Profile of natural Trypanosoma cruzi infection among dogs from rural areas of southern Espírito Santo, Brazil.

BACKGROUND: The emergence of Trypanosoma cruzi infection via oral transmission has a habitual character in its primitive endemic cycle. Recent findings revealed the first death by oral transmission of T. cruzi in the state of Espírito Santo, Brazil, in 2012, which was recorded in the rural area of Guarapari. This study evaluated the characteristics related to the occurrence of natural T. cruzi infection among dogs from the rural areas of Alegre and Iconha, municipalities of Espírito Santo. METHODS: Logistic regression analysis of factors contributing to serological detection of T. cruzi in dogs was performed in environments where Espírito Santo's Department of Health Surveillance had previously notified triatomines positive for Trypanosoma spp. from 2014 to 2017. RESULTS: A total of 36 dogs were analyzed, of which 10 (27.77%) tested positive, one was borderline (2.79%), and 25 tested negative (69.44%) for T. cruzi infection. São Caetano, a district from the Iconha municipality, presented a 25 times greater chance for the detection of positive tests (OR:25; 95% CI; 2.37->100). Dogs with updated mandatory vaccination presented with a lower risk of positive serodiagnosis (OR:0.12; 95% CI: 0.02-0.63). CONCLUSIONS: Our results highlight for the first time the occurrence of natural T. cruzi canine infection, detected in the municipality of Iconha, mainly among dogs with un-updated mandatory vaccines in the district of São Caetano.

Profile of natural Trypanosoma cruzi infection among dogs from rural areas of southern Espírito Santo, Brazil

ABSTRACT Background: The emergence of Trypanosoma cruzi infection via oral transmission has a habitual character in its primitive endemic cycle. Recent findings revealed the first death by oral transmission of T. cruzi in the state of Espírito Santo, Brazil, in 2012, which was recorded in the rural area of Guarapari. This study evaluated the characteristics related to the occurrence of natural T. cruzi infection among dogs from the rural areas of Alegre and Iconha, municipalities of Espírito Santo. Methods: Logistic regression analysis of factors contributing to serological detection of T. cruzi in dogs was performed in environments where Espírito Santo's Department of Health Surveillance had previously notified triatomines positive for Trypanosoma spp. from 2014 to 2017. Results: A total of 36 dogs were analyzed, of which 10 (27.77%) tested positive, one was borderline (2.79%), and 25 tested negative (69.44%) for T. cruzi infection. São Caetano, a district from the Iconha municipality, presented a 25 times greater chance for the detection of positive tests (OR:25; 95% CI; 2.37->100). Dogs with updated mandatory vaccination presented with a lower risk of positive serodiagnosis (OR:0.12; 95% CI: 0.02-0.63). Conclusions: Our results highlight for the first time the occurrence of natural T. cruzi canine infection, detected in the municipality of Iconha, mainly among dogs with un-updated mandatory vaccines in the district of São Caetano.

Detection of Leishmania (Viannia) braziliensis in bats from Espírito Santo, Brazil (2018-2019).

Studying the occurrence of Leishmania in bats would help toward clarifying the role of these animals in epidemiological cycles. Therefore, the objective of this study was to detect Leishmania spp. in bats from the Brazilian State of Espírito Santo. In total, 105 bats from 11 species which had been collected from 17 municipalities of Espírito Santo were obtained from the IDAF Rabies Diagnostic Laboratory between December 2018 and June 2019. DNA was extracted from the bone marrow, skin, and spleen samples and analysed using conventional PCR, with primers specific for the kinetoplastic DNA minicircle of the parasitic species Leishmania (Viannnia) braziliensis, Leishmania (Leishmania) infantum, and Leishmania (Leishmania) amazonensis. To identify the Leishmania species, the positive PCR products were analysed using PCR-RFLP with the HaeIII enzyme and 8% polyacrylamide gel electrophoresis. Positivity for L. (V.) braziliensis was observed in five (4.76%) bats: namely, one male Eumops glaucinus from Vitória (in a skin sample), one male Artibeus lituratus from Vila Velha and one female Eumops glaucinus from Vitória (both in bone marrow samples), and one male A. lituratus from Vitória and one male Molossus rufus from Guarapari (both in the spleen samples). To our best knowledge, this is the first report of the detection of Leishmania in bats from Espírito Santo as well as of Leishmania DNA in the bone marrow of bats.

