Risticii Infection Research Articles

Prevalence and sequence analyses of Neorickettsia risticii.

The presence of Neorickettsia (Ehrlichia) risticii DNA was confirmed by PCR amplification and sequence analysis from cercaria in snails collected from stream water in Chungcheong and Jeonra provinces. A total of 3,219 snails were tested for trematode cercariae and N. risticii infection. N. risticii 16S rRNA gene fragment was amplified in cercariae from Semisulcospira libertina and Radix auricularia coreana snails by nested PCR. More than four genus cercariae (Schistosomatidae, Microphallidae, Furcocercus, and Xiphidiocercaria) as well as unidentified cercariae were found from Semisulcospira libertina snails. Three species of cercariae (E. cinetorichis, E. hortense, and Fasciola sp.) were found from Radix auricularia coreana snails. The cercariae were present in 429 (13.3%) snails of 3,216 collected at the Chungcheong and Jeonra provinces. The prevalence of N. risticii in these 429 cercariae was 17.9% (77 of 429 cercariae-infected snails). The amplicons of N. risticii 16S rRNA gene fragment (527 bp) from cercariae DNA had two genotypes (NR-JA1 and NR-JA2) with an identity of 96.4% between their nucleotide sequences. However, when compared to the sequence of N. risticii Shasta strain these sequences showed a 94.3% and 96.4% homology, respectively. The comparison of N. risticii 51 kDa major antigen gene sequences (572 bp) from NR-JA1 and NR-JA2 were 100% identical to the sequence of the isolates from Juga sp., Caddisfly larvae, Shasta, Juga yrekaensis, and trematode of California. This study reports for the first time the detection of N. risticii from cercariae found in Radix auricularia coreana snail. These data also indicate that N. risticii could be widespread in Korea.

Seroprevalence of Ehrlichia canis, Ehrlichia equi, and Ehrlichia risticii in sick dogs from North Carolina and Virginia.

Ehrlichia canis, E. equi, and E. risticii seroprevalence was determined by microimmunofluorescent antibody testing (IFA) in a sequential population of 1,845 sick dogs admitted during a 1-year period to the North Carolina State University Veterinary Teaching Hospital. A seroreactor was defined by a reciprocal IFA titer of > or =80 to E. canis, E. equi, or E. risticii antigens. Of the 48 IFA seroreactors, 44 dogs were seroreactive to E. canis, 21 to E. equi, and 0 to E. risticii. Seventeen dogs reacted to both E. canis and E. equi antigens. There was concordance of E. canis IFA and western immunoblot (WI) test results for 36/44 dogs. Because of cross-reactivity of E. canis sera with E. equi antigens, WI was of less utility to confirm E. equi exposure. After elimination of E. canis seroreactors, there was concordance of 2/4 E. equi IFA and WI test results. Based upon a retrospective review of medical records, ehrlichiosis was diagnosed in 10/48 (21%) IFA seroreactive dogs, 9 of which were confirmed positive by WI. Of the remaining 38 IFA seroreactors, 29 also were confirmed by E. canis or E. equi WI. These results indicate that (1) ehrlichiosis was not diagnosed in the majority of serologically confirmed cases, (2) based upon E. canis and E. equi WI analysis, IFA testing was not specific (21% false positive), (3) E. canis sera cross-react with E. equi antigens, and (4) serologic evidence of E. risticii infection was lacking in the dog population studied.

