Cloning and expression of the porcine attaching and effacing-associated (paa) gene of enteropathogenic Escherichia coli.

Porcine enteropathogenic Escherichia coli (PEPEC) produce an outer membrane protein (intimin) called Paa (porcine attaching and effacing-associated), which is involved in the pathogenesis of E. coli in piglets with diarrhea. The paa gene of a PEPEC strain isolated in Paraná, Brazil, was amplified by polymerase chain reaction, sequenced, and cloned into the pTrcHisTOPO2 vector. The deduced amino acid sequence encoded by the paa gene of PEPEC from Paraná, Brazil, showed 99% homology to the sequences from other PEPEC strains. In this study, the overexpression of recombinant Paa (rPaa) using alternative induction strategies was attempted. The auto-induction protocol showed excellent results for rPaa protein production with 0.4% (w/v) lactose. The rPaa protein is insoluble and was purified with Triton X-100 wash as a total antigen. This method produced a relatively high yield of rPaa. rPaa was recognized by serum from pigs immunized with the PEPEC strain. These results suggest that rPaa could be included in the development of a vaccine against swine colibacillosis.

Neospora caninum: evaluation of vertical transmission in slaughtered beef cows (Bos indicus).

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.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil.

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7.

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Prevalence of Neospora caninum and Toxoplasma gondii in sheep and dogs from Guarapuava farms, Paraná State, Brazil.

Sheep and dog blood samples were collected from nine farms in the county of Guarapuava, Paraná, Brazil. The indirect fluorescent antibody test (IFAT) was used to detect Neospora caninum and Toxoplasma gondii antibodies. Herein, serum samples from 305 sheep were evaluated, being 29 (9.5%) and 157 (51.5%) seropositives to N. caninum and T. gondii, respectively. Seven (29.1%) and five (20.8%) out of 24 dogs were seropositives to N. caninum and T. gondii, respectively. There were no differences among the sheep serology for N. caninum and reproductive problems, management and animal feeding variables, neurological problems and presence of other animals species on the farm (P>or=0.05). The simultaneous frequency of antibodies between N. caninum and T. gondii was 5.2% in the herds. Age, breed, farm size, semi-intensive activity, mineral salt supplementation, water origin, stage of the pregnancy when reproduction problems occurred, neurological problems in lambs, presence of rodents in the food room and pasture cat access were identified as associated factors for the occurrence of toxoplasmosis in sheep (P<0.05). There were no differences among the seropositivity in dogs for N. caninum and T. gondii and breed, age and sex (P>or=0.05). The present work is the first report on serum prevalence of N. caninum in sheep from the state of Paraná, Brazil.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil.

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1.

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the Jaboticabal strain of Anaplasma marginale.

Anaplasma is a tick-borne ehrlichial pathogen of cattle that causes the disease, anaplasmosis. In the present study, a total of 11 Anaplasma marginale seronegative calves were assigned into two groups: one immunized (G1, n = 6) and one nonimmunized-control (G2, n = 5). Six calves were immunized by using a DNA vaccine containing the gene of a major surface protein, MSP1b, encoded by the plasmid identified as pcDNA3.1/MSP1b. Calves received three intramuscular inoculations of 100 microg of pcDNA3.1/MSP1b at a 20-day interval. The control group received buffer phosphate at the same schedule as the experimental group. The immune response elicited by immunization with pcDNA3.1/MSP1b was evaluated in mice and calves. Twenty days following initial immunization, specific serum antibody from four BALB/c mice bound MSP1b in immunoblots. Sixty days after the last immunization, all calves were challenged with cryopreserved A. marginale at a dose of 10(4) parasites/mL/animal by intravenous injection. Results of packed cell volume (PCV) and detection of infected erythrocytes in all experimental groups revealed that the decrease of PCV and detection of infected erythrocytes occurred at 28 to 42 days after challenge. Mean temperature values did not increase over 39.85 degrees C. Antibodies developed by immunized bovines from G2 were detected 14 days after challenge. MSP1b was characterized during the immunization period and MSP2 was the most predominant polypeptide at the challenge period. DNA of A. marginale was detected in all groups just after challenge by nested PCR assay. It can be concluded that all immunized bovines were partially protected against homologous challenge.

Use of repetitive DNA elements to define genetic relationships among Anaplasma marginale isolates.

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.

Antigenic characterization of Anaplasma marginale isolates from different regions of Brazil.

Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.

Enteroparasites prevalence among daycare and elementary school children of municipal schools, Rolândia, PR, Brazil.

Coproparasitological analyses were performed on 191 daycare children and 434 elementary school children from urban and rural areas in Rolândia, Parana State, Brazil. The overall prevalence of enteroparasites was 15.2 % for daycare children and 52.5% for elementary school children. Risk factors are discussed.

