Y.H. Schukken

Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum

Intestinal bacteria are implicated increasingly as a pivotal factor in the development of Crohns disease, but the specific components of the complex polymicrobial enteric environment driving the inflammatory response are unresolved. This study addresses the role of the ileal mucosa-associated microflora in Crohns disease. A combination of culture-independent analysis of bacterial diversity (16S rDNA library analysis, quantitative PCR and fluorescence in situ hybridization) and molecular characterization of cultured bacteria was used to examine the ileal mucosa-associated flora of patients with Crohns disease involving the ileum (13), Crohns disease restricted to the colon (CCD) (8) and healthy individuals (7). Analysis of 16S rDNA libraries constructed from ileal mucosa yielded nine clades that segregated according to their origin (P<0.0001). 16S rDNA libraries of ileitis mucosa were enriched in sequences for Escherichia coli (P<0.001), but relatively depleted in a subset of Clostridiales (P<0.05). PCR of mucosal DNA was negative for Mycobacterium avium subspecies paratuberculosis, Shigella and Listeria. The number of E. coli in situ correlated with the severity of ileal disease (ρ 0.621, P<0.001) and invasive E. coli was restricted to inflamed mucosa. E. coli strains isolated from the ileum were predominantly novel in phylogeny, displayed pathogen-like behavior in vitro and harbored chromosomal and episomal elements similar to those described in extraintestinal pathogenic E. coli and pathogenic Enterobacteriaceae. These data establish that dysbiosis of the ileal mucosa-associated flora correlates with an ileal Crohns disease (ICD) phenotype, and raise the possibility that a selective increase in a novel group of invasive E. coli is involved in the etiopathogenesis to Crohns disease involving the ileum.

Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment.

ABSTRACT A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.

Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs.

ABSTRACT The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a“ splash”-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohns disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohns disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile.

Risk factors for neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997)

A 2 to 1 matched case control study design was used to analyze herd level risk factors for Neospora caninum-associated abortion storms in 47 dairy herds. Data were obtained using a questionnaire regarding the state of affairs at the farms over the 2 years prior to the abortion storm. The questionnaire included 120 variables considered to be potential risk factors for either introduction of infection or recrudescence of chronic infection. The relationship between risk factors and case control pairs was analyzed by conditional logistic regression using a three-steps procedure. In addition, cross sectional serology was used to assess the possible role of concomitant infections. The main factors that were significant in the analysis and that were considered to have potential biological relevance were the presence of dogs, the presence of poultry, and the feeding of moldy maize-silage during summer. For both the presence of dogs and the presence of poultry on the farms, a linear relationship was found between the number of animals and the assessed risk for an abortion storm. These findings suggest a possible role of these species in the transmission of N. caninum. Further evidence for such a role of dogs was the significant association between the presence of dogs and the presence of seropositive cattle in the control herds. The feeding of moldy fodder is considered to be a factor which may induce recrudescence of a latent N. caninum-infection by mycotoxins causing immune suppression. We also found some evidence for a possible influence of management practices around calving and a high prevalence of retained afterbirths. No significant association was found for herd level prevalence of antibodies to bovine viral diarrhea virus, bovine herpesvirus 1, Leptospira hardjo or Salmonella dublin.

Host-response patterns of intramammary infections in dairy cows

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.

Abortion risk in progeny of cows after a neospora caninum epidemic

A study was done of the descendants of cows from 4 dairy herds in which there had been N. caninum abortion outbreaks. Precolostral antibodies to N. caninum were demonstrated in 34 of 50 (68%) F1 calves and in 14 of 17 (82%) F2 calves from cows that aborted during the outbreaks. In 214 F1 progeny, N. caninum seroprevalence was nearly 50%, and there was a significant association between serostatus of the offspring and serostatus of dams. These observations indicated that congenital infection was an important mode of transmission after abortion outbreaks in these herds. A total of 52 abortions was recorded in 293 pregnancies of F1 progeny cows (1 to 3 pregnancies per animal). It was found that seropositive F1 cows had a three-fold increased abortion risk compared with seronegative F1 cows. In 2 of 10 abortions in seronegative cows evidence for N. caninum infection was found, suggesting that a low level of postnatal infection may also have occurred. It is concluded that N. caninum-infected calves should not be used as replacement stock, to decrease the future risk of abortion in dairy herds.

