Linda S. Mansfield

Massive Microbiological Groundwater Contamination Associated with a Waterborne Outbreak in Lake Erie, South Bass Island, Ohio

Background A groundwater-associated outbreak affected approximately 1,450 residents and visitors of South Bass Island, Ohio, between July and September 2004. Objectives To examine the microbiological quality of groundwater wells located on South Bass Island, we sampled 16 wells that provide potable water to public water systems 15–21 September 2004. Methods We tested groundwater wells for fecal indicators, enteric viruses and bacteria, and protozoa (Cryptosporidium and Giardia). The hydrodynamics of Lake Erie were examined to explore the possible surface water–groundwater interactions. Results All wells were positive for both total coliform and Escherichia coli. Seven wells tested positive for enterococci and Arcobacter (an emerging bacterial pathogen), and F+-specific coliphage was present in four wells. Three wells were positive for all three bacterial indicators, coliphages, and Arcobacter; adenovirus DNA was recovered from two of these wells. We found a cluster of the most contaminated wells at the southeast side of the island. Conclusions Massive groundwater contamination on the island was likely caused by transport of microbiological contaminants from wastewater treatment facilities and septic tanks to the lake and the subsurface, after extreme precipitation events in May–July 2004. This likely raised the water table, saturated the subsurface, and along with very strong Lake Erie currents on 24 July, forced a surge in water levels and rapid surface water–groundwater interchange throughout the island. Landsat images showed massive influx of organic material and turbidity surrounding the island before the peak of the outbreak. These combinations of factors and information can be used to examine vulnerabilities in other coastal systems. Both wastewater and drinking water issues are now being addressed by the Ohio Environmental Protection Agency and the Ohio Department of Health.

In Vitro and In Vivo Characterization of Helicobacter hepaticus Cytolethal Distending Toxin Mutants

ABSTRACT Helicobacter hepaticus expresses a member of the cytolethal distending toxin (CDT) family of bacterial cytotoxins. To investigate the role of CDT in the pathogenesis of H. hepaticus, transposon mutagenesis was used to generate a series of isogenic mutants in and around the cdtABC gene cluster. An H. hepaticus transposon mutant with a disrupted cdtABC coding region no longer produced CDT activity. Conversely, a transposon insertion outside of the cluster did not affect the CDT activity. An examination of these mutants demonstrated that CDT represents the previously described granulating cytotoxin in H. hepaticus. Challenge of C57BL/6 interleukin 10−/− mice with isogenic H. hepaticus mutants revealed that CDT expression is not required for colonization of the murine gut. However, a CDT-negative H. hepaticus mutant had a significantly diminished capacity to induce lesions in this murine model of inflammatory bowel disease.

C57BL/6 and Congenic Interleukin-10-Deficient Mice Can Serve as Models of Campylobacter jejuni Colonization and Enteritis

ABSTRACT Campylobacter jejuni is a globally distributed cause of human food-borne enteritis and has been linked to chronic joint and neurological diseases. We hypothesized that C. jejuni 11168 colonizes the gastrointestinal tract of both C57BL/6 mice and congenic C57BL/6 interleukin-10-deficient (IL-10−/−) mice and that C57BL/6 IL-10−/− mice experience C. jejuni 11168-mediated clinical signs and pathology. Individually housed mice were challenged orally with C. jejuni 11168, and the course of infection was monitored by clinical examination, bacterial culture, C. jejuni-specific PCR, gross pathology, histopathology, immunohistochemistry, and anti-C. jejuni-specific serology. Ceca of C. jejuni 11168-infected mice were colonized at high rates: ceca of 50/50 wild-type mice and 168/170 IL-10−/− mice were colonized. In a range from 2 to 35 days after infection with C. jejuni 11168, C57BL/6 IL-10−/− mice developed severe typhlocolitis best evaluated at the ileocecocolic junction. Rates of colonization and enteritis did not differ between male and female mice. A dose-response experiment showed that as little as 106 CFU produced significant disease and pathological lesions similar to responses seen in humans. Immunohistochemical staining demonstrated C. jejuni antigens within gastrointestinal tissues of infected mice. Significant anti-C. jejuni plasma immunoglobulin levels developed by day 28 after infection in both wild-type and IL-10-deficient animals; antibodies were predominantly T-helper-cell 1 (Th1)-associated subtypes. These results indicate that the colonization of the mouse gastrointestinal tract by C. jejuni 11168 is necessary but not sufficient for the development of enteritis and that C57BL/6 IL-10−/− mice can serve as models for the study of C. jejuni enteritis in humans.

