First report on the isolation and genotyping of Toxoplasma gondii strains from free-range chickens in the state of Mato Grosso, Midwestern Brazil.

In the present study, 51 strains of Toxoplasma gondii (T. gondii) were isolated from free-range chickens in the state of Mato Grosso, Midwestern Brazil, upon conducting bioassays in mice, and genotyped them using PCR-restriction fragment length polymorphism (PCR-RFLP) and 11 markers, including SAG1, SAG2 (5'3'SAG2 and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. Fifty isolates were completely genotyped revealing 17 genotypes of T. gondii as follows: 12 matched using ToxoDB PCR-RFLP with the previously reported genotypes, including #6 type BrI (n = 4), #8 type BrIII (n = 7), #11 type BrII (n = 3), #14 (n = 1), #19 (n = 1), #41 (n = 1), #99 (n = 1), #109 (n = 4), #116 (n = 1), #140 (n = 2), #166 (n = 9), #190 (n = 1); and five genotypes have not been described before [#313 (n = 6), #314 (n = 1), #315 (n = 1), #316 (n = 1), #317 (n = 1)]. Moreover, mixed infections were identified in five isolates (TgCkBrMT8, TgCkBrMT9, TgCkBrMT33, TgCkBrMT38, and TgCkBrMT41). Additionally, genotype #190 was reported for the first time in chickens from Brazil. Our results corroborate with previous studies on T. gondii isolates identified in chickens from Brazil, thereby confirming their diversity, a typicality, and possibility of co-infection due to different T. gondii strains present in the country.

Isolation and genotyping of Toxoplasma gondii in the Midwestern Brazil revealed high genetic diversity and new genotypes.

We aimed to describe the genetic diversity of Toxoplasma gondii strains isolated from domestic animals, wildlife and humans in the Midwestern Brazil. For this purpose, fragments of tissue samples (heart, brain and lung) from 35 dogs, four cats, 105 wildlife, and amniotic fluids from eight pregnant women were collected and submitted to mouse bioassay test. In a total, 22 isolates from nine dogs, one cat, ten wild animals and two women were obtained. The DNA was extracted from T. gondii isolates (lungs and brains of infected mice) and from "primary samples" (aliquots of tissue homogenate from wild animals and amniotic fluids from pregnant women) in order to screen using a Polymerase Chain Reaction (PCR) targeting a repeated 529-base pairs fragment of the T. gondii genome. All positive PCR samples were genotyped using restriction fragment length polymorphism (RFLP) analysis. To the best of our knowledge, this was the first study to report isolates of T. gondii from Leopardus pardalis, Crax fasciolata, and Dasyprocta azarae. Moreover, multilocus PCR-RFLP revealed 11 T. gondii RFLP genotypes, comprising nine previously described, including the archetypal lineage #2 type III (n = 1); two clonal Brazilian lineages, #6 type BrI (n = 1) and #8 type BrIII (n = 5); #14 (n = 2), #41 (n = 1), #108 (n = 1), #140 (n = 2), #166 (n = 4), #190 (n = 1), one potentially mixed, and two new described genotypes in two isolates. Our results confirmed the high diversity of T. gondii strains in Brazil, including identical genotypes circulating among humans, domestic dogs and wildlife.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats.

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.

rROP2 from Toxoplasma gondii as a potential vaccine against oocyst shedding in domestic cats / rROP2 de Toxoplasma gondii como potencial vacina contra a eliminação de oocistos em gatos domésticos

Abstract 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.

