Disease features in horses with induced equine monocytic ehrlichiosis (Potomac horse fever)

Fifty-five horses were inoculated IV and/or SC with materials containing Ehrlichia risticii, ie, infected whole blood, buffy coat cells, or cell culture, to study clinical and hematologic features of equine monocytic ehrlichiosis (Potomac horse fever). Major clinical and hematologic features of induced E risticii infection were biphasic increase in rectal temperature with peak increases of 38.9 C and 39.3 C on postinoculation days (PID) 5 and 12, respectively; depression; anorexia; decreased WBC count (maximal decrease of 47% on PID 12); and diarrhea from PID 14 to PID 18. Increased WBC count was an inconsistent feature, with a maximal increase of 51.5% on PID 20. During times of decreased and increased WBC counts, lymphocyte/neutrophil ratios remained fairly constant. However, not all horses had all clinical and hematologic features, and these features were present in different degrees among horses. Increased rectal temperature, depression, anorexia, and decreased WBC count were more consistent features, whereas diarrhea developed in 73% of the horses. Of 55 horses, 39 (71%) had all clinical and hematologic features of the disease (classic disease), whereas 16 (29%) horses did not have greater than or equal to 1 of these features (nonclassic disease). The E risticii titer in the blood (ehrlichemia) was maximum during the peak increase in rectal temperature. In 55 horses, mortality was 9%. Significant differences (P greater than 0.5) in clinical and hematologic features were not detected between horses that survived and those that died of E risticii infection.     

Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report

Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.     

Investigation of the effect of black walnut extract on in vitro ion transport and structure of equine colonic mucosa

OBJECTIVE: To examine the secretory response (in the presence and absence of prostaglandin inhibition) in vitro and structural alterations of colonic mucosa in horses after intragastric administration of black walnut extract (BWE). ANIMALS: 14 adult horses. PROCEDURE: Seven horses were administered BWE intragastrically and monitored for 11 hours. Tissue samples were obtained from the right ventral, left ventral, and right dorsal colons (RVC, LVC, and RDC, respectively) of the 7 BWE-treated and 7 control horses. Tissue samples were examined via light microscopy, and the extent of hemorrhage, edema, and granulocytic cellular infiltration (neutrophils and eosinophils) was graded. Colonic mucosal segments were incubated with or without flunixin meglumine (FLM) for 240 minutes; spontaneous electrical potential difference and short-circuit current (Isc) were recorded and used to calculate mucosal resistance. RESULTS: Colonic tissues from BWE-treated horses (with or without FLM exposure) had an overall greater Isc during the 240-minute incubation period, compared with tissues from control horses. The resistance pattern in RVC, LVC, and RDC samples (with or without FLM exposure) from BWE-treated horses was decreased overall, compared with control tissues (with or without FLM exposure). Histologically, colonic mucosal tissues from BWE-treated horses had more severe inflammation (involving primarily eosinophils), edema, and hemorrhage, compared with tissue from control horses. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWE administration appears to cause an inflammatory response in colonic mucosal epithelium that results in mucosal barrier compromise as indicated by decreased mucosal resistance with presumed concomitant electrogenic chloride secretory response, which is not associated with prostaglandin mediation.     

Detection of Ehrlichia risticii using an avidin-biotin-immunoperoxidase staining system

An indirect immunoperoxidase procedure using a specific anti-Ehrlichia risticii monoclonal antibody and an avidin-biotin-peroxidase staining method was used to detect E. risticii antigen in infected P388D1 murine monocytes. Several different methods of cytological fixation were used, including acetone (15 min), 95% ethanol (15 min), Bouin's fixative (5 hr), and 10% buffered neutral formalin (24 hr). The E. risticii organisms were labeled effectively and identified in cells fixed with acetone and ethanol. However, infected P388D1 cells fixed in 10% formalin or Bouin's fixative required enzymatic digestion with 1.0% trypsin for 15 min at 37 C before positive results were evident. This indirect immunoperoxidase avidin-biotin staining procedure proved to be a sensitive assay for the detection of intracellular E. risticii and may be an effective diagnostic procedure for formalin-fixed paraffin-embedded tissue.     

