Molecular and pathogenic variability of Drechslera isolates from oats

Severe epidemics of leaf blotch and black leaf spot of oat (Avena sativa) caused by Drechslera avenae and Drechslera sp., respectively, are frequently observed in the State of Paraná, Brazil. Although some morphological differences between the isolates causing two different symptoms were noticed, the genetic relationship between them was not clear. Twenty-four isolates of D. avenae and Drechslera sp, collected between 1996-98, were assessed for the genetic variability by molecular and pathogenic analyses. The amplification products using primer pair ITS4/ITS5 showed a fragment length of approximately 600 bp for all the isolates except for one black spot isolate, where the fragment length was approximately 550 bp. Restriction enzymes Hinf I and Taq I, that cut in the ITS region, produced similar restriction patterns for all the isolates, whereas four others produced variable restriction patterns. RAPD analysis also showed distinctive patterns for some isolates. No clear difference between the black spot and the leaf blotch isolates was observed either by the molecular or by the pathogenicity analysis. Nonetheless, the rDNA analysis suggests that Drechslera probably comprises at least three distinct taxa. The results indicate that the difference observed between the isolates originating from two types of symptoms is due to intra-specific variants of D. avenae.

Potential of non-pathogenic Fusarium oxysporum isolates for control of Fusarium wilt of tomato

This study was done to evaluate the efficiency of non-pathogenic Fusarium oxysporum isolates (141/3, 233, 233/1, 245, 245/1, 251, 251/2, 251/5, and 257) in controlling vascular wilt caused by F. oxysporum f. sp. lycopersici, race 2 (isolates C-21A, TO11, and TO245) in tomato (Lycopersicon esculentum) cv. Viradoro seedlings. In order to determine the effect of non-pathogenic F. oxysporum isolates in tomato plants, the root system of 30-day-old seedlings was immersed in conidial suspensions (10(6) ml-1) of each isolate and the seedlings were transplanted to a cultivation substrate. Thirty-five days after transplanting it was observed that the non-pathogenic F. oxysporum isolates were not pathogenic to the cv. Viradoro nor did they affect seedling development. The efficiency of the non-pathogenic F. oxysporum isolates in controlling Fusarium wilt was determined by immersing the tomato seedling roots in the conidial suspension (10(6) ml-1) of each isolate and then transplanting them into substrates previously infested with isolates of F. oxysporum f.sp. lycopersici, race 2 (10(5) conidia ml-1 of substrate). Evaluations were performed 35 days after transplanting, for severity in scale with 1=healthy plant to 6=dead plant or plant showing vessel browning and wilted leaves up to the leader shoot and seedling height. The non-pathogenic F. oxysporum isolates were efficient in reducing the severity of the disease and maintaining normal plant development. These results provide evidence of the antagonistic activity of non-pathogenic F. oxysporum isolates in controlling vascular wilt caused by F. oxysporum f. sp. lycopersici race 2 in tomato.

The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

Swine manure post-treatment technologies for pathogenic organism inactivation

Swine manure agricultural use is a common practice in Brazil. Their physic-chemical characteristics favor its use as biofertilizer, but the presence of pathogens may become a risk to human health. This research presents a qualitative study of the main alternatives of pig manure disinfection, analyzing efficiency, advantages and limitations of each procedure. The disinfection studies reported in literature are based on the following treatments: alkaline, thermal, biological, chemical, and physical. The greater efficiencies are in thermal treatment (> 4 log: 60 °C), chemical treatment (3 to 4 log: 30mg Cl- L-1; 3 to 4 log: 40 mg O3 L-1) and physical treatment (3 a 4 log: 220 mJ UV radiation cm-2). The biological treatment (anaerobiosis) also promotes the pathogen reduction of swine manure, however with lower efficiency (1 to 2 log). The selection of the treatment should consider: implementation and operation cost, necessity of preliminary treatment, efficiency obtained and destination of the treated manure (agricultural use, water reuse). Brazilian regulation does not have specific guidelines for the microbiological quality of animal production effluents that is very important to be considered due to confined animal feeding operation transformation in the last years in the country.

