Corresponding author: Nicola Pusterla, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616; e-mail: firstname.lastname@example.org.
Background: Equine proliferative enteropathy (EPE) is an emerging disease of weanling foals.
Objectives: Describe clinical, hematologic, biochemical, serologic, molecular, and ultrasonographic findings in foals experimentally infected with Lawsonia intracellularis.
Animals: Eight foals.
Methods: Recently weaned foals were assigned to either the challenge (n = 3), the sentinel (n = 3), or the control (n = 2) group. Foals were experimentally challenged via intragastric inoculation of 3 × 1010L. intracellularis organisms grown in culture. Each experimentally infected foal was housed with a sentinel foal in order to assess feco-oral transmission. All foals were monitored daily for the development of clinical abnormalities and were weighed once weekly for the duration of the study (90 days). Abdominal ultrasound examination was performed weekly. Feces were collected every other day for 60 days, then weekly for an additional 30 days for the quantitative molecular detection of L. intracellularis. Blood was collected weekly for hematologic, biochemical, and serologic analysis.
Results: Only challenged foals developed transient clinical signs of EPE consisting of anorexia, lethargy, fever, loose feces, and peripheral edema. Two challenged foals developed transient hypoalbuminemia. Fecal shedding of L. intracellularis was first detected in the challenged foals between days 12 and 18 postinoculation and lasted for 7–21 days. Seroconversion was documented in all challenged foals and in 1 sentinel foal. The remaining sentinel and control foals remained unaffected.
Conclusions and Clinical Importance: Clinical EPE of variable severity was induced in all foals infected with L. intracellularis. Furthermore, L. intracellularis can be transmitted via the feco-oral route to susceptible herdmates.
Lawsonia intracellularis is the etiologic agent of the recently recognized intestinal disease in horses, equine proliferative enteropathy (EPE).1L. intracellularis is an obligate intracellular, curved, Gram-negative bacterium that resides freely within the apical cytoplasm of infected intestinal enterocytes. It causes proliferation of the affected enterocytes, resulting in a thickened small and sometimes large intestine.2L. intracellularis can only be grown in vitro in cell culture and requires a specific atmosphere for growth.2 Besides horses, L. intracellularis infects many species of domestic and wild animals, including pigs, hamsters, rabbits, fox, deer, ferrets, ostriches, and nonhuman primates.2 EPE was first reported in horses in 1982.3 Since 1996, many more reports of sporadic cases4–14 and outbreaks on breeding farms15–18 have been described. In the last few years, reported cases of EPE have increased in number, primarily in postweaning foals. The disease has almost reached a worldwide distribution and has been reported in the United States,5 Canada,15 South America,19 Europe,10 and Australia.9
Predisposing factors such as the stress of weaning, underlying diseases, and parasitism have been suggested in the development of proliferative enteropathy (PE) in foals.19 The route of infection for weanling foals remains unknown; however, similar to other species, a feco-oral route is suspected, via contaminated feed, water or both. In pigs, the incubation period is 2–3 weeks after exposure; however, this period has not been determined for horses. Because of the wide host range of PE, numerous potential reservoir hosts exist. L. intracellularis recently has been detected by polymerase chain reaction (PCR) in the feces of a variety of domestic and wild animals.20–22 Epidemiologic investigations on premises from which clinical cases have been identified indicate that 10–65% of clinically unaffected foals and adult horses are seropositive for L. intracellularis.17,23,24
A suspected diagnosis of PE in foals is based on age (4–7 months), clinical signs (lethargy, weight loss, subcutaneous edema, diarrhea, colic), and clinicopathologic findings (hypoproteinemia, hypoalbuminemia).1 Thickening of the small intestinal wall, as seen on abdominal ultrasonography, further supports the diagnosis. The diagnosis of EPE is further reinforced by the presence of PCR positive fecal results for L. intracellularis, the detection of specific antibodies against L. intracellularis by serology or both. Although these 2 diagnostic assays appear to be promising tools to indicate possible active infection and previous exposure, their sensitivity and specificity have not been evaluated for horses.
