Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis.

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a - Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1-40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

Subsistence swine farming: seroprevalence and risk factors associated with Lawsonia intracellularis infection in the state of Minas Gerais Brazil in 2016.

Although it is considered an economically relevant and prevalent disease, little information is available on the epidemiology and risk factors of porcine proliferative enteropathy (PPE) in commercial pigs, and no publication is available on subsistence pig farming. The objectives of this study were to estimate the seroprevalence of L. intracellularis and identify associated risk factors in backyard pigs in the 12 mesoregions of the state of Minas Gerais, Brazil. Blood from pigs between 2 months and 6 years of age were sampled; an epidemiological questionnaire was applied to 288 properties investigated in 2016. Serum samples were tested for the presence of anti-L. intracellularis antibodies using an immunoperoxidase monolayer assay. The seroprevalence of L. intracellularis was 97.7% (CI 95%: 96.7-98.4), and there was no statistical difference among the prevalence of the sampled mesoregions. Only 3 of the 12 risk factors were significant when samples were analyzed from strongly seropositive animals (≥ 1:120) in a Poisson multivariate regression model. There was an interaction between properties in peri-urban areas and extensive production systems. This interaction demonstrated an increase in prevalence rates by 3.7 times (95%CI: 2.4-5.8). Properties close to dumps demonstrated an increase in prevalence rates by 2.2 times (95%CI: 0.99-4.8). In conclusion, anti-L. intracellularis antibodies were widely dispersed in subsistence pig farming's in Minas Gerais, indicating a wide circulation of the agent in this type of production system. The interactions of animals raised close to peri-urban areas, extensively, and close to landfills are risk factors for spread of PPE.

Lawsonia intracellularis LI0666 is a new EPIYA effector exported by the Yersinia enterocolitica type III secretion system.

Lawsonia intracellularis is the causative agent of proliferative enteropathy. While it harbors genes encoding the entire apparatus required for the type III secretion system (T3SS) and the expression of some of these components has been detected during experimental infection, the identification of L. intracellularis T3SS substrates (effector proteins) has been hampered. The Yersinia T3SS and yeast growth inhibition assays are two important heterologous systems used for the characterization of effector proteins. Bacterial EPIYA effectors are a distinct class of bacterial effectors defined by the presence of EPIYA or the EPIYA-related motif. When delivered into host cells via a T3SS or type IV secretion system, these effectors undergo tyrosine phosphorylation of the EPIYA motif, which enables them to manipulate host cell signaling by promiscuously interacting with multiple SH2 domain-containing proteins. A previous study showed that L. intracellularis LI0666 contains two EPIYA motifs and speculated that this protein could be a T3SS effector. In this study, we show that LI0666 is secreted by Yersinia in a T3SS-dependent manner and inhibits yeast growth. LI0666 is phosphorylated at tyrosine residues in porcine intestinal epithelial cells and in human epithelial cells. Like the archetypal EPIYA effector CagA, the EPIYA-containing region is not required for LI0666 association with yeast and mammalian cell membranes. Our results indicate that LI0666 is an authentic bacterial EPIYA effector. Identification of the tyrosine kinases that are responsible for LI0666 phosphorylation and the SH2 domain-containing host proteins that LI0666 interacts with will help to explore the molecular mechanisms of LI0666 in disease development.

Lawsonia intracellularis infected enterocytes lack sucrase-isomaltase which contributes to reduced pig digestive capacity.

Lawsonia intracellularis is endemic to swine herds worldwide, however much is still unknown regarding its impact on intestinal function. Thus, this study aimed to characterize the impact of L. intracellularis on digestive function, and how vaccination mitigates these impacts. Thirty-six L. intracellularis negative barrows were assigned to treatment groups (n = 12/trt): (1) nonvaccinated, L. intracellularis negative (NC); (2) nonvaccinated, L intracellularis challenged (PC); and (3) L. intracellularis challenged, vaccinated (Enterisol® Ileitis, Boehringer Ingelheim) 7 weeks pre-challenge (VAC). On days post-inoculation (dpi) 0 PC and VAC pigs were inoculated with L. intracellularis. From dpi 19-21 fecal samples were collected for apparent total tract digestibility (ATTD) and at dpi 21, pigs were euthanized for sample collection. Post-inoculation, ADG was reduced in PC pigs compared with NC (41%, P < 0.001) and VAC (25%, P < 0.001) pigs. Ileal gross lesion severity was greater in PC pigs compared with NC (P = 0.003) and VAC (P = 0.018) pigs. Dry matter, organic matter, nitrogen, and energy ATTD were reduced in PC pigs compared with NC pigs (P ≤ 0.001 for all). RNAscope in situ hybridization revealed abolition of sucrase-isomaltase transcript in the ileum of PC pigs compared with NC and VAC pigs (P < 0.01). Conversely, abundance of stem cell signaling markers Wnt3, Hes1, and p27Kip1 were increased in PC pigs compared with NC pigs (P ≤ 0.085). Taken together, these data demonstrate that reduced digestibility during L. intracellularis challenge is partially driven by abolition of digestive machinery in lesioned tissue. Further, vaccination mitigated several of these effects, likely from lower bacterial burden and reduced disease severity.

