Lawsonia intracellularis, the causative agent of equine

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1 J Vet Intern Med 2008;22: Lawsonia intracellularis Infection in Horses: M.L. Frazer Background: Lawsonia intracellularis is an emerging equine pathogen that is a cause of equine proliferative enteropathy (EPE). Objective: To describe the signalment, month of presentation, common clinical signs, clinicopathologic values, diagnostic tests used, antimicrobial use, and survival status in horses affected with EPE; to evaluate how affected horses sold at public auction as yearlings; and to determine results of fecal polymerase chain reaction (PCR) and serum immunoperoxidase monolayer assay (IPMA) results in age matched, clinically normal herdmates. Animals: The study group was 57 horses treated for disease associated with L. intracellularis infection between August 2005 and January Methods: Retrospective study examined horses exhibiting evidence of infection with L. intracellularis and testing positive for fecal PCR or serum IPMA. Results: Horses ranged in age from 2 to 8 months with a median age of 6 months, and all were examined between August and January. Ventral edema was present in 81% of horses and hypoalbuminemia occurred in all horses. Only 50% of horses tested positive on both PCR and IPMA. Ninety-three percent of horses survived, and survival was unrelated to antimicrobial administered. Affected horses sold as yearlings an average of 68% less than other yearlings by the same sire. Age matched, clinically normal herdmates also tested positive for L. intracellularis on fecal PCR (6%) and IPMA (33%). Conclusion: L. intracellularis infection should be considered in young horses with ventral edema and hypoalbuminemia that are examined between August and January. Both fecal PCR and serum IPMA are needed to help determine disease status. Treated animals usually survive, although they do not sell for as high a price at public auction as other yearlings by the same sire. Age matched, clinically normal herdmates also test positive for L. intracellularis on fecal PCR and serum IPMA. Key words: Equine proliferative enteropathy; Lawsonia intracellularis; Protein losing enteropathy. Lawsonia intracellularis, the causative agent of equine proliferative enteropathy (EPE), has emerged as an important pathogen in horses over the last decade. The organism was associated with proliferative enteropathy in pigs. 1,2 The cause of proliferative enteropathy in pigs and horses is the same organism based on similarities between the 16S rrna. 1,3 L. intracellularis is typically found in the apical cytoplasm of the intestinal crypt cells resulting in crypt cell expansion and elongation. Hyperplasia of the ileum occurs from decreased MHCII expression leading to depressed immune function as well as the abundance of mitotic cells. Inflammatory cells and goblet cells are reduced or absent. The lack of normal crypt cells and a functional brush border leads to malabsorption. Fecal shedding occurs when infected cells from the epithelium are extruded to the intestinal lumen. 2,4,5 The spread and source of the disease in horses are still largely unknown, although a fecal-oral route is probable. 6,7 Whereas the main source of the disease in pigs is the mixing of chronic carriers with naïve pigs, 8 clinical cases in horses usually occur as isolated cases. 3,7,9 16 Four herd outbreaks are described in Canada 6,17 and personal communication indicates several herd outbreaks have occurred on farms in central Kentucky. From the Hagyard Equine Medical Institute, Lexington, KY. Part of the study was presented at the ACVIM meeting in Seattle, Washington in June 2007 and the 53rd Annual Convention of the AAEP in Orlando, FL in December Corresponding author: Michele L. Frazer, DVM Dipl. ACVIM, 4250 Iron Works Pike Lexington, KY 40511, ; mfrazer@hagyard.com. Submitted March 20, 2008; Revised May 7, 2008; Accepted June 16, Copyright r 2008 by the American College of Veterinary Internal Medicine /j x Possible sources of infection in horses include rodents, birds, insects, soil, 1 or dogs, 16 but the source is unknown. The organism is not believed to transmit directly between species. 