Resumo O objetivo do presente estudo foi avaliar a eliminação de oocistos de Toxoplasma gondii em gatos imunizados pela via nasal com proteínas ROP2 de T. gondii. Doze gatos sem raça definida (Felis catus) foram divididos em três grupos experimentais G1, G2 e G3 (n = 4). Os animais do G1 receberam 100 μg de proteínas de rROP2 mais 20 μg de Quil-A, G2 recebeu 100 μg de albumina de soro bovino (BSA) junto com 20 μg de Quil-A, e o G3 recebeu apenas solução salina (grupo de controle). Todos os tratamentos foram realizados pela via intranasal nos dias 0, 21, 42 e 63. O desafio foi realizado em todos os grupos no dia 70 com aproximadamente 800 cistos de tecido da cepa ME-49 por via oral. Os animais de todos os grupos tiveram as suas fezes examinadas e o número de oocistos foi determinado durante 20 dias após o desafio. Os animais de G1 eliminaram menos oocistos (86,7%) do que os grupos controles. O ELISA foi utilizado para detectar IgG e IgA anti-rROP2, no entanto, não houve correlação entre o número de eliminhação de oocistos com os níveis de anticorpos IgG ou IgA. No presente trabalho, apesar da menor produção de oocistos no grupo imunizado (G1) em relação aos grupos controles (G2 e G3), não foi possível associar o uso de rROP2 pela via nasal com proteção contra eliminação de oocistos de T. gondii. Para o futuro, a utilização de outras proteínas recombinantes, ou mesmo vacina de DNA, em combinação com rROP2 poderia ser utilizada para tentar melhorar a eficácia deste tipo de vacina.

Estudo epidemiológico e avaliação de fatores de risco da infecção por Toxoplasma gondii e achados clinico-patológicos da infecção aguda em cães admitidos em um Hospital Escola Veterinário / Epidemiological study and evaluation of risk factors for infection with Toxoplasma gondii and clinical and pathological findings of acute infection in dogs admitted at a Veterinary School Hospital

We aimed to verify the prevalence and risk factors for Toxoplasma gondii infection in dogs addimitted at the hospital. We also assessed the occurrence rates and the clinical and pathological repercussion of the acute infection by T. gondii in these animals. Antibodies were detected in 7% (26/386) of a population of 386 dogs of both genders and different breeds and ages. Only variables, eating offal, rural origin and contact with cattle have significant values of p 0.05. Dogs from rural areas showed higher risk (OD=7.00) of infection than those of urban origin. In 6.5% (25/386) contact titles were detected (between 16 and 256); these titles do not necessarily mean acute infection, but only prior exposure. The recognition of prior infection by T. gondii is vital in those hospital patients. Depending on the cause of admission, although not being toxoplasmosis the responsible, the patient should receive prophylactic anti-parasite treatment or be monitored for further treatment in case of further acute occurrence of the disease by recrudescence of encysted bradyzoites. Only one dog (3:44%, 1/386) was admitted with high titer, what may be suggestive of acute infection (title of 4096). Although the dog with acute infection has shown neurological signs, caution is required not to extrapolate a false interpretation that toxoplasmosis is the main responsible for neurological signs, since numerous other cases included in this study had neurological signs without title of acute infection, even title of prior contact. Acute toxoplasmosis was not a significant clinic disease in this hospital; however differential diagnosis should be made in ill patients, especially those from rural areas, and definitive diagnosis must be reached for the correct therapeutic approach.