PREVALENCE OF LAWSONIA INTRACELLULARIS INFECTION IN NONHUMAN PRIMATES AND PEST RODENTS IN A ZOOLOGICAL COLLECTION.

In 2016 and 2017, Lawsonia intracellularis was isolated from several pileated gibbons (Hylobates pileatus) presenting with diarrhea in Mulhouse Zoo (eastern France). To this day, infection with this bacterium has rarely been described in nonhuman primates (NHP) in captivity or in the wild and there are no data about the prevalence or transmission of the disease. This study focuses on finding the prevalence of this infection amongst Mulhouse Zoo's NHP collection and trying to identify a source of contamination responsible for this epizooty. Forty-eight real-time PCR were conducted on feces from all NHP species in the zoo and on small mammals trapped in the NHP housing structures. No NHP was experiencing symptoms at the time of the study, however test results showed that Lawsonia intracellularis can be found in 61.76% (21/34) of the group total (n = 34) and the prevalence even increases to 92.3% (12/13) in the Lemuriform infraorder (n = 13). In small mammals (n = 14), prevalence of the bacterium is 57.17% (8/14) including 77.78% in rodents (7/9). The results of this study show that several NHP species are healthy carriers and some species of small mammals can be considered as a potential source of contamination. Because of the difficulty encountered trying to isolate the bacterium, it is plausible that infections caused by Lawsonia intracellularis have been underdiagnosed to this day, and that it could be an emerging disease in Europe. Therefore, using real-time PCR to search for this bacterium seems essential in case of diarrhea occurring in nonhuman primates. Moreover, even though further studies on contamination sources need to be conducted, the issue of the presence of rodents in NHP housing structures has to be taken very seriously and tackled with the utmost care.

The dose regimen formulation of tilmicosin against Lawsonia intracellularis in pigs by pharmacokinetic-pharmacodynamic (PK-PD) model.

In this study, the rational dose regimens of tilmicosin against Lawsonia intracellularis (L. intracellularis) were studied using pharmacokinetic-pharmacodynamic (PK-PD) model approach to provide a maximal efficacy. The healthy and infected pigs were orally administrated the tilmicosin premix at a single dose of 10 mg/kg, and then the plasma and ileum content were collected at different time points. The time to peak (Tmax), the peak concentration (Cmax), the area under concentration time curve (AUC0-24h), the apparent volume of distribution by bioavailability (V/F), the body clearance rate by bioavailability (CL/F) and the mean residence time (MRT) of tilmicosin premix for plasma were 2.00 h, 1.08 ± 0.04 µg/mL, 9.61 ± 1.47 µg h/mL, 34.43 ± 1.02 L/kg, 0.71 ± 0.03 L/h/kg and 15.03 ± 0.04 h in healthy pigs, and 2.00 h, 0.99 ± 0.03 µg/mL, 9.30 ± 1.43 µg h/mL, 58.59 ± 1.81 L/kg, 0.44 ± 0.02 L/h/kg and 15.75 ± 0.03 h in infected pigs, respectively. The Tmax, Cmax, AUC0-24h, V/F, CL/F and MRT of tilmicosin premix for ileum content were 2.00 h, 3.78 ± 0.03 µg/mL, 20.41 ± 1.64 µg h/mL, 11.29 ± 0.97 L/kg, 0.44 ± 0.02 L/h/kg and 11.29 ± 0.09 h in healthy pigs, and 2.00 h, 3.41 ± 0.06 µg/mL, 22.65 ± 1.32 µg h/mL, 8.16 ± 1.51 L/kg, 0.41 ± 0.01 L/h/kg and 11.44 ± 0.05 h in infected pigs, respectively. Based on the intracellular minimum inhibitory concentration (MIC) of L. intracellularis isolate was 2 µg/mL, the results of the mutant prevention concentration (MPC), the post-antibiotic effect (PAE) and time-killing curves all showed strong concentration-dependenttendencies. Integrating the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data using the sigmoid Emax (Hill) equation to obtain the ileum content AUC0-24h/MIC values of 6.87, 26.80, and 36.02 h to achieve the bacteriostatic activity, bactericidal activity, and virtual eradication of bacteria, respectively. Based on these results, a dosage regimen of daily 14.39 mg/kg for 3 d could be sufficient in the treatment of L. intracellularis. This study will provide a guidance of dosage regimen formulation for drug against animal intracellular bacterial infections.

