Three Months of Composting Are Enough to Eliminate the Fowl Typhoid Bacteria

The composting technique has been increasingly highlighted in poultry production units, as an efficient and low-cost solution for the destination of carcasses. The process is based on the accelerated decomposition of organic material under high temperatures, associated with eliminating pathogenic microorganisms. This study aims to evaluate the effectiveness and the time necessary for the elimination of Salmonella Gallinarum in carcasses of poultry submitted to the composting process. The composting was carried out following the models used in the field, and microbiological analysis was performed in five different periods: 45, 90, 120, 150 and 180-days after closing the composter. After 90 days of experiment and in the subsequent analysis, the elimination of the bacteria in 100% of the samples was verified, validating the composting process as an effective method for eliminating S. Gallinarum in poultry carcasses, when respecting the period necessary for the elimination of the bacteria and the good quality of the structure adopted for the process.(AU)

Effect of doses and administration routes of 9R vaccine on protection of Japanese quails against experimental infection with Salmonella Gallinarum / Efeito das doses e vias de administração da vacina 9R na proteção de codornas japonesas contra infeccção experimental com Salmonella Gallinarum

Coturniculture has increased significantly in the last decades. There are several pathogens that can affect these birds. Among the diseases, fowl typhoid stands out as a disease with a potentially great impact to the poultry industry. The objective of this the study was to evaluate the effect of doses and administration routes of live 9R vaccine on protection of Japanese quails against experimental infection with Salmonella Gallinarum (SG). Two hundred and fifty birds were used, divided into five groups: G1, oral vaccination with one dose; G2, oral vaccination with 2 doses; G3, subcutaneous vaccination with one dose; G4, subcutaneous vaccination with two doses and G5 not vaccinated. All birds from all five groups were challenged with SG at an age of 45 days. SG was quantified in the periods of one, four, seven and twelve days after the challenge. The presence of clinical signs and macroscopic lesions of the disease were observed. The groups vaccinated by subcutaneous route had a higher egg production and lower mortality rate. Birds receiving a dose of the vaccine by subcutaneous route also showed lower amount of SG in the liver and spleen seven days after the challenge.(AU)

A coturnicultura tem aumentado significativamente nas últimas décadas. Existem vários patógenos que podem afetar essas aves. Entre as doenças, o tifo aviário se destaca como uma doença de grande impacto para a indústria avícola. O objetivo deste estudo foi avaliar o efeito de doses e vias de administração da vacina viva 9R na proteção de codornas japonesas contra infecção experimental por Salmonella Gallinarum (SG). Foram utilizadas duzentos e cinquenta aves, divididas em cinco grupos: G1, vacinação oral com uma dose; G2, vacinação oral com 2 doses; G3, vacinação subcutânea com uma dose; G4, vacinação subcutânea com duas doses e G5 não vacinado. Todas as aves dos cinco grupos foram desafiadas com SG aos 45 dias de idade. A SG foi quantificada nos períodos de um, quatro, sete e doze dias após o desafio. Foi observada a presença de sinais clínicos e lesões macroscópicas da doença. Os grupos vacinados por via subcutânea apresentaram maior produção de ovos e menor taxa de mortalidade. Aves recebendo uma dose da vacina por via subcutânea também apresentaram menor quantidade de SG no fígado e baço sete dias após o desafio.(AU)

Effect of doses and administration routes of 9R vaccine on protection of Japanese quails against experimental infection with Salmonella Gallinarum / Efeito das doses e vias de administração da vacina 9R na proteção de codornas japonesas contra infeccção experimental com Salmonella Gallinarum

Coturniculture has increased significantly in the last decades. There are several pathogens that can affect these birds. Among the diseases, fowl typhoid stands out as a disease with a potentially great impact to the poultry industry. The objective of this the study was to evaluate the effect of doses and administration routes of live 9R vaccine on protection of Japanese quails against experimental infection with Salmonella Gallinarum (SG). Two hundred and fifty birds were used, divided into five groups: G1, oral vaccination with one dose; G2, oral vaccination with 2 doses; G3, subcutaneous vaccination with one dose; G4, subcutaneous vaccination with two doses and G5 not vaccinated. All birds from all five groups were challenged with SG at an age of 45 days. SG was quantified in the periods of one, four, seven and twelve days after the challenge. The presence of clinical signs and macroscopic lesions of the disease were observed. The groups vaccinated by subcutaneous route had a higher egg production and lower mortality rate. Birds receiving a dose of the vaccine by subcutaneous route also showed lower amount of SG in the liver and spleen seven days after the challenge.(AU)