Resposta humoral, recuperação bacteriana e lesões histológicas em camundongos geneticamente selecionados para bons e maus produtores de anticorpos e Balb/c, frente à infecção por Leptospira interrogans sorovar icterohaemorrhagiae

O presente trabalho teve por finalidade associar a cinética da resposta humoral à recuperação de leptospiras viáveis e à intensidade das lesões teciduais em camundongos geneticamente selecionados para bons (High) e maus (Low) produtores de anticorpos (seleção IV-A), além de camundongos outbreed, Balb/c, inoculados com amostra patogênica de Leptospira interrogans sorovar icterohaemorrhagiae. As linhagens High e Low (seleção IV-A) apresentam modificações em alguns compartimentos da resposta imune, principalmente em relação à atividade macrofágica, representando fenótipos extremos encontrados em populações naturais heterogêneas. Os camundongos foram sacrificados em oito momentos após a infecção. A análise dos resultados revelou que a partir do 7º dia após a infecção, os camundongos da linhagem High apresentaram elevação nos títulos de anticorpos estatisticamente significantes quando comparados aos camundongos da linhagem Low, mantendo assim o efeito multiespecífico. Os camundongos Balb/c apresentaram resultados intermediários entre as duas linhagens. A produção de anticorpos colaborou como fator limitante à infecção, pois quando obteve-se maior recuperação de leptospiras, na fase inicial da infecção, os títulos de anticorpos encontravam-se em elevação. As lesões observadas nos órgãos de camundongos infectados consistiram basicamente nos mesmos processos inflamatórios e degenerativos, que não se alteraram, variando apenas o grau de comprometimento tecidual, de acordo com a linhagem. A linhagem high apresentou lesões mais extensas que as apresentadas pelas linhagens low e Balb/c, sendo que nesta última as lesões foram moderadas. De forma geral a linhagem High e Balb/c apresentaram um perfil de resposta Th2, com o maior índice de produção de anticorpos e gravidade das lesões, enquanto a linhagem Low apresentou um perfil de resposta Th1.

Typing of avian pathogenic Escherichia coli strains by REP-PCR

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Seroprevalence of Leptospira spp in cattle from Monte Negro municipality, western Amazon

The prevalence of anti-Leptospira spp antibodies was investigated in 2,109 female cattle from 86 herds of Monte Negro municipality, Rondônia, Brazil. Sera samples were evaluated by Microscopic Agglutination Test against 24 leptospira serovars. Titers =100 for at least one of 24 leptospira serovars were detected in 1,114 cows (52.8%) from 82 (95.3%) herds. The adjusted overall prevalence for Monte Negro municipality was 53.9% (49-58.7%; CI: 95%). The most prevalent serovars were Hardjo (14.5%), Wolffi (12.3%), Shermani (10.8%), Patoc (7.9%), and Hebdomadis (6.1%). Other serovars worldwidely reported like Bratislava, Pomona and Grippotyphosa were detected in low levels.

Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC)

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.

Virulence factors of avian pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.

Anticoccidial effects of acetic acid on performance and pathogenic parameters in broiler chickens challenged with Eimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of the different concentrations of the acetic acid in the broiler chickens in comparison with the amprolium anticoccidial. A total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations (1%, 2% and 3%) of acetic acid and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10-19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. All the groups were inoculated orally with 75,000 sporulated oocysts at the 12th day of age except non infected non medicated control. Anticoccidial effect was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality %age) parameters. Among acetic acid medicated groups, the maximum anticoccidial effect was seen in the group medicated with 3% acetic acid followed by 2% and 1% acetic acid medicated groups. Amprolium and 3% acetic acid were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, acetic acid has the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. Concentration-dependent anticoccidial effect of acetic acid suggests that further studies should be carried out to determine the possible maximum safe levels of acetic acid with least toxic effects to be used as anticoccidial.