Faecal composition in foal heat diarrhoea

Developmental changes of the gastrointestinal tract were probably responsible for the changes in faecal composition during the first week of the foals' life, which resembled small intestinal ingesta of adult horses, suggesting a minimal colonic modification. Faecal composition at the time of foal heat diarrhoea was suggestive of a secretory-type diarrhoea, in that the electrolyte concentration accounted for most of faecal osmolality and the faecal pH was alkaline. After foal heat diarrhoea faecal composition slowly approached that of adult horses. These data suggest that foal heat diarrhoea is most likely caused by hypersecretion in the small intestinal mucosa, which may overwhelm an immature colon that is unable to compensate by increased fluid and electrolyte absorption.     

Ehrlichial diseases.

Equine granulocytic and monocytic ehrlichiosis caused by Ehrlichia equi and E. risticii, respectively, are seasonal diseases in horses that occur throughout the United States E. equi is transmitted by lxodes ticks and causes high fever, depression, anorexia, limb edema, petechiation, icterus, ataxia, and stiffness in gait. E. risticii, also known as the agent of Potomac horse fever, causes a febrile illness with a colitis of variable severity. Its occurrence is associated with aquatic habitats. The natural route of transmission is oral, through the ingestion of E. risticii infected trematode stages either free in water or in an intermediate host, such as aquatic animals.     

Histopathological and ultrastructural changes in simulated large colonic torsion and reperfusion in ponies.

This investigation examines the histological and ultrastructural lesions of the colonic mucosa during terminal experimental infarction and subsequent reperfusion. Four ponies were anaesthetised and subjected to surgical torsion of the colon. Biopsies were collected at hourly intervals for 3 h, at which point the torsions were corrected. Circulation was re-established for 2 h and the bowel was re-biopsied at hourly intervals. The ponies were killed while under anaesthesia. During the 3 h experimental infarction, the bowel became macroscopically thickened and dark purple. Histologically, the mucosa degenerated from Grade 0 to Grade 3. Ultrastructurally, there was progressive micro-vascular distension with erythrodiapedesis and damage to the interstitial cells. Spaces developed between the bases and sides of the columnar epithelial cells and sloughing followed subsequently. During the 2 h reperfusion interval, the mucosa continued to degenerate rapidly to a Grade 5, and was characterised by extensive interstitial damage, oedema, cellular swelling, necrosis and mitochondrial damage. The results showed that the experimentally infarcted colonic mucosa degenerated sequentially. Following circulatory reestablishment, continued rapid mucosal degeneration characteristic of reperfusion injury occurred. Reperfusion injury is probably responsible, at least in part, for the often poor outcome of infarcted bowel in horses following surgical correction.     

Enterotoxigenic colibacillosis in colostrum-fed calves: pathologic changes.

Enterotoxigenic colibacillosis was experimentally produced in 8 of 9 colostrum-fed calves orally given 10(11) Escherichia coli. The eight calves developed profuse diarrhea accompanied by dehydration and depression. At 12 hours after exposure, all calves were euthanatized for necropsy and for collection of tissues for microscopic examination. Histopathologic changes included stunted villi in the jejunum and ileum, focal degeneration and exfoliation of absorptive epithelial cells at the tips of jejunal and ileal villi, and focal emigration of neutrophils which was especially prominent above the dome area of aggregated lymphatic follicles (Peyer's patches). A layer of E coli adhered to the epithelial surface of the jejunum and ileum. In the duodenum, lesions were minimal or absent and bacteria were not adhering to the mucosa. Histopathologic changes were not observed in other tissues. In two calves examined 24 hours after they were inoculated and in two calves euthanatized 24 to 36 hours after spontaneously developing enteric colibacillosis, lesions were similar to those observed in the calves at 12 hours after exposure.     

Serodiagnosis of equine monocytic ehrlichiosis in selected groups of horses in Minnesota

Antibody titer to Ehrlichia risticii was determined, in 2,549 equine serum samples, using an indirect fluorescent antibody assay. During 1986, samples were obtained from the Minnesota State-Federal Equine Infectious Anemia Diagnostic Laboratory, the Minnesota Racing Laboratory, from horses admitted to the University of Minnesota Veterinary Teaching Hospital, and as a result of field investigations of horses with acute diarrhea. Results of the study revealed antibody prevalence of 33, 24, 47, and 25% for the respective groups. There was no statistical association between seropositive status and age, sex, breed, or clinical problem of horses referred to the teaching hospital. There was an increase in the total percentage of seropositive samples over the duration of the sample collection period, suggesting a seasonal exposure pattern, and E risticii was associated with clinical and subclinical infections in horses of Minnesota.     

