Low frequency of itraconazole resistance found among Aspergillus fumigatus originating from poultry farms in Southwest Poland.

AIM: The aim of this study was to investigate the collection of avian Aspergillus fumigatus isolates for the presence of triazole resistance. MATERIAL AND METHOD: The study was performed on 60 A. fumigatus isolates cultured from lung tissue samples from chicken (25), geese (17), turkeys (13) and ducks (5). The samples were obtained from 40 different farms located in the Southwest Poland and were collected in the period of September 2015 to November 2016. The EUCAST microdilution method, with the use of three concentrations of itraconazole (ITR) (1, 0.5, and 0.25mg/L), was used to screen the susceptibility of all isolates. Additionally, the selected 20 isolates were tested with eleven concentrations ranging 0.015-16mg/L of ITR, voriconazole, posaconazole and isavuconazole. RESULTS: Most tested isolates (59/60) were susceptible to ITR (MIC≤0.5mg/L). One isolate showed elevated MIC for ITR (16mg/L), as well as voriconazole (4mg/L), izavuconazole (4mg/L), and posaconazole (0.5mg/L). This isolate was identified on the basis of DNA analysis as A. fumigatus carrying TR34/L98H mutation. All of the ITR-susceptible isolates under study were also susceptible to other triazoles. CONCLUSION: Obtained results indicated a low frequency (1.6%) of A. fumigatus resistant to triazoles among avian isolates from the Southwest regions of Poland.

Antimicrobial susceptibility of Salmonella spp. strains isolated from free-living birds.

Salmonella is one of the most common causes of food poisoning in the European Union and the United States of America. Free-living birds are known as a reservoir for the different serovars of Salmonella, including S. Typhimurium, S. Enteritidis, S. Infantis, S. Newport and S. Hadar, which may play an important role in the epidemiology of salmonellosis in farm animals, particularly poultry. Also, the antibiotic resistance of Salmonella spp. is a growing, public health emergency. In the present study, the authors examined 36 Salmonella spp. strains, which belonged to 3 subspecies; enterica, salamae and houtenae. All of them were obtained from 13 species of free-living birds in Poland. The antimicrobial susceptibility of these Salmonella strains was determined, using commercial SensititreTM Salmonella, MIC plates, for fourteen antimicrobials, from nine antimicrobial groups: sulfonamides, aminoglycosides, fluorochinolones, cephalosporines, beta-lactams, tetracyclines, phenicols, polymyxins and trimethoprim. The prevalence of selected genes which determine antimicrobial resistance; i.e. aadB, aacC, blaTEM, blaPSE-1, blaOXA, tetA, tetB, tetC, tetG, cat1, cat2, cat3 and floR was also tested. Among all of the examined strains, no resistance was detected in relation to gentamicin, cefotaxime and ceftazidime, while most strains (94.5%) were resistant to sulfamethoxazol. Among the 36 examined bacteria isolates, twenty were resistant to more than one antimicrobial agent. The antimicrobial resistant gene, floR was most frequently detected among all examined strains (50%).

Invasiveness of Listeria monocytogenes strains isolated from animals in Poland.

Animals are important reservoir of Listeria monocytogenes, a pathogen causing serious infections in both humans and livestock. However, data on invasiveness of L. monocytogenes strains of animal origin is very scarce. Ability of 18 L. monocytogenes strains of animal origin to invade HT-29 cells was investigated. Plaque forming assay was used to assess invasiveness and ability of the pathogen to spread in the cell line. Almost 40% of L. monocytogenes strains were weakly invasive. It was shown that strains from serogroup 4b exhibited the highest invasiveness, whereas serogroup 1/2b consisted of strains of invasiveness below 0.0001%. Analysis of translated inlA and inlB gene sequences revealed no premature stop codons. Lineage-specific mutations in low invasive strains were identified within inlA and inlB sequences. Our results demonstrate high incidence of low invasive animal L. monocytogenes strains, which may be at least partly explained by unique point mutations in the InlA and InlB.

The effects of subcutaneous and intraocular administration of class B ODN CpG in chicken on the expression of TLR21, IFN-γ and IL-1ß.