Seroprevalence and associated factors of infectious bovine rhinotracheitis and bovine viral diarrhea in dairy cows in the Caparaó region, Espírito Santo, Brazil / Soroprevalência e fatores associados à rinotraqueíte infecciosa bovina e diarreia viral bovina em vacas leiteiras na região do Caparaó, Espírito Santo, Brasil

ABSTRACT: This study evaluated the seroprevalence and associated factors of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD), and to analyze the possible relationship between IBR, BVD, and the occurrence of mastitis. For this purpose, 854 crossbred dairy cows were evaluated from 69 properties allocated in the 12 municipalities that make up the Caparaó region, Espírito Santo (ES), Brazil. The seroprevalence of IBR and BVD was determined using the indirect ELISA test. Associations between variables were estimated using the prevalence ratio (PR) with a 95% confidence interval. The chi-square test was used to verify the significance of the associations (P < 0.05). The average prevalence of IBR and BVD was 48.59% and 26.46%, respectively. Animals seroreactive for IBR were more likely to develop subclinical mastitis (P < 0.01; PR: 1.27), and cows that were seroreactive for BVD were more likely to develop clinical mastitis (P < 0.01; PR: 2.24). Mechanical milking was considered a factor associated with IBR (P < 0.01; PR: 1.36) and BVD (P < 0.01; PR: 1.25). Reproductive management by natural breeding was considered a factor associated with IBR (P < 0.01; PR: 1.22). Animals seroreactive for BVD were more likely to develop reproductive problems (P < 0.01; PR: 1.34). It was concluded that the agents that cause IBR and BVD are widely disseminated in dairy cattle herds in the municipalities of the Caparaó region, ES, Brazil. The presence of IBR and BVD increased the chances of cows developing subclinical mastitis and clinical mastitis, respectively, and the cows that were mechanically milked were more likely to be seroreactive for IBR and BVD.

RESUMO: O objetivo do presente estudo foi avaliar a soroprevalência e os fatores de risco associados à Rinotraqueíte Infecciosa Bovina (IBR) e Diarreia Viral Bovina (BVD), e analisar a possível relação entre IBR, BVD e à ocorrência de mastite. Para tanto, foram avaliadas 854 vacas leiteiras mestiças de 69 propriedades localizadas nos 12 municípios que compõem a região do Caparaó, Espírito Santo (ES), Brasil. A soroprevalência de IBR e BVD foram determinadas pelo teste ELISA indireto. As associações entre variáveis ​​foram estimadas pela razão de prevalência (PR) com intervalo de confiança de 95%. O teste do qui-quadrado foi utilizado para verificar a significância das associações (P < 0,05). A prevalência média de IBR e BVD foi de 48% e 26%, respectivamente. Os animais sororreagentes para IBR foram mais propensos a desenvolver mastite subclínica (P < 0,01; PR: 1,27), e as vacas sororreagentes para BVD foram mais propensas a desenvolver mastite clínica (P < 0,01; PR: 2,24). À ordenha mecânica foi considerada um fator associado a IBR (P < 0,01; PR: 1,36) e BVD (P < 0,01; PR: 1,25). O manejo reprodutivo por monta natural foi considerado um fator associado IBR (P < 0,01; PR: 1,22). Os animais sororreagentes para BVD foram mais propensos a desenvolverem problemas reprodutivos (P < 0,01; PR: 1,34). Concluiu-se que os agentes causadores de IBR e BVD estão amplamente disseminados em rebanhos leiteiros nos municípios da região de Caparaó, ES, Brasil. A presença de IBR e BVD aumentaram as chances das vacas desenvolverem mastite subclínica e mastite clínica, respectivamente, e as vacas que foram ordenhadas mecanicamente apresentaram maior probabilidade de serem sororreagente para IBR e BVD.

Physicochemical characterization and in vitro biological evaluation of solid compounds from furazolidone-based cyclodextrins for use as leishmanicidal agents.