Seroprevalence ofEhrlichia canis, Ehrlichia equi, andEhrlichia risticiiin Sick Dogs from North Carolina and Virginia

Ehrlichia canis, E equi, andE risticiiseroprevalence was determined by microimmunofluorescent antibody testing (IFA) in a sequential population of 1,845 sick dogs admitted during a 1‐year period to the North Carolina State University Veterinary Teaching Hospital. A seroreactor was defined by a reciprocal IFA titer of ≥80 toE canis, E equi, orE risticiiantigens. Of the 48 IFA seroreactors, 44 dogs were seroreactive toE canis, 21 toE equi, and 0 toE risticii.Seventeen dogs reacted to bothE canisandE equiantigens. There was concordance ofE canisIFA and western immunoblot (WI) test results for 36/44 dogs. Because of cross‐reactivity ofE canissera withE equiantigens, WI was of less utility to confirmE equiexposure. After elimination ofE canisseroreactors, there was concordance of 2/4E equiIFA and WI test results. Based upon a retrospective review of medical records, ehrlichiosis was diagnosed in 10/48 (21%) IFA seroreactive dogs, 9 of which were confirmed positive by WI. Of the remaining 38 IFA seroreactors, 29 also were confirmed byE canisorE equiWI. These results indicate that (1) ehrlichiosis was not diagnosed in the majority of serologically confirmed cases, (2) based uponE canisandE equiWI analysis, IFA testing was not specific (21% false positive), (3)E canissera cross‐react withE equiantigens, and (4) serologic evidence ofE risticiiinfection was lacking in the dog population studied.

Evidence for a high rate of false-positive results with the indirect fluorescent antibody test for Ehrlichia risticii antibody in horses

Objective— The original objective was to determine seroprevalence of Ehrlichia risticii antibody among horses in California. On the basis of the unexpected results of the survey, an investigation into the accuracy and reproducibility of results of the indirect fluorescent antibody ( ifa) test for E risticii was carried out. Design— Prospective, seroprevalence study. Animals— Healthy horses (n = 655) and horses with clinical signs of equine monocytic ehrlichiosis ( eme; n = 514) from various regions of California. Procedure— The ifa test was performed. Results were compared with results of an elisa and with results of western immunoblot analysis. Results— Overall, 104 of 655 (15.9%) healthy horses had evidence of an antibody response. However, 84 of 514 (16.3%) horses with clinical signs of eme also had positive test results, and of the 8 seropositive diseased horses for which paired (acute and convalescent) samples had been submitted, only 1 had a rise in antibody titers between the acute and convalescent samples. Comparison of results for the ifa test, elisa, and western immunoblot analysis revealed a high rate of false-positive results for the ifa test. Subsequent studies suggested that routine vaccination of horses with non-E risticii vaccines may have contributed to the false-positive reactions. Clinical Implications— The data failed to provide conclusive evidence of E risticii infection among California horses. Owing to the high percentage of false-positive test results, caution is advised when using the ifa test to diagnose eme in horses or to determine the necessity for E risticii vaccination.

Humoral antibody and lymphocyte blastogenesis responses in BALE/c, C3H/HeJ and AKR/N mice following Ehrlichia risticii infection

Three strains of mice, two susceptible to Ehrlichia risticii induced disease ( balb/c and C3H/HeJ) and one resistant ( akr/n), were evaluated for the development of Immoral and cell mediated immune responses following infection with E risticii. The production of serum antibody was determined by indirect fluorescent antibody testing of sera from mice of each strain challenged with one of three different dose levels of E risticii. Antibody was assayed on days 7, 9, 12, 15 and 20 after inoculation. Cell mediated immune responses were evaluated by measuring the blastogenesis response of spleen cells from E risticii infected mice 28 days after inoculation. All three strains of mice at the high challenge level responded with the production of antibody by day 9 after inoculation. Overall, the antibody response occurred earlier and was of greater magnitude in the susceptible balb/c and C3H/HeJ strains. A marked blastogenesis response occurred in splenocytes from E risticii infected mice of all three strains upon re-exposure to ehrlichial antigen. The findings of this study indicate that susceptibility to E risticii induced disease was not the result of deficient or delayed humoral immune responses and that E risticii infection induced the development of strong cell mediated immunity.