Esse trabalho teve como objetivo estudar a prevalência e respectivos fatores de risco para infecção do Toxoplasma gondii em cães provenientes de uma população hospitalar. Além disso, avaliou-se as taxas de ocorrência e as repercussões clínico-patológicas da infecção aguda pelo T. gondii nesses animais. Anticorpos foram detectados em 7% (26/386) da população estudada, composta de 386 cães de ambos os sexos e diferentes raças e idades. Somente as variáveis, ingestão de vísceras, origem rural e contato com bovinos apresentaram valores significativos com p 0.05. Adicionalmente os cães de origem rural apresentaram maiores risco (OD=7.00) de infecção do que aqueles de origem urbana. Em 6,5% (25/386) foram detectados títulos de contato (entre 16 e 256); esses títulos não significam necessariamente infecção aguda e sim apenas exposição prévia. É de fundamental importância o reconhecimento da infecção prévia por T. gondii nesses pacientes hospitalares. Dependendo da causa da admissão, mesmo não sendo a toxoplasmose a responsável, o paciente deve receber o tratamento anti-protozoário profilaticamente ou ser monitorado para posterior tratamento em caso de reagudização da enfermidade por recrudescência dos bradizoítos encistados. Apenas um animal (3.44%, 1/386) foi admitido com titulação elevada, o qual pode ser sugestivo de infecção aguda (titulo de 4096). Embora o animal com infecção aguda tenha sido apresentado com sinais neurológicos, cautela é necessária para não extrapolar uma falsa interpretação que a toxoplasmose é a grande responsável por quadros neurológico, uma vez que inúmeros outros casos incluídos nesse estudo tinham manifestações neurológicas e não tinham títulos de infecção aguda, nem mesmo título de contato prévio. A toxoplasmose aguda não foi uma afecção clínica expressiva nessa ambiência hospitalar, no entanto diagnóstico diferencial [...]

Estudo epidemiológico e avaliação de fatores de risco da infecção por Toxoplasma gondii e achados clinico-patológicos da infecção aguda em cães admitidos em um Hospital Escola Veterinário / Epidemiological study and evaluation of risk factors for infection with Toxoplasma gondii and clinical and pathological findings of acute infection in dogs admitted at a Veterinary School Hospital

Esse trabalho teve como objetivo estudar a prevalência e respectivos fatores de risco para infecção do Toxoplasma gondii em cães provenientes de uma população hospitalar. Além disso, avaliou-se as taxas de ocorrência e as repercussões clínico-patológicas da infecção aguda pelo T. gondii nesses animais. Anticorpos foram detectados em 7% (26/386) da população estudada, composta de 386 cães de ambos os sexos e diferentes raças e idades. Somente as variáveis, ingestão de vísceras, origem rural e contato com bovinos apresentaram valores significativos com p<0.05. Adicionalmente os cães de origem rural apresentaram maiores risco (OD=7.00) de infecção do que aqueles de origem urbana. Em 6,5% (25/386) foram detectados títulos de contato (entre 16 e 256); esses títulos não significam necessariamente infecção aguda e sim apenas exposição prévia. É de fundamental importância o reconhecimento da infecção prévia por T. gondii nesses pacientes hospitalares. Dependendo da causa da admissão, mesmo não sendo a toxoplasmose a responsável, o paciente deve receber o tratamento anti-protozoário profilaticamente ou ser monitorado para posterior tratamento em caso de reagudização da enfermidade por recrudescência dos bradizoítos encistados. Apenas um animal (3.44%, 1/386) foi admitido com titulação elevada, o qual pode ser sugestivo de infecção aguda (titulo de 4096). Embora o animal com infecção aguda tenha sido apresentado com sinais neurológicos, cautela é necessária para não extrapolar uma falsa interpretação que a toxoplasmose é a grande responsável por quadros neurológico, uma vez que inúmeros outros casos incluídos nesse estudo tinham manifestações neurológicas e não tinham títulos de infecção aguda, nem mesmo título de contato prévio. A toxoplasmose aguda não foi uma afecção clínica expressiva nessa ambiência hospitalar, no entanto diagnóstico diferencial deve ser feito nos pacientes enfermos, principalmente os da área rural, e diagnostico definitivo deve ser alcançado para a correta conduta terapêutica.(AU)