A coturnicultura tem aumentado significativamente nas últimas décadas. Existem vários patógenos que podem afetar essas aves. Entre as doenças, o tifo aviário se destaca como uma doença de grande impacto para a indústria avícola. O objetivo deste estudo foi avaliar o efeito de doses e vias de administração da vacina viva 9R na proteção de codornas japonesas contra infecção experimental por Salmonella Gallinarum (SG). Foram utilizadas duzentos e cinquenta aves, divididas em cinco grupos: G1, vacinação oral com uma dose; G2, vacinação oral com 2 doses; G3, vacinação subcutânea com uma dose; G4, vacinação subcutânea com duas doses e G5 não vacinado. Todas as aves dos cinco grupos foram desafiadas com SG aos 45 dias de idade. A SG foi quantificada nos períodos de um, quatro, sete e doze dias após o desafio. Foi observada a presença de sinais clínicos e lesões macroscópicas da doença. Os grupos vacinados por via subcutânea apresentaram maior produção de ovos e menor taxa de mortalidade. Aves recebendo uma dose da vacina por via subcutânea também apresentaram menor quantidade de SG no fígado e baço sete dias após o desafio.(AU)

A comparative survey between non-systemic Salmonella spp. (paratyphoid group) and systemic Salmonella Pullorum and S. Gallinarum with a focus on virulence genes / Uma investigação comparativa entre Salmonella spp. não-sistêmicas (grupo paratifoide) e sistêmicas Salmonella Pullorum e S. Gallinarum com enfoque nos genes de virulência

A comparative survey between non-systemic (paratyphoid Salmonellae) and systemic (S. Pullorum and S. Gallinarum) Salmonella strains was performed to produce a virulence gene profile for differentiation among the groups. The following virulence genes were evaluated: invA, spvC, sefC, pefA, fimY, sopB, sopE1, stn and avrA. There are substantial differences among paratyphoid Salmonellae, S. Pullorum, and S. Gallinarum regarding the genes sefC, spvC, sopE1 and avrA. A higher frequency of sefC, spvC, sopE1 and avrA genes were detected in S. Gallinarum and S. Pullorum when compared with strains from the paratyphoid group of Salmonella. These results may be useful for differentiating among different groups and serotypes.(AU)

Uma investigação comparativa entre amostras de Salmonella não-sistêmicas (grupo paratifoide) e sistêmicas (S. Pullorum and S. Gallinarum) foi desenvolvida para produzir um perfil de genes de virulência para diferenciação entre os grupos. Os seguintes genes de virulência foram avaliados invA, spvC, sefC, pefA, fimY, sopB, sopE1, stn e avrA. Detectou-se uma diferença substancial entre Salmonella do grupo paratifoide, S. Pullorum e S. Gallinarum considerando os genes sefC, spvC, sopE1 e avrA. Os genes sefC, spvC, sopE1 e avrA foram detectados, em maior número, em S. Gallinarum e S. Pullorum quando comparados com as amostras de Salmonella do grupo paratifoide. Estes resultados podem ser úteis para a diferenciação entre os diferentes grupos e sorotipos de Salmonella.(AU)

A comparative survey between non-systemic Salmonella spp. (paratyphoid group) and systemic Salmonella Pullorum and S. Gallinarum with a focus on virulence genes / Uma investigação comparativa entre Salmonella spp. não-sistêmicas (grupo paratifoide) e sistêmicas Salmonella Pullorum e S. Gallinarum com enfoque nos genes de virulência

A comparative survey between non-systemic (paratyphoid Salmonellae) and systemic (S. Pullorum and S. Gallinarum) Salmonella strains was performed to produce a virulence gene profile for differentiation among the groups. The following virulence genes were evaluated: invA, spvC, sefC, pefA, fimY, sopB, sopE1, stn and avrA. There are substantial differences among paratyphoid Salmonellae, S. Pullorum, and S. Gallinarum regarding the genes sefC, spvC, sopE1 and avrA. A higher frequency of sefC, spvC, sopE1 and avrA genes were detected in S. Gallinarum and S. Pullorum when compared with strains from the paratyphoid group of Salmonella. These results may be useful for differentiating among different groups and serotypes.(AU)

Uma investigação comparativa entre amostras de Salmonella não-sistêmicas (grupo paratifoide) e sistêmicas (S. Pullorum and S. Gallinarum) foi desenvolvida para produzir um perfil de genes de virulência para diferenciação entre os grupos. Os seguintes genes de virulência foram avaliados invA, spvC, sefC, pefA, fimY, sopB, sopE1, stn e avrA. Detectou-se uma diferença substancial entre Salmonella do grupo paratifoide, S. Pullorum e S. Gallinarum considerando os genes sefC, spvC, sopE1 e avrA. Os genes sefC, spvC, sopE1 e avrA foram detectados, em maior número, em S. Gallinarum e S. Pullorum quando comparados com as amostras de Salmonella do grupo paratifoide. Estes resultados podem ser úteis para a diferenciação entre os diferentes grupos e sorotipos de Salmonella.(AU)