Development of neutralizing antibody in horses infected with Ehrlichia risticii

The role of the humoral immune response in ehrlichial infection is unknown. Development of neutralizing antibodies during a course of Ehrlichia risticii infection in a pony was examined in vitro by determining the inhibition of E. risticii infection of P388D 1 cells in the presence of the sera. The pony experimentally infected with E. risticii developed significant neutralizing activity in the sera by 15 days postinfection when parasitemia started to decline. Neutralizing activity continued to rise after recovery from the disease up to 34 days postinfection at which time the experiment was terminated. In vitro neutralizing activities in the sera from 3 additional ponies infected with E. risticii were lower at 2 weeks than at 4 weeks postinfection. The sera from vaccinated/challenged ponies had comparable neutralizing activity to those of the recovered ponies at approximately 3 to 4 weeks postchallenge. Equine sera from infected or vaccinated/challenged ponies were also effective in protecting mice from E. risticii infection. These studies demonstrated the significant development of neutralizing activity in the sera of recovered or vaccinated/challenged ponies.

In vitro killing of Ehrlichia risticii by activated and immune mouse peritoneal macrophages

Normal resident murine peritoneal macrophages inoculated in vitro with Ehrlichia risticii readily phagocytized the organism but were unable to suppress ehrlichial replication as determined by indirect fluorescent-antibody staining of the inoculated cells. In contrast, macrophages from Corynebacterium parvum-inoculated and E. risticii-recovered mice rapidly eliminated the ehrlichiae. Macrophages from E. risticii-recovered mice were as effective as the C. parvum-activated cells in phagocytizing and eliminating the organism. Opsonization of E. risticii with homologous antiserum prior to inoculation of macrophage cultures resulted in enhancement of phagocytosis and greater suppression of E. risticii replication in all macrophage groups. These findings indicate that the pathogenesis of E. risticii infection centers on the ability of the organism to enter and replicate within the macrophage with avoidance of macrophage antimicrobial effects. An immune response results in macrophage activation with enhancement of the macrophage's ability to eliminate E. risticii. Opsonization of E. risticii with anti-E. risticii serum renders E. risticii more susceptible to macrophage destruction.

Detection of antigenemia by enzyme-linked immunosorbent assay in horses with experimental Ehrlichia risticii infection.

Four horses were inoculated with Ehrlichia risticii contained in either infected murine P388 D1 cells or heparinized blood from an infected horse. All 4 horses produced serum antibody, plasma antigen, and clinical signs of the disease. An enzyme-linked immunosorbent assay was used to detect antibody in the serum and was also used in conjunction with an anti-E. risticii monoclonal antibody to detect antigenemia. These laboratory and clinical findings were correlated to determine the efficiency of the antigen detection method for discerning E. risticii infection.

L-arginine-dependent killing of intracellular Ehrlichia risticii by macrophages treated with gamma interferon.

Thioglycolate-induced murine peritoneal macrophages infected with Ehrlichia risticii and treated in vitro with gamma interferon (IFN-gamma) developed antiehrlichial activity that eliminated the intracellular bacteria. This antiehrlichial activity was suppressed by NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthesis from L-arginine, but not by L-tryptophan. Increased levels of nitrite, an oxidative product of nitric oxide, were measured in cultures of infected macrophages treated with IFN-gamma. Sodium nitroprusside, which spontaneously releases nitric oxide, also showed the antiehrlichial activity. The antiehrlichial activity by reactive nitrogen intermediates was not mediated by elevation of the cellular concentration of cyclic GMP since the addition of 8-bromo-cyclic GMP itself had no influence on ehrlichial infection of macrophages. Addition of the intracellular iron chelator deferoxamine also inhibited E. risticii infection in vitro. These results suggest that intracellular E. risticii survival is iron dependent and that production of reactive nitrogen intermediates triggers iron loss from critical target enzymes of E. risticii, leading to lethal metabolic inhibition. However, addition of excess FeSO4, ferric citrate, or iron-saturated transferrin did not counteract the antiehrlichial effect induced by IFN-gamma.