18 In recent years, we have observed an increased number of L. intracellularis infections in foals and weanlings. This increased incidence prompted further examination of the affected horses. A retrospective study was performed with the goal of describing several factors in horses suspected to have L. intracellularis infection, including the signalment, month of presentation, presenting clinical signs, clinicopathologic values, diagnostic methods, antimicrobial options, if colloids were administered, survival status, and future sales price. Also, diagnostic results in age matched, clinically normal herdmates of affected horses were described. Materials and Methods The medical records of horses that were treated for L. intracellularis infection between September 2005 and January 2007 at Hagyard Equine Medical Institute in Lexington, Kentucky, were examined. Two criteria were used for inclusion in this study. First, each horse had presumptively been diagnosed with L. intracellularis infection based on physical examination findings and ruling out other diseases that cause enteric disease. Second, each horse with a presumptive diagnosis of EPE also had a positive fecal polymerase chain reaction (PCR), a positive serum immunoperoxidase monolayer assay (IPMA), or both. An IPMA titer 60 was considered positive for L. intracellularis infection (unpublished data). Fiftyseven horses met these criteria. The signalment, month of presentation, presenting clinical signs, blood cell counts, fibrinogen concentration, albumin concentration, hematocrit, antimicrobial choice, whether or not colloids were given, and survival status were evaluated on these horses. In addition, 14 of these 57 horses were sold at public auction as yearlings

2 1244 Frazer and their sales price was compared with the average sales price of all yearlings by the same stallion as the affected cases. One hundred and three age-matched, clinically normal herdmates of affected animals from 6 different farms (Farms A E) were evaluated via physical examination, albumin concentration, fecal PCR, and serum IPMA within 7 days of the index case from that farm being diagnosed with L. intracellularis infection. Herdmates were defined as those horses pastured with a horse or horses that had been diagnosed with L. intracellularis infection. These horses had direct contact with the infected horses and were of a similar age (o6 weeks age difference) as the index cases. Herdmates were considered clinically normal for L. intracellularis if they did not exhibit typical clinical signs (fever, lethargy, ventral edema, colic, or diarrhea) and had albumin concentrations within reference range. A paired t-test was used to evaluate the difference in sales price between affected horses and the average sales price for other yearlings by the same stallion. Descriptive statistics were used for the remainder of the data. Results Signalment Fifty-six horses diagnosed with L. intracellularis infection were Thoroughbreds, and 1 was an Oldenburg. Twenty-six horses were fillies and 31 were colts. Nineteen horses were foals still with the mare and 38 were weanlings. They ranged from 2 to 8 months of age with a median of 6 months. (Table 1) Month of Presentation All affected horses were diagnosed in August (3 of 57 horses), September (8/57), October (10/57), November (15/57), December (15/57), or January (6/57) (Table 1). Clinical Signs Five clinical signs (ventral edema, fever, colic, diarrhea, and lethargy) were evaluated in the affected horses. The most common clinical sign in horses examined was ventral edema (46/57) followed by lethargy (18/57) diarrhea (15/57), fever (11/57), and colic (4/57). All horses presented with at least 2 clinical signs. The severity of the ventral edema, diarrhea, and colic was variable. Horses with ventral edema ranged from mild (42/46) to severe (4/46). Mildly affected horses exhibited only slight swelling in the throatlatch or pectoral area. Two severely affected horses had profound ventral edema leading to rupture of the integument and purulent drainage from the pectoral and scrotal areas. Two horses presented with respiratory distress from a decreased functional airway secondary to severe edema in the throatlatch area. Horses with diarrhea also ranged from mild (7/15) to severe (8/15). Mildly affected horses had no electrolyte abnormalities and did not require IV administration of crystalloid fluids. Severely affected horses had profound dehydration and electrolyte and acid-base derangements, which required IV fluid therapy and acid-base stabilization. Colic was either treated medically (3/4) or surgically (1/4). Horses that were treated medically responded to analgesics, whereas the horse that required surgery had a level of pain that was not responsive to analgesics. Surgical findings were a thickened small intestine consistent with EPE as well as a necrotic cecum secondary to an infarct that required a cecal resection. A biopsy of the small intestine was not obtained at the time