Luiz Daniel de Barros

Genetic characterization of Toxoplasma gondii isolates from eared doves (Zenaida auriculata) in Brazil

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.

Humoral and cellular immune responses in pigs immunized intranasally with crude rhoptry proteins of Toxoplasma gondii plus Quil-A.

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.

Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2

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 cellular 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.

Oocyst shedding in cats vaccinated by the nasal and rectal routes with crude rhoptry proteins of Toxoplasma gondii

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.

Occurrence of gastrointestinal and renal helminths in Zenaida auriculata (Des Murs, 1847) trap-captured from Brazil

This study aimed to determine the prevalence of gastrointestinal and renal helminths from naturally infected Zenaida auriculata captured in Londrina, Paraná State. Two hundred and one Eared doves were trapped and the gastrointestinal and renal helminths were collected and identified according to morphological structures. One hundred and sixteen (57.71%) doves were parasitized by helminths with specific prevalences for Ornithostrongylus quadriradiatus in 50 doves (24.88%), Ascaridia columbae in 47 (23.38%), Paratanaisia bragai and P. confusa in 34 (16.92%), Tetrameres fissispina in 17 (8.46%), Synhimantus nasuta in 14 (6.47%), Brachylaima mazzantii in 4 (1.99%) and Raillietina allomyodes in 2 doves (1.00%). Seventy four/201 (37.00%) birds were infected with only one species, and 96/201 (48.00%) pigeons were infected with nematodes. The association between different classes of helminths occurred in 40/201 (20.00%) animals. The results showed statistically differences between the presence of nematode (p = 0.00001) and trematode species (p ≤ 0.05) in the doves, and there was an association between the local of capture and the presence of trematodes and A. columbae (p ≤ 0.05). This study is the first to report the infection of Z. auriculata from Brazil with O. quadriradiatus, A. columbae, T. fissispina, S. nasuta, R. allomyodes, P. bragai and P. confusa.

Neospora caninum: evaluation of vertical transmission in slaughtered dairy cows (Bos taurus).

Neospora caninum is a worldwide parasite recognized as one of the main parasites responsible for abortion in cattle. The objective of this study was to evaluate vertical transmission of N. caninum in dairy cows (Bos taurus) that were slaughtered at an abattoir in the state of Santa Catarina, southern Brazil. Blood samples (with and without EDTA) from 60 pregnant dairy cows and blood and tissue samples (brain, lung, heart and liver) from their fetuses were collected and used for PCR and serological evaluation. Blood samples from 60 non-pregnant cows were collected and used to detect antibodies. Anti-N. caninum antibodies were detected by indirect ELISA. Antibodies against N. caninum were observed in 41.6% (25∕60) of the pregnant cows and in 43.3% (26∕60) of the non-pregnant cows. Antibodies against the parasite were detected in sera from three fetuses (5.5%). PCR analysis revealed that 3.3% (2∕60) of the cows and 6.6% (4∕60) of the fetuses evaluated were positive for specific N. caninum primers. These positive fetuses were between 4-6 months of age. Therefore, considering PCR and serological tests to be indicative of vertical transmission in fetuses, 11.6% (7∕60) of the fetuses were infected by N. caninum during gestation.

Occurrence of antibodies to Toxoplasma gondii in rheas (Rhea americana) and ostriches (Struthio camelus) from farms of different Brazilian regions

This study aimed to verify the occurrence of antibodies against Toxoplasma gondii in rheas (Rhea americana) and ostriches (Struthio camelus) commercially breeding in Brazil. Blood samples from 20 rheas and 46 ostriches (young and adults) were serologically tested using a technique known as modified agglutination test (MAT) at an initial titration of 1:16 for ostriches and 1:25 for rheas. Antibodies against T. gondii were found in 50% (10/20) of the rheas, with titers ranging from 1:25 to 1:6,400. The incidence of antibodies against T. gondii in ostriches was 17.4% (8/46) with titers ranging from 1:16 to 1:256. Birds showing titers higher than 1:200 for T. gondii were mainly the young ones. Therefore, rheas and ostriches may be parasitized by T. gondii, showing high levels of antibodies against this parasite.

First isolation and RFLP genotyping of Toxoplasma gondii from crab-eating fox (Cerdocyon thous—Linnaeus, 1766)

Wild animals may play an important role in the transmission and maintenance of Toxoplasma gondii in the environment. The purpose of the present study was to isolate and genotype T. gondii from a free-ranging crab-eating fox (Cerdocyon thous-Linnaeus, 1766). A crab-eating fox in critical health condition was attended in a veterinary hospital in Recife, Pernambuco State, Brazil. The animal died despite emergency treatment. The brain was collected aseptically and destined for mouse bioassay. One isolate of T. gondii was obtained, and Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) was used to assess genetic variability at 11 markers (SAG1, SAG2, altSAG2, SAG3, BTUB, GRA6, c228, c292, L358, PK1 and APICO). A murine model was used to assess the virulence of the isolate. Using the PCR-RFLP, genotype ToxoDB #13 was identified, which is considered an atypical strain. The isolate was classified as avirulent in the murine model. This is the first study to report T. gondii infection in the crab-eating fox.

Atypical Toxoplasma gondii genotype in feral cats from the Fernando de Noronha Island, northeastern Brazil

Toxoplasma gondii isolates from Brazil have a different phenotypic and genotypic pattern, with predominance of virulent isolates and recombinant genotypes, compared to the North Hemisphere. Considering that a new T. gondii genotype, non-pathogenic to mice, was previously identified from free-range chickens from the Fernando de Noronha Island, Brazil, this study aimed to identify genotypes of this parasite in tissue samples of feral cats (Felis catus) from this Brazilian Island. Anti-T. gondii IgG antibodies were detected in 18/31 (58%) feral cats. Two non-virulent T. gondii isolates were obtained by mouse bioassay. Genotyping was performed by PCR-RFLP using 10 genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, PK1, L358 and Apico) and an atypical strain of T. gondii (ToxoDB #146) was identified. This is the first report of this genotype in feral cats.

First report of genotype #65 of Toxoplasma gondii in pigs.

The aim of the present study was to isolate and genotype Toxoplasma gondii from pigs slaughtered for human consumption in northeastern Brazil. Indirect immunofluorescence antibody test (IFAT) was used to screen positive pigs. Tissues samples of animals with antibody titers ≥64 were submitted to bioassay in mice. One isolate of T. gondii was obtained, and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, using 11 markers (SAG1, SAG2, altSAG2, SAG3, BTUB, GRA6, c228, c292, L358, PK1, and APICO), was applied to evaluate the genetic variability. DNA from reference strains was used as a positive control. By means of genetic analysis, genotype ToxoDB #65 was identified, which is considered an atypical strain. This is the first record of genotype #65 in pigs. Thus, further studies in this region are necessary to determine the genetic variability of T. gondii in pigs and possible impact on public health.