The purpose of the study reported here was to establish an animal model in order to study sequential clinical, laboratory, and ultrasonographic features of experimental EPE. Furthermore, a question of interest was whether experimentally infected foals would shed enough L. intracellularis in the environment to potentially infect sentinel herdmates. To our knowledge, there are no published data on the experimental challenge of weanling foals with an equine isolate of L. intracellularis.
Materials and Methods
Eight clinically healthy foals, between 4 and 5 months of age, were included in the study. The foals belonged to the research herd at the Center for Equine Health, University of California at Davis. The herd has had no history of EPE. Before beginning the study, foals were evaluated for any signs of illness by a full physical examination, CBC, and biochemical blood analysis; all results were within the reference range. Furthermore, whole blood was collected from all foals and their dams 7 days before the onset of the study and tested for L. intracellularis-specific antibodies by immunoperoxidase monolayer assay (IPMA)25 to document seronegative status for each animal. In addition, fecal samples were collected and tested for L. intracellularis DNA by real-time PCR in order to document PCR negative feces. The presence of Salmonella sp. and Clostridium difficile in feces of each study foal was ruled out before the study by conventional culture and ELISA,a respectively. The foals were randomly assigned to 1 of 3 groups. The experimental challenge and sentinel group each consisted of 2 colts and 1 filly. The control group included 1 colt and 1 filly. All procedures were approved by the Institutional Animal Care and Use Committee of the University of California.
Five days before challenge, all mare and foal pairs were brought into the barn and kept in individual stalls. At this time, the foals received oral omeprazoleb paste at 4 mg/kg BWT once daily for 5 consecutive days. Premedication with omeprazole was carried out to increase stomach pH and decrease the effect of low pH on the viability of the challenged L. intracellularis. One day before challenge, foals were separated from their dams and kept in pairs in individual stalls (12 × 12 feet) for the remainder of the study period (90 days). Three pairs comprised 1 challenge (foals 1, 2, 3) and 1 sentinel foal (foals 4, 5, 6) and 1 pair comprised the 2 control foals (foals 7 and 8). The 2 control foals were housed in a different barn than the challenge and sentinel pairs in order to minimize the risk of accidental exposure to L. intracellularis. The foals had free choice of grass and alfalfa hay and water and were supplemented daily with a commercial foal supplement. Hay was fed on the ground twice daily and the grain was given in individual buckets once daily. Stalls were cleaned once daily.
The challenge isolate of L. intracellularis originated from a foal that underwent routine necropsy at the Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota at St. Paul. The foal was diagnosed with EPE based on typical histopathological and immunohistochemical findings. The molecular identity of the isolate was determined by multiple-locus variable number tandem repeat analysis.26 Gut scrapings from the necropsied foal originally were grown in McCoy cells (mouse fibroblast cells) and the challenge organism was kept at −80°C until the study began. Bacterial numbers were assessed by direct microscopic count after indirect immunoperoxidase staining using L. intracellularis-specific antibody. Each of the 3 challenge foals was physically restrained and exposed via nasogastric intubation to 3 × 1010L. intracellularis organisms.
Monitoring and Sample Collection
All foals were observed daily for general attitude and appetite. Furthermore, a complete physical examination was performed every day for the 1st 60 days, then once weekly for an additional 30 days. Once weekly, the weight of each foal was recorded in order to determine average daily weight gain throughout the study period. Complete abdominal ultrasound examination was performed once weekly in order to assess intestinal wall thickness and motility and amount of free abdominal fluid. Briefly, after clipping of the hair over the entire abdominal cavity, each foal was manually restrained to allow systemic evaluation of the right and left paralumbar fossa regions, the right and left ventral intercostal spaces and the entire ventral abdomen with 3.5 curvilinear and 5.0 microconvex MHz transducers.c A total of 10 different measurements of colonic and small intestinal wall thickness chosen randomly during the examination were performed and recorded. Motility and diameter of small intestinal loops also were recorded. Evaluators were blinded to the assignment of each challenge and sentinel foal's group.