Infection by spotted fever rickettsiae in people, dogs, horses and ticks in Londrina, Parana State, Brazil.

Spotted fever is a disease caused by bacteria from the genus Rickettsia of the spotted fever group (SFG). Rickettsia rickettsii is likely the main agent of Brazilian spotted fever (BSF). With the objective of gathering information on the circulation of SFG rickettsiae in Londrina, Parana state, ticks from dogs and horses and also blood from dogs, horses and humans were collected in a neighbourhood of the city which presented potential for circulation of rickettsiae between hosts and vectors. Amblyomma cajennense, Dermacentor nitens, and Rhipicephalus sanguineus ticks were subjected to Polymerase Chain Reaction targeting a fragment of the Rickettsia gltA gene. This specific gene encodes the enzyme citrate synthase of Rickettsia spp., and results on all ticks were negative. Human and animal sera were tested by Indirect Immunofluorescence Assay in which R. rickettsii and R. parkeri were used as antigens. Sera from 4.7% human, 2.7% canine and 38.5% equine were positive for R. rickettsii. For R. parkeri, 0.9% human, 2.7% canine and 11.5% equine samples were positive. All samples reactive to R. parkeri also reacted to R. rickettsii. An epidemiological questionnaire was applied, but there were no statistically significant results. Comparison of our serological results with previous studies in Brazil, among BSF endemic and non-endemic areas, indicates that there is no established rickettsial infection in the study area, a statement corroborated with our molecular analysis. Nonetheless, as humans of the present study are highly exposed to tick infestations, health education within the population is needed to obtain efficient tick control.

Eficácia do dipropionato de imidocarb, da enrofloxacina e do cloridrato de oxitetraciclina no tratamento de bovinos naturalmente infectados por Anaplasma marginale / Efficacy of imidocarb dipropionate, enrofloxacin and oxytetracycline chlorydrate on the treatment of cattle naturally infected by Anaplasma marginale

O agente de maior importância, em relação à anaplasmose bovina, é o Anaplasma marginale. Os principais sinais clínicos dessa enfermidade são anemia hemolítica, icterícia, dispneia, taquicardia, febre, fadiga, lacrimejamento, sialorreia, micção frequente, anorexia, perda de peso, aborto e morte. A terapia antimicrobiana é o principal protocolo terapêutico. O objetivo do presente trabalho foi avaliar a eficácia do dipropionato de imidocarb, da enrofloxacina e do cloridrato de oxitetraciclina no tratamento de bovinos leiteiros naturalmente infectados por Anaplasma marginale. Para isso, foram avaliados 48 zebuínos mestiços que apresentavam os sinais clínicos sugestivos da doença. Os animais foram submetidos à coleta de sangue para a realização de hemograma e à extração de DNA para a confirmação da presença de A. marginale, por meio da reação em cadeia pela polimerase (PCR). Os animais foram divididos em três grupos experimentais, para realização dos protocolos terapêuticos, utilizando-se dipropionato de imidocarb, oxitetraciclina e enrofloxacina. Trinta e seis animais (75%) apresentaram reação positiva ao PCR. Os animais positivos não apresentaram diferenças significativas quanto ao hemograma e ao leucograma quando comparados com os negativos, no entanto os níveis de proteínas séricas foram inferiores nos animais positivos (P<0,05). Os três protocolos terapêuticos foram capazes de reduzir a infecção ao longo do tratamento (P<0,01), porém, após cinco dias de tratamento, a enrofloxacina apresentou maior efetividade em relação aos demais (P<0,01). Após o final do tratamento, nenhum protocolo foi capaz de eliminar totalmente a infecção pelo A. marginale em bovinos naturalmente infectados e manejados a campo.

Anaplasma marginale is the most important agent regarding cattle anaplasmosis. The main clinical signs of this disease are hemolitic anemia, jaundice, dyspnea, tachycardia, fever, fatigue, lacrimation, salivation, frequent urination, anorexia, weight loss, abortion and death. Antimicrobial therapy is the main therapeutic protocol. The aim of this paper was to assess the efficacy of this therapy frequently used in field conditions. In order to do so, 48 crossbred zebu cattle presenting suggestive clinical signs of the disease were assessed. The animals were submitted to blood sample collection to perform a CBC and DNA extraction to confirm the presence of A. marginale by the polymerase chain reaction (PCR) test. The animals were divided into three experimental groups to perform the therapeutic protocols, using imidocarb dipropionate, enrofloxacin and oxytetracycline. Thirty-six animals (75%) presented positive reaction to PCR. The positive animals do not present significant differences in the CBC and WBC when compared to the negative ones. However, the serum protein levels were lower in positive animals (P<0.05). All the treatments were able to reduce the infection throughout the treatment (P<0.01). However, in time 1, enrofloxacin presented greater effectiveness in relation to the other ones (P<0.01). After the end of the treatment no protocol was able to totally eliminate the infection by A. marginale in cattle naturaly infected and handled on the field.

Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice.

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n=11) received 12.5 microg of each recombinant protein plus 0.5 microg of cholera toxin, group 2 (G2, n=11) received phosphate buffer saline (PBS) plus 0.5 microg of cholera toxin, and group 3 (G3, n=11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRA7. Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P<0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P>0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T. gondii.

Phylogenetic analysis of Anaplasma marginale strains from Paraná State, Brazil, using the msp1alpha and msp4 genes.

Anaplasma marginale is an obligate intraerythrocytic rickettsial pathogen (order, Rickettsiales: family, Anaplasmataceae) that causes bovine anaplasmosis. This disease is widely distributed in tropical and sub-tropical regions of the world and causes important economic losses to cattle production. Major surface protein (MSP)1a (msp1alpha gene) is one of the six MSPs identified on A. marginale from cattle, whose sequence and size vary according to the number of tandem 28- to 29-amino acid repeats. This study characterized the msp1alpha and msp4 genes obtained from three distinct Brazilian herds from the State of Paraná. Three strains of the msp1alpha and one strain of the msp4 gene were sequenced. The strains evaluated revealed PCR products of different size, representing three, five and six internal repeats. Sequence analyses confirmed the number of tandem sequence copies and revealed a high degree of sequence identity with strains from other Brazilian States, as well as strains from the USA, Europe and Israel. The msp1alpha DNA and amino acid sequences from A. marginale and DNA sequences of msp4 strains did not reveal distinct phylogeographical segregation. However, the amino acid sequences of msp4 demonstrated definite phylogeographical relationship. These results suggest that the amino acid sequences of msp4 should be used for phylogenetic identification of A. marginale strains and may be an important tool for the epidemiology and control of anaplasmosis. Additionally, the close similarity of the Paraná strains of A. marginale with strains from USA, Europe and Asia may reflect the introduction of these genes during the development of the Brazilian bovine herd.

[Resistance of Toxoplasma gondii to sodium chloride and condiments in pork sausage]. / Resistência do Toxoplasma gondii ao cloreto de sódio e aos condimentos em linguiça de suínos.

The aim of this study was to determine the resistance of Toxoplasma gondii cysts to salt (sodium chloride) and condiments (black pepper and garlic) in fresh sausages prepared with experimentally infected pork. The sausages were treated with 1.25, 2.00, and 2.50% salt with condiments added, and were refrigerated for 2, 24, and 48 hours, after which they were artificially digested. Mice were then inoculated with the digested matter. After 42 days the surviving mice were sacrificed and their serum examined using indirect immunofluorescence to detect anti-T. gondii antibodies. This examination showed that the treatments with salt over periods of less than 24 hours did not eliminate the parasite, and that only after 48 hours did the salt, at concentrations of 2.00 and 2.50%, effectively kill the parasite. The results of this experiment also showed that the condiments had no effect on the viability of T. gondii.

Babesia bigemina: identification of B cell epitopes associated with parasitized erythrocytes.

Rhoptries are involved in host cell invasion and rhoptry polypeptides, including the Babesia bigemina rhoptry-associated protein-1 (RAP-1), are targets for protective immune responses. Polyclonal antisera produced against isolated rhoptries is directed predominantly against RAP-1 and reacts with both the merozoite and the membrane of parasitized erythrocytes. To determine whether these B cell epitopes associated with the parasitized erythrocyte are derived from RAP-1 or, alternatively, from previously undetected merozoite polypeptides, monoclonal antibodies (mAbs) were generated from mice immunized with rhoptries isolated from the JG-29 clone of the Mexico strain. The anti-RAP-1 mAbs bound only merozoites in a punctate immunofluorescence pattern. A second group of four mAbs, none of which were reactive with RAP-1, bound the parasitized erythrocyte. Two of these latter mAbs, 64/44.17.3 and 64/05.7.2, reacted only with parasitized erythrocytes that had been permeabilized. MAb 64/44.17.3 bound a 54-kDa merozoite polypeptide while 64/05.7.2 bound a > or = 225-kDa merozoite polypeptide. MAbs 64/32.8.5 and 64/38.5.3 recognized epitopes on 17.5- and 76-kDa polypeptides exposed on the external surface of intact parasitized erythrocytes. The results indicate that the identified RAP-1 epitopes are not associated with the erythrocyte cytoskeleton or membrane and that anti-RAP-1 immunity is most likely generated against the free merozoite. All new mAbs reacted with every B. bigemina strain tested (Mexico, Puerto Rico, St. Croix, Texcoco, Jaboticabal). The conservation of RAP-1 epitopes among these strains supports the continued testing of RAP-1 as a vaccine component. In addition, the identification of epitopes expressed on the surface of erythrocytes infected with all five strains provides new candidate immunogens.