The synthetic unmethylated oligodeoxynucleotides with CpG motifs (CpG ODN) were shown to activate Toll-like receptor 21 (TLR21) and stimulate the innate and adaptive immune system. In this study we tested the expression of TLR21, interferon (IFN)-γ and interleukin (IL)-1ß mRNA in the blood after subcutaneous and intraocular application of the class B CpG ODN in chicken. The relative expression of mRNA of TLR21, IFN-γ and IL-1ß were quantified at 3, 6, 12, 24 and 72 h post-stimulation. The study revealed that IFN-γ mRNA expression was significantly upregulated 12 h after subcutaneous stimulation with a high and low dose of ODN CpG, whereas the IL-1ß mRNA expression levels were significantly upregulated 3 and 72 h after subcutaneous administration. After intraocular administration, the IL-1ß mRNA levels were the highest at 24 h post-application, albeit not specifically. This data indicates that class B CpG ODN has the ability to induce TLR21 response in blood when administered parenterally in chicken. In contrast, intraocular administration of CpG ODN was not able to produce a significant increase in cytokine mRNA expression in blood. The data suggest that additional stimulus, e.g. the antigen, may be needed on the site of mucosal administration to activate systemic immune response.

Salmonella spp. as a cause of mortality and clinical symptoms in free-living garden bird species in Poland.

Some species of garden birds are considered to be sensitive to Salmonella (S.) spp. infections. The aim of this study was to determine the cause of mortality of six free-living birds in one private property in suburban area of Wroclaw (Poland). In 2013 Poland experienced prolonged winter, with low temperatures and snow precipitations. During March and April, two dead individuals of the Eurasian siskin (Carduelis spinus) and four dead individuals of the Greenfinch (Carduelis chloris) were found in proximity of the bird feeder. At the time of ringing procedure in the same area, faecal samples of all individuals belonging to these two species of birds were collected, regardless clinical symptoms. In total, twenty two faecal samples of birds belonging to both bird species were collected in the same property. All of them were Salmonella enterica subsp. enterica serovar Typhimurium positive. The visible illness among European siskins and Greenfinches, caused by S. Typhimurium, sug- gests that both Eurasian siskin and Greenfinch may be potential reservoirs of Salmonella spp. Therefore they might play a role in transmission of zoonotic pathogens to other garden bird species or to people.

Impact of CpG oligodeoxynucleotide stimulation on percentage of T and B cells in chicken.

TLR stimulation in chickens has been shown to play a role in the initiation and regulation of innate and adaptive immune responses. The aim of this study was to use flow cytometry to establish the percentage of T and B subset in blood and lymphoid organs in chicks after CpG oligodeoxynucleotide (ODN) stimulation. It was demonstrated that the percentages of CD3+, CD4+, TCRgamma delta+ cells and Bu-1+MHC class II+ cells in blood 24 h post-injection were significantly higher than in the control groups. It was also shown that the percentages of CD3+ and CD4+ cells in the spleen at 48 h post-injection were significantly higher than in control groups. The percentage of Bu-1+ cells in the bursa of Fabricius after CpG ODN stimulation (98.38 +/- 0.84) was significantly higher than that found in the non-CpG ODN control group (94.54 +/- 2.51) (p < or = 0.05). The results indicate that class B CpG ODN increases the percentage of both T (especially CD4+ cells) and B cells.

New insight into the structure, development, functions and popular disorders of bursa Fabricii.

Humoral immune responses in birds, contrary to mammals, depend on the normal functioning of bursa Fabricii. Recent studies have delivered new information about the structure, development and origin of cells that compose the bursa environment. Several viral infections affect bursa, causing lymphocyte depletion or excessive proliferation. This review summarizes data on the development and histology of healthy bursa and introduces some common disorders that affect this organ.

The influence of antibiotics on B-cell number, percentage, and distribution in the bursa of Fabricius of newly hatched chicks.

Antibiotics are commonly used to prevent and treat poultry microbial infections, but certain antibiotic families depress humoral immunity, such as antibody production. Poultry humoral immunity depends on the normal functioning of the bursa of Fabricius and the B lymphocytes that mature in that gland. In this study, recommended therapeutic doses of enrofloxacin, florfenicol, or ceftiofur were administered to 2-d-old chicks. On d 7 post-hatch, bursae were sampled for histological, immunohistochemical, and flow cytometric determination of Bu-1-positive (Bu-1+) cell number, percentage, and distribution. The bursa of Fabricius from all treatment and control groups had normal morphology. The administration of antibiotics significantly decreased the number of Bu-1+ cells in the bursal medulla, with a simultaneous increase of these cells in the cortex. Flow cytometry revealed a significant decrease in the percentage of bursal Bu-1+ cells from all of the studied antibiotics: enrofloxacin (93.91 ± 3.27), florfenicol (87.84 ± 7.14), and ceftiofur (89.16 ± 5.68) compared with that of the control (96.48 ± 2.60). The combination of reduced percentages of Bu-1+ cells and a decrease in these cells in the medullary region suggests lower B cell maturation.

