Alice J. Murphy

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.

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.

Ecological Characterization of the Colonic Microbiota of Normal and Diarrheic Dogs

We used terminal restriction fragment polymorphism (T-RFLP) analysis to assess (1) stability of the fecal microbiota in dogs living in environments characterized by varying degrees of exposure to factors that might alter the microbiota and (2) changes in the microbiota associated with acute episodes of diarrhea. Results showed that the healthy canine GI tract harbors potential enteric pathogens. Dogs living in an environment providing minimal exposure to factors that might alter the microbiota had similar microbiotas; the microbiotas of dogs kept in more variable environments were more variable. Substantial changes in the microbiota occurred during diarrheic episodes, including increased levels of Clostridium perfringens, Enterococcus faecalis, and Enterococcus faecium. When diet and medications of a dog having a previously stable microbiota were changed repeatedly, the microbiota also changed repeatedly. Temporal trend analysis showed directional changes in the microbiota after perturbation, a return to the starting condition, and then fluctuating changes over time.

Brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona and act as intermediate hosts.

We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Kochs postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.

Comparison of Sarcocystis neurona isolates derived from horse neural tissue

Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan. Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed that Sarcocystis spp. from the eight horses appeared the same, but different from the grackle isolate. One Michigan horse isolate (MIH6) had two bands at 72 and 25kDa that were more prominent than the UCD1 isolate and other Michigan horse isolates. Western blot analysis showed that merozoites of eight of eight equine-derived isolates, and the UCD1 S. neurona isolate had similar bands when developed with serum or CSF of an infected horse. Major bands were seen at 60, 44, 30, and 16kDa. In the grackle (Cornell) isolate, bands were seen at 60, 44, 29, and 16kDa. DNA from merozoites of each of the eight equine-derived isolates and the grackle-derived isolate produced a 334bp PCR product (Tanhauser et al., 1999). Restriction fragment length polymorphism (RFLP) analysis of these horse isolates showed banding patterns characteristic for S. neurona. The grackle (Cornell) isolate had an RFLP banding pattern characteristic of other S. falcatula species. Finally, electron microscopy examining multiple merozoites of each of these eight horse isolates showed similar morphology, which differed from the grackle (Cornell) isolate. We conclude that the eight Michigan horse isolates are S. neurona species and the grackle isolate is an S. falcatula species.

Genetic background of IL-10−/− mice alters host-pathogen interactions with Campylobacter jejuni and influences disease phenotype

We hypothesized that particular genetic backgrounds enhance rates of colonization, increase severity of enteritis, and allow for extraintestinal spread when inbred IL-10(-/-) mice are infected with pathogenic C. jejuni. Campylobacter jejuni stably colonized C57BL/6 and NOD mice, while congenic strains lacking IL-10 developed typhlocolitis following colonization that mimicked human campylobacteriosis. However, IL-10 deficiency alone was not necessary for the presence of C. jejuni in extraintestinal sites. C3H/HeJ tlr4(-/-) mice that specifically express the Cdcs1 allele showed colonization and limited extraintestinal spread without enteritis implicating this interval in the clinical presentation of C. jejuni infection. Furthermore, when the IL-10 gene is inactivated as in C3Bir tlr4(-/-) IL-10(-/-) mice, enteritis and intensive extraintestinal spread were observed, suggesting that clinical presentations of C. jejuni infection are controlled by a complex interplay of factors. These data demonstrate that lack of IL-10 had a greater effect on C. jejuni induced colitis than other immune elements such as TLR4 (C3H/HeJ, C3Bir IL-10(-/-)), MHC H-2g7, diabetogenic genes, and CTLA-4 (NOD) and that host genetic background is in part responsible for disease phenotype. C3Bir IL-10(-/-) mice where Cdcs1 impairs gut barrier function provide a new murine model of C. jejuni and can serve as surrogates for immunocompromised patients with extraintestinal spread.

Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10-/- mice

BackgroundCampylobacter jejuni infection produces a spectrum of clinical presentations in humans – including asymptomatic carriage, watery diarrhea, and bloody diarrhea – and has been epidemiologically associated with subsequent autoimmune neuropathies. This microorganism is genetically variable and possesses genetic mechanisms that may contribute to variability in nature. However, relationships between genetic variation in the pathogen and variation in disease manifestation in the host are not understood. We took a comparative experimental approach to explore differences among different C. jejuni strains and studied the effect of diet on disease manifestation in an interleukin-10 deficient mouse model.ResultsIn the comparative study, C57BL/6 interleukin-10-/- mice were infected with seven genetically distinct C. jejuni strains. Four strains colonized the mice and caused disease; one colonized with no disease; two did not colonize. A DNA:DNA microarray comparison of the strain that colonized mice without disease to C. jejuni 11168 that caused disease revealed that putative virulence determinants, including loci encoding surface structures known to be involved in C. jejuni pathogenesis, differed from or were absent in the strain that did not cause disease. In the experimental study, the five colonizing strains were passaged four times in mice. For three strains, serial passage produced increased incidence and degree of pathology and decreased time to develop pathology; disease shifted from watery to bloody diarrhea. Mice kept on an ~6% fat diet or switched from an ~12% fat diet to an ~6% fat diet just before infection with a non-adapted strain also exhibited increased incidence and severity of disease and decreased time to develop disease, although the effects of diet were only statistically significant in one experiment.ConclusionC. jejuni strain genetic background and adaptation of the strain to the host by serial passage contribute to differences in disease manifestations of C. jejuni infection in C57BL/6 IL-10-/- mice; differences in environmental factors such as diet may also affect disease manifestation. These results in mice reflect the spectrum of clinical presentations of C. jejuni gastroenteritis in humans and contribute to usefulness of the model in studying human disease.

Purification of Sarcocystis neurona sporocysts from opossum (Didelphis virginiana) using potassium bromide discontinuous density gradient centrifugation

This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.

Sarcoptic Mange in Raccoons in Michigan

Sarcoptic mange is a cause of pruritic skin disease in domestic dogs and a wide range of wildlife species. We describe sarcoptic mange in free-ranging raccoons (Procyon lotor). Three adult raccoons from upper Wayne County, Michigan (USA), were captured, killed, and submitted for diagnostic evaluation. The animals were intensely pruritic, and two had advanced alopecic and crusting lesions over their dorsum and hind limbs. Skin scrapings and skin biopsies revealed crusting and hyperkeratotic dermatitis with high numbers of Sarcoptes scabiei adults, larvae, nymphs, and eggs. These raccoons were not otherwise debilitated, with minimal internal parasites, good body condition, and no evidence of infectious bacterial or viral diseases. Because sarcoptic mange is highly contagious and affects many species, including humans, transiently, it is important that wildlife biologists and rehabilitators include sarcoptic mange in their differential list for raccoons exhibiting pruritus and alopecia.

Effects of temperature and host cell type on the in vitro growth and development of Sarcocystis falcatula

The growth and development of Sarcocystis falcatula in four different cultured cell lines [vero cells, bovine turbinate (BT) cells, equine dermal (ED) cells, and human Hep-2 cells] inoculated with culture-derived merozoites are described. Parasite yields, viability, and plaque forming efficiency were used to compare the growth between different cell lines. Additionally, each cell line was tested at two temperatures of incubation (35°C and 37°C). Based on yield, viability, and plaque forming efficiency, vero cells and BT cells supported growth of S. falcatula better than ED cells and Hep-2 cells. During an 18-day culture period, vero cells produced a mean total of 1.3×107 S. falcatula merozoites/T25 flask, BT cells 1.1×107, ED cells 0.9×107, and Hep-2 cells 0.7×107 merozoites/T25 flask. All experimental cell lines grew equally well at 35°C and 37°C. The type of host cells but not the temperature of incubation had a profound effect on the in vitro growth and proliferation of S. falcatula.