Evaluation of the hygienogram scores and related data obtained after cleaning and disinfection of poultry houses in Flanders during the period 2007 to 2014.

Cleaning and disinfection (C&D) of poultry houses is an essential aspect in farm hygiene management. Adequate performance of the different steps of a C&D protocol and the use of suitable products are key to prevent and control zoonoses and animal diseases. Hygiene monitoring on total aerobic flora through sampling with agar contact plates at different locations of the poultry house results in a hygienogram score that is used to evaluate the proper execution of C&D.This study analyzed the hygienogram scores of 19,739 poultry flocks in Flanders after C&D. Data relating to the C&D protocol, i.e., year, season, husbandry system, production type, cleaning product, sampler, active components of the disinfectant, disinfection time, disinfection temperature, and disinfection responsible, were collected.The average hygienogram score decreased significantly over time, suggesting a general improvement between 2007 and 2014. Differences in scores were found among the husbandry systems, with the barn/aviary system having a significantly better hygienogram score compared to the floor house, furnished cage, and battery. Significantly better scores also were found when a cleaning product was used in the C&D protocol. Disinfection with a peracetic acid and hydrogen peroxide combination or formaldehyde gave the best scores. In addition, C&D protocols using ≥2 different disinfectants showed improved results compared to the use of one single disinfectant. Finally, disinfection applied by a specialist contractor resulted in a better score compared to disinfection by the farmer.In conclusion, analysis of the hygienogram scores and related data allowed identifying several factors, resulting in an improvement, which may reduce the total bacterial load in poultry stables and, consequently, the number of zoonotic and pathogenic micro-organisms.

Seroprevalence of Toxoplasma gondii in domestic sheep in Belgium.

Even though infected sheep are a potential source of Toxoplasma gondii infection in humans, information is lacking concerning the seroprevalence of T. gondii infection in sheep in Belgium. We examined 3170 serum samples for anti-Toxoplasma IgG in sheep by total lysate antigen (TLA) enzyme-linked immunosorbent assay (ELISA). IgG to T. gondii was demonstrated in 87.4% of the tested sheep and in 96.2% of the 209 tested flocks. The seroprevalences in Antwerp (65.2%) and Wallonia (68.6%) are statistically lower than in the other regions in Belgium (96.7-97.8%) (P<0.05). The present study is the first report that analyzed the prevalence of T. gondii infection in sheep in Belgium and confirms the high prevalence of Toxoplasma-specific IgG antibodies in the sheep population.

Polyphasic characterization of Salmonella Enteritidis isolates on persistently contaminated layer farms during the implementation of a national control program with obligatory vaccination: a longitudinal study.

Since 2007, a national Salmonella control program including obligatory vaccination has been ongoing in Belgium. In this context, the aim of the present study was to investigate the diversity of Salmonella enterica serovar Enteritidis isolates on 5 persistently contaminated Belgian layer farms and to examine the potential sources and transmission routes of Salmonella Enteritidis contamination on the farms during successive laying rounds. A collection of 346 Salmonella isolates originating from the sampled farms were characterized using a combination of multilocus variable number of tandem repeat analysis (MLVA) and phage typing (PT). On each farm, one or 2 dominant MLVA-PT types were found during successive laying cycles. The dominant MLVA type was different for each of the individual farms, but some farms shared the same dominant phage type. Isolates recovered from hens' feces and ceca, egg contents, eggshells, vermin (mice, rats, red mites, and flies), and pets (dog and cat feces) had the same MLVA-PT type also found in the inside henhouse environment of the respective layer farm. Persistent types were identified in the layer farm inside environment (henhouse and egg collecting area). Furthermore, this study demonstrated cross-contamination of Salmonella between henhouses and between the henhouse and the egg collecting area. Additional isolates with a different MLVA-PT type were also recovered, mainly from the egg collecting area. A potential risk for cross-contamination of Salmonella between the individual layer farms and their egg trader was identified.

Persistent Salmonella Enteritidis environmental contamination on layer farms in the context of an implemented national control program with obligatory vaccination.