Isolation and in vitro antimicrobial susceptibility of porcine Lawsonia intracellularis from Brazil and Thailand.

BACKGROUND: Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control (antimicrobial-free). RESULTS: Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group (valnemulin and tiamulin) and carbadox were the most active against L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions. CONCLUSIONS: This is the first report of isolation of L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.

Effect of tetracycline treatment regimens on antibiotic resistance gene selection over time in nursery pigs.

BACKGROUND: The majority of antimicrobials given during the production of pigs are given to nursery pigs. The influence of antimicrobial use on the levels of antimicrobial resistant (AMR) genes is important to quantify to be able to assess the impact of resistance on the food chain and risk to human and animal health. RESULTS: This study investigated the response on the levels of nine AMR genes to five different treatment strategies with oxytetracycline, and the dynamics of gene abundance over time by following 1167 pigs from five different farms in Denmark. The results showed no significant difference between treatments and an increase in abundance for the efflux pump encoding tet(A) gene and the genes encoding the ribosomal protection proteins tet(O) and tet(W) tetracycline resistant genes following treatment, while tet(M) showed no response to treatment. However, it was also observed that the levels of tet(O), tet(W), and ermB in some farms would drift more over time compared to a single treatment-course with antibiotic. CONCLUSION: This study underlines the large variation in AMR levels under natural conditions and the need for increased investigation of the complex interactions of antimicrobial treatment and other environmental and managerial practices in swine production on AMR gene abundance.

Evaluation of mouse enteroids as a model for Lawsonia intracellularis infection.

Lawsonia intracellularis, an obligate intracellular bacterium, is an important enteric pathogen in pig herds and horse farms worldwide. The hallmark feature of L. intracellularis infection is the proliferation of epithelial cells in intestinal crypts. A major limitation to the study of L. intracellularis infection is the lack of an in vitro model that reproduces the changes observed in proliferative enteropathy. Here we investigated the suitability of mouse enteroids as a model to study L. intracellularis infection. Mouse enteroids were microinjected with L. intracellularis, filter-sterilized L. intracellularis culture supernatant, or sterile cell culture media (DMEM). L. intracellularis antigen was detected in mouse enteroids by immunohistochemistry and was located mostly in the basal region of the epithelium. There was no differential growth of enteroids among treatment groups, and cellular proliferation was not increased in L. intracellularis-infected enteroids in relation to non-infected enteroids based on immunofluorescence staining. L. intracellularis infection did not induce changes in gene expression of Ki-67 (proliferation marker), Sox9 (marker for transit amplifying cells) and Muc2 (marker for goblet cells). These results indicate that although L. intracellularis antigen is detectable in mouse enteroids, indicating susceptibility to infection, mouse enteroids fail to replicate the cellular proliferation and gene expression changes observed in proliferative enteropathy. Nevertheless, we have successfully demonstrated that mouse enteroids can be used to model days-long intracellular pathogen infection, serving as potential models for the study of other pathogens of interest in veterinary medicine.

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions.

The causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10-20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10-65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.

Rapid and visual detection of Lawsonia intracellularis with an improved recombinase polymerase amplification assay combined with a lateral flow dipstick.