Pesquisa de aglutininas, antígeno de leptospiras e apoptose em rim de suínos naturalmente infectados por Leptospira spp.

Soro e rim de 75 suínos mestiços sem raça definida, criados em sistema extensivo e abatidos em Timon (MA) e Teresina (Piauí), Brasil, duas cidades separadas pelo Rio Parnaíba e 75 suínos mestiços de um sistema de criação em confinamento, filhos de Landrace, Large White e/ou Duroc foram utilizados neste estudo. Das 150 amostras analisadas pela prova de Soroaglutinação Microscópica (SAM), sete foram reagentes e o sorovar Icterohaemorrhagiae (42,86%) foi o mais frequente. Uma comparação entre os dois sistemas para examinar uma predisposição para infecção para Leptospira spp. mostrou que a suscetibilidade foi maior nos animais criados extensivamente do que naqueles criados em confinamento (teste χ2, p<0,05). A presença de infiltrado inflamatório foi significantemente maior nos animais soropositivos comparados aos soronegativos (p<0,05, Teste U de Mann-Whitney). A análise morfométrica mostrou Leptospira spp. e o antígeno de leptospira apenas nos animais soropositivos (p<0,05, teste de U de Mann-Whitney). Apoptose em células epiteliais tubulares foi significantemente mais evidente nos animais infectados comparados aos não infectados (p<0,05, Teste U de Mann-Whitney). Uma eventual associação de antígeno de Leptospira e células epiteliais em apoptose sugere um provável mecanismo de lesão renal na leptospirose suína.

Cinética da resposta imune humoral de cães jovens imunizados contra Leptospira interrogans

Objetivou-se analisar a resposta imune humoral contra Leptospira interrogans mediante a utilização da prova de soroaglutinação microscópica (SAM) em 26 cães jovens, sendo 17 de raça definida (Grupo A) e nove sem raça (Grupo B), de ambos os sexos, pertencentes a canis e ambientes domiciliares em Uberlândia, Minas Gerais. Os cães foram vacinados com bacterina inativada comercial polivalente com os sorovares Canicola, Grippotyphosa, Icterohaemorrhagiae e Pomona. O esquema vacinal baseou-se em três imunizações. A primovacinação foi realizada aos quarenta e cinco dias de idade, considerado dia zero, e após dois reforços com intervalos de trinta dias cada. Sete colheitas sanguíneas de cada cão foram efetuadas do dia zero até aos 180 dias pós-vacinais, com intervalos de trinta dias cada. Não foram detectados títulos pré-vacinais para os sorovares Canicola, Icterohaemorrhagiae e Grippotyphosa no dia zero. Apenas um cão do grupo A foi reagente com título 1:100 contra o sorovar Pomona na primeira colheita. Não houve diferença estatística entre os títulos de anticorpos aglutinantes entre os Grupos A e B (p>0,05), induzidos pela bacterina comercial utilizada, exceto na colheita II (p<0,05), na qual o grupo B apresentou títulos para o sorovar Autumnalis em todos os cães avaliados, enquanto que, no grupo A, 64,7% dos cães não foram reagentes a nenhum sorovar testado. No dia 30, títulos para o sorovar Autumnalis que persistiram até os 180 dias pós-vacinais, em ambos os grupos, variando apenas a intensidade da resposta imunológica sem diferença estatística significativa. Para avaliação da eficiência vacinal da bacterina anti-Leptospira, a presente pesquisa alerta para os ricos à infecção que os cães vacinados anualmente estão submetidos.