of surgery. Clinicopathologic Values CBC and serum biochemistry showed that 42 horses had a fibrinogen concentration within the reference range (range, mg/dl), 11 had an increased fibrinogen (range, mg/dl), and 4 did not have fibrinogen concentration tested. Twenty-seven horses had a white blood cell count within reference range (range, K/ ml), 25 had leukocytosis (range, K/mL), 3 had leucopenia (range, K/mL), and 2 horses did not have a white blood cell count available. Twenty-five horses had a red blood cell count within reference range (range, M/mL) and hematocrit within reference range (range, 33 48%), 30 horses had an increased red blood cell count (range, M/mL) and increased hematocrit (range, 49 59%), and 2 horses did not have red blood cell count and hematocrit available. All 57 horses were hypoalbuminemic (range, mg/dl). Forty-two horses had an albumin concentration o2.0 mg/dl and 21 had a concentration o1.4 mg/dl. Hypoalbuminemia was the only consistent clinicopathologic abnormality of those evaluated. Table 1. Number of EPE horses that presented to HEMI arranged by age of horse in months and month of the year that clinical signs were first observed. Month Horse Was Diagnosed with EPE Age in Months of Horses Diagnosed with EPE August September October November December January November and December were the most common months, with nearly 50% of all horses presenting in one of those 2 months.

3 Lawsonia intracellularis in Horses 1245 Diagnosis All 57 horses in the retrospective group presented with clinical signs suggestive of EPE and were tested for other causes of enteric disease. Salmonella, Clostridium, and rotavirus were ruled out based on fecal samples taken as required per hospital biosecurity protocol. All horses included had at least 2 or 3 negative serial fecal Salmonella cultures and all horses o3 months of age (7 of 57 horses) had a negative rotavirus test. Horses with diarrhea (15/ 57) tested negative for Clostridium difficile toxins A and B via enzyme immunoassay and Clostridium perfringens via fecal culture. All horses had a fecal flotation for parasite eggs, and although 7 horses had Strongyle eggs, no horses had Ascarid eggs. No horses had physical examination findings or a history suggestive of other causes of enteric disease such as NSAID toxicity, sand impaction, gastric ulceration, or Rhodococcus equi infection. Second, each horse with a presumptive diagnosis also had a positive fecal PCR, a positive serum IPMA, or both. Fifty-one horses were tested for L. intracellularis by fecal PCR and 38 of those were positive. Forty-seven horses were tested by serum IPMA and 38 were positive. Twentysix horses had both a positive fecal PCR and a positive serum IPMA. Thirteen horses had a positive serum IPMA, but had a negative fecal PCR. Nine horses had a positive fecal PCR, but had a negative serum IPMA. Treatment Forty-six cases were treated with oxytetracycline a at a dose ranging between 6.5 and 18 mg/kg, q24h, IV. Nine were treated with chloramphenicol b at a dose of 44 mg/ kg, q6h to q8h, PO and 2 were treated with clarithromycin c at a dose of 7.5 mg/kg, q12h, PO. In addition, 16 cases that were treated with oxytetracycline were also treated with metronidazole d at a dose ranging between 10 and 15 mg/kg, q8h to q12h, PO. Colloids were administered to 17 horses. Hetastarch e was given to 13 horses at a dose range between 5 and 10 mg/kg. Thirteen horses received equine plasma at a dose ranging between 2.5 and 10 mg/kg. Nine horses received both plasma and hetastarch. Survival Status Fifty-three horses survived EPE, 3 horses died from secondary complications (severe azotemia and dehydration), and 1 horse was euthanized at owner s request because of a grave prognosis. EPE caused by L. intracellularis infection was confirmed in the 4 nonsurviving horses at postmortem examination by conventional nested, gel-based PCR of small intestinal scrapings. The gross description of the ileum and jejunum in these 4 cases was thickening of the intestinal walls. The histologic description was multifocal mucosal hyperplasia of the small and large intestine in 1 case, multifocal epithelial hyperplasia in the small intestine in 1 case, segmental mucosal ulceration in the small intestine in 1 case, and no histologic description was given in the 4th case. All four of the nonsurvivors had been treated with oxytetracycline. One of those four had also been treated with metronidazole. The 3 horses that died had received both plasma and hetastarch. The horse that was euthanized did not receive either plasma or hetastarch. Future Performance Horses