The effect of passively acquired antibodies on Lawsonia intracellularis infection and immunity in the horse.

REASONS FOR PERFORMING STUDY: Multiple hypotheses into the age-based susceptibility of animals to Lawsonia intracellularis exist, including the decline of passively acquired antibodies. OBJECTIVES: To determine whether the decline in passively acquired antibodies in horses is responsible for the age predilection of equine proliferative enteropathy (EPE). Additional objectives included examination of various risk factors for the development of EPE as well as the determination of naturally occurring attack rates for clinical and subclinical EPE. STUDY DESIGN: Prospective, multifarm field study. METHODS: A total of 369 mare and foal pairs from 15 central Kentucky Thoroughbred farms were used in this study, which took place from January 2012 to February 2013. Serum samples were collected from mares and foals within 48 h of parturition, and then monthly from foals to February of their yearling year. Lawsonia intracellularis-specific antibodies were measured using an enzyme-linked immunosorbent assay. RESULTS: No effect of passively acquired antibodies on the occurrence of presumptive clinical or subclinical EPE was noted. In total, 5.3% and 6.3% of seropositive horses developed presumptive clinical or subclinical EPE, respectively. In multiple logistic regression models, colts were at a significantly greater risk than fillies of developing presumptive clinical EPE (odds ratio [OR] 5.468, 95% confidence interval [CI] 1.134-26.362, P = 0.034) or a combination of either presumptive clinical or subclinical EPE (OR 3.861, 95% CI 1.461-10.206, P = 0.006) while foals that were weaned in September or beyond were at a lower risk of developing presumptive EPE (OR = 0.281, 95% CI 0.0807-0.981, P = 0.05). CONCLUSIONS: This is the first study to show that passively acquired antibodies to L. intracellularis do not have an effect on the occurrence of clinical or subclinical EPE. A number of novel findings, including identification of the disease rate among naturally exposed horses, warrant additional work as they may help to identify potential risk factors for L. intracellularis exposure and/or the reservoir host(s) of the bacterium.

Adaptation and validation of a bacteria-specific enzyme-linked immunosorbent assay for determination of farm-specific Lawsonia intracellularis seroprevalence in central Kentucky Thoroughbreds.

REASONS FOR PERFORMING STUDY: Lawsonia intracellularis is the causative agent of equine proliferative enteropathy (EPE), a disease for which no large-scale seroprevalence studies have been conducted. OBJECTIVES: To validate and use an equine-specific enzyme-linked immunosorbent assay (ELISA) for L. intracellularis to determine the seroprevalence of L. intracellularis on numerous farms. METHODS: An ELISA, in which purified antigen was used, was adapted from previous work in swine. A total of 337 Thoroughbreds from 25 central Kentucky farms were enrolled and monthly serum samples collected from August 2010 to January/February 2011. Samples were screened for L. intracellularis-specific antibodies using a modified ELISA. Farms were classified into one of 3 groups based on 3 year prior history with EPE. RESULTS: The ELISA intra-assay coefficient of variation (CV) was 6.73 and inter-assay CV was 9.60. An overall seroprevalence of 68% was obtained, with farm-specific seroprevalances ranging from 14 to 100%. A significant difference was found in the average seroprevalence (P<0.05) on farms with a confirmed recent history of EPE cases. Additionally, both lower average ELISA unit (EU) values (P = 0.079) and maximum EU values (P = 0.056) were detected on farms with no recent EPE history when compared to the other groups. A bimodal exposure distribution to L. intracellularis was detected in the fall and winter months. CONCLUSIONS: Recent history of EPE was associated with higher average seroprevalence indicating increased exposure on farms with prior cases of EPE. Seasonally bimodal exposure was also observed. POTENTIAL RELEVANCE: The adapted ELISA appears to be useful for determination of L. intracellularis-specific antibody levels. The high farm-specific seroprevalences and bimodal distribution of exposure to L. intracellularis were unexpected and suggest that farms with a previous history of EPE remain at risk due to heightened exposure levels beyond early winter.

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model.

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.

CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus.

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.

Improved model for teaching interventional EUS.