BACKGROUND: Lawsonia intracellularis (L. intracellularis) is the etiologic agent of porcine proliferative enteropathy (PPE), which is reported in many swine breeding countries all over the world, and has caused enormous economic losses in intensive pig production systems. Therefore, the aim of this study was to develop a simple and rapid method for on-site detection of Lawsonia intracellularis (L. intracellularis). As the isothermal recombinase polymerase amplification (RPA) can be performed at a constant temperature and its product is directly observed on a lateral-flow dipstick (LFD) with naked eyes without electrophoresis, the RPA-LFD assay should be useful for field diagnosis of L. intracellularis as well as its detection from clinical samples. RESULTS: The established RPA-LFD assay could be finished in 30 min at a wide temperature range of 25 to 40 °C, and the amplicons could be visualized by naked eyes. The developed RPA-LFD assay was specific to dnaA gene of L. intracellularis, and did not detect nucleic acids extracted from other common gastrointestinal pathogens. The minimum detection of this RPA-LFD method was 400 L. intracellularis per reaction, which was as sensitive as conventional PCR. Further, the RPA-LFD assay was performed with 150 clinical fecal samples and the detection results were compared with conventional PCR. Results showed that the coincidence rate of RPA-LFD and conventional PCR was 100%. CONCLUSIONS: The combined RPA with LFD assay provides a simple, rapid, specific and sensitive alternative for field diagnosis of L. intracellularis infection.

Antigenic and functional profiles of a Lawsonia intracellularis protein that shows a flagellin-like trait and its immuno-stimulatory assessment.

The obligate intracellular Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), is an economically important disease in the swine industry. Due to extreme difficulty of in vitro culture of the pathogen, molecular characterization of protein components of LI that are targets of the immune system, is difficult; thus, the scientific evidence to drive the development of preventive measures is lacking. In this work, we investigated the antigenic and functional characteristics of a putative flagellar-associated protein, LI0570, using in silico computational approaches for epitope prediction and an in vitro protein-based molecular assay. The amino acid sequence of LI0570 exhibited similarities to flagellar-associated proteins in four different bacterial strains. The presence of B cell linear confirmative epitopes of the protein predicted by a bioinformatics tool was validated by western blot analysis using anti-LI mouse hyperimmune serum, which implied that LI0570 induced production of antigen-specific antibodies in vivo. Further, TLR5-stimulating activity and IL-8 cytokine expression produced via downstream signaling were observed in HEK-Blue™-hTLR5 cells stimulated with LI0570. This result indicates that the LI0570 protein can trigger an innate immune response followed by a T-cell-related adaptive immune response in an infected host. Collectively, the data presented here support that the LI0570 protein which shows the antigenic potential could be a useful component of a recombinant vaccine against PE, providing progress toward an effective prevention strategy.

The effects of zinc amino acid complex supplementation on the porcine host response to Lawsonia intracellularis infection.

Lawsonia intracellularis is among the most important enteric pathogens of swine and antibiotic alternatives are needed to help mitigate the negative effects of infection. Zinc is an essential trace mineral known to be crucial for maintaining intestinal barrier function and proper immune response. In this study, we investigated the porcine host response to L. intracellularis infection when supplemented with a zinc-amino acid complex, a form of zinc that can lead to greater bioavailability when compared to traditional inorganic forms of zinc. Our results show that a zinc-amino acid complex supplementation with a final concentration of 125 ppm of zinc in feed significantly (p < 0.05) decreased the number of animals with lesions and severity of lesions caused by L. intracellularis. Animals supplemented with the zinc-amino acid complex also exhibited a significantly (p < 0.05) earlier onset of seroconversion as well as an increased number of T cells in infected and non-infected intestinal tissue. This study demonstrated that this zinc-amino acid complex aids the host in responding to L. intracellularis infection and may be a new approach to help minimize negative effects of disease.

Potent O-antigen-deficient (rough) mutants of Salmonella Typhimurium secreting Lawsonia intracellularis antigens enhance immunogenicity and provide single-immunization protection against proliferative enteropathy and salmonellosis in a murine model.

