M.A.P. van Bergen

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

ABSTRACT Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.

Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates

The presence and characteristics of Clostridium difficile were investigated in 839 faecal samples from seven different animal species in the Netherlands. The number of positive samples ranged from 3.4% (cattle) to 25.0% (dogs). Twenty-two different PCR ribotypes were identified. Among 96 isolates, 53% harboured toxin genes. All C. difficile isolates from pigs, cattle and poultry were toxinogenic, whereas the majority of isolates from pet animals consisted of non-toxinogenic PCR ribotypes 010 and 039. Ribotype 012 was most prevalent in cattle and ribotype 078 in pigs. No predominant ribotypes were present in horse and poultry samples. Overall, PCR ribotypes 012, 014 and 078 were the most frequently recovered toxinogenic ribotypes from animal samples. Comparison with human isolates from the Dutch Reference Laboratory for C. difficile at Leiden University Medical Centre (LUMC) showed that these types were also recovered from human hospitalized patients in 2009/2010, encompassing 0.8%, 11.4% and 9.8% of all isolates, respectively. Application of multiple-locus variable-number tandem-repeat analysis indicated a genotypic relation of animal and human ribotype 078 strains, but a clear genotypic distinction for ribotypes 012 and 014. We conclude that toxinogenic C. difficile PCR ribotypes found in animals correspond to PCR ribotypes associated with human disease in hospitalized patients in the Netherlands. Contrary to PCR ribotype 078, significant genetic differences were observed between animal and human PCR ribotype 012 and 014 isolates.

Genetic Relationships among Reptilian and Mammalian Campylobacter fetus Strains Determined by Multilocus Sequence Typing

ABSTRACT Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared ∼90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two groups. The reptile group represents a possible separate genomospecies capable of infecting humans.

Colonization of Campylobacter spp. in broiler chickens and laying hens reared in tropical climates with low-biosecurity housing.

ABSTRACT The onset and prevalence of Campylobacter colonization in broilers and layers at commercial farms with low biosecurity in tropical climates were tested. Despite the presence of positive animals at the same farms, the broiler flocks tested negative until, on average, 21 days. Prelaying flocks showed a higher prevalence than laying flocks.

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.

Cross-reaction of a Campylobacter fetus subspecies venerealis real-time PCR

Campylobacter fetus is a significant veterinary pathogen, which is divided into two subspecies: C fetus fetus and C fetus venerealis ([Veron and Chatelain 1973][1]). Differentiating between the two subspecies of C fetus and other Campylobacter species can be challenging due to their fastidious

Enterococcus hirae-associated endocarditis outbreaks in broiler flocks: clinical and pathological characteristics and molecular epidemiology.

Background: Enterococcus hirae-associated endocarditis, characterized by a peak in mortality during the second week of the grow-out, and occasionally lameness, was diagnosed at Dutch broiler farms. Objectives: Field cases were studied to increase knowledge on clinical and pathological characteristics, pathogenesis and epidemiology of these infections. Animals and methods. In total, 1266 birds of 25 flocks from 12 farms were examined. Post-mortem examinations, bacteriology, histopathology, PCR and DNA fingerprinting was carried out. Six flocks were followed longitudinally (n = 1017 birds). Results: Average mortality was 4.1% for the entire grow-out, of which 36% was attributed to endocarditis. Fibrinous thromboendocarditis of the right atrioventricular (AV) valve was found in 24% of hearts, compared to 7% and 4% with lesions of left and both AV valves, respectively. Thrombotic lesions were found in 24% (n = 432) of lungs, but only in larger branches of the Arteria pulmonalis. Occasionally, thrombi were found in the Arteria ischiadica externa and in liver and brain vessels. Enterococcus was cultured from 54% (n = 176) of heart and in 75% (n = 28), 62% (n = 106) and 31% (n = 16) of liver, bone marrow and lung samples, respectively. Further identification, using the Rapid ID Strep 32 API system and a PCR targeting mur-2 and mur-2ed genes was carried out on a subset of Enterococcus positive isolates (n = 65): both techniques identified the isolates as Enterococcus hirae. Pulsed-field gel electrophoresis did not indicate evidence of clonality between farms and flocks. Conclusions: The relevance of these findings for pathogenesis and epidemiology of E. hirae infections is discussed. Clinical importance. This study may facilitate diagnosis of field cases and may contribute to the design of further research and development of control measures.