Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Evidence of host adaptation in Lawsonia intracellularis infections

Background

Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate.

Materials and methods

Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (10⁹L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response.

Results

Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate.

Conclusions

Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.     

Species-specificity of equine and porcine Lawsonia intracellularis isolates in laboratory animals

Lawsonia intracellularis infection causes proliferative enteropathy (PE) in many mammalian species, with porcine and equine proliferative enteropathy (PPE and EPE) known worldwide. Hamsters are a well-published animal model for PPE infection studies in pigs. There is no laboratory animal model for EPE infection studies and it is not known whether there is species-specificity for equine or porcine isolates of L. intracellularis in animal models. The objective of this study was to determine whether it is possible to generate typical EPE lesions in hamsters after inoculation with an equine strain of L. intracellularis (EPE strain) and whether it is comparatively possible to generate PPE lesions in rabbits after inoculation with a porcine strain of L. intracellularis (PPE strain). In 2 separate trials, 4-week-old and 3-week-old weanling golden Syrian hamsters were challenged with EPE strains and compared to uninfected (both trials) and PPE-infected controls (Trial 2 only). Concurrently, 6 female New Zealand white juvenile rabbits were infected with PPE strain and observed concomitantly to 8 similar rabbits infected with EPE strain for a different experiment. Hamsters and rabbits were observed for 21 to 24 days post-infection (DPI), depending on the experiment. Neither infected species developed clinical signs. The presence of disease was assessed with diagnostic techniques classically used for pigs and horses: immune-peroxidase monolayer assay on sera; quantitative polymerase chain reaction (qPCR) detection of molecular DNA in feces; and hematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) on intestinal tissues. Our results showed that EPE-challenged hamsters do not develop infection when compared with PPE controls (IHC, P = 0.009; qPCR, P = 0.0003). Conversely, PPE-challenged rabbits do not develop typical intestinal lesions in comparison to EPE-challenged rabbits, with serological response at 14 DPI being significantly lower (P = 0.0023). In conclusion, PPE and EPE strains appear to have different host-specificities for hamsters and rabbits, respectively.     

Serologic follow-up of a repopulated swine herd after an outbreak of proliferative hemorrhagic enteropathy

Lawsonia intracellularis is an obligate intracellular organism that causes porcine proliferative enteropathy, a widespread infectious disease. Very little is known about the immune response and the epidemiologic features of the disease in the field. The aims of this study were to evaluate the duration and titers of antibody specific for L. intracellularis in gilts after an outbreak of proliferative hemorrhagic enteropathy (PHE), to evaluate maternal antibodies in piglets, and to evaluate seroconversion and fecal shedding in growing–finishing pigs. Thirty-six gilts in a herd that had recently experienced an outbreak of PHE, including 13 that had recovered, were bled 3 wk after the beginning of the outbreak and then every 3 wk until they became seronegative in 2 consecutive tests. Fourteen piglets from 5 gilts seropositive at farrowing and 5 piglets from 2 sows that remained seronegative were bled once or twice at the farrowing house and then every 3 wk until they reached market age. Fecal samples from these pigs were tested by polymerase chain reaction at 7 wk of age and then on the days of blood collection. After the PHE outbreak, the gilts had high serum antibody levels; the levels decreased over time, but antibody was still detectable for up to 3 mo in some animals. Four piglets from sows that were seropositive at farrowing had detectable passive antibodies up to 5 wk of age. Some nursery pigs started shedding L. intracellularis around 7 wk of age; peak shedding was observed between 13 and 16 wk. Antibody was not detected until 16 wk of age and was more often detected between 19 and 22 wk.     