in the retrospective group sold between 7 and 78%, with an average of 68%, of the average price of all yearlings by the same stallion as the affected animal. This was a significant difference in sales price (P value o.05) Prevalence One hundred and three age-matched, clinically normal herdmates of horses affected with EPE were examined by physical examination, albumin concentration, fecal PCR, and serum IPMA. In total, 6 had a positive fecal PCR and 34 had a positive serum IPMA result. Farm A had a herd size of 23 with 20 clinically normal horses and 3 horses affected with EPE. Four of the clinically normal horses had a positive fecal PCR and a negative IPMA, 8 had a positive IPMA and a negative PCR, and 8 were negative to L. intracellularis on both PCR and IPMA. Farm B had a herd size of 15 with 12 clinically normal horses and 3 affected animals. All of the clinically normal horses tested negative on PCR and IPMA. Farm C had a herd size of 22 with 21 clinically normal horses and 1 affected horse. Fourteen of the clinically normal horses had a positive IPMA, but PCR samples were not tested on this farm. Farm D had a herd size of 21 with 20 clinically normal horses and 1 affected animal. Two clinically normal horses had a positive PCR and negative IPMA, 4 had a positive IPMA and negative PCR, and 14 were negative to L. intracellularis on both PCR and IPMA. Farm E had a herd size of 14 with 10 clinically normal horses and 4 affected horses. Four clinically normal horses had a positive IPMA and negative PCR. Six had a negative PCR and IPMA. Farm F had a herd size of 24 with 20 clinically normal horses and 4 affected animals. Four clinically normal horses had a positive IPMA and negative PCR. Sixteen horses were negative on PCR and IPMA. Discussion There are characteristic signalment, month of presentation, clinical signs, and clinicopathologic values in horses with EPE. Also, this study emphasized the importance of testing animals with both PCR and IPMA to determine disease status. The study illustrated that animals usually survive infection after treatment, but affected animals may not bring as high a sale price at public auction as other yearlings by the same stallion. Also, the study indicated that age-matched, clinically normal herdmates can test positive to L. intracellularis with fecal PCR, serum IPMA or both. Age and breed were common factors in signalment between horses in the retrospective group, whereas a sex predilection was not apparent because a similar number of fillies and colts presented. Although all but 1 horse was a Thoroughbred and other literature has reported a herd outbreak in Thoroughbreds, 6 this was considered a re-

4 1246 Frazer flection of the breed population seen at this clinic and not a breed predilection for L. intracellularis infection. An age predilection was apparent because all horses in this retrospective study were foals or weanlings between 2 and 8 months of age. This is similar to previous literature stating that weanlings o7 months of age are most commonly affected. 6,7,9,10 L. intracellularis infection occurs more commonly in young horses because of the decline in maternal antibodies that occurs at several months of age. In addition, several management changes happen to weanlings that could cause stress and predispose them to disease, especially with this concurrent decrease in maternal antibodies. This age group must undergo weaning from the mare, which often results in movement of the animals to a new field or barn with different pasture mates. The farm can also implement new deworming and vaccination protocols as well as condition training. 6 Also, the more developed cell-mediated immunity of adult horses compared with younger animals may provide greater protection against L. intracellularis in adults. However, we still do not have a definitive answer to why this disease is predominately seen in younger animals. All horses in this study presented between August and January, with more than 50% of horses presenting in the months of November and December. Although this could indicate a seasonal component, it might reflect the time of year that horses reach the typical age of 2 8 months in the central Kentucky area. The month of presentation in this retrospective study also corresponds to previous literature that described a herd outbreak of L. intracellularis infection that occurred in December and January. 