The aim of this study was to closely examine the Salmonella enterica serovar Enteritidis environmental contamination on persistently positive layer farms in Belgium during successive laying cycles. All of the farms were required to vaccinate their layers under the national control program for Salmonella. Seven farms with previous or current Salmonella Enteritidis contamination were monitored during different stages of the laying period and after cleaning and disinfection (CD). Environmental samples, including from the equipment and vermin, were taken in the henhouse and egg-collecting area. Dilutions were performed to define the degree of Salmonella Enteritidis contamination. Eggshells, egg contents, and ceca were also tested for Salmonella. At the end of the first sampled laying period, 41.6% of the environmental samples were contaminated with Salmonella Enteritidis. After CD, the prevalence dropped to 11.4%. On average, the prevalence in the second laying period increased again: 17.8, 18.4, and 22.3% at the onset, middle, and end of the lay period, respectively. After CD before the third laying period, the prevalence decreased to 6.6% and stabilized at the onset of lay (6.3%). During lay, as well as after CD, a wide variety of contaminated environmental samples were found; for example, in the henhouse, in the egg-collecting area, on mobile equipment and in or on vermin. In the henhouse during laying, the most recurrent and highly contaminated sites were the overshoes, floor, manure belt, and hen feces. The egg-collecting area had a significantly higher number of contaminated samples compared with that of the henhouse. For both sites, the floor appeared to be the most suitable sampling site to estimate the Salmonella Enteritidis status of the farms. Eggshell and egg content contamination varied between 0.18 and 1.8% and between 0.04 and 0.4%, respectively. In total, 2.2% of the analyzed ceca contained Salmonella Enteritidis. This study revealed that Salmonella Enteritidis is present in the environment of persistently Salmonella Enteritidis-contaminated layer farms, demonstrated that in many cases Salmonella Enteritidis contamination was not eliminated after CD, and identified the egg-collecting area as a critical point on most farms.

Disk prediffusion is a reliable method for testing colistin susceptibility in porcine E. coli strains.

During the last few years, acquired resistance to colistin in Escherichia coli, but also in other bacterial species, has been reported. It has been shown that the disk diffusion test is not a reliable method for the detection of this resistance. Therefore, there is a need for a reliable and cheap test to determine colistin susceptibility of pathogenic E. coli strains. In the current research, the colistin susceptibility of E. coli isolated during the period 2005-2006 from pigs was determined. Results obtained with the Kirby Bauer disk diffusion test (Neosensitabs, Rosco), the disk prediffusion test (Neosensitabs, Rosco) and the E-test (AB Biodisk) were compared with the results of the reference agar dilution assay. The MIC values or inhibition zones showed a bimodal distribution for the results obtained by all test methods, except the disk diffusion assay, suggesting acquired resistance in 15 strains (9.6%). The E-test and disk prediffusion assay generated results within acceptable levels compared to the reference agar dilution assay. The categorical agreement with the results obtained by the agar dilution method were good to very good for all tests, except the disk diffusion assay. In conclusion, current results suggest that, in addition to the E-test, the disk prediffusion test is a reliable, alternative agar-based colistin susceptibility method for testing colistin susceptibility of E. coli isolates in diagnostic bacteriology.

Plasma biochemistry in pigeons experimentally infected with Salmonella.

Ten pigeons were crop inoculated with 1 x 10(9) colony-forming units of Salmonella typhimurium var. Copenhagen and observed during 28 days. Ten sham-inoculated pigeons served as noninfected controls. Clinical signs after Salmonella infection consisted of polydipsia, polyuria, and diarrhea. Morbidity was 90%, but there was no mortality. All inoculated pigeons showed fecal excretion of Salmonella for at least 7 days. Biochemical analysis of plasma samples taken at 3-day intervals indicated decreased concentrations of creatine kinase (CK)-MM and CK-MB isoenzymes and elevated total protein and alpha- and gamma-globulin values. No consistent changes in the level of 17 other blood parameters were observed. After 28 days, all pigeons were necropsied. Gross lesions and bacteriologic and histologic examination indicated septicemia in all Salmonella-inoculated pigeons. Results indicate that Salmonella septicemia in pigeons induces only limited changes in biochemical blood parameters. Decreased CK concentration was a consistent finding, however, and may therefore be a useful aid in the diagnosis of salmonellosis in pigeons.