Seroprevalence of Salmonella and Mycoplasma in commercial broilers, backyard chickens, and spent hens in the region of Triângulo Mineiro, State of Minas Gerais, Brazil

Avian salmonellosis and mycoplasmosis are infectious diseases that, in addition of causing lack of flock uniformity, represent a hazard to human health. The objective of the present study was to evaluate the seroprevalence of mycoplasmosis and salmonellosis in commercial broilers, backyard chickens, and spent hens slaughtered at a processing plant with local health inspection in Uberlândia, MG, Brazil. A total of 210 samples were randomly collected at the time of bleeding. Samples were submitted to rapid plate serum agglutination test (RSA) for the classification of Salmonella pullorum, Salmonella gallinarum, Mycoplasma gallisepticum and Mycoplasma synoviae. In order to increase result specificity, mycoplasmosis-positive samples were submitted to hemagglutination inhibition test (HI). No samples presented detectable antibodies against Salmonella pullorum or Salmonella gallinarum in the RSA test. Only Mycoplasma synoviae was detected in 14% of the backyard chickens and 0.74% in commercial broilers, whereas no antibodies were detected in spent hens. The seroprevalence rates found in the present study emphasize the need of keeping chicken flocks free from disease using effective biosafety systems.

Seroprevalence of Salmonella and Mycoplasma in commercial broilers, backyard chickens, and spent hens in the region of Triângulo Mineiro, State of Minas Gerais, Brazil

Avian salmonellosis and mycoplasmosis are infectious diseases that, in addition of causing lack of flock uniformity, represent a hazard to human health. The objective of the present study was to evaluate the seroprevalence of mycoplasmosis and salmonellosis in commercial broilers, backyard chickens, and spent hens slaughtered at a processing plant with local health inspection in Uberlândia, MG, Brazil. A total of 210 samples were randomly collected at the time of bleeding. Samples were submitted to rapid plate serum agglutination test (RSA) for the classification of Salmonella pullorum, Salmonella gallinarum, Mycoplasma gallisepticum and Mycoplasma synoviae. In order to increase result specificity, mycoplasmosis-positive samples were submitted to hemagglutination inhibition test (HI). No samples presented detectable antibodies against Salmonella pullorum or Salmonella gallinarum in the RSA test. Only Mycoplasma synoviae was detected in 14% of the backyard chickens and 0.74% in commercial broilers, whereas no antibodies were detected in spent hens. The seroprevalence rates found in the present study emphasize the need of keeping chicken flocks free from disease using effective biosafety systems.(AU)

Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica)

Salmonella Gallinarum is the agent of fowl typhoid in poultry and infects mainly adult galliforms, causing significant economic losses in poultry production. Because quails are susceptible to this disease and quail production is becoming increasingly important in Brazil, this study was carried out to evaluate the virulence of Salmonella Gallinarum strain to quails. The inoculum was prepared from S. Gallinarum strain resistant to nalidixic acid. Forty eight 16-week-old Japanese quails were randomly distributed in three groups. Before the experiment, cloacal swabs were collected from all birds in order to confirm they were free from Salmonella spp. Cloacal swabs and fecal samples were collected on days 03, 06, 09, 12, and 15 post-inoculation. Birds that died during the experiment were submitted to post-mortem examination, and had their organs aseptically collected for bacteriological examination. All eggs produced during the experiment were also examined. The mortality rate recorded during the experiment was 43.75% (21/48). S. Gallinarum was recovered from the organs of the birds that naturally died during the experiment, but the agent was not isolated from the organs of sacrificed birds. No egg sample was positive for Salmonella Gallinarum. It was concluded that S. Gallinarum may be recovered from the organs of experimentally-infected Japanese quails.

Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica)

Salmonella Gallinarum is the agent of fowl typhoid in poultry and infects mainly adult galliforms, causing significant economic losses in poultry production. Because quails are susceptible to this disease and quail production is becoming increasingly important in Brazil, this study was carried out to evaluate the virulence of Salmonella Gallinarum strain to quails. The inoculum was prepared from S. Gallinarum strain resistant to nalidixic acid. Forty eight 16-week-old Japanese quails were randomly distributed in three groups. Before the experiment, cloacal swabs were collected from all birds in order to confirm they were free from Salmonella spp. Cloacal swabs and fecal samples were collected on days 03, 06, 09, 12, and 15 post-inoculation. Birds that died during the experiment were submitted to post-mortem examination, and had their organs aseptically collected for bacteriological examination. All eggs produced during the experiment were also examined. The mortality rate recorded during the experiment was 43.75% (21/48). S. Gallinarum was recovered from the organs of the birds that naturally died during the experiment, but the agent was not isolated from the organs of sacrificed birds. No egg sample was positive for Salmonella Gallinarum. It was concluded that S. Gallinarum may be recovered from the organs of experimentally-infected Japanese quails.(AU)

Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica)

Salmonella Gallinarum is the agent of fowl typhoid in poultry and infects mainly adult galliforms, causing significant economic losses in poultry production. Because quails are susceptible to this disease and quail production is becoming increasingly important in Brazil, this study was carried out to evaluate the virulence of Salmonella Gallinarum strain to quails. The inoculum was prepared from S. Gallinarum strain resistant to nalidixic acid. Forty eight 16-week-old Japanese quails were randomly distributed in three groups. Before the experiment, cloacal swabs were collected from all birds in order to confirm they were free from Salmonella spp. Cloacal swabs and fecal samples were collected on days 03, 06, 09, 12, and 15 post-inoculation. Birds that died during the experiment were submitted to post-mortem examination, and had their organs aseptically collected for bacteriological examination. All eggs produced during the experiment were also examined. The mortality rate recorded during the experiment was 43.75% (21/48). S. Gallinarum was recovered from the organs of the birds that naturally died during the experiment, but the agent was not isolated from the organs of sacrificed birds. No egg sample was positive for Salmonella Gallinarum. It was concluded that S. Gallinarum may be recovered from the organs of experimentally-infected Japanese quails.

Salmonella GALLINARUM MULTIRRESISTENTES E FORMADORAS DE BIOFILMES EM CASCAS DE OVOS SÃO SENSÍVEIS A BACTERIÓFAGOS

O tifo aviário possui grande importância econômica em função dos altos custos com programas de controle, devido ao sacrifício sanitário das aves e descarte dos ovos nos incubatórios. A Salmonella Gallinarum, há algumas décadas, havia sido considerada erradicada em aves comerciais no Brasil. Porém, acredita-se que, devido às falhas nos controles, como biosseguridade, insumos, monitorias e programas sanitários, associados aos mecanismos de virulência do patógeno, essa doença vem reemergindo nos últimos anos. A S. Gallinarum causa infecção e manifestações clinicas, com alta concentração bacteriana nos tecidos e pode ser transmitida vertical e horizontalmente. A contaminação via transovariana torna os ovos inviáveis para a produção de aves, as quais podem se tornar portadoras assintomáticas e transmitir o patógeno logo após o nascimento. A transmissão do microrganismo por via horizontal pode se caracterizar por contaminar os ovos através da casca logo após a postura, nos ninhos ou camas de aviários. Os fatores de virulência influenciam nos tratamentos com drogas antimicrobianas e no controle ambiental com a utilização de desinfetantes. É de fundamental importância o estudo desses fatores para auxiliar na análise epidemiológica dos surtos ocorridos e, também, para estabelecer estratégias de novos controles. O objetivo deste estudo foi realizar a caracterização fenotípica e genotípica de diferentes isolados de S. Gallinarum, oriundos da produção agroindustrial, através da avaliação da sensibilidade aos antimicrobianos e a desinfetante, potencial de lise por bacteriófagos, pesquisa de genes de virulência, formação de biofilme em poliestireno e em cascas de ovos. Pode-se observar que as S. Gallinarum são multirresistentes aos antimicrobianos, sensíveis aos bacteriófagos testados, e potencialmente virulentas, de acordo com perfis genéticos obtidos. A maioria dos isolados formaram biofilme no poliestireno tanto a 22°C como a 42°C, sem diferença estatística (p = 0.0965) entre as temperaturas testadas. Houve maior formação de biofilme nas cascas de ovos na temperatura de 22°C (4,656 log10UFC/cm2), com diferença estatística quando comparado aos biofilmes formados a 36°C e 42°C. A ação do desinfetante comercial, na concentração 1,5% e tempo 5 minutos, promoveu remoção do biofilme, estatisticamente significativa, apenas na formação a 22°C, propiciando uma redução de 3,125 log10. A presença de genes de virulência, a multirresistência e a capacidade de formar biofilmes em diferentes superfícies e temperaturas pelas S. Gallinarum estudadas, isoladas de aves comerciais, nos faz supor que estes podem ser fatores relevantes para que a SG se mantenha presente nos plantéis avícolas e esteja envolvida em surtos sanitários intermitentes. Apesar do cenário preocupante, a possibilidade de controle biológico da SG por bacteriófagos nos traz uma alternativa promissora para a contenção deste microrganismo.