We aimed to verify the prevalence and risk factors for Toxoplasma gondii infection in dogs addimitted at the hospital. We also assessed the occurrence rates and the clinical and pathological repercussion of the acute infection by T. gondii in these animals. Antibodies were detected in 7% (26/386) of a population of 386 dogs of both genders and different breeds and ages. Only variables, eating offal, rural origin and contact with cattle have significant values of p<0.05. Dogs from rural areas showed higher risk (OD=7.00) of infection than those of urban origin. In 6.5% (25/386) contact titles were detected (between 16 and 256); these titles do not necessarily mean acute infection, but only prior exposure. The recognition of prior infection by T. gondii is vital in those hospital patients. Depending on the cause of admission, although not being toxoplasmosis the responsible, the patient should receive prophylactic anti-parasite treatment or be monitored for further treatment in case of further acute occurrence of the disease by recrudescence of encysted bradyzoites. Only one dog (3:44%, 1/386) was admitted with high titer, what may be suggestive of acute infection (title of 4096). Although the dog with acute infection has shown neurological signs, caution is required not to extrapolate a false interpretation that toxoplasmosis is the main responsible for neurological signs, since numerous other cases included in this study had neurological signs without title of acute infection, even title of prior contact. Acute toxoplasmosis was not a significant clinic disease in this hospital; however differential diagnosis should be made in ill patients, especially those from rural areas, and definitive diagnosis must be reached for the correct therapeutic approach.(AU)

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.

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.

Molecular detection of Hepatozoon canis and Babesia canis vogeli in domestic dogs from Cuiabá, Brazil.

The objective of this study was to report for the first time infection by Hepatozoon spp. and Babesia spp. in 10 dogs from the city of Cuiabá, State of Mato Grosso, central-western Brazil. A pair of primers that amplifies a 574 bp fragment of the 18S rRNA of Hepatozoon spp., and a pair of primers that amplifies a 551 bp fragment of the gene 18S rRNA for Babesia spp. were used. Six dogs were positive for Babesia spp., and 9 were positive for Hepatozoon spp. Co­infection of Babesia spp. and Hepatozoon spp. was seen in 5 dogs. Sequenced samples revealed 100% identity with B. canis vogeli, and H. canis. This is the first molecular detection of H. canis in domestic dogs from Cuiabá. Additionally, it is described for the first time the presence of B. canis vogeli circulating among dogs in Cuiabá.

Molecular detection of Hepatozoon canis and Babesia canis vogeli in domestic dogs from Cuiabá, Brazil / Detecção molecular de Hepatozoon canis e Babesia canis vogeli em cães domésticos de Cuiabá, Brasil

The objective of this study was to report for the first time infection by Hepatozoon spp. and Babesia spp. in 10 dogs from the city of Cuiabá, State of Mato Grosso, central-western Brazil. A pair of primers that amplifies a 574 bp fragment of the 18S rRNA of Hepatozoon spp., and a pair of primers that amplifies a 551 bp fragment of the gene 18S rRNA for Babesia spp. were used. Six dogs were positive for Babesia spp., and 9 were positive for Hepatozoon spp. Co-infection of Babesia spp. and Hepatozoon spp. was seen in 5 dogs. Sequenced samples revealed 100 percent identity with B. canis vogeli, and H. canis. This is the first molecular detection of H. canis in domestic dogs from Cuiabá. Additionally, it is described for the first time the presence of B. canis vogeli circulating among dogs in Cuiabá.

O objetivo deste estudo foi relatar pela primeira vez a infecção por Hepatozoon spp. e Babesia spp. em cães domésticos provenientes da cidade de Cuiabá, estado de Mato Grosso. Foram utilizados pares de primers que amplificam um fragmento de 574 pb do gene 18S rRNA de Hepatozoon spp., e 551 pb do gene 18S rRNA para Babesia spp. Dos 10 cães amostrados, 6 apresentaram-se positivos para Babesia spp., e 9 foram positivos para Hepatozoon spp. pela PCR. Co-infecção entre Babesia spp. e Hepatozoon spp. ocorreu em 5 cães. As amostras revelaram 100 por cento de identidade com B. canis vogeli, e as amostras que foram positivas para Hepatozoon spp. foram 100 por cento idênticas a H. canis. Esta é a primeira identificação molecular de H. canis em cães domésticos em Cuiabá. Adicionalmente, descrevemos pela primeira vez a presença de B. canis vogeli circulando entre cães em Cuiabá.

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.