BACKGROUND: A swine model was previously developed for teaching endoscopic ultrasound (EUS). The purpose of this study was to improve this model and develop a method for creating focal lesions for EUS imaging and intervention. METHODS: Experiments were performed in farm pigs (Sus scrofa) under general anesthesia. Under real-time EUS guidance attempts were made to create a submucosal lesion and a focal mediastinal lesion, to perform EUS-guided fine-needle aspiration of the pancreas, and to confirm the site of injection during "sham" EUS-guided celiac block. RESULTS: A hypoechoic, submucosal mass was created in the stomach, which was then imaged by EUS and punctured trans-gastrically. Injection of saline solution in the mediastinum created a pseudo-mediastinal lymph node. A needle was then advanced trans-esophageally into the mediastinum to mimic EUS-guided fine-needle aspiration of a mediastinal lymph node. Abdominal exploration of the pigs after euthanasia confirmed injection of the sham celiac block around the celiac ganglion. CONCLUSION: The swine model is not only useful for teaching normal EUS anatomy, but it may be a useful model for teaching EUS-guided intervention.

Evaluation of an agar gel immunodiffusion test kit for detection of antibodies to Mycobacterium paratuberculosis in sheep.

OBJECTIVE: To determine whether a commercially available agar gel immunodiffusion test approved for detecting antibodies to Mycobacterium paratuberculosis in cattle could be used for sheep. DESIGN: Experimental trial. SAMPLE POPULATION: Serum samples from 27 sheep confirmed to have paratuberculosis by means of acid-fast staining of smears of ileal mucosa, histologic examination of tissues, or bacteriologic culture; 7 sheep with clinical signs of paratuberculosis; and 55 sheep from 5 uninfected flocks. PROCEDURE: Serum samples were tested concurrently with the commercially available test and with a previously validated agar gel immunodiffusion test. Multiple samples collected from 13 infected sheep over a period of 6 years were also tested so that each test's ability to detect onset of seropositivity could be compared. RESULTS: For both tests, results for samples from all 55 uninfected sheep were negative, results for samples from 32 of the 34 sheep with paratuberculosis were positive, and results for the remaining 2 sheep with paratuberculosis were negative. Results of both tests were in agreement for 50 of 54 samples obtained from 13 infected sheep over time. The 4 samples for which results of the 2 tests disagreed were the fourth, eighth, and ninth of 10 samples from 1 sheep and the first of 6 samples from a second sheep. For all 4 samples, the commercially available assay yielded a weak-positive result, but the previously described test yielded a negative result. CLINICAL IMPLICATIONS: The commercially available agar gel immunodiffusion test approved for use in cattle may be useful in the differential diagnosis of paratuberculosis in sheep.

Evaluation of an enzyme-linked immunosorbent assay licensed by the USDA for use in cattle for diagnosis of ovine paratuberculosis.

A commercially available Mycobacterium phlei-absorbed enzyme-linked immunosorbent assay (ELISA) approved to detect antibodies to Mycobacterium paratuberculosis in cattle was evaluated for its applicability in sheep. The potential for interference with ELISA results from cross-reacting antibodies to Corynebacterium pseudotuberculosis was also investigated. Serum samples were randomly selected from a collection of samples obtained in 1986-1991 from 6 infected and 5 noninfected sheep flocks varying in breed, age, and geographic origin. Tests were performed on sera from 27 paratuberculous sheep, confirmed by histopathology, bacteriologic culture, and/or acid-fast staining of ileal mucosal smears, and on sera from 246 noninfected sheep. The optical density of each sample was expressed as a percentage of the optical density of a known positive sheep serum sample tested on the same plate. These values were log-transformed to achieve normality of distribution, and sensitivity and specificity estimates were calculated based on 2 and 3 standard deviations above the mean of the percent positive value (PPV) of the noninfected sheep. A cutoff value of PPV > or = 55.74 resulted in an estimated sensitivity of 0.48 and a specificity of 0.95. Sera from 10 noninfected sheep with PPV above the cutoff level of 55.74% were absorbed with heat-treated C. pseudotuberculosis organisms in addition to M. phlei antigens. Sera from 14 ELISA-positive paratuberculous sheep and 23 ELISA-negative noninfected sheep were similarly treated, and results were compared. Absorption with C. pseudotuberculosis resulted in a significant decrease in PPV in all 3 groups of sheep sera, but a greater decrease was observed in the noninfected sheep with PPV above the cutoff level when compared with noninfected sheep with PPV below that level. Results of this study suggest that ELISA may be of value in screening sheep flocks for paratuberculosis, but further experimentation is needed to optimize the sensitivity and specificity of the assay. Exposure to C. pseudotuberculosis may confound results obtained by M. phlei-absorbed ELISA for paratuberculosis.