Retrospective evaluation of factors associated with the risk of seropositivity to Ehrlichia risticii in horses in New York State.

A retrospective study was designed to determine the distribution of equine monocytic ehrlichiosis among the equine population in New York state, and to identify factors associated with risk of disease. Serum samples submitted to the diagnostic laboratory of the university during the period from January 1985 through December 1986 were examined for antibodies to Ehrlichia risticii, using the indirect fluorescent antibody technique. Factors evaluated included geographic origin and date of submission of the sample, and age, breed, and sex of the horse. Logistic regression analysis was used to identify which factors were significantly associated with the risk of seropositivity to E risticii, while simultaneously controlling for other factors. Of the 2,579 tested samples, 1,950 (76%) had positive results. Factors significantly associated with risk of seropositivity to E risticii were: breed of the horse (Thoroughbreds were 3 times more likely to have been exposed to E risticii, compared with non-Standardbred, non-Thoroughbred breeds); sex (female horses were 2.7 times more likely to have been exposed, compared with male horses); age of the horse (the risk of being exposed to E risticii increased with age, peaked at around 12 years, and decreased thereafter); and month of submission (horses tested during November and December had the highest odds of being seropositive [odds ratio = 2.1], and horses tested during March through April were least likely to be seropositive [odds ratio = 0.5], compared with horses tested during January and February).     

Monthly prevalence (in 1986) of antibody titers against equine monocytic ehrlichiosis in apparently healthy horses in Illinois

The seroprevalence and seasonal trend of antibody titers against equine monocytic ehrlichiosis (Potomac horse fever) were determined in apparently healthy horses in selected areas of Illinois in 1986. Sera from 1,367 horses (6 months to 29 years old) were evaluated for the presence of antibodies against Ehrlichia risticii with indirect immunofluorescence. The majority (88%) of the horses were Thoroughbred or Standardbred racehorses. The number of horses with antibodies against E risticii was 229/1,367 (16.75%). The titers in these horses ranged from 1:10 to 1:640. As the year progressed, the number of seropositive horses (titers greater than or equal to 1:10) and the magnitude of the titers increased significantly, both reaching a maximum in July and August, respectively (P less than 0.05). A relationship between seropositivity and gender was not detected. In the year prior to sampling, 56.8% of the seropositive horses had not been ill, whereas 0.8% had diarrhea, an episode of acute abdominal pain, or laminitis. It was concluded that a large number of horses in Illinois are exposed to E risticii, that maximal exposure occurs in July, and that the most common form of the disease in Illinois is not associated with clinical signs.     

Attempted transmission of Ehrlichia risticii by field-captured Dermacentor variabilis (Acari: Ixodidae)

The capability of field-collected American dog ticks, Dermacentor variabilis, to infect horses with Ehrlichia risticii, causative agent of Potomac horse fever (PHF), was examined by allowing adult ticks collected from horse farms with a history of PHF to feed on susceptible horses. More than 500 male and female ticks attached and fed on 3 test horses; however, no clinical or serologic evidence of PHF was observed in treated or control horses. All horses were challenge exposed with E risticii-infective blood by inoculation at 60 to 65 days after ticks fed, and all developed clinical PHF with subsequent seroconversion. The data, therefore, indicated that adult D variabilis, a common parasite of horses on Maryland premises where PHF is enzootic, may not serve as a vector of E risticii.     

Immunohistochemical and ultrastructural detection of intestinal spirochetes in Thoroughbred horses.