Seroprevalence of Toxoplasma gondii infection in swine matrices in Nova Mutum and Diamantino, Mato Grosso, Brazil.

This report aimed to assess the seroprevalence of Toxoplasma gondii infection in 708 swine matrices in Nova Mutum and Diamantino in the state of Mato Grosso, Central-West Brazil. Serum samples were examined by indirect fluorescent antibody test (IFAT). It was found a seroprevalence of 12.8%, considering titers ≥ 64. Therefore, the data reinforce the need for appropriate management of swine raising to minimize the risk of infection of pigs with T. gondii.

Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2 / Toxoplasma gondii: avaliação da resposta imune humoral e celular de camundongos BALB/c imunizados pela via nasal com 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 cell-ular immune response on day 62. Five (50 percent) and two (20 percent) 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.

TgROP2 é uma proteína localizada nas roptrias do Toxoplasma gondii, sendo um antígeno candidato a componente de uma vacina contra a toxoplasmose. O objetivo do presente estudo foi avaliar a eficácia da TgROP2 recombinante em estimular a resposta imune celular e humoral de camundongos BALB/c após estímulo intranasal. A sequência da TgROP2 foi amplificada pela PCR a partir da cepa RH e clonada em vetor de expressão pTrc-His. Após a transformação em Escherichia coli- Rosetta 2, a pTrcHis-TgROP2 exibiu alto nível de expressão após 4 horas de indução com IPTG. A proteína recombinante apresentou uma massa molecular aparente de aproximadamente 54 kDa. Para avaliar a imunogenicidade dessa proteína recombinante, 10 camundongos receberam, pela via intranasal, 10 µg da rROP2 associado a 10 µg de Quil-A. Três doses foram realizadas nos dias 0, 21 e 42. No dia 62 do experimento, três animais foram eutanasiados para avaliar as respostas imune celular e humoral. Cinco (50 por cento) e dois (20 por cento) dos 10 animais apresentaram níveis de IgG (DO média = 0,307; ponto de corte = 0,240) e IgA (DO média = 0,133; ponto de corte = 0,101) acima do ponto de corte no ELISA no dia 62. A proliferação de esplenócitos revelou altos Índices de Estimulação (SI), quando as células foram cultivadas com 5, 10 e 15 µg.mL-1 de rTgROP2. Os resultados obtidos indicam que a via nasal pode estimular tanto a resposta imune celular como a humoral.

Seroprevalence of Toxoplasma gondii infection in swine matrices in Nova Mutum and Diamantino, Mato Grosso, Brazil / Soroprevalência de infecção por Toxoplasma gondii em matrizes suínas em Nova Mutum e Diamantino, Mato Grosso, Brasil

This report aimed to assess the seroprevalence of Toxoplasma gondii infection in 708 swine matrices in Nova Mutum and Diamantino in the state of Mato Grosso, Central-West Brazil. Serum samples were examined by indirect fluorescent antibody test (IFAT). It was found a seroprevalence of 12.8 percent, considering titers >64. Therefore, the data reinforce the need for appropriate management of swine raising to minimize the risk of infection of pigs with T. gondii.

No presente trabalho, objetivou-se avaliar a soroprevalência da infecção por Toxoplasma gondii, em 708 matrizes suínas dos municípios de Nova Mutum e Diamantino do Estado de Mato Grosso, Brasil. As amostras de soro foram examinadas por meio da reação de imunofluorescência indireta (RIFI). Foi encontrada a frequência de 12,8 por cento de soros positivos, com diluições iguais ou superiores a 64. Portanto, os dados obtidos reforçam a necessidade de um manejo de criação adequado, visando à minimização do risco de infecção de suínos por T. gondii.

