Implementation of an Animal Sporotrichosis Surveillance and Control Program, Southeastern Brazil.

We report the implementation of an animal sporotrichosis surveillance and control program that evaluates strategies to identify suspected and infected cats in a municipality in southeastern Brazil. All adopted measures reinforced the program, although strategies had different abilities to detect the presence of infection.

Isolation and attempted cultivation of an Anaplasma marginale strain from Brazilian brown brocket deer (Mazama gouazoubira, Fisher, 1814) in the tick cell line IDE8.

The aim of the study was to isolate and establish an Anaplasma marginale strain from Brazilian brown brocket deer, Mazama gouazoubira, in the Ixodes scapularis cell line IDE8. Blood from a free-living adult female M. gouazoubira naturally infected with A. marginale (MGI5) was inoculated intravenously into a splenectomized calf. When A. marginale rickettsemia was 2.5%, blood was collected and cryopreserved in liquid nitrogen with dimethylsulfoxide (DMSO). IDE8 cell cultures were infected with calf blood inoculated with the A. marginale (MG15) isolate. The cultures were monitored by examination of Giemsa-stained cytocentrifuge smears. Light microscopy of stained IDE8 samples revealed the first inclusions of A. marginale (MGI5) at 48days post-inoculation (d.p.i). The IDE8-infected cells contained parasitophorous vacuoles with amorphous material and a few cocci-like organisms. A sample from IDE8-infected cells from the 16th subculture (336 d.p.i.) was analyzed by nPCR, nucleotide sequencing, electron microscopy, and an indirect fluorescent antibody test (IFAT). The IFAT highlighted some IDE8-infected cells with intense fluorescence in the parasitophorous vacuole, while in other cells, fluorescence was observed only at the periphery. DNA from a culture of the MG15 isolate was amplified with A. marginale msp4 gene primers, and nucleotide sequencing of the PCR product and BLAST software analysis further confirmed 100% identity with the MGI5 blood isolate (GenBank no. JN022558.1). Electron microscopy revealed increased numbers of lysosomes in the cytoplasm of IDE8 cells. Several cells exhibited large vacuoles containing cellular debris and amorphous material. After the 29th subculture, it was not possible to detect compatible Anaplasma structures by light microscopy, and subculture samples tested negative in nPCR. Despite the failure of the attempt to establish A. marginale (MGI5) in IDE8 cells, the results demonstrated the isolate's ability to infect, survive and multiply, although in limited numbers, in IDE8 cells.

Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses.

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.

In vitro establishment and propagation of a Brazilian strain ofAnaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells / Estabelecimento e propagação in vitro de uma amostra brasileira de Anaplasma marginale com apêndice em células IDE8 (Ixodes scapularis)

A Brazilian isolate of Anaplasma marginale with appendage was successfully established and maintained in vitro in a tick cell line (IDE8). Infection was confirmed by optical and transmission electron microscopy. In addition, primers MSP1aNF2 and MSP1aNR2 amplified products from DNA extracted from infected IDE8 cells. Comparisons with partial sequences of the msp1α gene and the complete genome of A. marginale confirmed that the sequences of amplified fragments were from the A. marginale genome. This is the first establishment of a Brazilian A. marginale isolate in tick cells, representing a new system for biological and molecular studies and also a new source of material for diagnosis and development of vaccines.

Uma amostra brasileira de Anaplasma marginale com apêndice foi estabelecida e mantida in vitro em uma linhagem de células de carrapatos (IDE8). A infecção foi confirmada através de microscopia ótica e eletrônica de transmissão. Além disso, os primers MSP1aNF2 e MSP1aNR2 amplificaram produtos do DNA extraído das células infectadas. Comparações de sequências parciais do gene msp1α e do genoma completo de A. marginale confirmaram que as sequências dos fragmentos amplificados pertenciam ao genoma de A. marginale. Este é o primeiro estabelecimento in vitro de uma amostra brasileira de A. marginale em células de carrapatos, representando um novo sistema para estudos biológicos e moleculares, além de ser uma nova fonte de material para o desenvolvimento de testes diagnósticos e de vacinas.

Babesia bigemina: in vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells.

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.

In vitro establishment and propagation of a Brazilian strain of Anaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells.

A Brazilian isolate of Anaplasma marginale with appendage was successfully established and maintained in vitro in a tick cell line (IDE8). Infection was confirmed by optical and transmission electron microscopy. In addition, primers MSP1aNF2 and MSP1aNR2 amplified products from DNA extracted from infected IDE8 cells. Comparisons with partial sequences of the msp1α gene and the complete genome of A. marginale confirmed that the sequences of amplified fragments were from the A. marginale genome. This is the first establishment of a Brazilian A. marginale isolate in tick cells, representing a new system for biological and molecular studies and also a new source of material for diagnosis and development of vaccines.

Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil.

Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PCR were sequenced and compared with each other and with known sequences. The serological results showed a total prevalence of 91.0% for T. equi and 83.0% for B. caballi, while by PCR, prevalences of 59.7% for T. equi and 12.5% for B. caballi were observed. However, no correlations were seen between positivity (neither by serology nor by PCR) and PCV values. As expected, the microscopic examination of blood smears showed low sensitivity in detecting the infections when compared to the PCR. Only 35 out of 570 blood smears were positive, with parasitemias below 0.1%. No congenital transmission was detectable. As far as sequencing is concerned, no differences were seen among the isolates of each species nor among them and known sequences available. These results confirm, by molecular methods, the high prevalence rates of T. equi and B. caballi infections in carrier horses in Brazil. However, no diversity was observed among the isolates within the studied regions.

Use of refrigeration as a practical means to preserve viability of in vitro-cultured IDE8 tick cells.

In vitro cultivation of the IDE8 cell line, derived from embryonic Ixodes scapularis ticks, constitutes an important system for the study of tick-borne pathogens, as these cells support growth of rickettsial species which are not normally transmitted by this tick. However, since cryopreservation of IDE8 cells is not always successful, there is a need to develop alternative ways to preserve these cells. In the present study, a suspension of IDE8 cells in culture medium was kept under refrigeration at 4 degrees C for up to 60 days. Every 15 days, the suspension was mixed and aliquots were re-cultured in 2-ml tubes, under standardized conditions. In addition, three techniques for cryopreservation, using two different cryoprotectants (DMSO and glycerol), were evaluated. Medium changes were carried out every week and subculturing every 2 weeks. The development of cultures and their respective subcultures, after returning to standard culture temperature, was evaluated by percentage viability and by cellular morphology evaluated in Giemsa-stained cytocentrifuge smears. All cultures and subcultures appeared healthy, showing growth rates comparable to cultures that had not been kept under refrigeration. The results demonstrated that storage under refrigeration at 4 degrees C is an efficient method for preservation of IDE8 cells for up to 60 days and that refrigeration may be preferable to cryopreservation for short-term preservation of IDE8 cells.