The obligate intracellular pathogen Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), poses a substantial economic loss in the swine industry worldwide. In this study, we genetically engineered an O-antigen-deficient (rough) Salmonella strain secreting four selected immunogenic LI antigens, namely OptA, OptB, LfliC, and Lhly. The genes encoding these antigens were individually inserted in the expression vector plasmid pJHL65, and the resultant plasmids were transformed into the ∆asd ∆lon ∆cpxR ∆rfaL Salmonella Typhimurium (ST) strain JOL1800. The individual expression of the selected LI antigens in JOL1800 was validated by an immunoblotting assay. We observed significant (P < 0.05) induction of systemic IgG and mucosal IgA responses against each LI antigen or Salmonella outer membrane protein in mice immunized once orally with a mixture of four JOL1800-derived strains. Further, mRNA of IL-4 and IFN-γ were highly upregulated in splenic T cells re-stimulated in vitro with individual purified antigens. Subsequently, immunized mice showed significant protection against challenge with 106.9 TCID50 LI or 2 × 109 CFU of a virulent ST strain. At day 8 post-challenge, no mice in the immunized groups showed the presence of LI-specific genomic DNA (gDNA) in stool samples, while 50% of non-immunized mice were positive for LI-specific gDNA. Further, all the immunized mice survived the virulent ST challenge, compared to a 20% mortality rate observed in the control mice. Collectively, the constructed rough ST-based LI vaccine candidate efficiently elicited LI and ST-specific humoral and cell-mediated immunity and conferred proper dual protection against PE and salmonellosis.

Nutrient digestibility, organ morphometry and performance in vaccinated or non-vaccinated Lawsonia intracellularis infected piglets.

BACKGROUND: Lawsonia intracellularis is one of the world's most important infectious diseases in pork production with regard to economic losses. So far, studies are missing that describe the effects of a natural infection of piglets on the digestibility of nutrients, possible effects on performance and the morphometrics of the intestine depending on whether piglets are vaccinated, clinically healthy or clinically affected with regard to Lawsonia intracellularis induced diarrhoea. RESULTS: Digestibility studies were performed on a total of 27 eight-week-old piglets with naturally occurring Lawsonia intracellularis infection in a trial with three repetitions. Nine out of 27 animals were vaccinated as suckling pigs with a commercial Lawsonia intracellularis vaccine (vac; Enterisol®Ileitis). Half of the remaining 18 animals were without clinical signs of infection (non-vac/cs-), half showed moderate clinical signs of Lawsonia intracellularis induced diarrhoea (non-vac/cs+). All three groups were fed one identical complete diet ad libitum. Faecal shedding of Lawsonia intracellularis was found in all groups (25 out of 27 animals). Numerically, the mean excretion in the group non-vac/cs + (7.69 ± 1.65 log10 copies/ g faeces) was higher in comparison to the group non-vac/cs- (5.83 ± 2.35 log10 copies/ g faeces) and vaccinated animals (vac: 6.00 ± 2.89log10 copies/ g faeces). The average daily weight gain (ADG; Ø 8.66 day period) differed significantly (vac: 894a ± 73.3, non-vac/cs-: 857ab ± 86.3, non-vac/cs+: 785b ± 137 g/day). The apparent total tract digestibility (ATTD) of nitrogen was significantly lower in clinically affected animals (vac: 83.0a ± 1.72, non-vac/cs-: 83.9a ± 2.03, non-vac/cs+: 80.7b ± 2.57).The total length of the small intestine in clinically affected animals increased significantly (vac: 15.9ab ± 1.57, non-vac/cs-: 14.6b ± 1.12, non-vac/cs+: 16.2a ± 1.37 m). The relative body weight depending on the length of the small intestine was lower for clinically affected animals (vac: 1.72a ± 0.21, non-vac/cs-: 1.83a ± 0.17, non-vac/cs+: 1.56b ± 0.12 kg/m). CONCLUSION: These studies show that clinically moderate L. intracellularis infections lead to significantly lower ADGs in comparison to vaccinated animals. The disease is also found in altered intestinal morphometry and reduced total N digestibility if clinical signs occur.

Ex vivo penetration of fosfomycin into healthy and Lawsonia intracellularis-colonized swine intestinal mucosa.

Fosfomycin (FOS) is an antibiotic used, mostly in Latin America, for the treatment of lung and enteric infections of pigs. Intracellular fluids of enterocytes can act as biophase for Lawsonia intracellularis, the causative agent of porcine proliferative enteropathy (PPE). The aim of this study was to determine whether the presence of L. intracellularis in the enterocytes modifies FOS penetration. Eight healthy pigs in growth-finishing stage were used to produce healthy (group A) and L. intracellularis-colonized (group B) intestinal explants. For both groups, treatment consisted of a 580 µg/ml concentration of calcium FOS, which was added to each explant (0.5-6 hr). For group B, the Enterisol Ileitis® vaccine was used as source of the micro-organism. Previously to the assay, the time necessary for L. intracellularis to colonize the enterocytes was defined. Also, a PCR protocol was optimized to determine the presence of the pathogen in the explants. There were nonstatistical differences for the penetration of the antibiotic into healthy and L. intracellularis-colonized enterocytes. MIC90 of FOS for L. intracellularis is unknown; nevertheless, MIC90 of various antibiotics ranges between 0.125 and 128 µg/ml. FOS reaches inside the enterocyte concentrations which surpass the MICs90 of other antibiotics that also act by the inhibition of cell wall synthesis; however, further studies should be carried out to determine fosfomycin MIC90 for L. intracellularis to discern the usefulness of this antibiotic in the treatment of PPE.

