Loss of expression of the glutamate dehydrogenase (gdh) of Streptococcus suis serotype 2 compromises growth and pathogenicity.

Streptococcus suis serotype 2 is a zoonotic agent that causes substantial economic losses to the swine industry and threatens human public health. Factors that contribute to its ability to cause disease are not yet fully understood. Glutamate dehydrogenase (GDH) is an enzyme found in living cells and plays vital roles in cellular metabolism. It has also been shown to affect pathogenic potential of certain bacteria. In this study, we constructed a S. suis serotype 2 GDH mutant (Δgdh) by insertional inactivation mediated by a homologous recombination event and confirmed loss of expression of GDH in the mutant by immunoblot and enzyme activity staining assays. Compared with the wild type (WT) strain, Δgdh displayed a different phenotype. It exhibited impaired growth in all conditions evaluated (solid and broth media, increased temperature, varying pH, and salinity) and formed cells of reduced size. Using a swine infection model, pigs inoculated with the WT strain exhibited fever, specific signs of disease, and lesions, and the strain could be re-isolated from the brain, lung, joint fluid, and blood samples collected from the infected pigs. Pigs inoculated with the Δgdh strain did not exhibit any clinical signs of disease nor histologic lesions, and the strain could not be re-isolated from any of the tissues nor body fluid sampled. The Δgdh also showed a decreased level of survival in pig blood. Taken together, these results suggest that the gdh is important in S. suis physiology and its ability to colonize, disseminate, and cause disease.

Meningoencephalitis, Vasculitis, and Abortions Caused by Chlamydia pecorum in a Herd of Cattle.

A cow dairy (n = 2000) in close proximity to a sheep flock had third-trimester abortions and fatalities in cows and calves over a 14-month period. Eighteen of 33 aborted fetuses (55%) had multifocal random suppurative or mononuclear meningoencephalitis with vasculitis. Seventeen of these affected fetuses had intracytoplasmic bacteria in endothelial cells, and 1 fetus with pericarditis had similar bacteria within mesothelial cells or macrophages. Immunohistochemistry for Chlamydia spp. or polymerase chain reaction (PCR) for Chlamydia pecorum or both, performed on brain or pooled tissue, were positive in all 14 tested fetuses that had meningoencephalitis and in 4/4 calves and in 3/4 tested cows that had meningoencephalitis and thrombotic vasculitis. In 1 calf and 11/11 fetuses, C. pecorum PCR amplicon sequences were 100% homologous to published C. pecorum sequences. Enzootic chlamydiosis due to C. pecorum was the identified cause of the late term abortions and the vasculitis and meningoencephalitis in fetuses, calves, and cows. C. pecorum, an uncommon bovine abortogenic agent, is a differential diagnosis in late-term aborted fetuses with meningoencephalitis, vasculitis, and polyserositis.

Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.

Functional characterization of glucosamine-6-phosphate synthase (GlmS) in Salmonella enterica serovar Enteritidis.

Salmonella is a threat to public health due to consumption of contaminated food. Screening of a transposon library identified a unique mutant that was growth and host cell binding deficient. The objective of this study was to determine the functional role of glucosamine-6-phosphate synthase (GlmS) in the biology and pathogenesis of Salmonella. To examine this, we created a glmS mutant (ΔglmS) of Salmonella and examined the effect on cell envelope integrity, growth, metabolism, and pathogenesis. Our data indicated ΔglmS was defective in growth unless media were supplemented with D-glucosamine (D-GlcN). Examination of the bacterial cell envelope revealed that ΔglmS was highly sensitive to detergents, hydrophobic antibiotics, and bile salts compared to the wild type (WT). A release assay indicated that ΔglmS secreted higher amounts of ß-lactamase than the WT in culture supernatant fractions. Furthermore, ΔglmS was attenuated in cell culture models of Salmonella infection. Taken together, this study determined an important role for GlmS in the pathogenesis and biology of Salmonella.

Evaluation of Candida spp. and Other Fungi in Feces from Dogs with Naturally Occurring Diabetes Mellitus.