The discovery of new drugs and dosage forms for the treatment of neglected tropical diseases, such as human and animal leishmaniasis, is gaining interest in the chemical, biological, pharmaceutical, and medical fields. Many pharmaceutical companies are exploring the use of old drugs to establishing new drug dosage forms and drug delivery systems, in particular for use in neglected diseases. The formation of complexes with cyclodextrins is widely used to improve the stability, solubility, and bioavailability of pharmaceutical drugs, as well as reduce both the toxicity and side effects of many of these drugs. The aim of this study was to characterize solid compounds obtained from the association between furazolidone (FZD) and ß-cyclodextrin (ß-CD) or hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The solid compounds were prepared in molar ratios of 1:1 and 1:2 (drug:CD) by kneading and lyophilization. Molecular docking was used to predict the preferred relative orientation of FZD when bound in both studied cyclodextrins. The resulting solid compounds were qualitatively characterized by scanning electron microscopy (SEM), thermal analysis (DSC and TG/DTG), X-ray diffraction (XRD), Raman spectroscopy with image mapping (Raman mapping), and 13C nuclear magnetic resonance spectroscopy (13C NMR) in the solid state. The cytotoxicity of the compounds against THP-1 macrophages and the 50% growth inhibition (IC50) against Leishmania amazonensis promastigote forms were subsequently investigated using in vitro techniques. For all of the solid compounds obtained, the existence of an association between FZD and CD were confirmed by one or more characterization techniques (TG/DTG, DSC, SEM, XRD, RAMAN, and 13C NMR), particularly by a significant decrease in the crystallinity of these materials and a reduction in the melting enthalpy associated with furazolidone thermal events. The formation of more effective interactions occurred in the compounds prepared by lyophilization, in a 1:2 molar ratio of the two CDs studied. However, the formation of an inclusion complex was confirmed only for the solid compound obtained from HP-ß-CD prepared by lyophilization (LHFZD1:2). The absence of cytotoxicity on the THP-1 macrophage lineages and the leishmanicidal activity were confirmed for all compounds. MHFZD1:2 and LHFZD1:2 were found to be very active against promastigote forms of L. amazonensis, while all others were considered only active. These results are in line with the literature, demonstrating the existence of biological activity for associations between drugs and CDs in the form of complexes and non-complexes. All solid compounds obtained were found to be promising for use as leishmanicidal agents against promastigote forms of L. amazonensis.

Physicochemical and in vitro biological evaluations of furazolidone-based ß-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 ß-cyclodextrin (ß-CD) to guarantee increased drug solubility and reduce the toxicity associated with high doses. The FZD:ß-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 ß-CD cavity after complexation by both methods. Biological in vitro evaluations demonstrated that free FZD and the FZD:ß-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.

The spiny rat Proechimys guyannensis (Rodentia: Echimydae) fails to respond to intradermal inoculation with Leishmania (Viannia) braziliensis / O rato espinhoso Proechimys guyannensis (Rodentia: Echimydae) falhou para inoculação intradérmica com Leishmania (Viannia) braziliensis

Leishmaniasis a disease of worldwide occurrence is caused by protozoa of the Leishmania genus. In Brazil, Leishmania (Viannia) braziliensis is the main parasite responsible for the American cutaneous leishmaniasis. Main hosts of this protozoa are small wild mammals particularly marsupials and rodents. The aim of this study was to evaluate if spiny rat Proechimys guyannensis (Rodentia: Echimydae) has role in the cycle of the American cutaneous leishmaniasis caused by L. (V.) braziliensis. Thus, promastigotes (the flagellate stage) of Leishmania (Viannia) braziliensis were used to inoculate seven spiny rats (Proechimys guyannensis). After inoculated intradermal at the ear pinna, nose and plantar pad, the rats were monitored for 180 days. Tissue samples collected at 90 and 180 days from the rats proved to be negative for the presence of genetic material from the parasite. After euthanasia, the protozoa also failed to growth in culture medium containing tissue samples collected from the rats showing that there was no infection. These results fail to prove that spiny rat has a role in the cycle of the American cutaneous leishmaniasis caused by L. (V.) braziliensis.

A leishmaniose é uma doença de ocorrência mundial causada por protozoários do gênero Leishmania. No Brasil, a Leishmania (Viannia) braziliensis é o principal parasita responsável pela leishmaniose tegumentar americana. Os principais hospedeiros deste protozoário são pequenos mamíferos selvagens em particular marsupiais e roedores. O objetivo deste estudo foi avaliar o papel do rato espinhoso Proechimys guyannensis (Rodentia: Echimydae) no ciclo da leishmaniose tegumentar americana. Para isto, formas promastigotas (estágio flagelado) de Leishmania (Viannia) braziliensis foram inoculadas em sete ratos espinhosos (Proechimys guyannensis). Após a inoculação intradérmica no pavilhão auricular, focinho e área plantar, os ratos foram monitorizados durante 180 dias. Amostras de tecido colhidas aos 90 e 180 dias dos ratos revelaram-se negativas para a presença de material genético do parasita. Após eutanásia, tecidos coletados dos ratos também falharam para crescimento em meio de cultura demonstrando que não houve infecção. Estes resultados demonstram que o rato espinhoso não tem papel no ciclo da leishmaniose tegumentar americana causada por L. (V.) braziliensis.

