Thales Quedi Furian

Natural infection of turkeys by infectious laryngotracheitis virus

The infectious laryngotracheitis virus (ILTV) is an important respiratory pathogen of chickens that also infects pheasants and peafowl. Epidemiologically non-related commercial turkey flocks with clinical signs such as tracheitis, swollen sinuses, conjunctivitis and expectoration of bloody mucus were examined for the presence of the virus. Laboratory ILTV detection was performed by virus isolation in embryonated eggs and cell cultures, PCR and sequencing of amplification products, histopathology, indirect immunofluorescence and electron microscopy. One ILTV turkey isolate was also experimentally inoculated into susceptible chickens and turkeys, reproducing a mild respiratory disease. This is the first description of natural infections with ILTV in turkeys.

Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR

The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC) is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25), the sodA and nanH genes were present in 96% (24/25), ptfA was present in 92% (23/25), and pfhA was present in 60% (15/25). Gene toxA was not identified in any of the samples studied (0/25). Five different genetic profiles were obtained, of which P1 (negative to toxA) was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil

Salmonella spp. are considered the main agents of foodborne disease and Salmonella Enteritidis is one of the most frequently isolated serovars worldwide. The virulence of Salmonella spp. and their interaction with the host are complex processes involving virulence factors to overcome host defenses. The purpose of this study was to detect virulence genes in S. Enteritidis isolates from poultry in the South of Brazil. PCR-based assays were developed in order to detect nine genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH and spvC) associated with the virulence in eighty-four isolates of S. Enteritidis isolated from poultry. The invA, hilA, sivH, sefA and avrA genes were present in 100% of the isolates; lpfA and sopE were present in 99%; agfA was present in 96%; and the spvC gene was present in 92%. It was possible to characterize the isolates with four different genetic profiles (P1, P2, P3 and P4), as it follows: P1, positive for all genes; P2, negative only for spvC; P3, negative for agfA; and P4, negative for lpfA, spvC and sopE. The most prevalent profile was P1, which was present in 88% of the isolates. Although all isolates belong to the same serovar, it was possible to observe variations in the presence of these virulence-associated genes between different isolates. The characterization of the mechanisms of virulence circulating in the population of Salmonella Enteritidis is important for a better understanding of its biology and pathogenicity. The frequency of these genes and the establishment of genetic profiles can be used to determine patterns of virulence. These patterns, associated with in vivo studies, may help develop tools to predict the ability of virulence of different strains.

Laryngotracheitis: reproducibility of the disease and comparison of diagnostic methods

Infectious laryngotracheitis virus (ILTV) cause mild to severe respiratory disease in chickens, the purpose of our study being to use Brazilian isolate of ILTV to reproduce ILTV disease in chickens by experimental infection and to compare three diagnostic methods (nested polymerase chain reaction (PCR), virus isolation, histopathology) for detection of ILTV. Forty-eight chickens intratracheally inoculated with ILTV and a further 48 with PBS, showing mild respiratory signs 48 hours post infection (PI) but no signs of infection after day 10 PI. Every 2 days PI, six birds were arbitrarily selected from the control and infected groups, sacrificed and the trachea collected. Both the nested PCR and virus isolation detected the virus from day 2 until day 12 PI. However, at day 12 PI, PCR detected ILTV DNA in 100% of the samples while the virus isolation method detected ILTV in only 33% of the samples. Tracheal histopathology showed intranuclear inclusion bodies on days 8 and 10 PI. The results indicate that the field-isolate of ILTV studied by us is of low pathogenicity and that our nested PCR protocol was able to detect positive samples over a longer infection period than many ILTV diagnostic test already described.

Detecção do vírus da laringotraqueíte das galinhas no Brasil

A study was carried out in search for evidences of infectious laryngotracheitis virus (ILTV) infections in some Brazilian chicken flocks. Tracheal tissues and swabs were collected from 10 different flocks of layers and broilers displaying respiratory signs of disease. Samples were processes for virus isolation in embryonated eggs and the membranes examined by histopathology. In addition, specimens were examined by polymerase chain reaction (PCR). Three flocks had ILTV positive chickens by virus isolation and PCR. These results confirm the occurrence of ILTV in chickens in Brazil.

Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine

Pasteurella multocida causes atrophic rhinitis in swine and fowl cholera in birds, and is a secondary agent in respiratory syndromes. Pathogenesis and virulence factors involved are still poorly understood. The aim of this study was to detect 22 virulence-associated genes by PCR, including capsular serogroups A, B and D genes and to evaluate the antimicrobial susceptibility of P. multocida strains from poultry and swine. ompH, oma87, plpB, psl, exbD-tonB, fur, hgbA, nanB, sodA, sodC, ptfA were detected in more than 90% of the strains of both hosts. 91% and 92% of avian and swine strains, respectively, were classified in serogroup A. toxA and hsf-1 showed a significant association to serogroup D; pmHAS and pfhA to serogroup A. Gentamicin and amoxicillin were the most effective drugs with susceptibility higher than 97%; however, 76.79% of poultry strains and 85% of swine strains were resistant to sulphonamides. Furthermore, 19.64% and 36.58% of avian and swine strains, respectively, were multi-resistant. Virulence genes studied were not specific to a host and may be the result of horizontal transmission throughout evolution. High multidrug resistance demonstrates the need for responsible use of antimicrobials in animals intended for human consumption, in addition to antimicrobial susceptibility testing to P. multocida.

Use of Molecular Pathogenicity Indices to Identify Pathogenic Strains of Pasteurella multocida.

SUMMARY In addition to being the causative agent of fowl cholera (FC), Pasteurella multocida is also one of the most prevalent opportunistic pathogens associated with respiratory diseases in various hosts. However, understanding of the traits that distinguish the virulent isolates that cause FC is still limited. The objective of this study was to characterize P. multocida isolates of Brazil by PCR–restriction fragment length polymorphism (PCR-RFLP) analysis in order to determine if strain-type correlates with virulence or with 22 previously studied virulence genes. The PCR-RFLP was used to classify the isolates into seven strain types, and the isolates in Profile II had a higher pathogenicity index (P < 0.05) than did those in Profiles I, V, and VI. The overall identity among the nucleotide sequences of the ompH was 89.8%. Furthermore, strains available in GenBank showed a high level of homology of the different bacterial serotypes with the groupings resulting from the PCR-RFLP. Strain Types I and II showed the highest identity with Serotypes 3 (100%) and 3-4 (99.1%), respectively. Detection of the pfhA gene indicated the presence of strains that are highly pathogenic. The screening detection of 22 virulence genes and inference through the decision tree models comparing the results of pathogenicity indices permitted the identification of the most highly pathogenic strains of P. multocida.

Classification of avian pathogenic Escherichia coli by a novel pathogenicity index based on an animal model

Background: Avian Pathogenic Escherichia coli is the main agent of colibacillosis, a systemic disease that causes considerable economic losses to the poultry industry. In vivo experiments are used to measure the ability of E. coli to be pathogenic. Generally, these experiments have proposed different criteria for results interpretation and did not take into account the death time. The aim of this study was to propose a new methodology for the classification of E. coli pathogenicity by the establishment of a pathogenicity index based in the lethality, death time and the ability of the strain to cause colibacillosis lesions in challenged animals. Materials, Methods & Results: A total of 293 isolates of E. coli were randomly selected to this study. The strains were isolated from cellulitis lesions, broiler bedding material or respiratory diseases and were previously confirmed through biochemical profile. The bacterial isolates were kept frozen at -20°C. The strains were retrieved from stocks and cultured in brain-heart infusion broth overnight at 37°C to obtain a final concentration of 109 UFC/mL. A total of 2940 one-dayold chicks from commercial breeding hens were randomly assigned to groups containing 10 animals and each group was subcutaneously inoculated in the abdominal region with 0.1 mL of the standard inoculum solution containing each of the strains. A control group of 10 broilers were inoculated with 0.1 mL of brain-heart infusion broth by the same route. The chicks were kept for seven days. They were observed at intervals of 6, 12 and 24 h post-inoculation during the first days. From the second day on, the chicks were observed at intervals of 12 h. According to the death time and to the scores of each lesion (aerosaculitis, pericarditis, perihepatitis, peritonitis and cellulitis), a formula to determine the Individual Pathogenicity Index was established. A value of 10 was established as the maximum pathogenicity rate for an inoculated bird. From this rate, 5 points corresponded to scores for gross lesions present at necropsy. For each lesion present, it represents 1 point. The remaining 5 points corresponded to the death time. To obtain the death time value, an index of 1, corresponding to the maximum value assigned to a death on the first day, was divided by the number of days that the birds were evaluated, resulting in a value of 0.1428, which corresponded to a survival bonus factor. It was possible to classify E. coli strains into four pathogenicity groups according to the pathogenicity index: high pathogenicity (pathogenicity index ranging from 7 to 10), intermediate pathogenicity (pathogenicity index ranging from 4 to 6.99), low pathogenicity (pathogenicity index ranging from 1 to 3.99) and apathogenic (pathogenicity index ranging from 0 to 0.99). The analysis of the strains according to their origin revealed that isolates from broiler bedding material presented a lower pathogenicity index. Discussion: It is possible that the source of isolation implies in different results, depending on the criteria adopted. This data reinforces the importance of use a more accurate mathematical model to represents the biological phenomenon. In the study, all avian pathogenic Escherichia coli strains were classified based on a pathogenicity index and the concept of the death time represents an interesting parameter to measure the ability of the strain to promote acute and septicemic manifestation. The use of a support method for poultry veterinary diagnostic accompanying the fluctuation of the bacteria pathogenicity inside the farms may indicate a rational use of antimicrobial in poultry industry.