The fowl typhoid has great economic importance due to the high costs with control programs, due to the sanitary sacrifice of the birds and discard of the eggs in the hatcheries. Salmonella Gallinarum, a few decades ago, had been considered to be eradicated in commercial birds in Brazil. However, due to the lack of controls, such as biosecurity, inputs, monitoring and health programs, associated with the virulence mechanisms of the pathogen, this disease has been reemerging in recent years. S. Gallinarum causes infection and clinical manifestations, with high bacterial concentration in the tissues and can be transmitted vertically and horizontally. The contamination via transovarian renders the eggs unviable for the production of birds, which can become asymptomatic carriers and transmit the pathogen soon after the birth. Transmission of the microorganism horizontally can be characterized by contaminating the eggs through the bark soon after laying, in the nests or beds of aviaries. Virulence factors influence the treatments with antimicrobial drugs and environmental control with the use of disinfectants. It is of fundamental importance the study of these factors to assist in the epidemiological analysis of the outbreaks occurred and, also, to establish strategies of new controls. The objective of this study was to characterize the phenotypic and genotypic characteristics of different S. Gallinarum isolates from agroindustry production through the evaluation of antimicrobial susceptibility and disinfectant, bacteriophage lysis potential, virulence genes, biofilm formation Polystyrene and eggshells. It can be observed that the S. Gallinarum are multiresistant to the antimicrobials, sensitive to the bacteriophages tested, and potentially virulent, according to the genetic profiles obtained. Most of the isolates formed biofilm in polystyrene at 22°C and 42°C, with no statistical difference (p = 0.0965) between the temperatures tested. There was a higher biofilm formation in eggshells at 22°C (4,656log10UFC / cm2), with a statistical difference when compared to biofilms formed at 36°C and 42°C. The action of the commercial disinfectant, in the concentration 1.5% and time 5 minutes, promoted the removal of the biofilm, statistically significant, only in the formation at 22°C, leading to a reduction of 3,125 log10. The presence of virulence genes, the multiresistance and the ability to form biofilms on different surfaces and temperatures by S. Gallinarum studied, isolated from commercial birds, makes us suppose that these can be relevant factors for the SG to remain present in poultry farms and is involved in intermittent outbreaks. Despite the worrying scenario, the possibility of biological control of SG by bacteriophages brings us a promising alternative for the containment of this microorganism.

RESSURGÊNCIA DO TIFO AVIÁRIO NA AVICULTURA INDUSTRIAL BRASILEIRA: NOVOS ESTUDOS EPIDEMIOLÓGICOS DE UMA ENFERMIDADE ANTIGA

Salmonella Gallinarum (SG) é o agente etiológico do tifo aviário (TA), uma doença sistêmica grave responsável por perdas econômicas para a indústria avícola em todo o mundo. TA foi considerado sob controle no Brasil e em países desenvolvidos. No entanto, nos últimos anos, inúmeros surtos dessa enfermidade têm sido identificados em lotes de aves de vários estados do Brasil. Com intuito de investigar fatores que pudessem ajudar a compreender a epidemiologia destes surtos, o presente estudo foi realizado. Foram avaliadas: (i) as possíveis alterações na patogenicidade de uma estirpe de SG isolada de um dos surtos recentes de TA, para as aves de linhagens comerciais utilizadas atualmente no Brasil; (ii) as possibilidades de transmissão de SG por via vertical e durante a incubação de ovos; (iii) a influência do uso antimicrobianos na persistência de SG na ave e consequente transmissão vertical; (iv) os perfis genéticos das estirpes isoladas anteriormente e, recentemente, por PFGE. No presente estudo, aves de linhagens mais suscetíveis infectadas por SG apresentaram alterações patológicas de maior intensidade e altas taxas de mortalidade, enquanto que as aves de linhagens mais resistentes apresentaram a mortalidade e sinais clínicos de forma mais branda. No entanto, foram capazes de manter SG por períodos mais longos que as aves de linhagens susceptíveis. SG não foi recuperada dos ovos produzidos por aves suscetíveis ou resistentes. A transmissão vertical não foi observada, embora aves recém-eclodidas foram infectadas por contato durante a incubação. Antibióticoterapia foi eficaz na redução da mortalidade, mas não foi capaz de eliminar a infecção e nem favoreceu a transmissão de SG via ovo. Estirpes de SG exibiram perfis genéticos muito semelhantes, sugerindo que eles vêm circulando nos lotes brasileiros há mais de 20 anos. Falhas em componentes do programa de biossegurança, provavelmente, podem ser responsáveis pela propagação FT no Brasil.