Diabetes mellitus is a common endocrinopathy in dogs and in most cases is analogous to type 1 diabetes mellitus (T1DM) in humans. Candida spp. is a common commensal fungi with higher prevalence and magnitude of growth in humans with T1DM. There is currently no published information about the fungal microbiome in diabetic dogs. Therefore, the objectives of this study were to (i) determine whether diabetic dogs were more likely to have Candida spp. or other types of fungi from feces compared to non-diabetic controls, and (ii) identify variables associated with fungi colonization. Fourteen diabetic dogs and 14 age, sex, and breed matched non-diabetic healthy control dogs were included in this prospective case-control study. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for fungal identification. Diabetic dogs had greater quantitative fungal growth compared to controls (p = 0.004). Moreover, female dogs were more likely to have fungi colonization than males (p = 0.02). All instances of Candida spp. and Aspergillus spp. colonization were exclusively identified in diabetic dogs. Serum fructosamine concentration was higher in diabetic dogs with fecal colonization of Candida spp. compared to diabetic dogs without growth (p = 0.03). Our results indicate that the fungal microbiome in feces is altered in diabetic dogs, which seem to favor an increased prevalence of Candida spp. and higher quantitative fungal growth. Moreover, female sex and glycemic control could affect the intestinal mycobiome.

Comparison of three methods for extraction of Mycobacterium avium subspecies paratuberculosis DNA for polymerase chain reaction from broth-based culture systems.

Conventional and real-time polymerase chain reaction (PCR) assays were used to measure the recovery of DNA from Mycobacterium avium subspecies paratuberculosis (MAP) extracted with 3 different methods (MagMAX, DNeasy(R), and phenol-chloroform) after growth in a broth-based culture system. Of the 304 samples tested, bacterial DNA was detected in 197 (65%) of samples after MagMAX, 156 (51%) after phenol-chloroform, and 123 (40%) after DNeasy extractions. By acid-fast stain, 177 (58%) of the samples yielded acid-fast-positive bacilli, of which 4 were PCR negative by the 3 extraction methods. The results demonstrated that the amplifiable MAP DNA, as evidenced by the number of PCR-positive cultures and amplicon intensity on ethidium bromide-stained agarose gel, was best for MagMAX, intermediate for phenol-chloroform, and least for DNeasy. When subjected to real-time polymerase chain reaction, the MagMAX extracts produced the best results, thereby making it an excellent kit for the efficient extraction of MAP DNA from the broth-based culture system.

Growth rate retardation and inhibitory effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis.

The effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis (MAP) inoculated into broth-based culture media was evaluated by using 84 fecal samples with known MAP status. Results showed that growth of the organism in samples inoculated into the broth without the para-JEM BLUE was detectable 1-35 days (average of 6 days) earlier in 35 of the samples (42%) compared with the same samples inoculated in broth with para-JEM BLUE. Four additional samples (5%) that were MAP positive in the culture broth that lacked the para-JEM BLUE gave negative results when the reagent was included. Of the remaining 45 samples, growth of MAP was detected 1-4 days (average of 3 days) earlier in 4 of the samples (5%) inoculated in the broth with para-JEM BLUE compared with the same samples inoculated in the broth without the para-JEM BLUE, whereas 41 samples (49%) yielded equivalent results with respect to time-to-growth detection and negative growth, regardless of whether para-JEM BLUE was present in the culture broth. However, exclusion of para-JEM BLUE from the broth increased the number of samples that produced false-positive instrument signals compared with the number that produced false-positive signals when the reagent was added. Modification of the sample processing step had no measurable effect. Observations indicated that, although elimination of para-JEM BLUE from the broth increased false-positive instrument signals, its inclusion has an adverse effect on the growth of certain MAP, which suggests that its elimination from broth cultures may increase sensitivity.

Draft Genome Sequence of a Streptococcus suis Isolate from a Case of Cattle Meningitis.

Streptococcus suis is primarily a pig pathogen and a zoonotic agent. Recently, the isolation of S. suis strain 10-36905 from a case of meningitis in cattle was reported. The draft genome sequence of this isolate demonstrates its divergent relationship with other S. suis strains.

Coccidioides posadasii in a Dog With Cervical Dissemination Complicated by Esophageal Fistula.