Genetic comparison of Campylobacter jejuni isolated from different cattle farms.

To compare the genotypes of Campylobacter jejuni, isolates of cattle origin were collected from 9 Polish farms and genotyped by ERIC-PCR. We identified 28 genotypes among the 43 C. jejuni isolates, and demonstrated high genomic diversity. The highest level of diversity was observed in strains isolated from stanchion-barn animals in opposition to those from the loose-housing system.

Optimization of Salmonella enteritidis recombinant heat shock protein 60 production.

The aim of the study was to optimize conditions for producing Salmonella Enteritidis recombinant heat shock protein 60 (rHsp60). Seven Escherichia coli host strains (Rosetta, Turner, C41, C43, Origami, BL21pLys, Rosetta pLys) were transformed by a recombinant plasmid containing Hsp60 gene from Salmonella Enteritidis, and then cultured and induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The highest S. Enteritidis rHsp60 yield was obtained using E. coli strain C41. Induction of this strain using IPTG allowed the yield 400 microg of S. Enteritidis Hsp60 protein/2L of culture, but by autoinduction the yield exceeded 800 microg/2L.

Antimicrobial resistance of thermophilic Campylobacter spp. isolated from cattle in Poland.

Campylobacter species are among the most frequently identified bacterial causes of human gastroenteritis. Because Campylobacter spp. harbored by cattle can be transmitted to humans, in this study we investigated antimicrobial resistance of thermophilic Campylobacter isolated from cows. Our study included 150 strains of Campylobacter (143 strains of C. jejuni and 7 strains of C. coli) isolated from cows in South-Western Poland. The minimal inhibitory concentration (MIC) to ciprofloxacin, erythromycin, gentamicin and tetracycline were determined using the agar dilution methodology. All strains of C. coli were susceptible to all four drugs studied. The most frequently detected resistance of C. jejuni was to ciprofloxacin (26 strains 18.2%). Resistance to tetracycline was observed in 5 strains (3.5%). All strains of C. jejuni were susceptible to erythromycin and gentamicin.

Feline herpesvirus 1 and Chlamydophila felis prevalence in cats with chronic conjunctivitis.

The prevalence of Chlamydophila felis and Feline Herpesvirus type 1 was investigated in 30 cats with chronic conjunctivitis, with use of conjunctival swabs and conventional polymerase chain reaction (PCR). In cats with chronic conjunctivitis the DNA of C. felis and FHV-1 was detected in 2 of 30 cats (6.7%) and in 10 of 30 animals (33.3%), respectively. One case of FHV-1 DNA, and none of C. felis was found in control group. There was no case of co-infection with both pathogens.

Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR-RFLP of the fimH gene.

In our studies on FimH adhesins expressed by different Salmonella serovars, we cloned and sequenced the fimH genes from Salmonella enterica ssp. Enterica ser. Gallinarum biovar Gallinarum and S. enterica ssp. Enterica ser. Gallinarum biovar Pullorum. Comparison of the nucleotide sequences revealed the presence of a single-nucleotide polymorphism (SNP) at position 544 bp from the A of the start codon of the fimH open reading frame (ORF). Further analysis of the restriction enzyme sites in fimH gene showed that the SNP at this position is responsible for a sequence specifically recognized by SacI in S. Gallinarum biovar Gallinarum only, making it possible to differentiate both biovars with the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digestion of PCR amplicons of the fimH gene from S. Gallinarum biovar Gallinarum strains with SacI gave two DNA fragments of 554 and 472 bp and only one fragment of 1026 bp for S. Gallinarum biovar Pullorum. This allows a clear differentiation between these two biovars.

Genotype analysis of Campylobacter jejuni isolated from broilers in Poland.

Twenty-six Campylobacter jejuni strains isolated from poultry were analyzed by genotypic typing including ITS-profiling, REP- and ERIC-PCR. ITS-profiling revealed the presence of 8 different genotypes. Amplification of REP sequences by PCR gave similar results with 10 different genotypes. ERIC-PCR was found to be the most discriminatory for typing C. jejuni. As many as 13 different DNA patterns were obtained with this technique. Based on data obtained it was found that C. jejuni isolates recovered from broilers at the slaughterhouses in southwest Poland are characterized by a high degree of genetic heterogeneity.