Ocorrência de anticorpos e fatores de risco associados à infecção por Leptospira spp. em Cebus spp. mantidos em cativeiro no Nordeste do Brasil

Estudou-se a ocorrência de anticorpos anti-Leptospira e os fatores de risco associados à infecção em primatas do gênero Cebus mantidos em cativeiro no Nordeste do Brasil. Foram analisadas 139 amostras de soro sanguíneo de diferentes espécies de primatas de ambos os sexos e idades variadas. Para a pesquisa de anticorpos empregou-se a técnica de Soroaglutinação Microscópica (SAM) e para estudo dos fatores de risco utilizou-se análise multivariada. Foram observados anticorpos anti-Leptospira, sorovar Patoc em 6,5% dos primatas (09/139). O fator de risco identificado nesse estudo foi a não higienização diária de bebedouros (Odds ratio=12,095; IC 95% = 1,73-84,52; p=0,012,). Conclui-se que a população de Cebus mantidos em cativeiros no Nordeste do Brasil está exposta à infecção por anticorpos anti-Leptospira e que medidas corretivas de manejo dos animais, especificamente aquelas relacionadas à higienização diária de bebedouros devem ser implementadas para reduzir o risco de infecção por sorovares de Leptospira spp.

Prevalence and risk factors for Leptospira spp. in cattle herds in the south central region of Paraná state

The aim of this study was to determine the prevalence of anti-Leptospira spp. antibodies and the risk factors for Leptospira spp. infection in breeding cattle herds in the south central region of Paraná state. It was based on the statistic delineation/serological samples and information regarding the selected farms employed in the study of bovine brucellosis for Paraná state in the context of National Program for Control and Eradication of Brucellosis and Tuberculosis. A total of 1.880 females aged >24 months from 274 non vaccinated herds were studied. Serum samples were tested for antibodies against Leptospira spp. using microscopic agglutination test (MAT) with 22 Leptospira serovars. The epidemiological questionnaire was applied on all the selected farms and aimed to obtain epidemiological data. Hundred eighty one of 274 herds were positive for Leptospira spp./presenting prevalence of positive herds of 66.06% (IC95%=60.12-71,65%). Presence of >43 cattle (OR=3.120; IC=1.418-6.867)/animal purchase (OR=2.010; IC=1.154-3.500)/rent of pastures (OR=2.925; IC=1.060-8.068) and presence of maternity paddock (OR=1.981; IC=1,068-3,676) were identified as risk factors for leptospirosis due to any serovar in the multivariate logistic regression. Risk factors for leptospirosis due to serovar Hardjo were presence of >43 cattle (OR=3.622; IC=1.512-8,677)/animal purchase (OR=3.143; IC=1.557-6.342)/rent of pastures (OR=4.070; IC=1.370-12.087) and presence of horses (OR=2.981; IC=1.321-6.726). These results indicate that Leptospira spp. infection is widespread in the south central region of Paraná state and that factors related to the herd characteristic and management are associated with the infection.

Frequency of Leptospira spp. in sheep from Brazilian slaughterhouses and its association with epidemiological variables

Leptospirosis is a worldwide anthropozoonosis that infects livestock, including sheep as the carriers to other animals and humans. The present study aimed to determine the prevalence of Leptospira spp. in sheep from two slaughterhouses in the state of São Paulo, Brazil and its association with epidemiological variables. Serum samples from 182 sheep were evaluated for Leptospira spp. antibodies by microscopic agglutination test (MAT). Results indicated 34/182 (18.68%; CI95% 13.70-24.98%) positive serum samples, mainly to the serovar Copenhageni (17/34; 50%; CI95% 33.99-66.01%). Bacterial growth in the Fletcher medium was detected for 13/34 (38.24%; CI95% 23.87-55.08%) animals, and confirmed by Polymerase Chain Reaction (PCR) and sequencing for only two kidney samples from two animals. Thus, treatment and vaccination of sheep, besides rodent control, can be useful to prevent the infection in the studied region since sheep are important Leptospira spp. carriers, and its transmission to slaughterhouse workers is mainly through the manipulation of visceral tissues.