A 5-year-old male, neutered mixed breed dog with a history of a mass with an associated draining tract on the ventral cervical region was diagnosed with an esophageal fistula. The dog exhibited serosanguinous discharge from the draining tract, with enlarged left superficial cervical and mandibular lymph nodes, and was reported to have difficulty with deglutition of solid foods. Computed tomography revealed a communication of the draining tract with the esophagus along with enlargement of the left lateral retropharyngeal, left medial retropharyngeal, and mandibular lymph nodes. This prompted surgical exploration and debridement of the site, with closure of the esophageal fistula. Histopathology of thyroid gland, skeletal muscle, and adipose tissue obtained during surgical exploration showed spherules consistent with Coccidioides spp. infection. Antibody titers performed post-operatively were consistent with an active Coccidioides spp. Infection. By fungal culture and subsequent PCR and DNA sequencing, C. posadasii was identified as the species infecting the dog. Over the course of 85 days of antifungal therapy, discharge from the draining tract, lymphadenomegaly, and cutaneous and subcutaneous nodules resolved. In conclusion, this is the first reported case of disseminated coccidioidomycosis to the cervical region of a dog with involvement of the thyroid gland, skeletal muscle, adipose tissue, connective tissue, and secondary esophageal fistula. Coccidioides spp. infections should be considered a differential diagnosis in unusual cases for dogs that live in or have traveled to endemic areas.

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Preadministration of Fermented Sorghum Diet Provides Protection against Hyperglycemia-Induced Oxidative Stress and Suppressed Glucose Utilization in Alloxan-Induced Diabetic Rats.

Sorghum bicolor grains are rich in phytochemicals known to considerably impact human health. Several health-promoting products such as flour, staple food, and beverages have been produced from sorghum grains. This study investigated the protective and modulatory effects of a sorghum diet on the genes of some antioxidant and glycolytic enzymes in alloxan-induced diabetic rats. The rats were randomly distributed into six groups: the control group received normal diet, while the other groups were pretreated with 12.5, 25, 50, 75, and 100% of the sorghum diets daily for 8 weeks before the administration of a dose of alloxan (100 mg/kg BW), after which blood was collected and the liver was excised. The effects of the diets on blood glucose levels, liver dysfunction indices, and markers of oxidative stress were assessed spectrophotometrically, while the gene expressions of key glycolytic enzymes and enzymatic antioxidants were assayed using reverse transcriptase polymerase chain reaction. It was observed that the pretreatment of the experimental animals with the diets normalized the blood glucose before and after the administration of alloxan. The sorghum-treated groups also showed statistically significant (p < 0.05) decrease in liver dysfunction indices and markers of oxidative damage compared with the control. In addition, statistically the diets significantly decreased (p < 0.05) the relative expression of superoxide dismutase, glutathione peroxidase, glucokinase, phosphofructokinase, and hexokinase genes in the experimental animals compared with the control. Overall, this study showed that the preadministration of fermented sorghum diet significantly protected against hyperglycemia and suppressed glucose utilization via glycolysis in the liver of alloxan-induced diabetic rats. Thus, the consumption of sorghum diet may protect against hyperglycemia and oxidative damage and may therefore serve as functional food for management of diabetic mellitus.

Investigation of the spread of Brucella canis via the U.S. interstate dog trade.

OBJECTIVES: The aim of this study was to illustrate and help address a growing need for regulatory or molecular tools to track and control the spread of canine brucellosis. Our study objectives were to first characterize Brucella canis outbreaks in Wisconsin kennels in the context of the dog trade in the USA, and then to identify a molecular technique that may be useful for strain differentiation of B. canis isolates. METHODS: Wisconsin Veterinary Diagnostic Laboratory (WVDL) B. canis serology data from 1995 to 2005 were reviewed, three canine brucellosis outbreaks in Wisconsin dog kennels were investigated, and eight B. canis isolates recovered from Wisconsin outbreaks and kennels in Missouri and Arkansas and four isolates received from outside sources were subjected to ribotyping, pulsed-field gel electrophoresis (PFGE), outer membrane protein analysis (OMPA), and cellular fatty acid profiling (CFAP). RESULTS: WVDL has received increasing numbers of B. canis positive samples from Wisconsin kennels, and Wisconsin outbreaks are associated with the interstate dog trade. All of the B. canis isolates we examined were genetically homogenous and as such could not be differentiated by ribotyping, PFGE and OMPA. However, dendrogram analysis of CFAP divided the isolates into two groups, indicating that CFAP methyl ester analysis has discriminatory power. CONCLUSIONS: CFAP methyl ester analysis has promise as a tool for epidemiological tracing of B. canis outbreaks and will be useful in comparison studies as isolation of B. canis continues to expand globally.