Studies of equine intestinal spirochetes have long focused on intestinal contents alone, but intestinal spirochetosis has been reported recently in a 21-month-old Thoroughbred colt in Japan. To define the clinical and pathological significances of intestinal spirochetosis in several horses, an epizootiologic survey with histologic, immunohistochemical, and ultrastructural methods was conducted for Brachyspira antigen-containing intestinal spirochetes in 12 diseased or injured Thoroughbred horses, aged from 35 days to 17 years. Brachyspira antigen-containing spirochetes were found in 7 of 12 horses (58.3%) and were more frequent in the cecum than in other parts of the bowel. It was not clear whether the infection was clinically related to diarrhea or dysentery, but histopathology revealed a close association between the bacterial infection and epithelial hyperplasia. Crypt epithelium consisted mainly of goblet cells and showed frequent mitosis throughout its length. Inflammatory cells and congestion were also present. There were numerous spirochetes in the crypts, and some invaded the cecal and colonic epithelia and underlying lamina propria. Ultrastructurally, the spirochetes were divided into 4 types. Three types were identified in degenerative epithelial cells or intracellularly. Brachyspira antigen-containing intestinal spirochetes invading the mucosa were capable of causing epithelial hyperplasia in the cecum and colon in the horses. The findings in this study will increase awareness of the importance of intestinal spirochetosis and may also be helpful for diagnosis and treatment of this condition.     

Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR

A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evaluated on horses with infectious colitis and on freshwater stream snails collected from regions with a history of PHF. E. risticii could be detected in 22 of 153 (14.4%) horses with infectious colitis and in 25 of 234 (10.7%) snails in the TaqMan PCR. The same results were obtained in the conventional nested PCR. The Ehrlichia-load was in the range of 10,000-9,000,000 and 35,000-680, 000,000 Ehrlichia equivalents per microg leukocyte DNA and snail DNA, respectively.     

Ultrastructural mucosal injury after experimental ischemia of the ascending colon in horses.

The ultrastructural injury that develops sequentially in the ascending colon during experimentally induced ischemia was examined in 6 halothane-anesthetized horses. Colonic ischemia was created by 2 types of vascular occlusion 24 cm proximal and distal to the pelvic flexure. In all horses, transmural vascular compression was created. The colonic venous circulation was obstructed in 3 horses, whereas in the other 3 horses, arterial and venous circulation was obstructed. Two additional horses were anesthetized as controls for determination of any morphologic alterations associated with the experimental protocol. Full-thickness colonic biopsy specimens were obtained from the antimesenteric border of the pelvic flexure at 0, 0.25, 0.5, 1, 1.5, 1.75, 2, 2.25, 2.5, 3, 3.5, 4, 4.5, and 5 hours during occlusion, and were studied by light and transmission electron microscopy. Morphologic alterations did not develop in the colon of control horses. Mucosal congestion was observed by light microscopy in the colon of horses with experimentally induced ischemia, but congestion developed early in those with obstructed colonic venous circulation, compared with those having arterial and venous obstruction. Inter- and intracellular vacuolation and loss of staining initially resulted in groups of 3 to 5 superficial luminal epithelial cells. Alterations in the glandular epithelium lagged behind those in the superficial epithelium, but were observed in both groups by 2 hours of obstruction. These changes progressed to 100% sloughing of all epithelium by 4.5 to 5 hours. The initial cellular alterations, which were observed by transmission electron microscopy, developed at 0.25 hour in horses with colonic venous obstruction and was characterized by inter- and intracellular edema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathophysiologic features of swine dysentery: cyclic nucleotide-independent production of diarrhea

Net electrolyte and water transport and unidirectional Na+ fluxes were examined in ligated colonic loops of clinically normal pigs and in pigs with swine dysentery (etiologic agent Treponema hyodysenteriae) in the presence or absence of theophylline. In normal pigs, theophylline abolished net Na+ absorption via a reduction in the lumen-to-blood flux, decreased Cl- absorption, and increased HCO3- accumulation in the lumen. In infected pigs, all net ion transport was abolished, with the addition of theophylline producing little effect. The absence of net Na+ absorption in infected pigs was also the result of a decreased lumen-to-blood flux. Seemingly, colonic malabsorption may be the primary transport alteration in swine dysentery. Concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in samples of colonic mucosa from normal and infected pigs after in vitro exposure to a Ringer's solution containing 0 or 20 mM theophylline. Basal values of cAMP or cGMP did not increase in infected colonic mucosa. There was a diminished capacity of the infected mucosa to respond to theophylline. Alterations in ion transport in conjunction with measurements of cAMP and cGMP indicated that the pathogenic mechanism(s) in swine dysentery were not similar to those of Salmonella, Shigella, Vibrio cholerae, or Escherichia coli diarrhea.     

