Fowl Typhoid Outbreak on a Commercial Turkey Farm in Croatia.

Fowl typhoid is a septicemic disease caused by Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum. It is a host-specific disease primarily affecting chickens and turkeys, although it has been reported in various animal species and sporadically in humans. Here, we present a case of a fowl typhoid outbreak on a turkey poult farm where the source of infection was the hatchery. The birds started showing symptoms of growth retardation at 21 days of age, after which the mortality rates gradually started to increase. Post mortem examination revealed that the main lesions were granulomatous proliferations in the small intestines. The results of the histopathological examination indicate that the severity of the infection was alleviated by the application of phytogenic mixtures and probiotics as a supportive treatment, even though the affected flock was eventually culled at 60 days of age. The farmer was advised to apply more strict biosecurity measures to prevent the spread of the disease on the farm and try to eradicate the pathogen from the barn. Since the outbreak, there have been no recurrent infections.

Autogenous Escherichia coli Vaccine Application as an Innovative Antimicrobial Therapy in Poultry Farming-A Case Report.

Escherichia coli (E. coli) is one of the most common bacterial causes of infection in poultry farming. Whether the infection is localized or systemic, a primary or secondary disease, it is most frequently treated through the application of wide-spectrum antimicrobials. Excessive use of antimicrobials in agriculture is significantly contributing to the worldwide rise of antimicrobial resistance, but is also very expensive and often ineffective in the long term. Here, we present a case where a colibacillosis outbreak on a family farm of laying hens was treated using an autogenous vaccine. The birds had septicemia, cellulitis, and severe skin wounds. They were not vaccinated against E. coli, and did not receive any antimicrobials previously. E. coli strains were isolated from the daily mortalities on the farm and used for preparation of the vaccine. Each bird was given an intramuscular injection of the autogenous vaccine. The immunogenicity of the vaccine was tested by the determination of specific antibody levels in the sera of the birds using the in-house ELISA. Shortly after vaccination, the morbidity and mortality rates significantly decreased, and egg production was improved. The application of the autogenous vaccine served as a curative and preventive measure, and has proven to be a very efficient method of antimicrobial therapy.

Genomic Analysis of Escherichia coli Longitudinally Isolated from Broiler Breeder Flocks after the Application of an Autogenous Vaccine.

Escherichia coli is the main bacterial cause of major economic losses and animal welfare issues in poultry production. In this study, we investigate the effect of an autogenous vaccine on E. coli strains longitudinally isolated from broiler breeder flocks on two farms. In total, 115 E. coli isolates were sequenced using Illumina technologies, and compared based on a single-nucleotide polymorphism (SNP) analysis of the core-genome and antimicrobial resistance (AMR) genes they carried. The results showed that SNP-based phylogeny corresponds to a previous multilocus-sequence typing (MLST)-based phylogeny. Highly virulent sequence types (STs), including ST117-F, ST95-B2, ST131-B2 and ST390-B2, showed a higher level of homogeneity. On the other hand, less frequent STs, such as ST1485, ST3232, ST7013 and ST8573, were phylogenetically more distant and carried a higher number of antimicrobial resistance genes in most cases. In total, 25 antimicrobial genes were detected, of which the most prevalent were mdf(A) (100%), sitABCD (71.3%) and tet(A) (13.91%). The frequency of AMR genes showed a decreasing trend over time in both farms. The highest prevalence was detected in strains belonging to the B1 phylogenetic group, confirming the previous notion that commensal strains act as reservoirs and carry more resistance genes than pathogenic strains that are mostly associated with virulence genes.

Comparison of Chicken Immune Responses to Immunization with Vaccine La Sota or ZG1999HDS Strain of Newcastle Disease Virus.

Newcastle disease (ND) is a highly contagious avian disease. Global control of ND is mainly based on vaccination of poultry; however, reported outbreaks of ND in vaccinated flocks indicate a constant need to re-evaluate the existing vaccines and a development of the new ones. In this study, 4-week-old male chickens of the layer commercial hybrid were immunized oculonasally with a commercial NDV live La Sota vaccine (LS group), a suspension of lyophilized NDV strain ZG1999HDS (ZG group), or saline (Control (K) group). Antibody response was determined by haemagglutination inhibition (HI) assay. Cell-mediated immunity (CMI) was characterized by immunophenotyping of leukocyte's and T-lymphocyte's subpopulations (flow cytometry). Applied NDV strains did not cause any adverse reaction in treated chickens. Both strains induced the significantly higher HI antibody response in comparison to the control group, and overall antibody titer was higher in ZG group than in LS group. CMI, manifested as a higher proliferation of B- and T-helper cells, yielded better results in the ZG groups than in the LS group. Based on the obtained results, we conclude that the strain ZG1999HDS is immunogenic and is a suitable candidate for further research and development of poultry vaccines.

Kurthia gibsonii, a novel opportunistic pathogen in poultry.