Isolation and partial characterization of Streptococcus suis from clinical cases in cattle.

Sixteen isolates of gram-positive, coccoid bacteria were obtained from clinical cases of diverse conditions in cattle and identified as Streptococcus suis using 16S ribosomal DNA gene sequencing and other bacterial identification methods. None of the isolates could be assigned to any of the known S. suis capsular types. Virulence-associated gene profiling that targeted muramidase-released protein, extracellular protein factor, suilysin, 89-kb pathogenicity island, and arginine deiminase ( arcA) genes were negative except for 1 isolate that was arcA positive. The arcA-positive isolate caused severe widespread lesions, including multiorgan suppurative and hemorrhagic inflammation in the meninges, lung, liver, spleen, lymph nodes, and serosae of heart and intestines. The other isolates were primarily associated with meningitis, bronchopneumonia, and multifocal acute necrotizing hepatitis. The isolates differed from each other by 4-6 fragments when examined by pulsed-field gel electrophoresis, indicating they are possibly related. The isolates were susceptible to ampicillin, penicillin, and tiamulin. Resistance was noted to sulfadimethoxine (93%), oxytetracycline (86%), chlortetracycline (86%), neomycin (67%), tilmicosin (47%), clindamycin (47%), enrofloxacin (33%), gentamicin (13%), florfenicol (7%), trimethoprim-sulfamethoxazole (7%), and spectinomycin (53%). Multi-drug resistance (defined as resistance to at least 1 agent in 3 or more antimicrobial classes) was detected in 67% of the isolates. The pathology observations provide evidence that S. suis may be an important pathogen of bovine calves. S. suis is an agent that clinical bacteriology laboratories should consider when dealing with cases involving cattle.

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases.

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase.

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.

Deletion of gene encoding methyltransferase (gidB) confers high-level antimicrobial resistance in Salmonella.

The glucose-inhibited division gene (gid)B, which resides in the gid operon, was thought to have a role in the modulation of genes similar to that of gidA. Recent studies have indicated that GidB is a methyltransferase enzyme that is involved in the methylation of the 16S ribosomal RNA (rRNA) in Escherichia coli. In this study, we investigated the role of GidB in susceptibility to antibiotics and the overall biology of Salmonella. A gidB isogenic mutant of Salmonella was constructed and subsequently characterized under different conditions. Our data indicated that growth and invasion characteristics of the gidB mutant were similar to those of the wild type (WT). The gidB mutant was outgrown by the WT in a competitive growth assay, indicating a compromised overall bacterial fitness. Under the stress of nalidixic acid, the gidB mutant's motility was significantly reduced. Similarly, the mutant showed a filamentous morphology and smaller colony size compared with the rod-shaped and large colonies of the WT in the presence of nalidixic acid. Most importantly, deletion of gidB conferred high-level resistance to the aminoglycoside antibiotics streptomycin and neomycin. A primer extension assay determined the methylation site for the WT to be at G527 of the 16S rRNA. A lack of methylation in the mutant indicated that GidB is required for this methylation. Taken together, these data indicate that the GidB enzyme has a significant role in the alteration of antibiotic susceptibility and the modulation of growth and morphology under stress conditions in Salmonella.

Ulcerative dermatitis in C57BL/6 mice lacking stearoyl CoA desaturase 1.

