vel observar nos ?cinos celulares a forma??o de esporoblastos e de
esporozo?tas ov?ides medindo 1,5 μm de di?metro e alongados medindo 3,8 a 5,2μm de
comprimento e 0,8 a 1,5μm de largura. A rea??o em cadeia de polimerase (PCR) confirmou a
presen?a de B. equi em DNA de gl?ndulas salivares extra?das de ninfas, machos, f?meas,
assim como dos ovos e larva. A infec??o natural foi observada a partir de ninfas e adultos de
B. microplus coletados de dois eq?inos naturalmente infestados e comprovadamente
infectados por B. equi, procedentes do munic?pio de Serop?dica, Rio de Janeiro. Foram
dissecados 324 esp?cimes (ninfas= 145, f?meas= 138 e machos= 41). O processamento das
gl?ndulas salivares dissecadas foi semelhante ao descrito para infec??o experimental. Das
gl?ndulas salivares submetidas a PCR, 70% apresentaram resultados positivos para B. equi.
As an?lises por microscopia ?ptica das gl?ndulas salivares das ninfas e dos adultos revelaram
a presen?a nos ?cinos celulares os est?gios de esporoblastos e o desenvolvimento de
esporozo?tas alongados. As an?lises morfol?gicas, morfom?tricas e moleculares confirmaram
a infec??o experimental e natural das gl?ndulas salivares de ninfas e adultos de B. microplus
por B. equi. Os resultados deste estudo demonstram a capacidade de multiplica??o de B. equi
em gl?ndulas salivares de ninfas e adultos de B. microplus. A detec??o de DNA de B. equi em
ovos e larvas de B. microplus tamb?m sugere a possibilidade da transmiss?o transovariana
nesta esp?cie de carrapato. Estes resultados sugerem que o carrapato B. microplus ? vetor
biol?gico de B. equi na regi?o estudada.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:tede/819 |
| Date | 26 February 2007 |
| Creators | Fernandes, K?tia Roberta |
| Contributors | Massard, Carlos Luiz, Almeida, Fernando Queiroz, Barreira, Jairo Dias |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Curso de P?s-Gradua??o em Ci?ncias Veterin?rias, UFRRJ, Brasil, Parasitologia Veterin?ria |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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