Feces were collected from every foal directly from the rectum every other day for 60 days, then weekly for an additional 30 days for the quantitative molecular detection of L. intracellularis. Blood sample collections to perform serum biochemical analysis, CBC determination, and serologic analysis were done at weekly intervals.
Case definition of EPE included the development of any of the main clinical signs of EPE, hypoalbuminemia, or both with concurrent fecal shedding of L. intracellularis and detectable antibodies to L. intracellularis in peripheral blood. Foals showing PCR positive feces after the 1st 7 days postinoculation and measurable antibody to L. intracellularis by IPMA were considered infected.
Fecal samples were processed for nucleic acid purification within 2 hours of collection. Two milliliters of phosphate-buffered saline were added to 10 g of feces in a conical tube. Thereafter, each sample was vortexed for 10 seconds and centrifuged at 13,000 ×g for 10 seconds. Nucleic acid purification from 200 μL of supernatant fluid was performed with an automated nucleic acid extraction systemd according to the manufacturer's recommendations. The purified DNA then was analyzed by real-time PCR for the presence of the aspartate ammonia lyase (AAL) gene of L. intracellularis, as reported previously.22 Positive (DNA from cell-grown L. intracellularis) and negative (L. intracellularis-free DNA from fecal samples) DNA controls were used with each run. Absolute quantitation was calculated by a standard curve for L. intracellularis and expressed as copy numbers of the AAL gene of L. intracellularis per gram of feces. Furthermore, a real-time PCR assay targeting a universal sequence of the bacterial 16S rRNA gene was used as quality control (ie efficiency of DNA purification and amplification) and as an indicator of fecal inhibition.27
Whole blood samples collected from the foals were used to perform a CBC and biochemistry, as well as to measure anti-L. intracellularis-specific IgG by IPMA, as reported previously.25 All serum samples were screened at a dilution of 1 : 60, which is considered the standard cut-off titer for L. intracellularis testing by IPMA. Positive serum samples (titer ≥ 60) were tested to endpoint dilution and titers were reported as the reciprocal of the dilution.
Because of the small number of study animals, mainly descriptive analyses were used to describe clinical and laboratory findings among the different groups. The Mann-Whitney U-teste was used to detect differences in mean daily weight gain between infected (challenged and sentinel exposed) and uninfected (control and sentinel nonexposed) foals. Infection was based on the presence of PCR positive feces to L. intracellularis, on detectable antibody response to L. intracellularis by IPMA or both. Results were considered significant at values of P≤ .05.
Development of clinical signs was only observed in the 3 challenged foals. Foal 1 developed fever (T > 101.5°F) on days 19 and 20 postinoculation. The foal became mildly lethargic with partial anorexia on day 24 postinoculation, which lasted for 4 days. Peripheral edema in the distal extremities and throatlatch was first noticed on days 28 and 29 postinoculation, respectively. The edema lasted for 27 and 5 days in the distal extremities and throatlatch, respectively. Fecal character remained normal in this foal. The additional 2 challenged foals only displayed transient changes in fecal character from formed to loose feces, which lasted for 3 days (postinoculation days 15–17) in foal 2 and for 4 days (postinoculation days 16–19) in foal 3. Salmonella sp. and C. difficile were not detected in the feces of foals 2 and 3 at the onset of loose feces by culture and ELISA,a respectively. The 3 sentinel and the 2 control foals did not display any abnormal clinical signs during the entire study period. Mean daily weight gain during the study period ranged from 649 to 987 g/d. The mean daily weight gain of 4 infected foals (3 challenged and 1 sentinel foal) was significantly lower (Mann-Whitney U-test, P= .0286) than the mean daily weight gain of 4 noninfected foals (2 control and 2 sentinel foals; Fig 1).
CBC remained in the normal reference range with the exception of blood collected from foal 1 on day 19 postinoculation, which showed leukopenia of 4,930 cell/μL (reference range 5,300–14,000 cells/μL) because of neutro-penia of 1,400 cells/μL (3,400–11,900 cells/μL). The CBC of that foal was normal on the next blood sample. Biochemical blood abnormalities were observed in foals 1 and 2 and consisted of mild and transient decreases in serum albumin concentration observed on day 14 postinoculation (foal 2, albumin 2.5 g/dL; reference range, 2.7–4.2 g/dL) and on days 21 (foal 1, 2.5 g/dL) and 28 postinoculation (foal 1, 2.3 g/dL).