Diagnosis of Helicobacter spp. infection in canine stomach

A total of 75 biopsied samples of cardia, fundus, body, and pyloric antrum from necropsied dogs that were submitted to the Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University from April 2003 to June 2004 were investigated. The objectives of this study were to determine the prevalence of Helicobacter spp. in canine stomach by polymerase chain reaction (PCR) in comparison to histochemistry versus immunohistochemistry (IHC), and to correlate these diagnostic methods with the clinical significance in infected dogs. Histopathological results revealed 60.0% (45/75) of samples to be positive, and consisted of mild gastritis in 64.44% (29/45), moderate gastritis in 11.11% (5/45), and severe gastritis in 24.44% (11/45). The proportion showing no histopathological lesions was 40.0% (30/75). Helicobacter spp. were localized to the luminal crypt in 18.67% (14/75), gastric pit in 22.67% (17/75), gastric gland in 21.33% (16/75), and gastric epithelium in 8% (6/75). The percentages of positive samples of Helicobacter spp. diagnosed by hematoxylin and eosin stain (H&E), Warthin Starry stain (WSS), IHC with rabbit polyclonal anti-H. pylori antibody, and PCR were 17.3% (13/75), 46.7% (35/75), 30.7% (23/75), and 10.7% (8/75), respectively. No significant differences weree observed in histopathological changes in portions of the stomach (p>0.05). The diagnosis of Helicobacter spp. by PCR in comparison to that by WSS and IHC was not significantly different (p>0.05). There were no relationships between pathological studies using H&E, WSS, and IHC, and especially between PCR and clinical signs of Helicobacter spp. infections in canine stomachs (p>0.05). The present study revealed significantly different levels of correlation for Helicobacter spp. detection between H&E and WSS (p<0.001). Results indicate that the method of choice for diagnosis of Helicobacter spp. infection in canine stomach is dependent on the purpose of study and appropriate specimen collection.     

Association between average daily gain,faecal dry matter content and concentration of Lawsonia intracellularis in faeces

Background

The objective of this study was to investigate the association between average daily gain and the number of Lawsonia intracellularis bacteria in faeces of growing pigs with different levels of diarrhoea.

Methods

A longitudinal field study (n = 150 pigs) was performed in a Danish herd from day 29 to 47 post weaning. Every third day all pigs were weighed, subjected to a clinical examination and faecal samples were obtained. Faecal samples were subjected to dry matter determination and absolute quantification by PCR for L. intracellularis and porcine circovirus type 2 (PCV2). Association between average daily gain, faecal dry matter content, numbers of L. intracellularis bacteria and PCV2 genome copies in faeces was investigated in a multilevel mixed-effects linear model.

Results

Increasing numbers of L. intracellularis log₁₀ bacteria/g faeces were significantly associated with decreasing average daily gain (P < 0.001). The association was decreasing with increasing faecal dry matter content (P < 0.01). The number of PCV2 log₁₀ copies/g faeces was not significantly associated with average daily gain of the pigs (P > 0.5).

Conclusion

The results suggest a potential application of a PCR quantifying L. intracellularis in growing pigs. Faecal dry matter content must be taken into consideration in interpretation of such test results.     

A Lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model

Transmission of Lawsonia intracellularis from experimentally inoculated pigs to naive swine was demonstrated in this study. The study was conducted using conventional pigs divided into three groups as follows: principles inoculated with L. intracellularis, sentinels, and controls. The pigs were inoculated and paired on 13 and 9 days post-inoculation with a sentinel pig for 7 days. Fecal samples and serum samples were collected throughout the study for polymerase chain reaction (PCR) and antibody testing by indirect fluorescent antibody techniques. After co-mingling, the inoculated group was necropsied; sentinel and control pigs were necropsied 7-14 days later. The intestinal tracts were evaluated grossly and microscopically for lesions. PCR was performed on intestinal mucosal scrapings and feces. Warthin-Starry and fluorescent antibody staining procedures were conducted to confirm colonization with L. intracellularis. Gross and microscopic lesions typical of porcine proliferative enteropathy (PPE) were observed in both the inoculated and sentinel groups. Transmission was demonstrated from inoculated principle pigs to sentinel pigs. PCR results detected cyclical shedding of L. intracellularis in the feces. Seroconversion occurred in pigs that were exposed to L. intracellularis. From this study, it was demonstrated that transmission of L. intracellularis can occur easily in an environment with experimentally infected pigs and that PCR can be a useful tool to monitor fecal shedding of the organism.     