Suppression of Ia antigen expression on gamma interferon treated macrophages infected with Ehrlichia risticii

Ehrlichia risticii is an obligate intracellular bacterium of monocytes/macrophages. In this report, using immunofluorescence staining, flow cytometry, and Kolmogorov-Smirnov analysis of histograms, the response of P338D 1 and peritoneal macrophages stimulated with recombinant murine interferon-γ (rIFN-γ) was examined for the expression of major histocompatibility complex Class II gene product (Ia) and effect of E. risticii infection on induction of Ia surface expression. Maximal expression of Ia by sham-infected P388D 1 cells was observed 2 days post rIFN-γ addition followed by a progressive decline. These stimulatory effects of rIFN-γ were dose dependent. Relative to sham-infected P388D 1 cells, the induction of Ia by rIFN-γ (200 U ml −1) on E. risticii-infected P388D 1 cells was significantly suppressed at each time point tested through Day 5 with maximal suppression of 88% occurring on Day 2. Similarly, the induction of Ia by rIFN-γ on E. risticii-infected peritoneal macrophages was significantly suppressed by 77% (fluorescent microscopy) when compared to sham-infected peritoneal macrophages. The higher dose of rIFN-γ (2000 U ml −1) failed to restore Ia surface expression by E. risticii-infected P388D 1 cells. The suppression of Ia on P388D 1 cells in response to RIFN-γ was not related to the degree of infection of these cells by E. risticii. A soluble inhibitor substance was not demonstrable in the supernatant from E. risticii-infected cells, nor were inhibitor levels of prostaglandin E 2 levels found in the supernatant. Suppression of surface Ia expression on the macrophage suggests a mechanism whereby I. risticii may evade T-lymphocyte recognition, hinder antigen-specific T-lymphocyte activation, and promote their own survival.

Loss of absorptive capacity for sodium and chloride in the colon causes diarrhoea in Potomac horse fever

Ehrlichia risticii, an obligate intracellular bacterium in the family Rickettsiaceae, causes Potomac horse fever which is often associated with severe watery diarrhoea. The mechanism of the diarrhoea is unknown. The aim of this study was to determine whether sodium and chloride transport, morphology and cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) content of colonic mucosa was altered in E risticii-infected horses. Mucosa-submucosa sheets from the large and small colon of nine infected and seven to nine uninfected horses were set up in Ussing chambers for measurement of short-circuit current and transepithelial 22Na and 36Cl fluxes Uninfected tissues absorbed both sodium and chloride whereas absorption of sodium and chloride was abolished in infected tissues. Bethanechol and histamine evoked a concentration-dependent increase in short-circuit current in both groups, but the responses were attenuated at all concentrations in infected horses. Slight focal degeneration of colonic epithelial cells and loss of microvilli from glandular epithelial cells occurred in infected horses. There was a significant increase in cyclic amp content in colonic mucosa of infected animals. The results suggest that E risticii infection induces focal microscopic degeneration of epithelial cells and an increase in intracellular cyclic AMP in colonic mucosa. These alterations are associated with malabsorption of sodium and chloride and could cause diarrhoea.

Inhibition of Ehrlichia risticii infection in murine peritoneal macrophages by gamma interferon, a calcium ionophore, and concanavalin A

Ehrlichia risticii incubated with mouse peritoneal macrophages elicited with thioglycolate broth survived and replicated, thereby allowing examination of the effects of several immunopotentiating agents. Treatment of the macrophages with recombinant murine gamma interferon (rMuIFN-gamma) in vitro at 1 day before or 3 h after infection made the macrophages resistant to infection with E. risticii, and macrophages treated with rMuIFN-gamma at 1 to 3 days after infection developed the capacity to eradicate intracellular E. risticii. Similar effects were seen with macrophages treated with the Ca2+ ionophore A23187 before or after E. risticii infection in vitro. Concanavalin A treatment before or 3 h after infection caused the macrophages to become resistant to infection with E. risticii but could confer neither ehrlichiacidal nor ehrlichiastic activity to them once infection had been established for more than 1 day. Bacterial products such as lipopolysaccharide and muramyl dipeptide were less or not at all effective, respectively, in conferring antiehrlichial activity to macrophages. Finally, protein kinase C activator, phorbol myristate acetate, and recombinant tumor necrosis factor did not induce any antiehrlichial activity in macrophages when the macrophages were treated either before or after infection.