Protection against murine potomac horse fever by an inactivated Ehrlichia risticii vaccine

Ehrlichia risticii propagated in a murine macrophage cell line were freed from the host cell by hypotonic lysis of the infected cells. The cell-free ehrlichiae were inactivated with beta-propiolactone and combined or not combined with polymyxin-B. The vaccines were administered to mice with Quil-A (saponin) as an adjuvant twice at 2 to 3 week intervals and the mice were challenged with live E. risticii 2 to 3 weeks after the last vaccination. With or without the addition of polymyxin-B, the vaccine preparations protected mice from developing clinical signs and gross pathologic changes such as thymic atrophy, splenomegaly, and increase in whole intestinal weight. Mice vaccinated with or without polymyxin-B developed high titer IgG antibody against E. risticii before and after the challenge with live E. risticii. Spleen lymphocyte proliferative response assay at 11 days post challenge revealed that with polymyxin-B a higher lymphocyte proliferation occurred as compared with that of the mice which received polymyxin-B-free vaccine. Spleen lymphocytes of the placebo (polymyxin-B and Quil-A) pretreated/challenged mice showed no proliferative activity. Western blot analysis revealed that vaccinated mice reacted mainly with 110, 57 and 33 kDa antigen bands before and after challenge. The placebo (polymyxin-B and Quil-A)/challenged mice showed a very weak response to ehrlichial antigens at day 10 to 11 post challenge. Comparison with inactivated Renografin-purified E. risticii or 0.25% SDS-insoluble fraction of E. risticii with the inactivated host cell-free vaccine revealed no increased protection. These results indicate that inactivated host cell-free E. risticii can protect mice from murine Potomac horse fever. The presence of polymyxin-B appeared to be not harmful but rather beneficial for lymphocyte proliferation response upon challenge with live E. risticii.     

Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:16558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.     

Pathogenesis of bovine viral diarrhoea-mucosal disease: distribution and significance of BVDV antigen in diseased calves

The distribution of bovine viral diarrhoea virus (BVDV) antigen in tissues of animals with acute and chronic bovine viral diarrhoea-mucosal disease (BVD-MD) was examined using improved indirect immunofluorescence and indirect immunoperoxidase staining methods on cryostat and paraffin wax tissue sections. In lymphoid tissues the antigen was principally located in cells belonging to the mononuclear located in cells belonging to the mononuclear phagocyte system and in other cells with antigen-retaining capacity. The distribution of the infected cells within a particular organ varied with the clinical stage. In the non-lymphoid organs the antigen was detected in cells of the mononuclear phagocyte system as well as in epithelial cells. An apparent time lag between initial antigen-detection and progression of pathological manifestations was noticed in the intestinal mucosa and in the keratinized epithelia of the upper digestive system and in the skin. Only limited involvement of blood vessels was observed in the tissues investigated. In the light of the mononuclear phagocyte system being an apparent common denominator for all the different tissues involved in the morphological alterations which characterise BVD-MD, a possible pathogenesis of BVDV-MD is discussed.     

Neorickettsia risticii surface-exposed proteins: proteomics identification, recognition by naturally-infected horses, and strain variations

ABSTRACT: Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrated universal and strong recognition of recombinant P51 over other Neorickettsia recombinant proteins. Comparisons of amino acid sequences for predicted secondary structures of P51, as well as Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3) among N. risticii strains from horses with PHF during a 26-year period throughout the United States revealed that the majority of variations among strains were concentrated in regions predicted to be external loops of their β-barrel structures. Large insertions or deletions occurred within a tandem-repeat region in Ssa3. These data demonstrate patterns of geographical association for P51 and temporal associations for Nsp2, Nsp3, and Ssa3, indicating evolutionary trends for these Neorickettsia surface antigen genes. This study showed N. risticii surface protein population dynamics, providing groundwork for designing immunodiagnostic targets for PHF.