Dietary approaches reducing boar taint-Importance of Lawsonia intracellularis colonisation for interpreting results.

In the fattening of male pigs, boar odour is a major problem with regard to the acceptance of the meat by consumers. Skatole can be one cause. Tryptophan from non-digested feed ingredients and intestinal cell debris can be the precursor in skatole formation. Lawsonia intracellularis, one of the most widespread pathogens in swine, promotes the epithelial cell turnover and might favour the tryptophan influx into the hindgut. Therefore, the question arises how far the severity of a Lawsonia intracellularis infection has an effect on results of dietary experiments with specific issues. Fifty finishing boars from a specific pathogen-free farm were randomly allotted to ten boxes in five feeding groups. Natural developing Lawsonia intracellularis colonisation was monitored serologically (twice individually) and molecular biologically (weekly individually). Over 4 weeks, animals were fed either a finely ground pelleted diet (FP), a coarsely ground meal diet (CM), a meal diet either with 22% cracked corn (CORN), 16.9% dried whey (WHEY) or 30% raw potato starch (RPS). Fifty % of animals showing lower differences in serological Lawsonia intracellularis values between the start and the end of the trial were characterised by a higher dry matter content in faeces (256 ± 29.4 vs. 239 ± 23.6 g/kg). Lawsonia intracellularis-negative caecal samples showed the highest butyrate concentrations (27.2 ± 7.53 mmol/kg). Lawsonia intracellularis-negative faecal samples of group FP showed the highest DM levels in faeces (neg: 290 ± 46.1/pos: 250 ± 52.2 g/kg); negative samples from group RPS had the lowest values (217 ± 24.4 g/kg). Lawsonia intracellularis-negative faecal samples from the group CM were lower in skatole than positive samples (82.8 ± 32.8 vs. 119 ± 29.3 µg/g DM). RPS group samples without pathogen detection had the lowest skatole concentrations (30.5 ± 36.3 µg/g DM). This study provides first evidence that clinically unremarkable colonisation with intestinal pathogens might influence the results of dietary approaches.

Effect of Tetracycline Dose and Treatment Mode on Selection of Resistant Coliform Bacteria in Nursery Pigs.

This study describes the results of a randomized clinical trial investigating the effect of oxytetracycline treatment dose and mode of administration on the selection of antibiotic-resistant coliform bacteria in fecal samples from nursery pigs. Nursery pigs (pigs of 4 to 7 weeks of age) in five pig herds were treated with oxytetracycline for Lawsonia intracellularis-induced diarrhea. Each group was randomly allocated to one of five treatment groups: oral flock treatment with a (i) high (20 mg/kg of body weight), (ii) medium (10 mg/kg), or (iii) low (5 mg/kg) dose, (iv) oral pen-wise (small-group) treatment (10 mg/kg), and (v) individual intramuscular injection treatment (10 mg/kg). All groups were treated once a day for 5 days. In all groups, treatment caused a rise in the numbers and proportions of tetracycline-resistant coliform bacteria right after treatment, followed by a significant drop by the time that the pigs left the nursery unit. The counts and proportions of tetracycline-resistant coliforms did not vary significantly between treatment groups, except immediately after treatment, when the highest treatment dose resulted in the highest number of resistant coliforms. A control group treated with tiamulin did not show significant changes in the numbers or proportions of tetracycline-resistant coliforms. Selection for tetracycline-resistant coliforms was significantly correlated to selection for ampicillin- and sulfonamide-resistant strains but not to selection for cefotaxime-resistant strains. In conclusion, the difference in the dose of oxytetracycline and the way in which the drug was applied did not cause significantly different levels of selection of tetracycline-resistant coliform bacteria under the conditions tested.IMPORTANCE Antimicrobial resistance is a global threat to human health. Treatment of livestock with antimicrobials has a direct impact on this problem, and there is a need to improve the ways that we use antimicrobials in livestock production. We hypothesized that antibiotic resistance development following treatment of diarrhea in nursery pigs could be reduced either by lowering the dose of oxytetracycline or by replacing the commonly used practice of flock treatment with individual or small-group treatments, since this would reduce the number of pigs treated. However, the study showed no significant difference between treatment groups with respect to the number or proportion of tetracycline-resistant coliforms selected. The most important conclusion is that under practical field conditions, there will be no added value, in terms of lowering resistance development, by exchanging flock treatment for individual or small-group treatment of nursery pigs. The reason for the lack of an effect of single-animal treatment is probably that such animals share the environment with treated animals and take up resistant bacteria from the environment.