Sonographic abnormalities were observed only in foal 1 on days 21 and 28 postinoculation and consisted of mild increase in abdominal free fluid and moderately thickened small intestinal loops with wall thickness ranging from 3.2 to 3.8 mm (reference range ≤ 3 mm).28
DNA was successfully extracted from all fecal samples based on positive PCR signals for the universal bacterial 16S rRNA gene, ruling out inadequate nucleic acid purification and inadequate amplification efficiency. Fecal shedding determined by real-time PCR was detected in all 3 challenged foals. PCR detection of L. intracellularis started between days 12 and 18 postinoculation and lasted for 7–21 days after initial detection (Fig 2). The multiple-locus variable number tandem repeat profile of L. intracellularis from the feces of all challenge foals was identical to the profile of the original challenge organism. Foal 1 with the more severe clinical signs displayed a 2–3 times longer duration and a 7–16 times higher maximal load of pathogen shedding compared with the 2 other challenged foals. Fecal shedding was not detected in any of the sentinel and control foals.
The 3 challenged foals had measurable antibodies against L. intracellularis at 14 (foal 1) and 21 (foal 2 and 3) days postinoculation. The highest titers measured in these foals ranged from 240 to 3,840 and positive titers persisted up to the end of the study (Fig 3). Foal 4 (sentinel to foal 1) had a positive titer of 60 against L. intracellularis on days 49, 56, and 63 postinoculation. The remaining sentinel and control foals remained seronegative for L. intracellularis for the entire study period.
This pilot study represents the 1st attempt at inducing EPE in weanling foals using an equine isolate of L. intracellularis. The study was designed to maximize disease in challenged foals by mimicking naturally occurring stress factors such as the weaning process. Previous studies in piglets have determined that large group size, weaning, transportation, diet change, and mixing are stress factors associated with increased susceptibility to PE.2 Furthermore, in an attempt to increase viability of the challenged microorganisms, all challenged foals were premedicated with a proton-pump inhibitor in order to increase stomach pH. This approach has previously been use to study the immune response and fecal shedding in weanling foals vaccinated with an avirulent vaccine strain.29
Challenge studies in pigs have used L. intracellularis either from scraped intestinal mucosa of affected animals or from in vitro grown organisms at a dose ranging from 105 to 1010 organism per pig.30,32 In the present study, the authors elected to use a high challenge dose in order to enhance the potential pathogenic effect of administered L. intracellularis. It remains to be determined if L. intracellularis induces dose-dependent severity of disease in foals as shown for pigs,32–33 and titration studies will be needed in the future in order to improve the equine animal model. Clinical signs commonly seen in affected piglets vary with the clinical presentation of PE and may include acute onset of intestinal blood loss and death in pigs affected by proliferative hemorrhagic enteropathy, or more commonly the clinical signs are restricted to transient anorexia, diarrhea, and poor growth in pigs affected by proliferative intestinal adenomatosis (PIA).2 The abnormal clinical signs seen in the 3 challenged foals were mild to moderate in nature, self-limiting, and more consistent with PIA in pigs. Abnormal clinical signs first observed in the challenged foals occurred between days 15 and 19 postinoculation. In pigs, changes in fecal character after experimental infection generally are observed as early as 4–7 days postinoculation.30 When compared with pigs, the study foals developed clinical signs at a later time. This may have been associated with a slower development of proliferative lesions in the distal small intestine of affected foals. The debilitating effect of natural infection with L. intracellulair in foals is well recognized, but its lasting effect on future performance such as weight gain has mainly been investigated in pigs. The decreased average weight gain in clinically and subclinically infected pigs with L. intracellularis has a substantial financial impact on herd economics.34 This is the reason why prophylactic measures directed to prevent this disease are routine practice in the swine industry. A recent retrospective study from central Kentucky determined that yearlings that had been clinically affected by EPE sold for an average of 68% of the average price of all yearlings by the same stallion.17 From the present study results, it is interesting to note that the only subclinically infected sentinel foal also showed decreased weight gain during the study period. Longitudinal studies are needed to investigate the long-term effect of subclinical disease on future performance and health.
