Utilização da vacina Escherichia coli J5 na imunização de vacas leiteiras contra mastites causadas por E. coli / Use of an Escherichia coli J5 vaccine on immunization of dairy cows against mastitis caused by E. coli

Avaliou-se a utilização da vacina Escherichia coli J5, na imunização de vacas leiteiras, para prevenção e controle da mastite causada por E. coli através da análise da prevalência das infecções intramamárias (IMM) no pós-parto, ocorrência e intensidade dos casos clínicos de mastite nos primeiros 100 dias de lactação, influência na contagem de células somáticas (CCS) e produção de leite. O grupo experimental foi composto de 187 animais, divididos em 2 grupos, vacas vacinadas e não vacinadas. As imunizações ocorreram 60 dias antes do parto, 30 dias antes do parto e na primeira semana pós-parto. No dia da secagem e sete dias após o parto foram coletadas amostras para diagnóstico microbiológico dos patógenos causadores de mastite. A ocorrência de casos clínicos foi verificada pelo teste da caneca durante as ordenhas sendo registrados os dados relacionados à intensidade. Amostras foram coletadas mensalmente, a partir do décimo dia de lactação, para avaliação da CCS. A produção de leite foi registrada mensalmente nos primeiros 100 dias de lactação. Verificou-se no grupo vacinado, redução na prevalência de E. coli no pós-parto, na ocorrência de casos clínicos por E.coli nos primeiros 100 dias de lactação bem como na intensidade destes casos clínicos. Não foram observadas diferenças estatísticas significativas na CCS, entretanto vacas vacinadas apresentaram maior produção de leite, comparadas às vacas não vacinadas. A vacinação com E. coli J5 foi eficaz em reduzir a prevalência de infecções intramamárias (IMM) ao parto, ocorrência e intensidade dos casos clínicos e aumento na produção de leite nos primeiros 100 dias de lactação.

We evaluated the use of Escherichia coli J5 vaccine immunization in dairy cows for the prevention and control of mastitis caused by E. coli. Were analyzed the prevalence of postpartum intramammary infections (IMM), occurrence and severity of clinical cases of mastitis in the first 100 days of lactation, influence on somatic cell count (SCC) and milk production. The experimental group consisted of 187 animals that were divided into two groups, vaccinated and unvaccinated cows. Immunizations occurred 60 days before calving, 30 days before calving and the first week postpartum. On the day of drying and seven days after birth, samples were collected for microbiological diagnosis of mastitis pathogens. The occurrence of clinical cases was verified by testing during the milking and data was recorded related to the intensity and duration. Samples were collected monthly starting at day ten of lactation to evaluate the CCS. Milk production was recorded monthly for the first 100 days of lactation. It was found in the vaccinated group reduction in the prevalence of E. coli in the postpartum period, the occurrence of clinical cases of E. coli in the first 100 days of lactation as well the intensity of these clinical cases. There were no statistically significant differences in CCS, however vaccinated cows had higher milk production, compared to unvaccinated cows. Vaccination with E. coli J5 was effective in reducing the prevalence of intramammary infections (IMM) at calving, occurrence and intensity of clinical cases and an increase in milk production in the first 100 days of lactation.

From cholera to enterotoxigenic Escherichia coli (ETEC) vaccine development.

It was shown earlier that immune responses against cholera toxin (CT) as well as Vibrio cholerae lipopolysaccharide (LPS) or whole bacterial cells (WC) were protective and that these different antibody specificities co-operated synergistically for protection against experimental cholera. Similarly, antibodies against the heat-labile toxin (LT) and major colonization factors (CFs) of enterotoxingenic Escherichia coli (ETEC) co-operated synergistically for protection against LT-producing ETEC expressing homologous CFs. Studies in humans revealed that repeated oral antigen administration was optimal in inducing intestinal immune responses. Based on these findings oral inactivated vaccines consisting of toxin antigen and whole cells, i.e. the licensed recombinant cholera B subunit (rCTB)-WC cholera vaccine Dukoral®, and candidate ETEC vaccines have been developed. In different trials the rCTB-WC cholera vaccine has provided very high (85-100%) short term protection, which was significantly higher than that induced by the WC component alone, whereas rCTB-WC and WC alone provided comparable (50-60%), long term protection. An oral ETEC vaccine consisting of rCTB and formalin-inactivated E. coli bacteria expressing major CFs was shown to be safe and immunogenic in adults and children in different countries. The vaccine also induced significant protection against non-mild ETEC diarrhoea, i.e. diarrhoea interfering with daily activity in American travellers but not against ETEC diarrhoea in young children in Egypt. Against this background, a modified ETEC vaccine consisting of recombinant E. coli strains overexpressing the major CFs and a more LT like hybrid toxoid (LCTBA) has been developed. This vaccine will be tested soon alone and together with a mucosal adjuvant, i.e. dmLT, in clinical trials.

Construction and immunization of an attenuated vaccine candidate enteropathogenic Escherichia coli O45 / 生物工程学报

In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(deltaler). The culture supernatant of PEPEC O45(deltaler) deletion mutant was inoculated in vero cell culture. PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(deltaler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(deltaler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(deltaler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.

Cloning, expression and immunity of pilA gene and ompC gene from avian pathogenic Escherichia coli / 生物工程学报

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.