Salmonella Gallinarum (SG) causes fowl typhoid (FT) a severe systemic disease responsible for economic losses to the poultry industry worldwide. FT was considered under control in Brazil and in developed countries. Nonetheless, in recent years has been identified on farms in several states of Brazil. In order to investigate the epidemiological reasons behind this large FT outbreak, the present study was carried out to assess: (i) possible changes in the patogenicity of a strain of SG isolated from a recent outbreak to chickens of white and brown lines of layers; (ii) transmission through eggs and hatching; (iii) the interference of antibiotic to the persistence of SG in the bird and to vertical transmission; (iv) the genetic profiles of strains isolated previously and recently by PFGE. Susceptible lines showed extensive pathological changes and high mortality rates, whereas in resistant lines mortality and clinical signs were almost absent. Although, they could maintain SG for longer periods than the susceptible lines did. SG was not recovered from any egg laid by birds from susceptible or resistant either. Vertical transmission was not observed although newborn chicks were infected due the contact during incubation. Antibiotic therapy was effective at reducing mortality but was not able to clear the infection and neither to favour vertical transmission. Strains did exhibit very similar genetic profiles, suggesting they have been circulating in Brazilian flocks for more than 20 years. Failures in components of biosecurity programs, probably, would be the responsible for the FT spread in Brazil

Reversion to virulence evaluation of a 9R vaccine strain of Salmonella enterica serovar gallinarum in commercial brown layers

The live vaccine Cevac S. Gallinarum, made from a rough strain of Salmonella enterica subspecies enterica serotype Gallinarum is used for preventing fowl typhoid, a disease that still causes considerable economic losses in countries with a developing poultry industry. The objective of this paper was to evaluate a possible reversion to virulence of the strain used in a vaccine in commercial brown layers. Only Salmonella-free chicks were utilized. One hundred twenty (120) 12-day-old Dekalb brown layers divided in two trials were used. The first trial had six groups of 15 birds each. Birds of group 1 were vaccinated with 10 doses of Cevac S. Gallinarum subcutaneously and 10 doses orally, in a total of 20 doses of vaccine. Then the birds of groups 2, 3, 4, and 5 received inocula that contained feces and a pool of organs with fragments of liver, heart, spleen, and cecal tonsils obtained from the immediately previous group. The second trial had three groups with 10 birds each. Birds in group 7 received inocula containing a pool of organs from birds of group 5 from trial 1, whilst the birds in group 8 were vaccinated subcutaneously with one dose of vaccine. Both trials included negative control groups (6 and 9). Throughout the experimental period, birds were monitored for reactions to the vaccination on the site of administration, clinical signs, and post-mortem lesions. In each passage, in addition to the birds euthanized to provide the inocula material, two birds from each group were euthanized for assessment of possible lesions, and their organs (liver, heart, spleen and cecal tonsils) were cultured in an attempt to isolate the vaccine strain. Except for one bird from group 1, that had a local reaction on the site of vaccination - a small vesicle with less that 0.5 mm that persisted until the third day post vaccination -, no other bird had any local reaction to the vaccine or any visible clinical alteration. Birds in group 8 did not present any reaction or clinical alteration because of the vaccine. We only managed to re-isolate the vaccine strain in the inocula made from organs of birds in group 1. We confirmed the isolation by means of biochemical tests, serology, and acriflavine agglutination test. All other cultures made from organs or feces, from all the other experimental groups did not show any growth of the vaccine strain or any other Salmonella serovar, suggesting that the vaccinated birds did not shed the SG9R vaccine strain. No bird presented any clinical symptoms or died during the trials, and no gross lesions were observed in the post-mortem examinations. Under the controlled conditions and time-frame of the present experiment, it was possible to conclude that the rough 9R strain of Salmonella Gallinarum present in the vaccine Cevac S. Gallinarum (Ceva Campinas Ltda. - Campinas, SP - Brazil) did not revert to virulence.

Reversion to virulence evaluation of a 9R vaccine strain of Salmonella enterica serovar gallinarum in commercial brown layers