Spread of a Major Clone of Salmonella enterica Serotype Enteritidis in Poultry and in Salmonellosis Outbreaks in Southern Brazil

Salmonella spp. are among the most important agents of foodborne diseases all over the world. Human Salmonella outbreaks are often associated with the consumption of poultry products (meat and eggs), and one of the most prevalent serotypes associated with these products is Salmonella Enteritidis. Brazil is one of the most important poultry exporters in the world. In southern Brazil, three closely related clones of Salmonella Enteritidis have been responsible for the majority of foodborne Salmonella outbreaks over the past decade. However, until now, there has been little information regarding the clonal relationship among the Brazilian Salmonella strains of avian origin and those involved in foodborne outbreaks. Therefore, the aim of the present study was to complete the molecular characterization of Salmonella Enteritidis strains isolated from poultry and food sources involved in Salmonella outbreaks. PCR ribotyping was performed to discriminate the strains into different ribotype profiles according to the banding pattern amplification. This technique was able to differentiate the Salmonella Enteritidis strains into two banding patterns: R2 and R4. R2 accounted for 98.7% of the strains. DNA sequencing of the 600-bp fragment, present in all ribotypes, was applied to confirm this result. The sequences generated showed high levels of similarity, ranging from 99.7 to 100%, and were grouped into a single cluster. These results suggest that there is a clonal relationship among the Salmonella Enteritidis strains responsible for several salmonellosis outbreaks and the strains collected from poultry sources.

Establishment of a Pathogenicity Index for One-day-old Broilers to Pasteurella multocida Strains Isolated from Clinical Cases in Poultry and Swine

Although Pasteurella multocida is a member of the respiratory microbiota, under some circumstances, it is a primary agent of diseases , such as fowl cholera (FC), that cause significant economic losses. Experimental inoculations can be employed to evaluate the pathogenicity of strains, but the results are usually subjective and knowledge on the pathogenesis of this agent is still limited. The objective of this study was to establish a new methodology for classifying the pathogenicity of P. multocida by formulating a standard index. Strains isolated from FC cases and from swine with respiratory problems were selected. One hundred mL of a bacterial culture of each strain, containing 106 CFU, was inoculated in 10 one-day-old broilers. Mortality after inoculation, time of death (TD), and the presence of six macroscopic lesions were evaluated over a period of seven days post-inoculation (dpi). A Pathogenicity Index Per Bird (IPI), ranging 0 to 10, was calculated. Liver and heart fragments were collected to reisolate the bacteria. Blood was collected from the surviving birds, and an ELISA test was carried out to detect specific antibodies. The median of the pathogenicity indices, the number of lesions and the rate of bacteria reisolation were significantly different (p<0.05) among the origins of the isolates (p<0.05). The pathogenicity index developed in this study allows the classification of Pasteurella multocida pathogenicity and may be an alternative to the pathogenicity models currently used for screening.