Induced immune response of Escherichia coli BL21 expressing recombinant MSP1a and MSP1b proteins of Anaplasma marginale

This work aims to evaluate the potential of immunization with E. coli BL21 expressing the recombinant rMSP1a and rMSP1b proteins of Anaplasma marginale. E. coli BL21 was transformed with recombinant plasmids pET102/msp1α and pET101/msp1β, and rMSP1a and rMSP1b were expressed after induction by IPTG. BALB/c mice were vaccinated with formolized BL21/rMSP1a and BL21/rMSP1b, and the production in mice sera of whole IgG was determined by ELISA. The mice immunized with BL21/rMSP1a showed a better humoral response for whole IgG when compared to the mice immunized with BL21/rMSP1b; these mice exhibited a small response after the second vaccination. Sera of mice immunized with BL21/rMSP1a reacted via western blot with BL21 and rMSP1a, with molecular masses varying from 70 to 105 kDa. Sera of mice immunized with BL21/rMSP1b reacted with BL21 and rMSP1b with a molecular mass of 100 kDa. These results demonstrate that BL21 containing rMSP1a and rMSP1b in the outer membrane were able to produce an immune response in mice, reinforcing its use in vaccine models against bovine anaplasmosis.

Esse trabalho avaliou o potencial de imunização de Escherichia coli BL21 expressando as proteínas recombinantes rMSP1a e rMSP1b de Anaplasma marginale. A E. coli BL21 foi transformada com os plasmídios recombinantes pET102/msp1α e pET101/msp1β e as proteínas rMSP1a e rMSP1b foram expressas após indução com IPTG. Camundongos BALB/c foram vacinados com BL21/rMSP1a e BL21/rMSP1b formolisadas, e a produção de IgG total foi determinada pelo teste de ELISA nos soros dos camundongos imunizados. Os camundongos imunizados com a BL21/rMSP1a mostraram uma melhor resposta humoral para IgG total, comparada à resposta apresentada pelos camundongos imunizados com BL21/rMSP1b; estes camundongos exibiram uma menor resposta após a segunda vacinação. Soros de camundongos imunizados BL21/rMSP1a reagiram pelo western blot com BL21 e rMSP1a, com massa molecular variando de 70 a 105 kDa. Soro de camundongos imunizados com BL21/rMSP1b reagiram com BL21 e rMSP1b com massa molecular de 100 kDa. Esses resultados demonstram que BL21 contendo rMSP1a e rMSP1b na membrana externa foram capazes de produzir resposta imune em camundongos, reforçando o seu uso em modelos de vacina contra a anaplasmose bovina.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs.

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n=10) and G2 (uninfected group, n=8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r=0.62, P=0.05), MAT(s) x MAT (ah) (r=0.97, P<0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r= 0.14, P=0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis.

Detection of Anaplasma marginale DNA in larvae of Boophilus microplus ticks by polymerase chain reaction.

Boophilus microplus larvae from two different sources were used for the detection of Anaplasma marginale DNA: larvae A, which were collected from a pasture of an endemic farm, and larvae B, which originated from engorged female ticks fed on calves with no clinical signs of disease and with low rickettsemia (approximately 0.01 to 1.0%). Larvae A were collected monthly, from January to May in 2001. Two hundred engorged female ticks fed on calves that provided larvae B were divided into groups of 10 and kept in a controlled environment at either 18 degrees C or 28 degrees C. Fifty larvae were used from each sample for DNA extraction, and 5 muL of DNA were submitted to amplification of the sequence of msp5 gene of A. marginale by polymerase chain reaction (PCR). Seven out of 50 samples of larvae A (14%) were positive for the presence of DNA of A. marginale showing amplified product of 457 bp. Ten out of 91 samples of larvae B (11%) kept at 18 degrees C were positive, and all larvae B at 28 degrees C were negative. Thus, this study confirmed the presence of A. marginale DNA in B. microplus larvae by PCR. The EcoRI restriction enzyme analysis confirmed the specificity of the amplicon, which resulted in two fragments: 265 bp and 192 bp. The sequencing analysis of the amplicon from larvae demonstrated 98% homology with the msp5 sequence from Florida A. marginale strain.