Comparative analysis of the 16S rRNA gene sequence of the putative agent of proliferative ileitis of hamsters.

Proliferative ileitis of hamsters is consistently associated with the presence of intracellular bacteria in affected ileal epithelial cells. The 16S rRNA gene sequence of the putative etiologic agent of proliferative ileitis was determined by using cell culture-maintained organisms. The highest level of relatedness (98.4%) was observed with a newly described obligately intracellular bacterium obtained from porcine intestines, and the level of homology with Desulfovibrio desulfuricans was 87.5%.

Detection of flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA)--new prospects for diagnosis.

A new diagnostic procedure was developed to detect the flagellar antigen of Campylobacter jejuni and Campylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected all C. jejuni or C. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative for C. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens of C. jejuni and C. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.

Comparison of the major outer-membrane protein (MOMP) gene of mouse pneumonitis (MoPn) and hamster SFPD strains of Chlamydia trachomatis with other Chlamydia strains.

Restriction fragments containing the major outer-membrane protein (MOMP) gene from two nonhuman (rodent) strains of Chlamydia trachomatis, the mouse pneumonitis (MoPn) strain and the SFPD strain isolated from hamsters with transmissible proliferative ileitis, were cloned and sequenced. The MOMP genes of both MoPn and SFPD encode an identical 22-amino acid leader peptide and mature polypeptides of 365 and 382 amino acids, respectively. Alignment of the MOMP genes of the two rodent strains revealed 91% identity. By comparison with other known chlamydial MOMP gene sequences, there was 80%-83% identity with human biovars strains of C. trachomatis, and there was 69%-70% identity with C. psittaci and C. pneumoniae strains. The main differences in these sequences were clustered into four variable domains. A minimum-length evolutionary tree was constructed on the basis of the MOMP gene variable positions by using PIMA package software. The minimum mutation distances indicated that (i) the MOMP genes of all chlamydial strains may have evolved from a common ancestor; (ii) all the strains of C. trachomatis compose one of the subtrees, and strains of C. psittaci and C. pneumoniae compose the other subtree; and (iii) in the C. trachomatis subtree, the human and the rodent strains are divided into two clusters. The branching pattern of this evolutionary tree is generally consistent with current classification based on serological, morphological, and other biological characteristics.

Evaluation of the skeletal effects of combined mild dietary calcium restriction and ovariectomy in Sinclair S-1 minipigs: a pilot study.

A pilot study was conducted to investigate the combined effects of ovariectomy (OVX) with preceding and concomitant mild dietary calcium restriction on the minipig skeleton. Minipigs 4 months old were fed diets containing 0.9, 0.75, or 0.5% calcium (Ca). At 10 months, the 0.75 and 0.5% pigs were OVX and the 0.9% were either sham operated or OVX. All pigs were maintained on their respective diets for an additional 6 months. Excised lumbar vertebrae and long bones were evaluated by densitometry and histomorphometry, and vertebral cancellous bone samples were tested biomechanically. In pigs fed the 0.9% Ca diet, OVX alone effected decreases of 6% in vertebral bone mineral density (BMD), 15% in trabecular bone volume (BV/TV), and 13% in trabecular number (Tb.N), an increase of 15% in trabecular separation (Tb.Sp), and a nonsignificant increase (p < 0.056) in vertebral cancellous final erosion depth (F.E.De) compared with the 0.9% Ca sham-operated group. Decreasing dietary Ca to 0.5% in combination with OVX effected an 8% reduction in vertebral BMD that was not associated with any significant alterations in parameters of vertebral cancellous bone microstructure or remodeling compared with the 0.9% Ca sham-operated pigs. Increases in serum PTH noted in the 0.5% Ca OVX group were generally paralleled by increases in calcitriol. In OVX pigs fed a diet containing 0.75% Ca, a 10% reduction in vertebral BMD was observed. This was associated with significant increases in F.E.De and vertebral marrow star volume (Ma.St.V) compared with the 0.9% Ca sham-operated pigs and the other OVX groups. In addition, Tb.Sp was increased and Tb.N decreased compared with the 0.9% Ca sham-operated pigs. Increases in serum PTH in this group were not accompanied by increases in calcitriol. Midradial and midfemoral BMD values were reduced in the 0.75 and 0.5% Ca OVX groups compared with the 0.9% Ca sham-operated pigs. Histomorphometric analyses of cortical bone suggested the reduction in cortical bone mass in the 0.75% Ca OVX group may have been largely due to net loss on the endocortical surface versus possible failure to accrue bone in the 0.5% Ca OVX group. Ash density and biomechanical parameters for vertebral cancellous bone decreased progressively in the 0.9% sham-operated, 0.9% Ca OVX, and 0.75% Ca OVX groups and then increased in the 0.5% Ca OVX group. After normalization for bone mass (ash), mechanical changes were still apparent, particularly for the 0.75% Ca OVX group compared with other OVX groups, reflecting that structural changes had taken place in the trabecular network.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigenic specificity and morphologic characteristics of Chlamydia trachomatis, strain SFPD, isolated from hamsters with proliferative ileitis.