Adhesion of Streptococcus gallolyticus strains to extracellular matrix proteins.

Fourteen pigeon Streptococcus gallolyticus strains of differing virulence, were tested for their ability to adhere to immobilised fibronectin, collagen types I, III and IV. Eight, 2 and 13 strains were able to bind fibronectin, collagen types III and IV, respectively. None of the strains adhered to collagen type I. Heat treatment, proteolytic digestion or periodate treatment reduced the binding of S. gallolyticus to fibronectin and collagen type IV, suggesting that surface receptors contain proteins and carbohydrates. Although binding to these extracellular matrix proteins can play a role in the pathogenesis of streptococcosis in pigeons, binding properties could not be related to virulence, indicating that other factors determine differences in virulence among pigeon S. gallolyticus strains. Adhesion to collagen type IV may account in part for the distribution pattern of the lesions observed in naturally and experimentally infected pigeons.

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains.

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.

Genomic fingerprinting of pigeon Streptococcus gallolyticus strains of different virulence by randomly amplified polymorphic DNA (RAPD) analysis.

Randomly amplified polymorphic DNA (RAPD) analysis was performed on 95 pigeon S. gallolyticus strains of different virulence and belonging to different biotypes and different culture supernatant phenotypes as determined by SDS-PAGE. Four distinct RAPD patterns, designated A, B, C and D, were distinguished using primer OPM6 (5'CTGGGCAACT). All 76 strains generating RAPD pattern A or B were designated highly virulent on the basis of their SDS-PAGE pattern. Five of seven strains generating RAPD pattern C and 11 of 12 strains generating RAPD pattern D belonged to the moderately virulent and low virulent culture supernatant phenotype groups, respectively. Only one RAPD group C strain belonged to a highly virulent culture supernatant phenotype group. There was a correlation between biotype and RAPD patterns. These findings indicate that there is a high correlation between RAPD pattern and virulence for pigeons. Therefore, RAPD typing seems a rapid, reliable method to distinguish pigeon S. gallolyticus strains of high, moderate and low virulence.

Efficacy of inactivated whole-cell vaccines against streptococcosis in pigeons.

Two experimental vaccines were developed and evaluated for their efficacy against Streptococcus gallolyticus septicaemia in pigeons. Both vaccines contained whole-cell formaldehyde-inactivated S. gallolyticus serotype 1 bacteria and a mineral oil adjuvant. The supernatant of a S. gallolyticus broth culture was also added to one of the vaccines. Four groups of 10 pigeons were inoculated either once, or twice, with a 4-week interval, with one of the vaccines. Four weeks after the last vaccination, pigeons were challenged by intravenous inoculation with S. gallolyticus serotype 1. Morbidity after infection was not significantly different between groups of pigeons vaccinated with the two vaccines. In groups of pigeons vaccinated once, morbidity after infection ranged from 50 to 70%; in pigeons vaccinated twice, morbidity was 10 to 30%. In a non-vaccinated inoculated control group, the morbidity was 80%. It was concluded that double vaccination can result in some clinical protection against streptococcosis in pigeons.

Differentiation between Streptococcus gallolyticus strains of human clinical and veterinary origins and Streptococcus bovis strains from the intestinal tracts of ruminants.

Strains formerly identified as Streptococcus bovis were allotted to two groups by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell proteins. Strains from humans with infections, mostly patients with endocarditis, and strains from pigeons with septicemia clustered with the recently described species Streptococcus gallolyticus. The original S. bovis type strain and strains exclusively from ruminants formed the second cluster. The findings indicate that S. gallolyticus is more likely to be involved in human and animal infections than S. bovis. Growth characteristics and several biochemical reactions were found to be useful in the differentiation of S. gallolyticus from S. bovis.

Typhlitis caused by intestinal Serpulina-like bacteria in domestic guinea pigs (Cavia porcellus).