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.

Zoological techniques parameters of Proechimys guyannensis(Rodentia, Echimydae) bred in captivity / Parâmetros zootécnicos de Proechimys guyannensis (Rodentia, Echimydae) criados em cativeiro

The rodents Proechimys guyannensis have led a great interest because they are considered as hosts to several pathogens causing zoological diseases. Therefore, it is important to know their biological and physiological parameters. The age at first cubs delivery and the average of birth interval per female were evaluated. The estimated age at puberty was 83 days. The number of pups born per delivery did not differ significantly when associated with the female mating age. However, an increase in the average mortality rate correlated with the increase in delivery number per female was detected. There is no statistical difference neither in the litter size in different seasons of the year, nor in the body weight between sexes for all ages. Differences were found in the daily water consumption and in the feed intake in animals with approximately 100 days of age, perhaps owing to the increased need for energy intake due to sexual activity or even because they had not reached adult weight yet. The parameters assessed in the present study are of relevance for maintaining this species in captivity as a laboratory animal.

Roedores da espécie Proechimys guyannensis têm motivado grande interesse por serem frequentementeconsiderados como hospedeiros de diversos agentes patogênicos causadores de doenças. É importanteestabelecer seus parâmetros biológicos e fisiológicos; neste contexto, foram avaliadas a idade ao primeiroparto e a média de intervalo de natalidade por fêmea. A idade à puberdade foi estimada em 83 dias.O número de filhotes nascidos por parto não diferiu significativamente com a idade das fêmeas aoacasalarem. Observou-se aumento da taxa média de mortalidade de acordo com o aumento do númerode partos por fêmea. Não houve diferença estatística no tamanho da ninhada em diferentes estações doano; tampouco houve diferença de peso entre os sexos. Foram detectadas diferenças no consumo diáriode água e no consumo de ração em animais com menos e mais de 100 dias de idade, talvez em funçãode aumento da necessidade de consumo de energia pela atividade sexual ou mesmo por ainda não teremalcançado o peso corporal de adulto. Os parâmetros apresentados são relevantes para a futura manutençãodesta espécie em cativeiro como animal de laboratório.

Óleo de aroeira vermelha e de suplementação de vitamina E em substituição aos promotores de crescimento sobre a microbiota intestinal de frangos de corte / Supplementation of pink pepper oil and vitamin E in replacement to antibiotic growth promoters in the broilers diet on response of ileal microbiota

Objetivou-se avaliar o uso de óleo de aroeira-vermelha (OAV) e da suplementação de vitamina E (VitE) sobre a microbiota intestinal de frangos de corte. Utilizou-se 400 pintos de corte distribuídos em um delineamento inteiramente casualizado, em grupos de cinco tratamentos e cinco repetições: dieta sem antimicrobiano; dieta com antimicrobiano; dieta com 0,4% OAV; dieta com 200mg de VitE kg-1; dieta com 0,4% OAV e 200mg de VitE kg-1. A adição de OAV reduziu o peso relativo dos intestinos (P<0,05). Verificou-se que o controle negativo teve a menor contagem de bactérias do gênero Lactobacillus spp. em contraste com os demais grupos (P<0,05). Observou-se que o uso de OAV com ou sem adição de VitE reduziu significativamente a contagem de bactérias do gênero Staphylococcus spp e E. coli, quando comparado com o grupo não tratado com o promotor de crescimento (P<0,05). Para os Staphylococcus spp coagulase-positivos, verificou-se que a concentração inibitória mínima foi de 14,72-117,75mg mL-1 para amostras isoladas de frangos de corte tratados com OAV + VitE, enquanto que para tratados com ou sem promotor de crescimento foi de 117,75 e de 29,44-235,5mg mL-1. Concluiu-se que a inclusão de OAV resultou em modulação da microbiota intestinal.

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.

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

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-ß-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 72 h 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.

