Connie J. Gebhart

Characterization of Lawsonia intracellularis gen. nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy.

A novel obligately intracellular bacterium, ileal symbiont intracellularis, which was obtained from the intestines of pigs with proliferative enteropathy disease, was grown in pure cocultures with tissue cultures of rat cells. An examination of the 16S ribosomal DNA gene sequence revealed that the isolates which we obtained are members of the delta subdivision of the Proteobacteria and that the sequences of these organisms exhibit a level of similarly of 91% with the sequence of Desulfovibrio desulfuricans ATCC 27774. These isolates were homogeneous and differed in cellular morphology, acid fastness, phenotype, electrophoretic protein profile, and habitat from Desulfovibrio species. On the basis of the results of an integrated study of the phenotype and genotype of a consistent morphological entity found in particular porcine cells and associated with a well-defined clinical condition, we concluded that these bacteria belong to a previously undescribed genus and species, for which we propose the name Lawsonia intracellularis gen. nov., sp. nov. A species-specific recombinant DNA probe was cloned previously, and this probe was used to identify the bacterium in tissue culture cells and in the ileal epithelia of pigs with proliferative enteropathy disease. Coculture of the organism with a rat enterocyte cell line allowed us to designate strain NCTC 12656 the type strain and to describe the new genus and species. The organism which we cultured is pathogenic for pigs and causes proliferative enteropathy lesions in their ilea and colons, and Kochs postulates were fulfilled for this organism.

Equine proliferative enteropathy: a cause of weight loss, colic, diarrhoea and hypoproteinaemia in foals on three breeding farms in Canada

Proliferative enteropathy (PE) is a transmissible enteric disease caused by Lawsonia intracellularis. An outbreak of equine PE was diagnosed in foals from 3 breeding farms. Most foals had been weaned prior to the appearance of clinical signs, which included depression, rapid and marked weight loss, subcutaneous oedema, diarrhoea and colic. Poor body condition with a rough haircoat and a potbellied appearance were common findings in affected foals. Respiratory tract infection, dermatitis and intestinal parasitism were also found in some foals. Haematological and plasma biochemical abnormalities included hypoproteinaemia, transient leucocytosis, anaemia and increased serum creatinine kinase concentration. Postmortem diagnosis of PE was confirmed on 4 foals based on the presence of characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa, using silver stains, and by results of PCR analysis and immunohistochemistry. Antemortem diagnosis of equine PE was based on the clinical signs, hypoproteinaemia and the exclusion of common enteric infections. Faecal PCR analysis was positive for the presence of L. intracellularis in 6 of 18 foals tested while the serum of all 7 foals with PE serologically evaluated had antibodies against L. intracellularis. Most foals were treated with erythromycin estolate alone or combined with rifampin for a minimum of 21 days. Additional symptomatic treatments were administered when indicated. All but one foal treated with erythromycin survived the infection. This study indicates that equine PE should be included in the differential diagnosis of outbreaks of rapid weight loss, diarrhoea, colic and hypoproteinaemia in weanling foals.

Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis

Little is known about the humoral and, especially, cell-mediated immune response in pigs exposed to Lawsonia intracellularis. The objectives of this study were to investigate the onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or a commercial live vaccine strain of L. intracellularis. Twenty-four 5-week-old pigs were exposed to 4.4x10(9) organisms of a pathogenic L. intracellularis isolate PHE/MN1-00 (10 pigs), a L. intracellularis live attenuated vaccine strain (10 pigs) or sham inoculum (4 pigs). Fecal, serum and whole blood samples were collected from all animals before exposure and weekly up to 13 weeks post inoculation and tested by PCR, immunoperoxidase monolayer assay serology and an interferon-gamma assay, respectively. One animal from each group was euthanized on day 22 post exposure to confirm infection. Humoral and cell-mediated immune responses were initially detected 2 weeks after exposure in pigs challenged with the pathogenic isolate, and 5 and 4 weeks, respectively, in pigs exposed to the modified-live vaccine group. Humoral and cell-mediated immune responses were still detected in some pigs from both L. intracellularis exposed groups 13 weeks after exposure. Fecal shedding was initially detected 1 week and lasted, intermittently, 12 weeks post exposure in pigs challenged with the pathogenic isolate, while fecal shedding was first detected 2 weeks and lasted, also intermittently, 9 weeks after exposure to the vaccine. In summary, both pathogenic isolate challenged and vaccine exposed pigs demonstrated long-term shedding of and immune responses to L. intracellularis.

Diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a Lawsonia intracellularis-specific multiplex PCR assay.