Attempted Ehrlichia risticii Transmission with Dermacentor variabilis (Acari: Ixodidae)

Larval Dermacentor variabilis (Say) (n = 327) were fed on Balb/C mice inoculated with Ehrlichia risticii, the etiologic agent of equine monocytic ehrlichiosis (Potomac horse fever). All mice displayed clinical signs of E. risticii infection at the time of feeding. After molting, resulting nymphs (n = 74) were fed on susceptible mice. No clinical signs were observed, and the mice remained seronegative for 6 wk after feeding.

Effect of antibiotics on clinical, pathologic and immunologic responses in murine Potomac horse fever: Protective effects of doxycycline

Effects of three antibiotics on clinical, pathologic and immunologic responses in murine Potomac horse fever caused by Ehrlichia risticii infection were examined. When antibiotics were given after the development of clinical signs, antibiotics ranked in the order of reducing clinical signs and in preventing body weight loss and an intestinal enlargement were doxycycline, demeclocycline and rifampin. Infected mice treated with doxycycline and demeclocycline developed greater splenomegaly than rifampin-treated or untreated infected mice. All antibiotics used prevented thymic atrophy due to E. risticii infection. Indirect fluorescent antibody titers were highest with doxycycline treatment. Mice treated with demeclocycline and rifampin produced higher antibody titer than those without treatment. Ehrlichia risticii was reisolated from the spleens of both untreated and rifampin-treated infected mice. The effects of administering single doses of doxycycline at different times after infection were examined. Body weight loss was prevented by the drug given at every treatment day examined, i.e. Days 3, 5 and 7 post-infection (PI). Thymic atrophy was minimum in mice treated at Day 5 PI, while splenomegaly was found on every treatment day. Splenocyte proliferative response to concanavalin A and lipopolysaccharide, and specific antibody development against E. risticii was best in mice treated at Day 5 PI followed by those treated at Day 3 and Day 7 PI.

Reduced immune responsiveness and lymphoid depletion in mice infected with Ehrlichia risticii.

The histopathology of the thymus and spleen and the response of spleen cells to mitogenic stimuli were evaluated in Sprague-Dawley CF-1 mice infected with Ehrlichia risticii. Intraperitoneal injection of 10(4) or 10(6) E. risticii-infected U-937 cells into mice resulted in 100% morbidity and partial mortality. Thymic atrophy became significant between 1 and 2 weeks postinfection and remained for the duration of the study. The atrophy appeared associated with antecedent destruction and rarefaction of lymphocytes, resulting in the loss of corticomedullary demarcation. Splenomegaly was prominent; significantly increased weights were detected 7 days postinfection. Histopathologic examination revealed rarefaction of lymphocytes around central arteries, the presence of necrotic debris in histiocytes, and replacement of erythropoiesis by granulopoiesis in the red pulp. Marked and acute reduction of in vitro proliferative responses of spleen cells to concanavalin A (ConA) and phytohemagglutinin were observed in mice infected with 10(4) or 10(6) E. risticii-infected U-937 cells. Interleukin-2 activity in the supernatant of ConA-stimulated spleen cells was also severely reduced. Both changes were time- and dose-dependent and were not associated with decreased spleen cell viability. Neither morbidity nor mortality occurred in mice infected with 10(2) E. risticii-infected U-937 cells. Although there was temporal reduction in phytohemagglutinin-driven lymphocyte proliferation, reduction in neither ConA-driven lymphocyte proliferation nor interleukin-2 activity was observed with this dosage. All E. risticii-inoculated mice seroconverted between days 18 and 25, as detected by the indirect fluorescent-antibody procedure. The findings indicate for the first time the hypoimmune responsiveness and histopathologic changes in lymphoid organs associated with E. risticii infection.