Marcos Santos Zanini

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Intestinal mucosa structure of broiler chickens infected experimentally with Eimeria tenella and treated with essential oil of oregano

In the first trial a total of 250 day-old male chicks were distributed into five treatments and given the following diets: a diet with growth promoter; a diet without added growth promoter; a diet added with avilamycin only; diet supplemented with 0.5g of oregano oil kg diet-1; 1.0g of oregano oil kg diet-1. In other trial a total of 288 day-old chicks was used and distributed into four treatments, which were given the following diets: a diet with anticoccidial agent; a diet without anticoccidial agent; a diet supplemented with 0.5g of oregano oil kg diet-1; a 1.0g of oregano oil kg diet-1. In the first trial the nonmedicated group had the highest crypt depth which differs from chickens fed with growth promoter or with 0.5 and 1.0g of oregano oil kg diet-1. The broilers fed with positive control (antibiotic and anticoccidial) had the highest villous: crypt ratio compared with the negative control that had the lowest villous:crypt ratio and the highest oocyst excretion in litter (P<0.05) In the second trial it was observed that broilers fed with non anticoccidial agent had the highest cecal lamina propria thickeness which differ from chickens fed with anticoccidial agent in diet or supplemented with 1.0 of oregano oil kg diet-1 (P<0.05).

Evaluation of the ratio of ω6 : ω3 fatty acids and vitamin e levels in the diet on the reproductive performance of cockerels

Three hundred and twenty 30-week old White Leghorn cockerels were housed in individual cages and distributed in a completely randomized factorial design of 5 × 3, with five oil sources (sunflower, soybean, canola, linseed and fish/soybean) and three levels of antioxidant (30, 200 and 400 mg of vitamin E/kg). The aim of this study was to evaluate the effect of the ratio of ω6 : ω3 fatty acids by the inclusion of different oil sources and of dietary supplementation with vitamin E on the reproductive performance of cockerels. The use of the fish/soybean combination determined the lowest total antioxidant status of the semen. However, the addition of vitamin E to the fish/soybean-oil-based diet resulted in a linear increase in semen volume, motility and sperm vigour in the 38th week and again in the 52nd week for motility and for sperm vigour and fertility rate in the periods from 50 – 53 and 41 – 53 weeks of age. The use of canola oil in the diet resulted in the highest fertility rate during 50 – 53 and 41 – 53 weeks of life. Animals receiving the soybean oil based diet showed the smallest fertility rate in the range from 50 – 53 weeks of age and concomitantly the highest level of cholesterol in the spermatozoa in the range from 47 – 51 weeks. An interaction between the vitamin E level and soybean oil was verified by the linear increase in motility and sperm vigour at 38 weeks of age. Later, the contrary was shown by a linear reduction in fertility in the periods from 44 – 46, 47 – 49 and 41 – 53 weeks of age. Cockerels that had been fed on the sunflower-oil-based diet showed the highest content of saturated fatty acids in the spermatozoa from 48 – 51 weeks. An interaction effect was observed between the vitamin E level and sunflower oil shown by a linear increase in the content of saturated fatty acids in the spermatozoa and a linear reduction in the C18 : 1ω9, C18 : 2ω6 and PUFA (C18 : 2ω6 + C20 : 4ω6) contents in the spermatozoa at 48 – 51 weeks and in sperm volume at 52 weeks of age.

Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification

Abstract Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L. infantum). Biomarkers in the canine immune system is an important technique in the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4+ and CD8+) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-β-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient for CD4 or CD8 T-cell purification (≥90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4+ and/or CD8+ T cells.

