Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle.

Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.

Experimental infection of pregnant queens with two major Brazilian clonal lineages of Toxoplasma gondii.

Toxoplasma gondii isolates from Brazil are biologically and genetically different from European and North America isolates. Recently, four genotypes were considered the common clonal lineages in Brazil and were designated as types BrI, BrII, BrIII, and BrIV. The pathogenicity of two major Brazilian lineages was investigated after oral inoculation of queens in the middle third of their pregnancies with T. gondii cysts. Twelve pregnant queens without T. gondii antibodies were distributed in group A (infected with a type BrI isolate); group 2 (infected with type BrIII isolate), and group 3 (non-infected control). Infection with type BrI isolate caused toxoplasmosis manifestations and abortion from one litter. Toxoplasmosis manifestations besides premature stillbirth of one litter were observed in queens infected with type BrIII isolate. Indirect fluorescence antibody test showed T. gondii antibodies in all eight infected queens at 30 days after inoculation. In two 10-day-old kittens of the same litter (group 1), titers of 16 and 64 were detected. At the same time, titers of 16, 32, and 32 were detected in three kittens from the same litter (group 2). Experimental infection with tissue cysts from a type BrI and type BrIII isolates of T. gondii developed similar reproductive disturbance in primary infected pregnant queens.

Preferential infection sites of Cysticercus bovis in cattle experimentally infected with Taenia saginata eggs.

The preferential sites of infection of Cysticercus bovis were evaluated in the skeletal muscle and entrails of 25 cattle that were experimentally infected with Taenia saginata (2×10(4) eggs). Two other animals were not inoculated (control). Ninety days after inoculation, all the cattle were euthanized. The carcasses were deboned and dissected into 26 anatomical sections (masseter muscles, brain, tongue, esophagus, heart, diaphragm, lungs, liver, kidneys, spleen, top sirloin butt, bottom sirloin butt, outside round, top (inside) round, transversus abdominus, top sirloin cap, strip loin, full tenderloin, eye of round, knuckle, shoulder clod, foreshank, shank, chuck, back ribs, and tail muscles). The dissected tissues were sliced into 5mm sections. From the 25 cattle, 9258 C. bovis (cysticerci) were recovered; 75.02% (6946) of these were recovered from skeletal muscles and 24.98% (2312) from the entrails. A high parasitism level was found in the shoulder clod (12.55%), heart (11.02%), liver (9.48%), masseter muscles (8.51%), chuck (8.25%), strip loin and full tenderloin (7.26%), knuckle (6.63%), and back ribs (5.53%), totaling 69.23% (5738) of all of the detected cysticerci. On the other hand, there was a low C. bovis parasitism level in the brain, spleen, tail muscles, kidneys, esophagus, and diaphragm, representing just 3.9% of the total number of cysticerci. Given these results, we conclude that specific skeletal musculature regions, such as the shoulder blade, chuck, strip loin and full tenderloin, knuckle, back ribs and top round, which are not officially examined in many countries, are effective sites to efficiently screen C. bovis infection. To date, these regions have not been considered as preferential sites of C. bovis infection. Based on our work, however, these regions deserve greater attention from health inspectors because they contained a greater number of Cysticercus than the other regions of carcasses that are parasitized by T. saginata larvae.

Weak phenotypic reversion of ivermectin resistance in a field resistant isolate of Haemonchus contortus by verapamil / Reversão fenotípica da resistência a ivermectina em isolado de campo de Haemonchus contortus pelo verapamil

Recent advances in anthelmintic resistant phenotype reversion by Pgp modulating drugs in ruminant nematodes indicate that this can be a useful tool to helminth control. The aim of the present study was to evaluate the efficacy of ivermectin (IVM) in combination with verapamil (VRP), in oil or water-based vehicle, against an IVM-resistant field isolate of Haemonchus contortus through a larval migration assay and experimental infection trial. In the in vitro assay was observed a phenotypic reversion of H. contortus resistance to ivermectin at a high concentration of VRP, increasing IVM efficacy from 53.1 percent to 94.3. In the in vivo trial, IVM + VRP demonstrated 36.02 percent efficacy compared to the 7.75 percent of IVM alone. The vehicle formulation showed no influence in efficacy. These are the first results demonstrating the effect of VRP as a partial IVM-resistance phenotype reverser in a field isolate of IVM-resistant H. contortus experimentally inoculated in sheep.

