No abstract

During this study, cats were immunized by the intranasal and rectal routes with crude rhoptry proteins of Toxoplasma gondii admixed with Quil-A. Twenty-five domestic short hair cats divided into five groups (n=5) were used during this evaluation: G1 and G3 cats received 200 μg of the rhoptry proteins with Quil-A (20 μg) by the intranasal and rectal routes, respectively; G2 and G4 cats received bovine serum albumin (BSA, 200 μg/dose) with Quil-A (20 μg); and G5 animals served as unvaccinated controls. All treatments were performed at days 0, 21, 42, and 63. The challenge was done with 800 cysts of the ME49 of T. gondii strain at day 70 (challenge day). The serum IgG, IgM, IgA, and fecal IgA antibody levels were evaluated by using the indirect enzyme-linked immunosorbent assay (ELISA). Some animals produced antibody levels beyond cut-off; however, two animals from G1 (OD(mean)=0.308, OD(cut-off)=0.200) and three from G3 (OD(mean)=0.254) demonstrated IgG levels on being challenged, with similar results occurring in two cats from G1 to IgM (OD(mean)=0.279, OD(cut-off)=0.200). Fecal IgA levels were detected in all G1 cats (OD(mean)=0.330, OD(cut-off)=0.065), and in one cat from G3 (OD(mean)=0.167). The serum and fecal humoral immune responses did not correlate with oocyst shedding. Oocyst shedding varied from 98.4% (G1), 87.5% (G2), 53.0% (G3), to 58% (G4), and was lower than that of G5 cats. The prepatent period of cats vaccinated intranasally (G1) was reduced from 6-9.6 to 2.8 days, suggesting protection of environmental contamination, considering cats as the primary source of contamination. The intranasally and rectally administered rhoptry vaccines were able to partially protect cats against T. gondii cysts on being challenged; however, the intranasal method of vaccination yielded better results relative to the rectal route.

Neospora caninum is a protozoan parasite that causes the most important reproductive problems in cattle worldwide. The objective of this study was to evaluate the possibility of vertical transmission of N. caninum in zebus breed beef cows (Bos indicus) submitted for slaughter at an abattoir in the northern region of the State of Paraná, southern Brazil. One hundred and fifty-nine cows were evaluated: 83 pregnant (in different stages of gestation) and 76 non-pregnant. Serum determination of N. caninum was evaluated by indirect ELISA (Idexx). Blood (with EDTA) from pregnant cows and tissue samples (brain and heart) from their fetuses were collected and used for PCR analyses. Antibodies against N. caninum were observed in 14.6% (12/83) of pregnant and in 15.8% (12/76) of non-pregnant cows. Antibodies against the parasites were detected in one fetus (1.4%). The PCR analyses revealed that 6.0% (5/83) of cows and 4.8% (4/83) of fetuses evaluated were positive to specific N. caninum primers. These positive fetuses were between 4 and 6 months of age. Thus, considering PCR and serology as an indicative of vertical transmission in fetuses, 4.8% of fetuses were infected by N. caninum during gestation.

We evaluated the humoral and cellular immune responses in pigs immunized intranasally with crude rhoptry proteins of Toxoplasma gondii plus Quil-A. The experiment used 13 mixed-breed pigs divided into the following three groups: G1 (vaccinated-challenged, n=6), which received the rhoptry vaccine (200(g/dose); G2 (adjuvant-challenged, n=4), which received PBS plus Quil-A; and G3 (unvaccinated-challenged, n=3), which was the control group. The treatments were performed intranasally at days 0, 21, and 42. Three pigs from G1 produced IgG and IgM antibody levels above the cut-off in the ELISA on the challenge day. Partial protection was observed in G1 at the chronic phase of infection when compared with G3. The preventable fractions were 41.6% and 6.5%, in G1 and G2, respectively. The results of this study suggest that rhoptry proteins plus Quil-A stimulated humoral, local, and systemic immune responses, which were able to partially protect the brain from cyst formation.

