Alessandra Taroda

Occurrence of Ehrlichia canis and Anaplasma platys in household dogs from northern Parana

Canine monocytic ehrlichiosis caused primarily by Ehrlichia canis and canine thrombocytic anaplasmosis induced by Anaplasma platys are important emerging zoonotic tick-borne diseases of dogs. There is evidence that these pathogens can also affect humans. This study evaluated the presence of E. canis and A. platys in blood samples collected from 256 domiciled dogs in the municipality of Jataizinho, located in north region of the State of Parana, Brazil, by PCR assay. The occurrence of E. canis and A. platys was 16.4% (42/256) and 19.4% (49/256), respectively; while 5.47% (14/256) of the dogs evaluated were co-infected by these two organisms. The presence of E. canis and A. platys was not significantly associated with the variables evaluated (sex, age, outdoor access, and presence of ticks during blood collection). Infection of dogs by E. canis was associated with anemia and thrombocytopenia, while infection induced by A. platys was related only to thrombocytopenia. Canine monocytic ehrlichiosis and canine thrombocytic anaplasmosis should be included in the differential diagnoses when these hematological alterations are observed during routine laboratory evaluation of dogs.

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.

Isolation and genotyping of Toxoplasma gondii from pregnant dairy cows (Bos taurus) slaughtered

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.

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.

Anti-Neospora caninum antibody detection and vertical transmission rate in pregnant zebu beef cows (Bos indicus): Neospora caninum in pregnant beef cows (Bos indicus)

The aim of the present study was to evaluate anti-Neospora caninum antibodies and the vertical transmission rate in naturally infected pregnant zebu beef cows (Bos indicus) reared on pasture. The present study began with 200 cows from four farms (50 cows from each farm), and these animals were submitted to timed artificial insemination (TAI). After ultrasonography, 76 pregnant cows were selected, 22, 15, 22, and 17, respectively, from farms 1, 2, 3, and 4. Blood samples were taken from cows thrice during the first, second, and third trimester of gestation, and a blood sample was collected from 31 calves before colostrum milking. From 76 cows 23 (30.3%) had anti-N. caninum antibodies detected by indirect ELISA (Idexx), and 53 (69.7%) did not. Sixty-four cows that initiated the experiment were negative to N. caninum and 11 became positive either during the second or third trimester of gestation, this mean an infection incidence of 17.2% (11/64). OD for ELISA was higher (OD=2.08) during the second and third (OD=2.10) trimesters of pregnancy when compared with the first (OD=1.81), however, there were no statistical differences (P=0.45). The vertical transmission was calculated to be 29.0% (9/31), and the risk of vertical transmission of N. caninum in seropositive dams was 26.25 times higher than seronegative animals (OR=26.25, 2.38<OR<289, P=0.007). In conclusion, the rate of vertical transmission of N. caninum in pregnant zebu beef cows was 29%, and the risk was 26.25 higher in seropositive dams relative to than seronegative animals.

Serum occurrence of anti-Toxoplasma gondii antibodies in dairy cows slaughtered in an abattoir for human consume

Toxoplasma gondii is a worldwide parasite recognized as one of the main zoonosis in human beings. The present study aimed to evaluate serology of T. gondii from dairy cows slaughtered in an abattoir for human consume. Serum samples from 120 dairy cows (60 pregnant and 60 non-pregnant) were collected, and indirect fluorescent antibody test (IFAT) was performed to detect anti-T. gondii antibodies by considering positive animals with titers ≥50. Serologic results from cows showed 29.1% (35/120), which 29 (48.3%) e 6 (10,0%) were from pregnant and non-pregnant cows, respectively. This revealed a risk 8.4 times-higher of positively in pregnant than non-pregnant cows (OR=8.4, 2.91<OR<25.6, P<0.0001). There was a statistical difference in the anti-T. gondii antibody frequency between Jersey and Holstein breed cows, 38.3% (23/60) and 20% (12/60) of prevalence, respectively (OR=2.49, 1.02<OR<6.13, P=0.04). Titers for cows were 50 (n=23), 100 (n=10), 200 (n=1) and 400 (n=1). There was no difference among age of gestation and anti-T. gondii antibody occurrence (P=0.74) in pregnant cows. The potential role of beef in epidemiology of T. gondii for human beings is yet enigmatic, and more studies are necessary to elucidate the real risk of this food for consumers.