Ulcerative dermatitis (UD) is a common cause of morbidity and euthanasia in mice with a C57BL/6 (B6) background. The purposes of the current study were to determine whether UD lesions could be reliably produced in B6 mice lacking stearoyl-CoA desaturase 1 (SCD1(-/-) mice), to ascertain whether the UD lesions in SCD1(-/-) mice were similar to those found in other B6 mice, and to characterize the cell invasion phenotype of Staphlococcus xylosus cultured from the lesions. S. xylosus isolates from the environment and human skin were used as controls. SCD1(-/-) (n = 8 per group) and nontransgenic B6 control mice (n = 22 mice pooled from 3 groups that received different concentrations of conjugated linoleic acid) were fed standard rodent chow or a semipurified diet (NIH AIN76A) for 4 wk. Samples from other B6 mice with UD (field cases; n = 7) also were submitted for histology and culture. All of the SCD1(-/-) mice developed UD lesions by 4 wk on NIH AIN76A. None of SCD1(-/-) fed standard rodent chow and none of the wildtype B6 mice fed NIH AIN76A developed UD. Supplementation with conjugated linoleic acid did not affect ulcerogenesis. UD lesions in SCD1(-/-) mice and field cases were grossly and histologically similar. S. xylosus was isolated from SCD1(-/-) mice with UD (71%) and field cases of UD (43%). These isolates were the most cell-invasive, followed by the environmental isolate, and finally the human skin isolate. Our results provide a basis for further pathologic and clinical study of UD.

The pathogenicity island-like DNA segment associated with Chinese outbreak strain of Streptococcus suis serotype 2 is absent in the United States isolates.

To determine if the 2005 Chinese outbreak strain of Streptococcus suis is circulating in the United States, three different PCR primer-pairs derived from the nucleotide sequences surrounding and internal to the unique pathogenicity island -like DNA segment of the Chinese outbreak strain (strain 05ZYH33) were used to screen 290 swine isolates of S. suis obtained from different locations. The first primer pair amplified an approximately 1000-bp fragment from 47 (16%) of the United States isolates and the second amplified an 1800-bp fragment from 23 (8%) of the isolates. Nucleotide sequences of the amplicons shared identity with those of strain 05ZYH33. The third primer pair amplified a 716-bp amplicon from the DNA of strain 05ZYH33 only. These observations demonstrated that the PAI homologue of strain 05ZYH33 is absent in the United States isolates tested and suggested that the PCR method may be useful for active surveillance to monitor possible spread of the highly invasive strain.

Identification of the gene encoding a 38-kilodalton immunogenic and protective antigen of Streptococcus suis.

In our continued effort to search for a Streptococcus suis protein(s) that can serve as a vaccine candidate or a diagnostic reagent, we constructed and screened a gene library with a polyclonal antibody raised against the whole-cell protein of S. suis type 2. A clone that reacted with the antibody was identified and characterized. Analysis revealed that the gene encoding the protein is localized within a 2.0-kbp EcoRI DNA fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 445 amino acid residues with a calculated molecular mass of 46.4 kDa. By in vitro protein synthesis and Western blot experiments, the protein exhibited an electrophoretic mobility of approximately 38 kDa. At the amino acid level the deduced primary sequence shared homology with sequences of unknown function from Streptococcus pneumoniae (89%), Streptococcus mutans (86%), Lactococcus lactis (80%), Listeria monocytogenes (74%), and Clostridium perfringens (64%). Except for strains of serotypes 20, 26, 32, and 33, Southern hybridization analysis revealed the presence of the gene in strains of other S. suis serotypes and demonstrated restriction fragment length differences caused by a point mutation in the EcoRI recognition sequence. We confirmed expression of the 38-kDa protein in the hybridization-positive isolates using specific antiserum against the purified protein. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic and may serve as an antigen of diagnostic importance for the detection of most S. suis infections. Pigs immunized with the recombinant 38-kDa protein mounted antibody responses to the protein and were completely protected against challenge with a strain of a homologous serotype, the wild-type virulent strain of S. suis type 2, suggesting that it may be a good candidate for the development of a vaccine that can be used as protection against S. suis infection. Analysis of the cellular fractions of the bacterium by Western blotting revealed that the protein was present in the surface and cell wall extracts. The functional role of the protein with respect to pathogenesis and whether antibodies against the antigen confer protective immunity against diseases caused by strains of other pathogenic S. suis capsular types remains to be determined.