Avanços recentes na reversão fenotípica da resistência anti-helmíntica por drogas moduladoras de Pgp em nematódeos de ruminantes indicam que esta pode ser uma ferramenta útil no controle de helmintos. O objetivo do presente estudo foi avaliar a eficácia da ivermectina (IVM), em combinação com o verapamil (VRP), em veículo oleoso ou à base de água, contra um isolado de campo de H. contortus resistente por meio de teste de migração de larvas e infecção experimental em ovinos. No teste in vitro, observou-se reversão fenotípica da resistência de Haemonchus contortus à ivermectina com alta concentração de VRP, aumentando a eficácia da IVM de 53,1 por cento para 94,3. No teste in vivo, IVM + VRP demonstrou 36,02 por cento de eficácia em relação a 7,75 por cento de IVM sozinha. O veículo da formulação não apresentou influência na eficácia. Estes são os primeiros resultados que demonstram o efeito da VRP como reversor parcial do fenótipo da resistência de IVM-fenótipo em um isolado de campo de H. contortus resistente, inoculado experimentalmente em ovinos.

Toxoplasma gondii in semen of experimentally infected swine

Eight reproductive boars were divided into three groups and inoculated with Toxoplasma gondii [GI (n=3) 1.5x10(4) oocysts strain P; GII (n=3) 1.0x10(6) tachyzoites strain RH; and GIII (n=2) non-inoculated control]. Clinical, hematological, parasitemia and serological tests and studies of the parasite in the semen through bioassay and PCR, and in reproductive organs (Bioassay and immunohistochemical analyses) were conducted to evaluate the toxoplasmic infection. Blood and semen were collected on day -2, -1, 1, 3, 5, 7, 9, 11, 14 and weekly up to 84 days post-inoculation (DPI). No clinical or hematimetric alteration was observed in the boars. Parasitemia was detected in one boar inoculated with oocysts at the 7th DPI and in another boar infected with tachyzoites (GII) at the 3rd and 49th DPI. Serological tests revealed antibodies against T. gondii in animals inoculated with oocysts or tachyzoites at the 7th DPI with dilutions of 1:256 and 1:64, which reached peaks of 1:4096 at day 11 and 9, respectively. The bioassays revealed the presence of the parasite in semen samples of a boar inoculated with oocysts (GI) at 3, 49 and 56 DPI and from two boars infected with tachyzoites (GII), one animal at 5 and two animals at 49 days DPI. Mice inoculated with semen from the control group (GIII) remained serologically negative. PCR analysis showed T. gondii DNA in the semen of Boar 1 and Boar 3 inoculated with tachyzoites and oocysts, respectively. The immuno-histochemical tests showed T. gondii in the reproductive organs of Boar 1 and Boar 2, inoculated with tachyzoites and oocysts, respectively. These findings suggest the possible occurrence of venereal transmission of T. gondii in swine.

Oito reprodutores suínos foram divididos em três grupos e inoculados com Toxoplasma gondii [GI (n=3) 1.5x10(4) oocistos cepa P; GII (n=3) 1.0x10(6) taquizoítos cepa RH, e GIII (n=2) controle, não inoculados]. Exames clínicos, hematológicos, de parasitemia e sorológicos foram realizados para avaliar a infecção toxoplásmica. Pesquisa do parasito no sêmen, por meio do bioensaio e pela técnica da PCR, e em órgãos do sistema reprodutor (bioensaio e imunohistoquímica) foi realizada. Sangue e sêmen foram colhidos nos dias -2, -1, 1, 3, 5, 7, 9, 11, 14, e semanalmente até o 84º dia pós-infecção (DPI). Nenhuma alteração clínica ou hematimétrica foi observadda nos animais. Parasitemia foi detectada em um animal inoculado com oocistos no 7º DPI e em outro inoculado com taquizoítos (GII) nos 3º e 49º DPI. A sorologia revelou a presença de anticorpos contra T. gondii nos animais inoculados com oocistos ou taquizoítos no 7º DPI com títulos de 1:256 e 1:64, que atingiram picos de 1:4096 nos dias 11 e 9, respectivamente. O bioensaio revelou a presença do parasita em amostras seminais de um animal inoculado com oocistos (GI) nos 3º, 49º e 56º DPI, e de dois animais infectados com taquizoítos (GII), um deles no 5º DPI e os dois ao 49º DPI. Pela PCR, o DNA de T. gondii foi detectado no sêmen dos Suínos 1 e 3 inoculados com taquizoítos e oocistos, respectivamente. A imunohistoquímica revelou T. gondii em órgão do aparelho reprodutor dos Suínos 1 e 2, inoculados com taquizoítos e oocistos, respectivamente. Esses achados sugerem a possibilidade da ocorrência da transmissão venérea do T. gondii em suínos.