The live vaccine Cevac S. Gallinarum, made from a rough strain of Salmonella enterica subspecies enterica serotype Gallinarum is used for preventing fowl typhoid, a disease that still causes considerable economic losses in countries with a developing poultry industry. The objective of this paper was to evaluate a possible reversion to virulence of the strain used in a vaccine in commercial brown layers. Only Salmonella-free chicks were utilized. One hundred twenty (120) 12-day-old Dekalb brown layers divided in two trials were used. The first trial had six groups of 15 birds each. Birds of group 1 were vaccinated with 10 doses of Cevac S. Gallinarum subcutaneously and 10 doses orally, in a total of 20 doses of vaccine. Then the birds of groups 2, 3, 4, and 5 received inocula that contained feces and a pool of organs with fragments of liver, heart, spleen, and cecal tonsils obtained from the immediately previous group. The second trial had three groups with 10 birds each. Birds in group 7 received inocula containing a pool of organs from birds of group 5 from trial 1, whilst the birds in group 8 were vaccinated subcutaneously with one dose of vaccine. Both trials included negative control groups (6 and 9). Throughout the experimental period, birds were monitored for reactions to the vaccination on the site of administration, clinical signs, and post-mortem lesions. In each passage, in addition to the birds euthanized to provide the inocula material, two birds from each group were euthanized for assessment of possible lesions, and their organs (liver, heart, spleen and cecal tonsils) were cultured in an attempt to isolate the vaccine strain. Except for one bird from group 1, that had a local reaction on the site of vaccination - a small vesicle with less that 0.5 mm that persisted until the third day post vaccination -, no other bird had any local reaction to the vaccine or any visible clinical alteration. Birds in group 8 did not present any reaction or clinical alteration because of the vaccine. We only managed to re-isolate the vaccine strain in the inocula made from organs of birds in group 1. We confirmed the isolation by means of biochemical tests, serology, and acriflavine agglutination test. All other cultures made from organs or feces, from all the other experimental groups did not show any growth of the vaccine strain or any other Salmonella serovar, suggesting that the vaccinated birds did not shed the SG9R vaccine strain. No bird presented any clinical symptoms or died during the trials, and no gross lesions were observed in the post-mortem examinations. Under the controlled conditions and time-frame of the present experiment, it was possible to conclude that the rough 9R strain of Salmonella Gallinarum present in the vaccine Cevac S. Gallinarum (Ceva Campinas Ltda. - Campinas, SP - Brazil) did not revert to virulence.

The contribution of genes required for anaerobic respiration to the virulence of Salmonella enterica serovar Gallinarum for chickens

Salmonella enterica serovar Gallinarum (SG) is an intracellular pathogen of chickens. To survive, to invade and to multiply in the intestinal tract and intracellularly it depends on its ability to produce energy in anaerobic conditions. The fumarate reductase (frdABCD), dimethyl sulfoxide (DMSO)-trimethylamine N-oxide (TMAO) reductase (dmsABC), and nitrate reductase (narGHIJ) operons in Salmonella Typhimurium (STM) encode enzymes involved in anaerobic respiration to the electron acceptors fumarate, DMSO, TMAO, and nitrate, respectively. They are regulated in response to nitrate and oxygen availability and changes in cell growth rate. In this study mortality rates of chickens challenged with mutants of Salmonella Gallinarum, which were defective in utilising anaerobic electron acceptors, were assessed in comparison to group of bird challenged with wild strain. The greatest degree of attenuation was observed with mutations affecting nitrate reductase (napA, narG) with additional attenuations induced by a mutation affecting fumarate reductase (frdA) and a double mutant (dmsA torC) affecting DMSO and TMAO reductase.

Efficacy of several Salmonella vaccination programs against experimental challenge with Salmonella gallinarum in commercial brown layer and broiler breeder hens

The protective effect of various Salmonella vaccines regimens against an experimental Salmonella Gallinarum challenge (SGNalr strain at 12 wk of age) was evaluated in two experiments. In Experiment 1 commercial brown layers were vaccinated according to one of the following programs: (i) two doses of a SE bacterin (Layermune SE; group 1); (ii) a first dose of a live SG9R vaccine (Cevac SG9R) followed by a SE bacterin (Layermune SE; group 2); (iii) one dose of each of two different multivalent inactivated vaccines containing SE cells (Corymune 4 & Corymune 7; group 3) or (iv) not vaccinated (group 4). In Experiment 2, broiler breeders were given the same vaccination treatments except for the group vaccinated with the multivalent vaccines. Overall, in both experiments, all vaccination schemes were effective in reducing mortality after challenge with a SG field strain. Primary vaccination with an initial dose of a live SG9R vaccine followed some weeks later by a dose of an inactivated SE bacterin was the most effective (p 0.05) vaccination program against mortality induced by field SG experimental challenge in both experiments. In conclusion, Salmonella vaccination programs containing SE bacterins alone or in combination with a live SG9R vaccine are effective in preventing mortality induced by infection of field SG. Nevertheless, it is important to emphasize that any vaccination program against any Salmonella serotype will only be effective if it is part of a sound and comprehensive biosecurity program designed for Salmonella control in poultry farms.