A review of methods used to adjust for cluster effects in explanatory epidemiological studies of animal populations

This paper reviews explanatory studies in the veterinary epidemiology literature, in which clusters (herds) were sampled and individual responses were measured. The studies were taken from the first ten volumes of Preventive Veterinary Medicine. The objectives of the review were: (1) to describe the statistical methods used to adjust for cluster effects; (2) to provide estimates of intracluster correlations for various health and production outcomes; and (3) for studies ignoring cluster effects, to estimate the proportion of significant inferences that would be rejected if cluster effects were included. Of the 67 papers reviewed, 36 (54%) used some form of adjustment for clustering. Using a fixed effect for herd was employed most frequently (27 papers). Four of the 67 papers used a matched design, two used a weighted analysis, and three papers included a random herd effect. An estimate of the intracluster correlation (^ϱ) was calculated when sufficient data were present. This estimate ranged from 0.0017 (lamb mortality data), to 0.46 (prevalence of brucelence serotiter). Most intracluster correlations ranged form 0.01 to 0.15. In the papers where ϱ was estimated from the data, the variance inflation factor ranged from 1.1 to 13. In 36 of the 67 (54%) papers reviewed, all inferences were accepted. In 30 of these 36, the authors correctly accounted for clustering, and in the other six the inference would not have changed after adjustment for clustering. In the remaining 31 (46%) papers, changes in inference were predicted. The proportion of incorrect inferences could be estimated in 20 of these 31 papers.

Multilocus Sequence Typing of Intercontinental Bovine Staphylococcus aureus Isolates

ABSTRACT A total of 258 bovine-associated Staphylococcus aureus isolates from the United States, Chile, and the United Kingdom, plus the reference isolate S. aureus Newbould 305 (NCIMB 702892), were analyzed by multilocus sequence typing (MLST). A collection of previously characterized United Kingdom isolates were also included in the analysis. The results demonstrated that MLST is suitable for the differentiation of bovine S. aureus isolates from various sites (milk, teat skin, milking machine unit liners, hands, and bedding) and countries. The theory of the host specificity of S. aureus is supported by the detection of a previously undescribed clonal complex that comprised 87.4% of the isolates studied, with representatives from all geographic locations investigated. This suggests that a single clonal group has achieved a widespread distribution and is responsible for the majority of infections. Some sequence types (STs; ST25, ST115, ST124, and ST126) demonstrated site specificity, as they were significantly (P < 0.05) associated with milk or teat skin.

Molecular Epidemiology of Mastitis Pathogens of Dairy Cattle and Comparative Relevance to Humans

Mastitis, inflammation of the mammary gland, can be caused by a wide range of organisms, including gram-negative and gram-positive bacteria, mycoplasmas and algae. Many microbial species that are common causes of bovine mastitis, such as Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae and Staphylococcus aureus also occur as commensals or pathogens of humans whereas other causative species, such as Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae or Staphylococcus chromogenes, are almost exclusively found in animals. A wide range of molecular typing methods have been used in the past two decades to investigate the epidemiology of bovine mastitis at the subspecies level. These include comparative typing methods that are based on electrophoretic banding patterns, library typing methods that are based on the sequence of selected genes, virulence gene arrays and whole genome sequencing projects. The strain distribution of mastitis pathogens has been investigated within individual animals and across animals, herds, countries and host species, with consideration of the mammary gland, other animal or human body sites, and environmental sources. Molecular epidemiological studies have contributed considerably to our understanding of sources, transmission routes, and prognosis for many bovine mastitis pathogens and to our understanding of mechanisms of host-adaptation and disease causation. In this review, we summarize knowledge gleaned from two decades of molecular epidemiological studies of mastitis pathogens in dairy cattle and discuss aspects of comparative relevance to human medicine.

The Cost of Generic Clinical Mastitis in Dairy Cows as Estimated by Using Dynamic Programming

The objective of this study was to estimate the cost of generic clinical mastitis (CM) in high-yielding dairy cows given optimal decisions concerning handling of CM cases. A specially structured optimization and simulation model that included a detailed representation of repeated episodes of CM was used to study the effects of various factors on the cost of CM. The basic scenario was based on data from 5 large herds in New York State. In the basic scenario, 92% of the CM cases were recommended to be treated. The average cost of CM per cow and year in these herds was