Standing genetic variation in contingency loci drives the rapid adaptation of Campylobacter jejuni to a novel host

The genome of the food-borne pathogen Campylobacter jejuni contains multiple highly mutable sites, or contingency loci. It has been suggested that standing variation at these loci is a mechanism for rapid adaptation to a novel environment, but this phenomenon has not been shown experimentally. In previous work we showed that the virulence of C. jejuni NCTC11168 increased after serial passage through a C57BL/6 IL-10-/- mouse model of campylobacteriosis. Here we sought to determine the genetic basis of this adaptation during passage. Re-sequencing of the 1.64Mb genome to 200-500X coverage allowed us to define variation in 23 contingency loci to an unprecedented depth both before and after in vivo adaptation. Mutations in the mouse-adapted C. jejuni were largely restricted to the homopolymeric tracts of thirteen contingency loci. These changes cause significant alterations in open reading frames of genes in surface structure biosynthesis loci and in genes with only putative functions. Several loci with open reading frame changes also had altered transcript abundance. The increase in specific phases of contingency loci during in vivo passage of C. jejuni, coupled with the observed virulence increase and the lack of other types of genetic changes, is the first experimental evidence that these variable regions play a significant role in C. jejuni adaptation and virulence in a novel host.

The pathogenesis of necrotic proliferative colitis in swine is linked to whipworm induced suppression of mucosal immunity to resident bacteria

Mucohemorrhagic enteritis syndrome in swine has a complex etiology with largely unknown pathogenesis. We have observed that inoculation of pigs with swine whipworm, Trichuris suis, initiates an interaction with resident bacterial flora to induce mucohemorrhagic enteritis. The role of bacteria in this mixed infection was demonstrated using 4 treatment groups. One group of pigs was inoculated with 2500 embryonated T. suis eggs alone, while a second group received T. suis eggs along with broad spectrum antibiotic treatment. Two other control groups of pigs were uninoculated and were either treated with antibiotic or untreated. Pigs inoculated with T. suis eggs exhibited diarrhea, mucosal edema, inflammatory cell infiltration, bacterial accumulation at the site of worm attachment in the proximal colon, and intestinal adenomatosis associated with the intracellular Ileal symbiont intracellularis bacteria. In addition, enlarged lymphoglandular complexes (LGCs) containing numerous extracellular bacteria, eosinophils, lymphocytes, macrophages, and neutrophils were observed in the distal colon. The other group of pigs that was inoculated with T. suis but treated with antibiotics had lesions localized to the site of worm attachment and histologically normal LGCs with no invasive bacteria in the distal colon. The groups of uninoculated pigs, with or without antibiotic treatment, exhibited no pathology or bacterial invasion. It appears that the complex pathogenesis of necrotic proliferative colitis in pigs may be linked to worm induced suppression of mucosal immunity to resident bacteria. Further, the association between bacteria,lymphocytes and macrophages in the LGCs of pigs infected with T. suis suggests an antigen-processing role for these structures in the colon. Further, the complex pathogenesis of necrotic proliferative colitis in pigs may be linked to worm induced suppression of mucosal immunity to resident bacteria.

Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice.

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

Campylobacter jejuni-Induced Activation of Dendritic Cells Involves Cooperative Signaling through Toll-Like Receptor 4 (TLR4)-MyD88 and TLR4-TRIF Axes

ABSTRACT Campylobacter jejuni is an important cause of human enteritis and has been linked to the development of autoimmune diseases. Recently we showed that infection of murine dendritic cells (DCs) with C. jejuni resulted in DC activation and induction of Campylobacter-specific Th1-effector responses. Toll-like receptor (TLR) signaling through myeloid differentiation factor 88 (MyD88) and/or Toll-interleukin 1 (IL-1) receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) is critical in inducing immunity against pathogens. In this study, we investigated the role of TLR2, TLR4, MyD88, and TRIF signaling in C. jejuni-induced inflammatory activation of DCs. DC upregulation of major histocompatibility complex class II and costimulatory molecules after C. jejuni challenge was profoundly impaired by TLR2, TLR4, MyD88, and TRIF deficiencies. Similarly, C. jejuni-induced secretion of IL-12, IL-6, and tumor necrosis factor alpha was significantly inhibited in TLR2−/−, TLR4−/−, MyD88−/−, and TRIF−/− DCs compared to that in wild-type DCs; however, the magnitude of inhibition was greater in MyD88−/−, TRIF−/−, and TLR4−/− DCs than in TLR2−/− DCs. Furthermore, C. jejuni induced interferon regulatory factor 3 phosphorylation and IFN-β secretion by DCs in a TLR4-TRIF-dependent fashion, further demonstrating activation of this pathway by C. jejuni. Importantly, TLR2, TLR4, MyD88, and TRIF deficiencies all markedly impaired the Th1-priming ability of C. jejuni-infected DCs. Thus, our results show that cooperative signaling through the TLR4-MyD88 and TLR4-TRIF axes represents a novel mechanism mediating C. jejuni-induced inflammatory responses of DCs. To our knowledge, such a mechanism has not been demonstrated previously for an intact bacterium.