Efficacy of several Salmonella vaccination programs against experimental challenge with Salmonella gallinarum in commercial brown layer and broiler breeder hens

The protective effect of various Salmonella vaccines regimens against an experimental Salmonella Gallinarum challenge (SGNalr strain at 12 wk of age) was evaluated in two experiments. In Experiment 1 commercial brown layers were vaccinated according to one of the following programs: (i) two doses of a SE bacterin (Layermune SE; group 1); (ii) a first dose of a live SG9R vaccine (Cevac SG9R) followed by a SE bacterin (Layermune SE; group 2); (iii) one dose of each of two different multivalent inactivated vaccines containing SE cells (Corymune 4 & Corymune 7; group 3) or (iv) not vaccinated (group 4). In Experiment 2, broiler breeders were given the same vaccination treatments except for the group vaccinated with the multivalent vaccines. Overall, in both experiments, all vaccination schemes were effective in reducing mortality after challenge with a SG field strain. Primary vaccination with an initial dose of a live SG9R vaccine followed some weeks later by a dose of an inactivated SE bacterin was the most effective (p 0.05) vaccination program against mortality induced by field SG experimental challenge in both experiments. In conclusion, Salmonella vaccination programs containing SE bacterins alone or in combination with a live SG9R vaccine are effective in preventing mortality induced by infection of field SG. Nevertheless, it is important to emphasize that any vaccination program against any Salmonella serotype will only be effective if it is part of a sound and comprehensive biosecurity program designed for Salmonella control in poultry farms.

Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates

Although Salmonella Pullorum and Salmonella Gallinarum cause different diseases in poultry, they are very similar. Both are non-motile and present the same somatic antigenic structure. They are differentiated by biochemical tests. Certain atypical strains are very difficult to distinguish. They do not produce the expected results when dulcitol and ornithine descarxboxylase tests are performed. Therefore, additional tests could be helpful. Many studies have chose the part I of the gene that encodes flagellin (fliC) to differentiate serotypes. Most Salmonella strains have two structural genes (fliC and fliB) that encode flagellins. Non-motile strains generally present these structural genes, but are not able to build a functional flagellum. It was demonstrated that enzymatic restriction of the amplified fliC gene using Hinp1I enzyme can differentiate SG from SP. In the present study, this method was adopted to analyze 14 SP and 22 SG strains, including some strains with atypical results in biochemical tests assessing the utilization of dulcitol and ornithine. The results showed that all SG strains were broken by the enzyme, whereas the 14 SP strains were not.

Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates

Although Salmonella Pullorum and Salmonella Gallinarum cause different diseases in poultry, they are very similar. Both are non-motile and present the same somatic antigenic structure. They are differentiated by biochemical tests. Certain atypical strains are very difficult to distinguish. They do not produce the expected results when dulcitol and ornithine descarxboxylase tests are performed. Therefore, additional tests could be helpful. Many studies have chose the part I of the gene that encodes flagellin (fliC) to differentiate serotypes. Most Salmonella strains have two structural genes (fliC and fliB) that encode flagellins. Non-motile strains generally present these structural genes, but are not able to build a functional flagellum. It was demonstrated that enzymatic restriction of the amplified fliC gene using Hinp1I enzyme can differentiate SG from SP. In the present study, this method was adopted to analyze 14 SP and 22 SG strains, including some strains with atypical results in biochemical tests assessing the utilization of dulcitol and ornithine. The results showed that all SG strains were broken by the enzyme, whereas the 14 SP strains were not.

A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain.

Salmonella enterica serovar Gallinarum (SG) é o agente do tifo aviário, doença severa que provoca mortalidade em até 80% do plantel de aves. SG encontra-se entre os poucos sorotipos de Salmonella que são agentes etiológicos de enfermidade específica, à semelhança de Salmonella Typhi em seres humanos podendo, portanto, servir de modelo experimental para outras salmoneloses hospedeiro especíifcas. Além dos mecanismos de virulência, a bactéria utiliza mecanismos de sobrevivência para permanecer no hospedeiro. A ativação desses mecanismos pode ou não estar associada à ativação dos mecanismos de virulência. Entre os mecanismos fisiológicos, está a produção de vitamina B12 que Salmonella spp. realiza em ambientes anaeróbicos, como quando encontra-se intracelularmente no organismo hospedeiro. Neste estudo, analisou-se a infecção de aves por cepas de SG, que tiveram genes alterados que participam da biossíntese de vitamina B12. Foram produzidos mutantes de SG contendo os genes cbiA e cobS alterados e um terceiro, contendo ambos os genes alterados. A sobrevivência e a ação patogênica de SG não foi modificada pela alteração simples de um dos genes, mas tornou a cepa de SG completamente atenuada quando os dois foram modificados. A mortalidade provocada pela cepa selvagem de SG foi de 64,52%, enquanto que não observou-se mortalidade alguma no grupo de aves infectadas com SGNal r"cobs"cbiA. Estudos futuros deverão ser realizados para elucidar este processo fisiológico bacteriano e para avaliar a utilização desta cepa de SG como cepa vacinal.