Although the laboratory abnormalities observed in 2 challenged foals were mild and transient, they were comparable to abnormalities seen in naturally infected foals.17 The exact mechanisms by which hypoalbuminemia develop in affected foals has not been investigated. It appears that a combination of decreased feed intake, coupled with malabsorption and protein-losing enteropathy because of the proliferative nature of the disease may represent likely mechanisms by which low serum albumin concentration occurs.35
Thickened small intestinal wall determined by abdominal ultrasonography often is used to support a diagnosis of EPE. The study results showed that 1 of 3 experimentally infected foals had ultrasonographic findings compatible with EPE. As recently reported, the lack of ultrasonographic abnormalities in a suspected case should not rule out the disease.17
Molecular detection of L. intracellularis was only documented in the challenge foals. In these foals, the shedding started between days 12 and 18 postinoculation and lasted for 11 to 21 days. Onset and duration of fecal shedding were comparable to results from experimentally infected pigs,32,36 although intermittent shedding lasting up to 12 weeks has been reported in infected pigs.30 The lack of detectable L. intracellularis in the 1st few days postchallenge is likely caused by the dilution of the challenge organisms within the gastrointestinal content and its slow intracellular replication. To our knowledge, this is the 1st time that quantitative PCR of L. intracellularis in feces has been used in an experimental study. Rapid increase in fecal shedding was followed by a steady decline in daily fecal shedding of L. intracellularis in the study foals. The foal with more pronounced clinical signs had a 7–16-fold higher peak fecal shedding than the 2 additional challenge foals. The quantitative differences in fecal shedding among the challenge foals may represent individual variability or may reflect severity of disease. It would have been interesting to include histopathologic data in the present study. However, because of the longitudinal nature of the experiment, necropsy of affected foals at the peak of disease was not considered. When the clinical and molecular results were compared with each other, fecal shedding preceded development of clinical signs as previously shown for experimentally infected pigs.32
Serologic responses were detected in all challenge and 1 sentinel foal. Onset and duration of measurable serologic response were similar among the challenge foals, although the highest serological response was determined in the foal with more severe clinical signs. The serologic response seen in the foals is similar to the response reported for experimentally infected pigs25,32,36 and foals with natural disease.15 Immunologic responses against L. intracellularis during infection have remained mostly uncharacterized.37 Secretory IgA and cell-mediated immune response are likely to play a role in overcoming natural infection with L. intracellularis. Although the cell-mediated immune response was not investigated in this study, detectable serum IgG against L. intracellularis should be interpreted as indicating previous infection to the bacteria and gross lesions in the intestine.
To the authors' knowledge, this study is the first to determine that naïve foals may acquire subclinical L. intracellularis infection through the feco-oral route when housed with diseased foals. This is exemplified by the seroconversion observed in 1 sentinel foal that was housed with the most severely affected challenge foal. Previous work in pigs has shown that L. intracellularis may survive in feces or the environment for 1–2 weeks38 and that seroconversion may occur without PCR-detectable shedding in sentinel pigs exposed to seeders.31 Together these studies show that low levels of bacteria may cause an animal to seroconvert without detectable shedding of bacteria or observable clinical signs. From a biosecurity standpoint, the present study results re-emphasize the need to institute infectious disease control measures in order to prevent spread of L. intracellularis from an infected to a susceptible animal.
aTox A/B and C Diff Quick Check, Techlab, Blacksburg, VA
bGastrogard 37% Paste, Merial Ltd, Duluth, GA
cMyLab Five, Biosound Esoate, Indianapolis, IN
dCAS-1820 X-tractor Gene, Corbett Life Science, Sydney, Australia
ePrism, Version 5.0, GraphPad Software, La Jolla, CA
The authors thank the staff of the Center for Equine Health, University of California at Davis for assistance with sample collection.