Improvement of Western Blot Test Specificity for Detecting Equine Serum Antibodies to Sarcocystis Neurona

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12–20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62–, 30–, 16–, 13–, 11–, 10.5–, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30-and 16-kD bands (P < 0.001, Fishers exact test). The S. cruzi antibody-blocked Western blot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

Use of tick surveys and serosurveys to evaluate pet dogs as a sentinel species for emerging Lyme disease.

OBJECTIVE To evaluate dogs as a sentinel species for emergence of Lyme disease in a region undergoing invasion by Ixodes scapularis. SAMPLE POPULATION 353 serum samples and 78 ticks obtained from dogs brought to 18 veterinary clinics located in the lower peninsula of Michigan from July 15, 2005, through August 15, 2005. PROCEDURES Serum samples were evaluated for specific antibodies against Borrelia burgdorferi by use of 3 serologic assays. Ticks from dogs were subjected to PCR assays for detection of pathogens. RESULTS Of 353 serum samples from dogs in 18 counties in 2005, only 2 (0.6%) contained western blot analysis-confirmed antibodies against B burgdorferi. Ten of 13 dogs with I scapularis were from clinics within or immediately adjacent to the known tick invasion zone. Six of 18 I scapularis and 12 of 60 noncompetent vector ticks were infected with B burgdorferi. No ticks were infected with Anaplasma phagocytophilum, and 3 were infected with Babesia spp. CONCLUSIONS AND CLINICAL RELEVANCE Serosurvey in dogs was found to be ineffective in tracking early invasion dynamics of I scapularis in this area. Tick chemoprophylaxis likely reduces serosurvey sensitivity in dogs. Ticks infected with B burgdorferi were more common and widely dispersed than seropositive dogs. In areas of low tick density, use of dogs as a source of ticks is preferable to serosurvey for surveillance of emerging Lyme disease. IMPACT FOR HUMAN MEDICINE By retaining ticks from dogs for identification and pathogen testing, veterinarians can play an important role in early detection in areas with increasing risk of Lyme disease.

LOCAL TH1 AND TH2 RESPONSES TO PARASITIC INFECTION IN THE INTESTINE : REGULATION BY IFN-GAMMA AND IL-4

Control of parasitic infections is dependent on the production of cytokines that activate mechanisms which limit invasion, reproduction or survival of the parasite. In contrast, conditions that induce inappropriate cytokine responses facilitate the spread of infection and ultimately exacerbate the level of disease. Measurement of local cytokine responses to different gastrointestinal parasites, such as the intracellular protozoan, Cryptosporidium parvum, and luminal dwelling nematodes like Nippostrongylus brasiliensis and Heligmosomoides polygyrus, reveal stereotype response patterns. In general, intracellular parasites stimulate type 1 responses where IFN-gamma is the predominant immune activator, while extracellular parasites stimulate type 2 responses where IL-4 plays a prominent role in elevating humoral immune mechanisms. Cytokines alter cellular function and the milieu of the intestinal lumen to affect the outcome of an infection. The importance of a particular response during the course of an infection can be studied by selective enhancement with an excess of exogenous recombinant cytokine or cytokine antagonists. For example, exogenous IL-12 enhances resistance to C.parvum, but suppresses the normally rapid cure of an infection with N. brasiliensis. Both mechanisms are dependent on expression of IFN-gamma. At the molecular level, exogenous IL-12 stimulates IFN-gamma production which elevates a protective type 1 response to C. parvum but converts the normally anti-worm type 2 response to a type 1 response that inappropriately regulates the infection. Alternatively, excess IL-4 plays a prominent role in modulating effector elements that change intestinal physiology to create a hostile environment for worm parasites. Exogenous IL-4 can cure chronic worm infection, while IL-4 antagonists interfere with protective responses to infection. These observations provide a paradigm for analysis of stereotype responses to different gastrointestinal parasites, and demonstrate how cytokine-induced immune system-dependent and independent effector mechanisms can limit parasitic infection, while inappropriate cytokine responses can exacerbate the state of disease.