Construction and immunization of a enterohemorrhagic Escherichia coli O157 attenuated / 生物工程学报

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important pathogen. One of the important virulence traits of EHEC O157:H7 is the capacity to produce attaching and effacing (A/E) lesions on enterocyte. This property encoded by a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE contains ler (LEE-encoded regulator) gene. The product of ler is a central up-regulator of many virulence genes of the LEE. Another important virulence factor of EHEC O157: H7 is Shiga toxin (Stx), encoded by a prophage integrated into the chromosome of O157:H7. In order to obtain an attenuated vaccine candidate, a ler deletion mutant of O157: H7 was constructed by use of suicide vector pCVD442. Meanwhile, due to potential instability of the prophage carrying the stx gene, the prophage was cured with serial passages of bacteria and confirmed by PCR and DNA sequencing. A ler/stx deletion mutant of EHEC O157:H7 was constructed, termed as O157:H7(deltaler/deltastx). The cultural supernatant of O157 ler/stx deletion mutant was inoculated in vero cell culture, and the result indicating that O157 ler/stx deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the O157 ler/stx deletion mutant and the virulent strain O157:H7 EDL933, respectively. Mice were observed daily for clinical signs and weight changes. After inoculation of the deletion mutant, test group of mice (inoculated with O157:H7(deltaler/deltastx)) gained weight normally and experienced no clinical signs. In contrast, control group of mice (inoculated with O157: H7) exhibited weight loss and all died in four days. In another experiment, pregnant mice were orally vaccinated by O157:H7(deltaler/ deltastx) twice at interval of 14 days. Subsequently, the suckling mice were orally challenged with O157:H7 EDL933 at 7 days of age. The results showed that 78.34% of the sucking mice born by vaccinated mice were survival and 12.73% of the sucking mice born by non-vaccinated mice were survival. This study demonstrated that O157 ler/stx deletion mutant lost the toxigenicity to vero cell and to be safety to mice. Oral immunization can induce specific immune responses, and this mutant strain could be used as an attenuated vaccine candidate against EHEC O157.

Síndrome Urémico Hemolítico: manifestaciones clínicas: tratamento / Hemolytic uremic syndrome: clinical manifestations: Treatment

Clinical manifestation are described in children with epidemic HUS. The intestinal involvement in the prodromic period, is outlined and the most common disturbances such acute renal failure, thrombocytopenia, hemolytic anemia, leucocitosis hypertension, neurological, pancreatic and cardiac manifestations are described. We discuss the acid-base and electrolyte disturbances, metabolic acidosis, hyponatremia, hyperkalemia. The etiopathogenic treatment and the control of renal sequelae are also discussed.

Papel de la Toxina Shiga en el sindrome urémico hemolítico / Role of the Shiga toxin in the hemolytic uremic syndrome

In the last years, infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolitic-Uremic Syndrome (HUS) became relevant as a public health since it was considered as one of the most important emergent patogen present in the food contaminated by cattle feces. STEC infection may be asymptomatic or begins with a watery diarrhea that may or may not progress to bloody diarrhea (hemorrhagic colitis) and HUS. In Argentina, HUS is the most common pediatric cause of acute renal insufficiency and the second cause of chronic renal failure. Up to now, STEC infection lacks of known effective treatment strategies that diminish risk of progression to HUS. The mechanisms by which Shiga toxin (Stx) induce HUS may help to find strategies to prevent or ameliorate HUS. In this article, recent progress that has contributed to understanding the disease pathogenesis of STEC is reviewed. New strategies to prevent further uptake of Shiga from the gut, either during the diarrheal phase or once HUS has developed are discussed.

Control de Escherichia coli enterohemorrágico (EHEC) en el ganado bovino / Control of enterohemorrhagic Escherichia coli (EHEC) in cattle

Cattle are recognized as the major reservoir of STEC and the source of infection for human beings. Until recently, intervention strategies to decrease the contamination of meat products have been focused on the slaughter plant with the application of practices to reduce the contamination and proliferation of STEC. This has now changed following the development of intervention strategies in the farm. This could be one of the most important points of intervention to lower the incidence of human infection. Vaccines, probiotics, bacteriophages, and changes in production practices may be useful as strategies to control EHEC in the cattle. The application of such intervention measures could be difficult due to the fact that this zoonotic agent rarely causes disease in bovines. The HUS is endemic in Argentina, and the factors leading to this epidemiological situation remain unknown. However, intervention strategies undoubtedly will contribute to reduce the incidence of this zoonosis.

Vacunas contra Escherichia coli causante de diarrea en humanos / Vaccines against scherichia coli causing of diarrhea in humans

En este capítulo se describen las interacciones entre diferentes tipos de cepas de Escherichia coli con las células del epitelio intestinal, lo cual lleva a que el hospedero presente diarrea. El conocimiento de estas interacciones, emanado de veinte años de investigación, han permitido en fechas recientes plantear el uso de vacunas como medida de control de la enfermedad diarreica. La asociación constante entre los serotipos E. coli con procesos diarreicos, ha permitido plantear medidas de control a través de la búsqueda de los mecanismos comunes de patogenicidad que comparten la mayoría de los serotipos de E. coli. Estos mecanismos corresponden fundamentalmente a tres tipos: 1) adhesivos, que permiten a las bacterias acercarse, pegarse y colonizar el epitelio de ciertas áreas del intestino; 2) producción de proteínas bacterianas (toxinas) que estimulan la secreción de agua y electrolitos al interactuar con mecanismos bioquímicos de las células del huésped y 3) invasión y reproducción dentro del citoplasma de células epiteliales del intestino para evadir los mecanismos de protección del hospedero