Clinical treatment of cutaneous leishmaniasis in dogs with furazolidone and domperidone

Cutaneous leishmaniasis (CL) is a zoonosis and a public health problem in countries of subtropical America. The aim of this study was to investigate the efficacy of furazolidone and domperidone treatment of dogs naturally infected with Leishmania (Viannia) braziliensis. Infection was confirmed by PCR and parasite culture of tissue collected from skin scrapings of the lesion borders of dogs. Naturally infected animals were divided into control (n=4) and treatment (n=8) groups. The treatment group was administered furazolidone for 21 days interspersed with domperidone for 10 days by oral gavage. Dogs that showed no lesion healing during this period were administered the same treatment cycle for up to 93 days. Among the eight treated animals, seven were clinically cured without recurrence of skin lesions during the 12-month study period. However, during lesion healing, skin scrapings were positive for L. (V.) braziliensis by PCR; no growth of the protozoan in NNN-LIT medium occurred until the end of follow-up. These results suggest that treatment with furazolidone and domperidone is effective for epithelialisation and lesion healing of dogs with clinical CL caused by L. (V.) braziliensis.

Leishmania (Viannia) braziliensis : immunoblotting analysis for the detection of IgG subclasses in the diagnosis of symptomatic and asymptomatic dogs.

During a seroepidemiological survey 2004-2006 from areas in Brazil endemic for American cutaneous leishmaniasis (ACL), serum samples from 10 dogs with ulcerated cutaneous lesions (S-ACL) and 52 asymptomatic dogs (AS-ACL) of unknown age and breed living in areas endemic for ACL were monitored for 1 year for ulcerated cutaneous lesions and immunoblotting using peroxidase-conjugated secondary anti-IgG, anti-IgG1 and anti-IgG2 dog antibodies. We reported that antibodies against Leishmania (Viannia) braziliensis in the sera of 22/52 dogs with asymptomatic disease showed intense reactivity to peptides larger than 66 kDa. We believe that dogs harboring subclinical amastigotes show an immunoblotting profile similar to that of symptomatic animals because a dog with self-healing presented antigens greater than 66 kDa. Such patterns can be exploited for diagnostic and epidemiological research for leishmaniasis.

Óleo de aroeira vermelha e de suplementação de vitamina E em substituição aos promotores de crescimento sobre a microbiota intestinal de frangos de corte

The aim was to evaluate the supplementation of pink pepper oil (PPO) and vitamin E on the intestinal microbiota of broiler chickens. A total of 400 day-old male chicks distributed in a randomized design in groups of five treatments and five replicates: diet without antimicrobial; diet with antimicrobial; diet with 0.4% PPO; diet with 200mg vitamin E kg-1; diet with 0.4% PPO and 200mg vitamin E kg-1 The supply of PPO in the diet reduced the relative weight of the intestines (P<0.05). It was verified that the negative control group had the lowest bacteria count of Lactobacillus in contrast to the other groups (P<0.05). It was also observed that the use of PPO with or without vitamin E supplementation significantly reduced the bacterial count of Staphylococcus spp and E. coli when compared with the untreated group with the growth promoter (P<0.05). For Staphylococcus spp. coagulase-positive, it was found that the minimum inhibitory concentration was 14,72-117,75mg mL-1 for isolates from broilers treated with PPO + vitamin E, while for treated with or without growth promoter was 117.75 and 29,44-235,5mg mL-1. It was concluded that the inclusion of PPO resulted in modulation of intestinal microbiota.

Mecânica e microbiologia de placas produzidas a partir de osso cortical bovino, conservadas em diferentes meios

The objective of this study was to evaluate the mechanical strength of bone plates yielded from bovine cortical bone, conserved in different solutions, and the efficiency of these solutions in the inhibition of microorganisms growth. A hundred and sixty eight plates yielded from bovine tibiae were conserved in 98% glycerin, 150% saline solution, 300% sugar solution, Dakin solution, frozen in N2L at - 196°C, or sterilized in ethylene oxide gas. After rehydration in NaCl 0.9% solution for six hours, plates were subjected to tensile, compression, bending and twisting testing. The microbiological evaluation of bone plates was s carried out before and immediately after rehydration, with or without enrofloxacin 0.5% addition. There was no significant difference (P<0.01%) on resistance to the rupture point in plates conserved in different solutions, frozen-thawed or sterilized. Microorganisms were isolated from plates conserved in satured salt solution, sugar satured solution, Dakin solution or frozen in N2L, before and after rehydraion However, after the use of NaCl 0.9% solution added of enrofloxacin 0.5%, microorganisms were not isolated. Therefore, bovine bone plates conserved in sugar or salt satured solution, 98% glycerin, Dakin solution, frozen in N2L or sterilized in ethylene oxide gas present similar biomechanical tests results, and rehydrated with a solution of NaCl 0.9% plus enrofloxacin 0.5% is effective on microorganism growth control.