Maternal short-chain fructo-oligosaccharide supplementation increases intestinal cytokine secretion, goblet cell number, butyrate concentration and Lawsonia intracellularis humoral vaccine response in weaned pigs.

Prebiotic supplementation modulates immune system development and function. However, less is known about the effects of maternal prebiotic consumption on offspring intestinal defences and immune system responsiveness. We investigated the effects of maternal short-chain fructo-oligosaccharide (scFOS) supplementation on mucin-secreting cells, ileal secretory IgA and cytokine secretion of weaned offspring and their humoral response to an oral vaccine against obligate intracellular Lawsonia intracellularis. Sows were fed a control diet (CTRL) or scFOS-supplemented diet during the last third of gestation and throughout lactation. At weaning, each litter was divided into two groups receiving a post-weaning CTRL or scFOS diet for a month. Pigs from the four groups were either non-vaccinated (n 16) or vaccinated (n 117) at day 33. Biomarkers related to intestinal defences and immune parameters were analysed 3 weeks later. SCFA production was assessed over time in suckling and weaned pigs. Maternal scFOS supplementation improved ileal cytokine secretions (interferon (IFN)-γ, P<0·05; IL-4, P=0·07) and tended to increase caecal goblet cell number (P=0·06). It increased IgA vaccine response in the serum (P<0·01) and ileal mucosa (P=0·08). Higher bacterial fermentative activity was observed during lactation (total faecal SCFA, P<0·001) and after weaning (colonic butyrate, P=0·10) in pigs from scFOS-supplemented mothers. No synergistic effect between maternal and post-weaning scFOS supplementation was observed. Therefore, maternal scFOS supplementation has long-lasting consequences by strengthening gut defences and immune response to a vaccine against an intestinal obligate intracellular pathogen. Prebiotic consumption by gestating and lactating mothers is decisive in modulating offspring intestinal immunity.

Lawsonia intracellularis in the feces of wild rodents and stray cats captured around equine farms.

BACKGROUND: Proliferative enteropathy is a global enteric disease of particular importance in pigs. The causative bacterium, Lawsonia intracellularis, has a wide range of susceptible host species. Recently, L. intracellularis has been recognized as an etiologic agent of an emerging enteric disease in foals called equine proliferative enteropathy (EPE). The presence of L. intracellularis in nonruminant wildlife has raised questions regarding the role of these species in EPE transmission. RESULTS: This study investigated exposure to L. intracellularis in wild rodents and feral cats from eight farms with confirmed EPE. Serum (42) and fecal (40) samples from resident foals and fecal samples (131), intestinal mucosa tissues (14), and mesenteric lymph nodes (14) from wild and feral animals were collected for the evaluation of the farm status and the molecular detection of L. intracellularis following the diagnosis of EPE in index cases. Fresh feces from wild rodents and feral cats were collected from the ground while walking the premises or after trapping the animals using live traps. A total of 3 brown rats, 7 house mice, 1 striped field mouse, 2 grey red-backed voles, and 3 feral cats showed evidence of prior exposure to L. intracellularis. CONCLUSIONS: Our data add to increasing evidence demonstrating the potential for L. intracellularis transmission and infection in wild rodents and feral cats and provide possible evidence of interspecies transmission. The exposure of wild rodents and feral cats provides potential evidence for the spillover of L. intracellularis to wildlife species and raises the question of spillback to horses. Additionally, these animals may represent an indicator of environmental exposure or may be actively involved in the transmission of L. intracellularis to foals by acting as potential reservoir/amplifier hosts. This study is the first to demonstrate the magnitude of L. intracellularis shedding in the feces of wild rodents and feral cats and to indicate the significant infection risk that wild rodents and feral cats pose to naïve horses in South Korea.