17 The most common clinical sign in the affected horses was ventral edema. Ventral edema, colic, lethargy, fever, and diarrhea were evaluated in horses because these are typical clinical signs observed with EPE and they were easily identified in the medical records. Other clinical signs reported in the literature such as weight loss and rough hair coat were not evaluated because of the subjective nature. Poor body condition was not evaluated because this information was not sufficiently available in the medical records. The only consistent clinicopathologic abnormality of those evaluated was hypoalbuminemia. Anemia as a cause of the hypoalbuminemia was ruled out because no horses had a decreased hematocrit or red blood cell count. This corresponds to previous literature describing hypoalbuminemia and hypoproteinemia as a consistent finding. 6,9 Antimicrobials described in the literature for treatment of EPE include erythromycin, clarithromycin, azithromycin, chloramphenicol, rifampin, tetracycline, penicillin, enrofloxacin, ampicillin, and metronidazole. 2,6,13 Oxytetracycline, clarithromycin, chloramphenicol, and metronidazole were the antimicrobials used in the cases examined. Antimicrobial choice in this retrospective group was not associated with survival. However, we were unable to evaluate if antimicrobial choice affected duration of treatment or duration before resolution of clinical signs. Also, this retrospective study examined only the initial antimicrobial administered to each horse. Many horses were switched to a different antimicrobial for continued treatment on the farm. For example, horses that were initially given oxytetracycline were often switched to doxycycline after several days of treatment. Also, we could not determine if survival status was related to the use of colloids. Other treatment modalities for EPE, such as crystalloids, sodium bicarbonate, parenteral nutrition, gastroprotectants, antidiarrheals, and nonsteroidal antiinflammatory medications, were not evaluated in this study. Fecal PCR and serum IPMA are the 2 laboratory diagnostic tests that are practical for the antemortem case, but interpreting the results can be difficult. Fecal PCR identifies chromosomal DNA and is an excellent method of determining the presence of the organism. This test is considered very specific, although sensitivity is questionable and false negative results are possible. 1,6,7,13,19 Fecal PCR confirms the presence of the L. intracellularis bacteria and does not necessarily indicate EPE as evidenced by the fact that 6 horses had a positive PCR without any clinical evidence of EPE. Also, PCR testing does not differentiate between viable and nonviable DNA, so these 6 horses could have had a previous infection and still be passing L. intracellularis DNA in the feces. False positive results cannot be ruled out in these 6 animals. Conventional nested gel-based PCR was used as opposed to real-time PCR, so cross contamination during laboratory testing and subsequent false positive results could occur. Thirteen horses that had clinical signs, albumin concentration, and serum IPMA results all suggestive of EPE tested negative for L. intracellularis on fecal PCR. One possible explanation for this negative PCR in horses presumed to have EPE is that these horses may have received antimicrobials on the farm before testing the fecal sample, so horses were not passing DNA in the feces. Three of these 13 horses had received antimicrobials before fecal sampling, but it was unknown in the other 10 horse if they had been treated before referral. Another possible explanation for a negative PCR in a horse affected with EPE is that horses may shed the organism intermittently in the feces, so multiple fecal samples might be required to obtain a positive result. Serum IPMA confirms the presence of antibodies and has been considered a more reliable indicator of active or recent infection. 6,19 Personal communication with Dr Gebhart indicates that an IPMA titer 60 is suggestive of a positive result for L. intracellularis infection. Thirtyfour horses that did not exhibit typical signs of EPE had a positive IPMA result. These horses may have had EPE in the past and still had a high concentration of antibody, although typical clinical signs had not been observed in these animals. Also, in the pig, serum IPMA for L. intracellularis has a sensitivity of 100% and a specificity of only about 90%, 20 so if the sensitivity and specificity are similar in the equine IPMA test, false positives may occur with the laboratory testing. Nine horses that were believed to have EPE had a negative IPMA. These horses may have been in the early stages of EPE and had not seroconverted yet. Serum IPMA results might have been higher if horses had been rechecked several days later.