Between January 1992 and December 1996, Serpulina-like bacteria were demonstrated in intestinal tract lesions from 37 of 88 guinea pigs submitted to the University of Ghent in Ghent, Belgium, for necropsy because of disease and death from different unknown causes. All infected animals had a history of sudden death with minimal introductory clinical signs. Occasionally, they produced yellow, slimy feces or showed nervous signs, but the condition always had a fatal outcome within 24 h. When larger colonies of guinea pigs were involved, the disease spread very rapidly unless treatment with ronidazole was initiated. Lesions consisted of a catarrhal or hemorrhagic inflammation of the colon and cecum (typhlitis). Electron microscopy demonstrated the presence of large numbers of Serpulina-like organisms adhering to the cecal mucosae of these animals. Attempts to isolate the agents failed. The organisms did not stain by an immunofluorescence technique for the detection of Serpulina hyodysenteriae. The present data provide evidence that intestinal Serpulina-like organisms can be important as a cause of disease in guinea pigs.

Extracellular proteins and virulence in Streptococcus bovis isolates from pigeons.

The association between virulence and the occurrence of the extracellular proteins A, T1, T2 and T3 in the culture supernatant of pigeon Streptococcus bovis strains, was examined in experimental infection studies. Fourteen groups of 10-17 pigeons were inoculated intravenously with 1 x 10(9) CFU of S. bovis strains that belonged to the phenotypes A + T1, A - T1, A + T2, A - T2, A + T3 and A - T3, respectively. The overall postinoculation morbidity in the phenotype groups was 85%, 87%, 70%, 5%, 100% and 37%, respectively. These results indicate that strains producing A or T1 are of high virulence, those producing T3 only are of moderate virulence and those producing T2 are of low virulence. Virulence of S. bovis for pigeons was more clearly correlated with supernatant-phenotype than with serotype.

Secreted antigens as virulence associated markers in Streptococcus bovis strains from pigeons.

SDS-PAGE and Western blot analysis were performed on the culture supernatant of 7 pigeon S. bovis reference strains belonging to the serotypes 1, 2, 3 and 5. The culture supernatant of highly virulent serotype 1, 2 and 5 strains contained four antigens that were absent in low virulent serotype 3 strains, notably a 185 kDa minor protein band (A) and a triplet (T1) of 70 kDa. The less virulent serotype 3 strains on the other hand contained protein triplets, that had a molecular mass of either 68 kDa (T2) or 74 kDa (T3). The prevalence of A, T1, T2 and T3 was examined in 68 S. bovis strains isolated from healthy pigeons and in 68 S. bovis strains isolated from pigeons that died of S. bovis septicaemia. Six supernatant phenotypes were identified: A-T1 (32 strains), A- T2 (10 strains), A- T3 (7 strains), A+ T1 (84 strains), A+ T2 (1 strain) and A+ T3 (2 strains). Ninety-four percent of the strains lacking the A and T1 proteins were isolated from healthy pigeons, and only 6% were isolated from septicaemia. Strains expressing A and/or T1, however, were isolated form septicaemia in 57% of the cases. These observations may indicate that the A and/or T1 proteins are associated with virulence.

Effect of antimicrobial treatment on the course of an experimental Yersinia pseudotuberculosis infection in canaries.

The efficacy of five antimicrobials administered via the drinking water was compared for the treatment of experimental Yersinia pseudotuberculosis infection in canaries. Groups of eight to 10 canaries were treated with ampicillin (2 g/1), doxycycline (500 mg/1), enrofloxacin (150 mg/1), chloramphenicol (1 g/1) or sulphamerazine-trimethoprim (1 g/1-200 mg/1) for 10 days, commencing 2 days after experimental infection. No clinical signs were observed in the group treated with enrofloxacin and there were no deaths and no isolations of Y. pseudotuberculosis from the spleen and liver at necropsy carried out at 35 days post-inoculation. In the groups treated with the other antibiotics morbidity varied between 50 and 100%, and mortality between 30 and 44%. Mortality and morbidity in an untreated infected control group of 12 canaries were 100%. Results indicate that enrofloxacin offers good possibilities for the treatment of pseudotuberculosis in recently infected canaries.