Eared doves (Zenaida auriculata), which are common in urban, rural and wild areas in many regions of Brazil, are frequently prey for domestic cats. Therefore Toxoplasma gondii isolates obtained from doves may reflect greater environmental diversity than those from other hosts. The aim of the present study was to evaluate T. gondii seroprevalence, isolate and genotype strains from Z. auriculata. Serum and tissue samples were collected from 206 doves for use in the modified agglutination test (MAT) and mouse bioassay. The prevalence of T. gondii antibodies in the doves was 22.3% (46/206), with titers ranging from 16 to 4096, and T. gondii strains were isolated from 12 of these doves. Five genotypes were detected by means of PCR-RFLP, including ToxoDB genotypes #1, #6, #17 and #65, and one genotype that had not previously been described (ToxoDB#182). This was the first report on isolation of T. gondii from Z. auriculata. This study confirmed the genetic diversity of T. gondii isolates and the existence of clonal type II (ToxoDB genotype #1) in Brazil.Keywords: Toxoplasma gondii, eared doves, genotyping, PCR-RFLP, MAT, Biossay. ResumoPombos silvestres (Zenaida auriculata), comuns em áreas urbanas, rurais e selvagens em muitas regiões do Brasil, são frequentemente predados por gatos domésticos. Sendo assim, os isolados de T. gondii obtidos de pombos podem refletir uma maior diversidade ambiental do que os outros hospedeiros. O objetivo do presente estudo foi avaliar a soroprevalência, isolar e genotipar T. gondii de Z. auriculata. Amostras de soro e tecido foram coletadas de 206 pombos para o teste de aglutinação modificado (MAT) e o bioensaio em camundongos. A prevalência de anticorpos contra T. gondii em pombos foi 22,3% (46/206), com títulos variando de 16 a 4096, e T. gondii foi isolado de 12 pombos. Cinco genótipos foram detectados por PCR-RFLP, incluindo os genótipos ToxoDB #1, #6, #17, #65 e um genótipo não descrito anteriormente (ToxoDB#182). Esse é o primeiro relato de isolamento de T. gondii de Z. auriculata. Este estudo também confirmou a diversidade dos isolados de T. gondii e a presença de tipo clonal II (ToxoDB #1) no Brasil.

The current study aimed to evaluate serology, and isolate and genotype Toxoplasma gondii strains from pregnant dairy cows, slaughtered in an abattoir for human consumption, and their fetuses. Blood from 60 pregnant dairy cows and blood and tissue samples (brain, lung, heart, and liver) from their fetuses were collected and analyzed in a mouse bioassay. Antibodies against T. gondii were observed in 48.3% of cows and 3.7% of fetuses (IFAT, titers ≥ 50 for cows and 25 for fetuses were considered positive). Fourteen fetuses (23.3%) and six cows (10.0%) were identified as positive in the bioassay. T. gondii was isolated from a blood sample of a cow older than 4 years old in the 6th month of pregnancy, and from a blood sample of a fetus in the 6th month of gestation. These isolates were identified by polymerase chain reaction (PCR) as being of T. gondii and both strains showed type II alleles for all PCR-restriction fragment length polymorphism (PCR-RFLP) markers tested. T. gondii type II strain from cattle was isolated for the first time in Brazil. The current study also showed that transplacental transmission of T. gondii naturally occurs in dairy cows (23.3%) from Southern Brazil.

TgROP2 is an intracellular protein associated with rhoptries of Toxoplama gondii and an antigen component of a candidate vaccine for toxoplasmosis. The purpose of the present study was to evaluate the efficacy of rTgROP2 to stimulate humoral and cellular immune responses in BALB/c mice via intranasal injection. TgROP2 partial coding sequence was (196-561) amplified by PCR from genomic T. gondii RH strain DNA and cloned into the pTrcHis expression vector. Escherichia coli Rosetta 2 cells transformed with pTrcHis-TgROP2 showed high levels (~1 mg.mL-1) of recombinant protein after 4 hours of IPTG induction. Recombinant TgROP2 exhibited an apparent Mr equal to 54 kDa. In order to test immunogenicity of the recombinant protein, 10 BALB/c mice received 10 µg of rROP2 protein + 10 µg of Quil-A via intranasal injection. Doses were administered at days 0, 21, and 42. Three animals were euthanized and used to evaluate cell-ular immune response on day 62. Five (50%) and two (20%) out of ten animals produced IgG (DO mean = 0.307; cut-off = 0.240) and IgA (DO mean = 0.133, cut-off = 0.101), respectively, by ELISA on day 62. The proliferation of splenocytes revealed high stimulation index (SI) when co-cultured with 5, 10 and 15 µg.mL-1 of rTgROP2. These results indicate that intranasal immunization with recombinant protein ROP2 plus Quil-A can elicit both cellular and humoral immune responses in BALB/c mice.

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.