Proliferative enteritis (PE) is an enteric disease that has been reported in a variety of animals. It is caused by an obligate intracellular bacterium identified in swine as Lawsonia intracellularis. The organism can be detected ante-mortem in swine with PE using molecular diagnostic methods. The disease can be diagnosed post-mortem in all species by gross examination of tissues and special histologic staining procedures. In this study we extracted total DNA from frozen or formalin-fixed, paraffin-embedded tissues from cases of pig, hamster, horse, deer and ostrich PE. The samples were subjected to a multiplex PCR reaction using primers specific for a swine isolate of L. intracellularis. Identical sized PCR products were detected in samples from all animals with PE and the specificity of the PCR reaction for L. intracellularis was demonstrated by Southern-blotting and hybridization using specific probes. These results suggest that the intracellular organism of PE in these species are all very closely related to the causative agent of PE in swine, L. intracellularis. In addition, this multiplex PCR assay can be used to detect the organism in frozen or archival tissues, facilitating retrospective diagnosis of PE.

Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species

A new genus and species of obligate intracellular-bacteria found in porcine intestines are described. Growth on any bacteriological medium deprived of living cells has not been demonstrated. The organism has been grown intracellularly in cell culture. The 16S rRNA gene sequence data, DNA probe results, and microscopic observations provide evidence that these bacteria differ from those in other described genera and that they belong to the delta subdivision of the class Proteobacteria. We have amplified and sequenced the 16S ribosomal DNA of four preparations of the intracellular bacterium from pigs. For this, intracellular organisms were released and purified from the infected cells without culture techniques. After DNA purification, the polymerase chain reaction with primers complementary to highly conserved eubacterial sequences was used to amplify regions of 16S ribosomal DNA which were subsequently cloned (in some cases) and sequenced directly by standard techniques. The sequences obtained from each preparation were identical and were most similar to that of a sulfate-reducing proteobacterium, Desulfovibrio desulfuricans ATCC 27774 (91% similarity). An oligonucleotide probe complementary to a hypervariable region of the 16S rRNA sequence of the bacterium hybridized with intracellular organisms obtained from porcine intestines. The bacterium is a gram-negative, curved rod with tapered ends. It multiplies intracellularly in the cytoplasm of ileal epithelial cells by septation. The vernacular name Ileal symbiont (IS) intracellularis is proposed for this bacterium. The type strain of IS intracellularis is strain 1482/89 grown in cell culture from a pig affected by proliferative enteropathy. It is deposited in the National Collection of Type Cultures, Colindale, London, as NCTC 12656.

Validation of an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy

The sensitivity and specificity of an immunoperoxidase monolayer assay (IPMA) was evaluated in a blind serologic study of a group of disease-free pigs and a group of pigs experimentally infected with intestinal homogenate containing Lawsonia intracellulars organisms. Sixty pigs from the control group were kept in the source farm, and another 60 animals were transferred to an isolation unit and challenged intragastrically. All animals were bled before and 21 days after challenge. Fecal samples were collected on the same dates. The IPMA results were tested for sensitivity and specificity in a 2 × 2 table using the challenged and nonchallenged status as gold standard. Sensitivity and specificity were evaluated for different cutoff points (serum dilutions). Specificities of 100% were obtained for all the serum dilutions tested (1:15, 1:30, 1:60, and 1:120). The sensitivity levels were 90.7%, 88.9%, 81.5%, and 75.9% for the serum dilutions 1:15, 1:30, 1:60, and 1:120, respectively. The sensitivity of the dilution 1:15 was slightly, but not significantly, higher than the dilution currently used as the cutoff point (1:30). Cross-reactivity of the IPMA test was evaluated using sera from pigs experimentally inoculated with Brachyspira pilosicoli and various Campylobacter species. All these samples were negative. Sera samples from 3 porcine proliferative enteropathy known negative populations, 40 growing pigs from 2 commercial farms and a group of 6 cesarean-derived and colostrum-deprived pigs, also tested negative by IPMA. The IPMA serologic test with the cutoff point of 1:30 showed specificity of 100% and sensitivity close to 90% and, therefore, is an appropriate diagnostic test for herd screening but not for diagnosing PPE on the individual level.

Proliferative enteropathy in a foal caused by Lawsonia intracellularis-like bacterium

Proliferative enteropathy (proliferative enteritis, proliferative ileitis, intestinal adenomatosis) has been reported in several animal species including the pig,1 dog, foal, blue fox, guinea pig, ferret, hamster, and rabbit. The disease is characterized by adenomatous hyperplasia of crypt epithelial cells in the ileum and colon with intracytoplasmic curved bacteria resembling Campylobacter species. In the single case previously reported in a foal, Campylobacter-like organisms were demonstrated within the cytoplasm of enterocytes by spirochete stains and electron microscopy. In a study using cloned DNA probes to isolated Campylobacterlike organisms, there was failure of the probes to hybridize with common porcine Campylobacter species, suggesting the causative agent to be an unidentified or uncultured species. This finding, along with DNA sequencing and structural characteristics, resulted in this organism being named ileal symbiont intracellularis. Subsequently, the organism was described and classified as a new genus and species, Lawsonia intracellularis. 1 An unweaned 5-month-old mixed-breed female foal with a history of anorexia, lethargy, and profuse watery diarrhea of greater than 1-week duration was presented to the University of Kentucky, Livestock Disease Diagnostic Center, for necropsy. Treatment had not been attempted. At necropsy, the foal was thin with readily apparent skeletal muscle atrophy. Gross lesions were confined to the small intestine. There was irregular thickening of the jejunum and ileum with the ileum being more severely affected. Lesions in the midjejunum were multifocal in nature and consisted of areas of discoid mucosal thickening, whereas the distal jejunum and ileum contained diffuse mucosal thickening resulting in a rugose pattern (Fig. 1). Samples of the small intestine, stomach, cecum, colon, brain, heart, lung, kidney, and spleen were placed in 10% neutral buffered formalin and, following fixation, were embedded in paraffin, sectioned at 5 μm, and stained with hematoxalin and eosin. In addition, selected sections of small intestine were stained by the Warthin-Starry silver impregnation method for bacteria. Pieces of formalinfixed small intestine were postfixed in osmium tetroxide and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate. Histopathologically, the affected mucosa was thickened and consisted of hyperplastic glandular structures lined by immature epithelial cells (Fig. 2). The hyperplastic epithelium resulted in distortion of the normal villous structure. Some

Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated "Brachyspira hampsonii".

Since 2007, outbreaks of severe bloody diarrhea and hemorrhagic colitis have been reported in the United States and Canada. Though the primary causative agent of swine dysentery is Brachyspira hyodysenteriae, which is strongly hemolytic, the current report describes the isolation of a novel strongly hemolytic Brachyspira sp. This novel Brachyspira sp. was identified from clinical submissions at the Minnesota Veterinary Diagnostic Laboratory, and 40 of such isolates were obtained from 22 clinical submissions representing 5 states. Isolates were confirmed to be different from any known Brachyspira sp. on the basis of phylogenetic analysis of nucleotide sequences of nox and 16S ribosomal RNA (rRNA) genes. Phylogenetic analyses grouped all isolates into 2 clades (clades I and II), and grouping patterns were similar for both nox and 16S rRNA gene sequence analyses. Phenotypically, all isolates were indole and hippurate negative, and enzymatic profiling indicated 2 types of profiles, irrespective of the phylogenetic grouping, differing only in the production of β-glucosidase. The results suggest that a potentially virulent new species of Brachyspira sp., provisionally named “Brachyspira hampsonii ”, is circulating among swine herds in the United States.

Detection of Lawsonia intracellularis by Real-time PCR in the Feces of Free-living Animals from Equine Farms with Documented Occurrence of Equine Proliferative Enteropathy

The objective of this study was to determine whether Lawsonia intracellularis was present in the feces of free-living animals collected on two equine premises with documented occurrence of equine proliferative enteropathy (EPE). Fresh feces from black-tailed jackrabbits (Lepus californicus, n=100), striped skunks (Mephitis mephitis, n=22), feral cats (Felis catus, n=14), Brewers Blackbirds (Euphagus cyanocephalus, n=10), Virginian opossums (Didelphis virginiana, n=9), raccoons (Procyon lotor, n=4), California ground squirrels (Spermophilus beecheyi, n=3), and coyotes (Canis latrans, n=2) were collected from August 2006 to January 2007 either from the ground while walking the premises or after trapping the animals using live traps. Nucleic acid purified from feces was directly processed for polymerase chain reaction (PCR) analysis using a real-time PCR assay targeting the aspartate ammonia lyase gene of L. intracellularis. Purified DNA samples were also precipitated, preamplified for L. intracellularis, and analyzed using the same real-time PCR assay, to increase the detection limit to one L. intracellularis organism per extracted sample. Feces from jackrabbits, striped skunks, Virginian opossums, and coyotes tested PCR positive for L. intracellularis, whereas all feces from feral cats, Brewers Blackbirds, raccoons, and ground squirrels tested PCR negative for L. intracellularis. PCR testing on DNA extracted directly from feces was positive for L. intracellularis in six of 164 fecal samples. When DNA purification from feces was followed by a precipitation and preamplification step, five additional fecal samples tested PCR positive for L. intracellularis (11/164). The largest number of PCR positive L. intracellularis fecal samples was observed in striped skunks, followed by Virginian opossums, jackrabbits, and coyotes. This is the first description of L. intracellularis in these four species. Because the fecal samples were collected at equine farms with confirmed cases of EPE, striped skunks, Virginian opossums, jackrabbits, and coyotes may act as potential sources of infection to susceptible weanlings.

Polymerase chain reaction for diagnosis of porcine proliferative enteropathy

A polymerase chain reaction (PCR) assay for detection of the intracellular bacteria, ileal symbiont intracellularis of porcine proliferative enteropathy is described. The test is based on specific DNA primers and gave positive PCR product from samples of preserved intestinal mucosa and faeces from affected pigs. Mucosa and faeces from normal pigs gave no positive PCR products. The identity of the PCR product was confirmed by DNA-DNA hybridization with a probe, pCLO78, specific for IS intracellularis. Positive results were only observed in animals with active lesions of proliferative enteropathy. PCR is probably the most useful method for diagnosis of proliferative enteropathy that is currently available for live animals.