Physicochemical and in vitro biological evaluations of furazolidone-based β-cyclodextrin complexes in Leishmania amazonensis

Recently, there have been numerous cases of leishmaniasis reported in different Brazilian states. The use of furazolidone (FZD) to treat leishmaniasis has been previously described; however, the drug is associated with adverse effects such as anorexia, weight loss, incoordination, and fatigue in dogs. Thus, in the present study, we prepared and evaluated inclusion complexes between FZD and β-cyclodextrin (β-CD) to guarantee increased drug solubility and reduce the toxicity associated with high doses. The FZD:β-CD complexes were prepared by two different techniques (kneading and lyophilization) prior to incorporation in an oral pharmaceutical dosage form. Formation of the complexes was confirmed using appropriate physicochemical methods. Antileishmanial activity against L. amazonensis was tested in vitro via a microplate assay using resazurin dye and cytotoxicity was determined using the fibroblast L929 lineage. Solubility studies showed the formation of complexes with complexation efficiencies lower than 100%. Physicochemical analysis revealed that FZD was inserted into the β-CD cavity after complexation by both methods. Biological in vitro evaluations demonstrated that free FZD and the FZD:β-CD complexes presented significant leishmanicidal activity against L. amazonensis with IC50 values of 6.16 μg/mL and 1.83 μg/mL for the complexes prepared by kneading and lyophilization, respectively. The data showed that these complexes reduced the survival of promastigotes and presented no toxicity for tested cells. Our results indicate that the new compounds could be a cost-effective alternative for use in the pharmacotherapy of leishmaniasis in dogs infected with L. amazonensis.

Duration of spermatogenesis and daily sperm production in the rodent Proechimys guyannensis.

The spiny rat (Proechimys guyannensis) is a neotropical rodent that is used in biomedical research, particularly research related to chronic resistance to epilepsy and infectious diseases. To our knowledge, there are few reports concerning the reproductive biology of this species. Therefore, besides providing basic biometric and morphometric data, in the present study we investigated testis function and spermatogenesis in adult spiny rats. The mean testis weight and gonadosomatic index obtained were 1.63 ± 0.2 g and 1.15 ± 0.1% respectively. Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized. Stages VI and VII presented the highest frequencies (~17-19%), whilst stages II to V showed the lowest frequencies (~2-4%). The most advanced germ cell types labelled at 1 h or 20 days after BrdU injections were respectively preleptotene/leptotene spermatocytes at stage VII and elongated spermatids at stage III. The mean duration of one cycle was 7.5 ± 0.01 days and the entire spermatogenic process lasted 33.7 ± 0.06 days (~4.5 cycles). The seminiferous tubules (ST) occupied ~96 ± 1% of the testis parenchyma, whereas Leydig cells comprised only 1.5 ± 0.4%. The number of Sertoli cells (SC) per testis gram and the SC efficiency (spermatids/SC) were respectively 78 × 106 ± 11 × 106 and 7.9 ± 1. The daily sperm production per testis gram (spermatogenic efficiency; daily sperm production (DSP)/g/testis) was 78 × 106 ± 8 × 106. To our knowledge, this spermatogenic efficiency is among the highest found for mammals investigated to date and is probably related to the very short duration of spermatogenesis and the very high ST percentage and SC number obtained for this species.