Profound diarrhea associated with proliferating intestinal cells containing intraepithelial campylobacter-like organisms (ICLO) occurs in a variety of mammalian hosts, particularly swine and hamsters. Recently, intracellular bacteria were isolated from proliferative intestinal tissue of hamsters and propagated in intestine cell line 407. Oral inoculation of hamsters with cell culture lysates containing these organisms reproduced the disease in susceptible hamsters. In the present study, an intracellular bacterium from the INT 407 cell line was shown by a variety of techniques to be a member of the genus Chlamydia and has been designated Chlamydia sp. strain SFPD. McCoy cells infected with Chlamydia sp. strain SFPD demonstrated bright fluorescent-stained intracytoplasmic inclusions when examined with fluorescein-labeled species-specific C. trachomatis monoclonal antibodies. The organism also reacted to fluorescein-labeled polyclonal but not monoclonal ICLO "omega" antisera. Ultrastructural examination of the Chlamydia sp. strain SFPD from McCoy cells revealed electrondense elementary bodies and a less electron-dense reticulate-like body that was circular; both features are consistent in morphology to developmental forms of Chlamydia and do not conform to ICLO morphology. Molecular studies, 16S ribosomal sequence analysis, and sequencing of the outer membrane protein confirmed that the isolate is a C. trachomatis closely related to the mouse pneumonitis strain of C. trachomatis.

Calcium-restricted ovariectomized Sinclair S-1 minipigs: an animal model of osteopenia and trabecular plate perforation.

The ovariectomized rat model has now been generally accepted as a useful model for screening different therapeutic agents, but there is a major requirement to identify reliable large animal models for osteoporosis research. In this study, the calcium restricted, ovariectomized minipig has been thoroughly investigated in order to define a large animal model with trabecular and cortical bone remodeling which would be reliable for further testing of agents that had shown promise of efficacy during the screening procedure. Twenty six female, 4-month old minipigs were randomized into four groups and fed either normal diet (0.90% calcium (Ca.)) or diet with restricted calcium content (0.75%, 0.50%). At the age of ten months, 3 groups were ovariectomized (OVX) while one group on normal diet was shamoperated. The groups were followed for six months after the operation. At death, bone mass was determined by densitometry and by ashing. Additionally, biomechanical competence was assessed in trabecular bone cores from the vertebral bodies. Finally, histomorphometry (static and dynamic parameters) and structural analyses (star volume) were performed on the vertebral bodies. The study revealed an OVX-related decline of 6% in vertebral bone mineral density (BMD) and a decline of 15% in trabecular bone volume (BV/TV). In contrast, a 15% increase in mean trabecular plate separation (Tb.Sp.) and a small increase in marrow space star volume (Ma. Star V.) were detected. The structural changes became more pronounced when OVX was combined with mild Ca. restriction (0.75% Ca.) with an increase in Ma. Star V. to 164%.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of an intracellular bacterium from hamsters (Mesocricetus auratus) with proliferative ileitis and reproduction of the disease with a pure culture.

An intracellular bacterium was isolated from hamsters (Mesocricetus auratus) with proliferative ileitis. The organism was isolated in Intestine 407 and GPC-16 cell cultures (incubated in a microaerophilic atmosphere) from isolated and lysed epithelial cells from hamsters with proliferative ileitis. The bacterium measured 1.4 to 1.7 microns in length by 0.26 to 0.34 microns in width, was slightly curved, and had an irregular trilaminar cell wall. Inoculation of hamsters with a cell culture lysate containing the organism or a 0.65-microns-pore-size filtrate of an infected-cell lysate resulted in the typical lesions of proliferative ileitis in approximately 50% of the animals in 28 days. Hamsters inoculated with uninfected cells or a 0.2-microns-pore-size filtrate of an infected-cell lysate remained uninfected. Attempts to propagate the organism on cell-free media have been unsuccessful.