Identificação bioquímica e molecular de Lactobacillus spp. isolados do íleo de frangos de corte tratados ou não com antimicrobianos / Biochemical and molecular characterization of Lactobacillus spp. isolated from the ileum of broilers treated with or without antimicrobials

Objetivou-se caracterizar bioquimicamente e molecularmente as espécies de Lactobacillus spp. isoladas do íleo de frangos de corte tratados ou não com antimicrobianos. Utilizou-se 400 pintos de corte alojados em 25 boxes de 2m² (16 aves/boxe), distribuídos em um delineamento inteiramente casualizado, em grupos de cinco tratamentos e cinco repetições: dieta sem promotor de crescimento; dieta com promotor de crescimento; dieta com 0,4% de óleo de aroeira-vermelha (OAV); dieta com 200mg de vitamina E kg-1; dieta com 0,4% OAV e 200mg de vitamina E kg-1. Após a caracterização fenotípica do gênero Lactobacillus, foram identificadas, em ambas as metodologias, 100 amostras de Lactobacillus spp. sendo 20 amostras por tratamento. Os resultados bioquímicos identificaram L. acidophilus, L. fermentum, L. plantarum, L. delbrueckii subsp. delbrueckii além de Lactococcus lactis subp. lactis. Para as amostras padrão ATCC, a identificação bioquímica suscitou algumas dúvidas em relação aos seus resultados. Os resultados da identificação molecular mostraram que os Lactobacillus que amplificaram ambos os iniciadores (LU-1'/ Lac-2) e (Laci-1 / 23-10C) são os da espécie L. acidophilus. As amostras que amplificaram apenas com o iniciador (LU-1'/ Lac-2) tratam-se das demais espécies que compõe o grupo L. acidophilus. Já as amostras com o iniciador L. fermentum (Fer 3/Fer 4) amplificaram um fragmento de 192pb padrão para essa espécie. Conclui-se que a identificação das espécies de Lactobacillus spp. isoladas do íleo a partir da PCR apresentou-se mais sensível que o método bioquímico.

This study aimed to characterize biochemically and molecular species of Lactobacillus spp. isolated from the ileum of broiler chickens treated with or without antimicrobial. A total of 400 day-old male chicks, Cobb, distributed in a randomized design in groups of five treatments and five replicates: diet without antimicrobials; diet with antimicrobials; diet with 0.4% Brazilian red pepper oil (BRP); diet with 200mg vitamin E kg-1; diet with 0.4% BRP and 200mg vitamin E kg-1. The biochemical results have identified L. acidophilus, L. fermentum, L. plantarum, L. delbrueckii subsp. delbrueckii besides Lactococcus lactis subp. Lactis. For samples ATCC, the biochemical identification raised some doubts about their results. The molecular identification showed that Lactobacillus that amplified both primers (LU-1 '/ Lac-2) and (Laci-1 / 23-10C) are the species L. acidophilus. However, samples that amplified only with primer (LU-1 '/ Lac-2) are other species that constitute the group L. acidophilus. However, the samples with the primer L. fermentum (Fer 3/Fer 4) amplified a 192pb fragment pattern for this species. We conclude that the identification of species of Lactobacillus isolated from the ileum of PCR was more sensitive than the biochemical method.

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.

Mecânica e microbiologia de placas produzidas a partir de osso cortical bovino, conservadas em diferentes meios / Mechanic and microbiology of plates produced from bovine cortical bone, conserved in several means

Objetivou-se com este trabalho avaliar a resistência mecânica de placas ósseas produzidas a partir de osso cortical bovino, conservadas em diferentes meios, e a eficiência desses meios em inibir o crescimento de microrganismos. Foram utilizadas 168 placas confeccionadas a partir de tíbia bovina e conservadas em glicerina a 98 por cento, solução salina a 150 por cento, solução de açúcar a 300 por cento, líquido de Dakin, congeladas em N2L a -196°C, ou esterilizadas em óxido de etileno. Após reidratação em solução de cloreto de sódio a 0,9 por cento por seis horas, elas foram submetidas a ensaios mecânicos de tração, compressão, flexão e torção. Foi realizada avaliação microbiológica das placas anterior e imediatamente após a reidratação, com ou sem adição de enrofloxacina a 0,5 por cento. Não se observou diferença significativa (P<0,01 por cento) da resistência ao ponto de ruptura entre as placas conservadas nos diferentes meios, congeladas-descongeladas ou esterilizadas. Micro-organismos foram isolados das placas conservadas em solução saturada de sal, açúcar, Dakin ou congeladas em N2L, anterior e após a reidratação. Entretanto, após o uso da solução de cloreto de sódio 0,9 por cento, acrescida de enrofloxacina a 0,5 por cento, não foram isolados microrganismos. Portanto, placas de osso bovino, conservadas em solução saturada de açúcar ou sal, glicerina a 98 por cento, solução de Dakin, congeladas em N2L ou esterilizadas em óxido de etileno assemelham-se em relação à biomecânica e a reidratação com solução de cloreto de sódio a 0,9 por cento, acrescida de enrofloxacina a 0,5 por cento, é eficaz no controle do crescimento microbiano.