The clinical course of the Infectious Laryngotracheitis (ILT) field case in hens and estimation of immunoprophylaxis efficiency.

The ILT case was observed in laying hen farm where birds of different age (from 40 to 107 weeks) were kept in 10 flocks. A rapid spread of the disease, the decrease in egg production (in flock No. 1 it reached 58%) and higher mortality (the highest in 76 and 77 week-old birds, accounting for 0.11% and 0.36%, respectively ) was recorded during first 2 weeks of disease. Antibodies against ILT virus were detected in serum of the examined birds during the whole observation period (50 weeks after the disease outbreak). The laying hens were vaccinated at 8 weeks of age and boosted after 5 weeks. The vaccine was applied in drinking water, in a dose twice as high as usually recommended per one bird. Immunopropylaxis efficiency was estimated on the basis of immunological response in birds (serum samples, ILT ELISA kit, Guildhay Ltd.) and general health status of hens in flocks. Postvaccinal immunity, the presence of specific antibodies against ILT, was observed in all birds during the observation period (51 weeks). During that time GMT value ranged from 8261,3 (week 10) to 5196 (week 51) after the second vaccination, and CV amounted in this period to 41.1% and 51%, respectively. Subsequently, clinical symptoms of the disease disappeared and the egg production, as well as mortality, returned to the level of technological norms for laying hens.

Prevalence of infectious diseases in ring-necked pheasant flocks in Poland.

The health status of ring-necked pheasants in view of the prevalence of infectious diseases was estimated in Polish pheasantries in the years 1997-2000. Anatomicopathological, microbiological and serological examinations were carried out on birds derived from 26 pheasantries, including birds randomly selected from 18 flocks and sick or dead birds sent from 8 pheasantries. Antibodies specific to the following viruses were detected in serum blood samples: HE, AE, AP, REO, AI, Adeno group 1, MD, ND, as well as Mycoplasma gallisepticum specific antibodies. However, in none of the examined flocks was the presence of antibodies against reticuloendoteliosis virus found. Marble spleen disease and salmonellosis proved to be the most frequent cause of death during the growing period.

Comparison of ITS profiling, REP- and ERIC-PCR of Salmonella Enteritidis isolates from Poland.

Thirty-one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP-PCR. ERIC-PCR and ITS profiling (PCR-ribotyping). Analysis of DNA banding patterns generated by REP-PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC-PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC-PCR fingerprints. REP- and ERIC-PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S-23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south-west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP- and ERIC-PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.

[Occurrence of Campylobacter and salmonellas in relation to liver changes in slaughtered poultry]. / Vorkommen von Campylobacter und Salmonellen im Zusammenhang mit Leberveränderungen bei Schlachtgeflügel.

On the occasion of slaughter, 97 laying chickens, 100 broilers, 48 geese and 36 ducks were examined for Campylobacter spp. and Salmonella spp. in liver, small intestine and caeca. Pathological changes in the liver were recorded. Campylobacters and salmonellas were isolated from 61 and 18% of the laying chickens, 63 and 3% of the broilers, 54 and 15% of the geese and from 81 and 39% of the ducks, respectively. Birds with diseased liver were found more often infected with campylobacters and salmonellas than those without. This correlation could be gathered from the isolation rates from the liver and from the intestine. The occurrence of campylobacters and salmonellas together in the liver was only observed in individuals with pathological changes of the liver. The own findings suggest that besides salmonellas also campylobacters can be responsible for diseases of the liver.

[The pathogenicity of Campylobacter jejuni as a monoinfection and as a mixed infection with Escherichia coli 078:K80 in broilers]. / Zur Pathogenität von Campylobacter jejuni als Monoinfektion und als Mischinfektion mit Escherichia coli O78:K80 bei Broilern.

Two week old broilers (n = 61) with a monoinfection with Campylobacter jejuni (0.5 ml of suspension containing 10(5) CFU/ml per os) showed reduced increase in weight during week 3 after infection compared to the control group. An other group of chickens (n = 31) was additionally infected with a suspension of Escherichia (E.) coli O78:K80 via drinking water from day 4 to 6 after the primary infection. This mixed infection provoked clinical signs of a disease and reduced increase in weight during the first two weeks of the experiment. Seven broilers of this group showed a fibrinous pericarditis and/or perihepatitis. Four of these chickens died. It can be concluded from the experiment that an infection with Campylobacter causes reduced weight gain and supports a systemic infection with E. coli.