Kurthia gibsonii [H. Kurth, 1883. Ueber Bacterium zopfii, eine neue Bakterienart. Berichte der Deutschen Botanischen Gesellschaft, 1, 97-100] was isolated on 10 unrelated laying hen farms over a period of 15 months. Farmers reported elevated morbidity and mortality rates, and suspected colibacillosis based on the necropsy findings. The most frequently found lesions were perihepatitis, fibrinous peritonitis, salpingitis and oophoritis. Necropsy findings and bacteriological results allowed the diagnosis of colibacillosis. In addition, K. gibsonii was isolated from the ovarian follicles (44.44%), liver (22.22%), peritoneum (16.67%), bone marrow (5.56%), spleen (5.56%), and duodenum (5.56%). On all farms, coinfection with E. coli was detected, while on some farms other common avian pathogens were found as well. In total, 18 K. gibsonii strains were identified and phylogenetically analysed based on the 16S rRNA gene sequences. The results showed some variability of the strains originating from the same farm, although the overall phylogenetic diversity was low, regardless of the geographical location of the farm, age of the flock or date of collection. Embryo lethality assay showed K. gibsonii is not able to cause a primary infection. We conclude that Kurthia gibsonii may play a role as an opportunistic pathogen for poultry. This is the first report of coinfection of Kurthia gibsonii and E. coli in laying hens. RESEARCH HIGHLIGHTSKurthia gibsonii is a novel opportunistic pathogen in poultry.Phylogenetic analysis showed low genetic diversity of Kurthia gibsonii isolates.Embryo lethality assay showed K. gibsonii does not cause primary infections.

Longitudinal study on the effect of autogenous vaccine application on the sequence type and virulence profiles of Escherichia coli in broiler breeder flocks.

Colibacillosis is one of the most common problems in the poultry industry. Escherichia coli strains on farms are often genetically diverse and therefore commercial vaccines provide little protection to the flocks. Here, we investigated the effect of the autogenous E. coli vaccines on the prevalence of 84 virulence-associated genes in E. coli isolated from four and five consecutive flocks on two broiler breeder farms, respectively. 115 E. coli isolates were sequenced using Illumina technologies, and compared based on both their set of housekeeping genes and their virulence profiles, defined through the composition of virulence genes. Predominantly, phylogenetic analysis showed obvious distinction between the isolates originating from different farms suggesting spatial-dependent transmission of pathogenic strains. We detected 23 sequence types, while 52.58 % of the isolates belonged to two clonal complexes. Analysis of the virulence genes showed highest prevalence (>85 %) of feoB, uspA, uspB, uspG, uspE, fimH, ompA, astA, focA, hlyE, uspC, crl, csgA, ompT and iss, of which 50 % are toxin associated genes, demonstrating the importance of competition in the pathogenesis process. Interestingly, usp genes, which are primarily associated with uropathogenic E. coli strains, were detected in all investigated isolates. The heatmap analysis demonstrated that strains belonging to same phylogenetic groups often share similar virulence profiles, confirming the usefulness of quick tests for phylogenetic typing. However, our results suggest the need to update the list of the minimal predictors used for the identification of avian pathogenic strains. Overall results indicate that continuous application of autogenous vaccines led to lower genetic diversity of E. coli housekeeping genes, but not virulence genes.

Phylogenetic characterization of avian pathogenic Escherichia coli strains longitudinally isolated from broiler breeder flocks vaccinated with autogenous vaccine.

Escherichia coli is the most common bacterial cause of infections in poultry farms. It is known for its genetic heterogenicity that complicates the protection of poultry health through immunoprophylaxis. In farms with continuous problems with colibacillosis, autogenous E. coli vaccine was implemented to the vaccination program instead of commercial vaccines. In this study, we investigated the effect of the autogenous vaccine on E. coli phylogroup diversity on 2 broiler breeder farms with 4 and 5 flocks, respectively. The first flocks on both farms were vaccinated with commercial vaccines, while application of autogenous vaccine was introduced in the second flock on both farms. In total, 113 strains were selected based on the target organs and age of chickens. Targeted organs were the peritoneum, liver, oviduct, and bone marrow, and analyzed strains were isolated from chickens older than 21 wk of age when problems with colibacillosis start emerging. The strains were phylotyped by PCR and allocated to phylogroups A, B1, B2, C, D, E, F or clades I-V. The results showed that autogenous vaccine could significantly affect the phylogroup shift of the strains. On farm A, application of the autogenous vaccine induced significantly lower prevalence (P = 0.01) of the phylogroups represented in the vaccine among the strains later isolated from the vaccinated flock, while on farm B, the results showed a decrease in the phylogenetic diversity with a dominant prevalence of group B2 despite the vaccine application. The results indicate that implementation of the autogenous vaccine can repress the majority of the strains, but also be unable to eliminate the presence of certain phylogroups, and thus lead to strain shift. Further detailed analyses of multilocus sequence typing and virulence genes will elucidate the pathogenic potential and selection of certain strains, with emphasis on B2 phylogroup.