5 Lawsonia intracellularis in Horses 1247 Also, the number of positive and negative horses may have been influenced by the titer used as the cut-off point. If a titer of 30 instead of 60 had been used for the cut-off point, the number of horses with a positive IPMA would have increased. Interestingly, only 50% of horses with clinical signs of EPE tested positive with both fecal PCR and serum IPMA, which stresses the importance of running both of these diagnostic tests on all suspected horses. If only fecal PCR samples had been submitted, 19% of horses may have been misdiagnosed. If only serum IPMA titers had been submitted, 25% of horses may have been misdiagnosed. This retrospective study indicated that clinically normal horses test positive for L. intracellularis, but it could not confirm or refute a correlation between number of affected animals in a herd and number of clinically normal horses that tested positive from that herd. Farm C had only 1 horse affected with EPE, but had 14 clinically normal horses test positive with IPMA, which suggests no correlation. However, Farm A had the highest number of positive PCR results in clinically normal horses and also had 3 horses affected with EPE. This would suggest that herds with higher numbers of horses affected with EPE may have higher numbers of clinically normal horses testing positive. Testing with PCR and IPMA on more farms is needed to determine if a link exists between the number of clinically normal horses testing positive and the number of horses affected with EPE in a herd. The biggest problem with performing this retrospective study was case definition. Laboratory confirmation of L. intracellularis requires a silver stain such as a Warthin Starry stain, 1,20 or PCR of a small intestinal biopsy or scraping; however, intestinal samples are rarely available in the antemortem horse. Although intestinal samples may be obtained with a duodenal biopsy via endoscopy or a rectal biopsy, these tissue samples do not typically harbor the organism. Ileal or jejunal samples obtained at postmortem examination or during exploratory laparotomy are typically the only methods to obtain affected intestinal tissue. Thus, fecal PCR and serum IPMA are the 2 diagnostic tests available in the antemortem horse, which makes establishing a definitive diagnosis a challenge. Neither test is definitive for infection as evidenced in this study by clinically normal horses testing positive and affected horses testing negative for L. intracellularis. Another recommended diagnostic procedure to aid in identifying horses with EPE is visualization of thickened small intestinal walls with abdominal ultrasound. In this study, abdominal ultrasonographic results were not evaluated because they had only been recorded in detail in 25 of the 57 medical records examined. Interestingly, 7 of those 25 horses had small intestinal wall thickness within the reference range. Thus, although abdominal ultrasound can be a very useful diagnostic tool, a normal ultrasound should not rule out EPE. In conclusion, L. intracellularis should be a differential diagnoses for young horses with hypoalbuminemia that present in the fall or early winter. Based on this retrospective study of 57 horses, ventral edema was the most common clinical sign and hypoalbuminemia was present in all affected horses, whereas blood cell counts, hematocrit, and fibrinogen concentration were inconsistent. When testing for L. intracellularis infection, both fecal PCR and serum IPMA should be evaluated. Observation of horses for typical clinical signs, abdominal ultrasound for thickened intestinal wall, presence of hypoalbuminemia, and ruling out other differential diagnoses for enteric disease allows early initiation of treatment and aids in interpreting fecal PCR and serum IPMA results. Treatment should be initiated with an appropriate antimicrobial and possibly synthetic colloids, plasma transfusions, or both. Horses in this retrospective study responded well to treatment based on survival rate. However, affected horses sold for significantly less at public auction than other yearlings by the same sire as the affected horse. Many aspects of L. intracellularis infection in horses are still unknown. Research continues today in horses to provide a better antemortem diagnosis as well as to determine the source of the organism and the prevalence in horse populations. Footnotes a Liquamycin LA 200, Pfizer Animal Health, Exton, PA b Chloramphenicol Oral Paste (300 mg/ml), Hagyard Pharmacy, Lexington, KY c Clarithromycin Tablets UPS, Rabaxy Pharmaceuticals Inc, Jacksonville, FL d Metronidazole Tablets, USP, Pliva Inc, East Hanover, NJ e Hetastarch, Hospira Inc, Lake Forest, IL References 1. Cooper DM, Gebhart CJ. Comparative aspects of proliferative enteritis. 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