Efficacies of erythromycin and chloramphenicol in extinguishing fecal shedding of Campylobacter jejuni in dogs.

Oral treatment regimens of erythromycin stearate and chloramphenicol were evaluated in naturally infected laboratory colony dogs for their efficacies in extinguishing fecal shedding of Campylobacter jejuni. Of the 25 Campylobacter-infected English Foxhounds in the study, 9 were assigned to erythromycin treatment, 9 to chloramphenicol treatment, and 7 to no treatment. Antimicrobials were administered for 12 days. All of the dogs that received erythromycin stearate ceased shedding C jejuni by the fourth day of treatment and remained negative throughout the treatment period. Chloramphenicol was associated with a reduction in shedding from 100% to 57% by the ninth day of treatment. Within 9 days of the discontinuation of antimicrobial treatment, C jejuni was isolated from all chloramphenicol-treated dogs and 89% erythromycin-treated dogs.

Isolation of a Campylobacter-like organism from healthy Syrian hamsters (Mesocricetus auratus).

A Campylobacter-like organism was isolated from the ilea of normal hamsters. The organism was isolated from an ileal homogenate which was passed through a filter (0.65-micron pore size) and cultured on blood-agar plates in a microaerophilic atmosphere at 37 degrees C. Pinpoint translucent colonies were first observed after 120 h of incubation. The isolated organism measured 2.0 to 3.5 microns in length (excluding flagella) by 0.17 to 0.25 micron in width and typically had a single terminal sheathed flagellum. The organism was oxidase, catalase, and urease positive, reduced nitrates, and was susceptible to nalidixic acid (30-micrograms disk) and resistant to cephalothin (30-micrograms disk). Unlike Campylobacter pylori subsp. mustelae, this organism did not hydrolyze indoxylacetate. Immunofluorescence studies with a Campylobacter species-specific monoclonal antibody (8322-2E6) revealed the presence of numerous positively stained organisms within the crypt epithelial cells of the hamsters from which this organism was isolated. The role of this organism in the pathogenesis of proliferative ileitis in hamsters is uncertain, as is the taxonomic relationship of this organism to other members of the genus Campylobacter.

Effects of sample holding time, temperature, and atmosphere on the isolation of Campylobacter jejuni from dogs.

Stool specimens were collected from 39 dogs, inoculated onto Campylobacter blood agar plates, and divided into four subsamples. Subsamples were held at 4 and 25 degrees C in room air and in a microaerobic environment and were reinoculated at 1, 2, 3, 4, 6, and 8 h. C. jejuni survived at least 3 h when it was held at 4 degrees C, but less than 2 h when it was held at 25 degrees C. The holding atmosphere was not associated with a difference in isolation rates.

Experimental production of proliferative ileitis in Syrian hamsters (Mesocricetus auratus) by using an ileal homogenate free of Campylobacter jejuni.

The role of Campylobacter jejuni in the pathogenesis of proliferative ileitis of Syrian hamsters (Mesocricetus auratus) has been uncertain. C. jejuni has been implicated as the etiologic agent on the basis of the campylobacter-type morphology of the intracellular organism and the repeated microbiologic isolation of C. jejuni from hamsters with proliferative ileitis. The inability to reproduce the disease with pure culture inocula, coupled with immunohistochemical studies, however, has suggested that although C. jejuni may be present in the ilea of infected hamsters, its involvement in the pathogenesis of proliferative ileitis is questionable. In this study hamsters were inoculated with infective ileal homogenates prepared from ilea which were extensively washed to remove the ileal contents before grinding. The ilea from hamsters inoculated with this homogenate were also washed before being ground and used to experimentally inoculate a second group of hamsters. Of the 20 hamsters from this second group, 12 developed lesions typical of proliferative ileitis. Extensive microbiologic cultures from these hamsters were negative for C. jejuni. Immunofluorescence studies with a C. jejuni-specific monoclonal antibody were also negative. The use of a Campylobacter genus-specific monoclonal antibody, however, revealed numerous campylobacter-type organisms within the ileal epithelial cells of the crypts and villi. The presence of C. jejuni is therefore apparently not necessary for the production of proliferative ileitis in hamsters, and the intracellular campylobacter-type organism present in the ileal epithelial cells of infected hamsters is probably not C. jejuni.