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 microorganism's growth. A hundred and sixty eight plates yielded from bovine tibiae were conserved in 98 percent glycerin, 150 percent saline solution, 300 percent sugar solution, Dakin solution, frozen in N2L at - 196°C, or sterilized in ethylene oxide gas. After rehydration in NaCl 0.9 percent 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 percent addition. There was no significant difference (P<0.01 percent) 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 percent solution added of enrofloxacin 0.5 percent, microorganisms were not isolated. Therefore, bovine bone plates conserved in sugar or salt satured solution, 98 percent 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 percent plus enrofloxacin 0.5 percent is effective on microorganism growth control.

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.

Intestinal mucosa structure of broiler chickens infected experimentally with Eimeria tenella and treated with essential oil of oregano / Morfometria intestinal de frangos de corte infectados experimentalmente com Eimeria tenella e tratados com óleo essencial de orégano

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).

Inicialmente, foram utilizados, neste estudo, 250 pintos de um dia de idade distribuídos em cinco tratamentos: dieta com promotor de crescimento; dieta sem promotor de crescimento; dieta contendo somente antibiótico; dieta com 0,5g de orégano óleo kg de ração-1 ou com 1,0g de orégano óleo kg de ração-1. No outro ensaio, foram utilizados 288 pintos de um dia de idade distribuídos em quatro grupos: dieta com anticoccidiano; dieta sem anticoccidiano; dieta com 0,5g de orégano óleo kg dieta-1 ou 1,0g de orégano óleo kg de ração-1. No primeiro ensaio, o grupo tratado sem promotor de crescimento apresentou a maior profundidade de cripta quando comparada com os animais tratados com promotor de crescimento ou com 0,5 e 1,0g de orégano óleo kg de ração-1. Os frangos que receberam a dieta com promotor de crescimento (antibiótico+anticoccidiano) apresentaram uma maior relação vilo:cripta em comparação com os frangos do controle negativo, os quais tiveram a menor relação vilo:cripta e uma maior excreção de oocistos por grama de fezes (P<0.05). No segundo ensaio, observou-se que os frangos alimentados com dieta sem anticoccidiano tiveram uma maior espessura de lâmina própria cecal, diferindo dos frangos tratados com anticoccidiano ou com 1,0 de orégano óleo kg de ração-1 (P <0,05).

Identification of an IS6110 insertion site in plcD, the unique phospholipase C gene of Mycobacterium bovis.

The IS6110 repetitive element is present in multiple copies in most Mycobacterium tuberculosis complex bacteria, except for Mycobacterium bovis strains, which usually contain a single copy of IS6110 located on a 1.9 kb PvuII fragment of the direct repeat region. IS6110 transposition can disrupt coding regions and is a major force of genomic variation. In a previous work it was demonstrated that phospholipase C genes are preferential loci for IS6110 transposition in M. tuberculosis clinical strains. Bacterial phospholipase C enzymes participate in pathogenic mechanisms used by different organisms, and have been implicated in intracellular survival, cytolysis and cell-to-cell spread. Four phospholipase C genes (plcA, plcB, plcC and plcD) were detected in the genomes of M. tuberculosis, Mycobacterium africanum, Mycobacterium microti and 'Mycobacterium canettii'. M. bovis and the vaccine strain M. bovis Bacillus Calmette-Guérin contain only the plcD gene. In the present work, the existence of IS6110 insertions within plcD, the unique phospholipase C gene of M. bovis, has been investigated by PCR, Southern blot hybridization and sequencing analysis. In 18 (7.3 %) of 245 isolates analysed, the plcD gene was interrupted by the insertion of one copy of IS6110, which in all cases was transposed in the same orientation and at the same position, 1 972 894, relative to the genome of M. bovis AF2122/97. These 18 isolates were distributed in 6 different spoligotype patterns and contained 4 to 8 IS6110 copies. In contrast, strains showing an intact plcD gene contained one (87 %), two (9.4 %) or three (2.4 %) IS6110 copies, and only a single isolate (1.2 %) had four IS6110 copies. The implications of plcD gene disruption in M. bovis have not been fully investigated, but no differences in the organ distribution of the disease were detected when animals infected with strains from the same spoligotype patterns bearing plcD : : IS6110 and intact plcD were compared.