Immunogenicity of Newcastle disease virus strain ZG1999HDS applied oculonasally or by means of nebulization to day-old chicks.

Newcastle disease (ND) is one of the classic viral infections of poultry which resists all the efforts of eradication. Newcastle disease virus (NDV) strain ZG1999HDS was isolated during the outbreak in 1,999 at a broiler farm in Croatia. Previous trials in chickens confirmed it to be a lentogenic pathotype and immunogenic by stimulating humoral and cell mediated immunity. Further characterization by deduced amino acid sequence at the cleavage site of fusion protein confirmed its lentogenic nature, and in vitro tests its oncolytic capacity. Owing to its immunogenicity, strain ZG1999HDS is considered for vaccine development. In this study, 1-day-old chicks were vaccinated using strain ZG1999HDS oculonasally or by nebulization. Strain ZG1999HDS induced humoral immune response in both immunized groups The cell-mediated immune response occurred earlier in the group immunized by nebulization, as shown by a higher frequency rate of T and B lymphocytes, and significantly higher expression of IFN-α in respiratory organs and IFN-γ expression in the spleen. Viral genomic RNA was not detected in investigated organs. Thus, NDV strain ZG1999HDS is immunogenic when administered by means of nebulization or oculonasally without any adverse effects and is therefore suitable for further research and vaccine development. Further research is needed regarding its tropism.

High phylogenetic diversity of Gallibacterium anatis is correlated with low biosecurity measures and management practices on poultry farms.

Gallibacterium anatis is considered one of the most common bacterial causative agents of reproductive tract disorders in poultry. In this study, phylogenetic analysis of partial rpoB sequences and biotyping using MALDI-TOF MS was done in order to investigate the genetic diversity of Gallibacterium isolates from 13 farms with different biosecurity measures and management practices. Sampling was done as a part of regular monitoring, except for Farms 9-13 that were included in the study to represent extensive production systems with lowest biosecurity levels. Pharyngeal and cloacal swabs were taken from live birds, while swabs from trachea, liver, peritoneum and oviduct were taken during necropsies. After cultivation and identification, strains from each farm were randomly selected for sequencing and biotyping. Both results showed high level of heterogeneity among the isolates originating from farms with low biosecurity levels, unlike isolates from farms with higher biosecurity levels and proper management that were more closely related and clustered together. Such correlation was statistically significant. Low biosecurity levels enable horizontal transmission of the pathogens, as well as gene transfer. The results confirm the importance of adequate biosecurity measures and management on poultry farms as they greatly affect the genetic diversity of the pathogens. Therefore, implementation of basic biosecurity measures could help control the heterogeneity of Gallibacterium strains, which would alleviate control of the infection prevalence on farms through immunoprophylaxis, and consequently improve poultry production. Also, the genetic diversity of G. anatis on poultry farms could be a good bioindicator of management practices and biosecurity measures used. RESEARCH HIGHLIGHTS High correlation between low biosecurity and high diversity of Gallibacterium anatis. Diversity of Gallibacterium is a good bioindicator of management practices on farms.

Enrichment of eggs in n-3 polyunsaturated fatty acids by feeding hens with different amount of linseed oil in diet.

The production of n-3 polyunsaturated fatty acid enriched eggs by addition of linseed oil to the laying hens' diet has been evaluated in terms of production parameters and n-6/n-3 ratio. A total of 150 18weeks old Lohmann Brown laying hens were housed in cages and fed with basal diet and four experimental diets containing 1%, 2%, 3% or 4% of linseed oil added to the basal diet. The effect of the altered level of linseed oil on hens laying performance, fatty acid content and composition and cholesterol content in egg yolk has been evaluated during 13weeks of experiment. Egg weight, yolk fat content, yolk weight, yolk percentage and shape index were not influenced by dietary treatment. The ratio between n-6 and n-3 polyunsaturated fatty acids in eggs decreased in first 5weeks and then remained stable until the end of the experiment for all experimental groups. Different contents of linseed oil in feed highly influenced the n-6/n-3 ratio (P<0.0001). Addition of linseed oil did not influence the cholesterol content in yolks (P=0.5200) while the only factor affecting the cholesterol content was the hens age (P<0.0001).

Immunogenicity and safety of Queensland V4 and Ulster 2C strains of Newcastle disease virus given to maternally immune, newly hatched chickens by nebulization.

Commercial chickens with a high level of maternal antibodies for Newcastle disease were vaccinated when newly hatched with Queensland V4 or Ulster 2C Newcastle disease virus (NDV) strains by nebulization. The exposure time to a fine aerosol of vaccine produced with an ultrasonic nebulizer was 60 sec. The chickens were challenged oculonasally with virulent NDV strain Texas GB in weekly intervals up to the 49th day of life. Although protected for several weeks by maternal antibody, they were sufficiently protected thereafter by active immune response to the vaccines. Vaccinal reactions were not observed. Queensland V4 produced higher titers than Ulster 2C and provided better protection to challenge.