The use of in-situ hybridization for the detection of Brachyspira spp. in pigs

Diagnosis of Brachyspira infections in swine and the differentiation of the involved bacteria is time-consuming and in most cases unsatisfactory. Detecting Brachyspira directly in the damaged Brachyspira of the large intestine could provide a direct correlation between histological lesionsa and bacterial growth.In this study we investigated whether in-situ hybridization (ISH) with a digoxigenin-labeled RNA-probe is a suitable method for detecting Brachyspira in the mucosa of the large intestine. Formalin-fixed and paraffin-embedded tissue sections of the large intestine from 78 pigs, which showed macroscopic and histological findings of Brachyspira-associated colitis, were stained with hematoxylin and eosin and Warthin-Starry silver impregnation and subjected to ISH. We used a RNA-probe with a length of 334bp, complementary to a part of the 23S rRNA of all members of the genus Brachyspira. All sections were treated with this anti-sense probe and with a sense control probe. 64 samples (82%) showed clearly positive ISH signals. Thus ISH is a suitable method for detecting Brachyspira directly within the lesions of the large intestine. The quantity of Brachyspira identified by ISH was always lower than by Warthin-Starry staining. Whether this reflects lower sensitivity of the ISH technique, or the fact that other bacteria with morphological similarities to Brachyspira were also stained by Warthin-Starry is unknown as yet. The present investigations provide a basis of further research developing specific probes to distinguish between pathogenic and non pathogenic Brachyspira species and probes detecting other bacteria with morphological similarity to Brachyspira.     

Demographic and Environmental Risk Factors for Exposure to Lawsonia intracellularis in Horses in Israel

Equine proliferative enteropathy (EPE) is an enteric disease of foals that is caused by Lawsonia intracellularis. Clinical cases have been reported worldwide; however, data regarding the epidemiology of L. intracellularis in horses are scarce. Thus far, L. intracellularis has not been reported in the Middle East. The objectives of this study were to determine whether the causative agent of EPE exists in horses in Israel and in the Palestinian Authority and to define environmental and demographic risk factors for exposure. Fecal and serum samples were collected from horses from various regions of the country. The presence of L. intracellularis in horses in Israel was determined by real-time polymerase chain reaction (PCR), and seroprevalence was determined by enzyme-linked immunosorbent assay. One fecal sample of 136 tested (0.7%), was PCR positive. Sixty-seven sera samples (30.5%) of 220 horses in sentinel farms had anti-L. intracellularis antibodies. Low seroprevalence was found in foals both from Israel and from the Palestinian Authority (4.2% and 13.3%, respectively). In logistic regression models, geographical locations, management type, and age were found to be significant risk factors associated with seroprevlaence to L. intracellularis. No significant correlation was found between environmental variables and L. intracellularis seroprevalence after controlling for management type. These results support the existence of L. intracellularis in horses in both Israel and the Palestinian Authority. The reasons for the relatively low prevalence are currently not known and may be the result of different management, low exposure to free-living animals, and differences in environmental variables affecting the bacterial burden.     

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.     

Comparative genome sequencing identifies a prophage-associated genomic island linked to host adaptation of Lawsonia intracellularis infections

Lawsonia intracellularis is an obligate intracellular bacterium and the causative agent of proliferative enteropathy (PE). The disease is endemic in pigs, emerging in horses and has also been reported in a variety of other animal species, including nonhuman primates. Comparing the whole genome sequences of a homologous porcine L. intracellularis isolate cultivated for 10 and 60 passages in vitro, we identified a 18-kb prophage-associated genomic island in the passage 10 (pathogenic variant) that was lost in the passage 60 (non-pathogenic variant). This chromosomal island comprises 15 genes downstream from the prophage DLP12 integrase gene. The prevalence of this genetic element was evaluated in 12 other L. intracellularis isolates and in 53 infected animals and was found to be conserved in all porcine isolates cultivated for up to 20 passages and was lost in isolates cultivated for more than 40 passages. Furthermore, the prophage region was also present in 26 fecal samples derived from pigs clinically affected with both acute and chronic forms of the disease. Nevertheless, equine L. intracellularis isolates evaluated did not harbor this genomic island regardless of the passage in vitro. Additionally, fecal samples from 21 clinically affected horses and four wild rabbits trapped in horse farms experiencing PE outbreaks did not show this prophage-associated island. Although the presence of this prophage-associated island was not essential for a virulent L. intracellularis phenotype, this genetic element was porcine isolate-specific and potentially contributed to the ecological specialization of this organism for the swine host.     

Equine Proliferative Enteropathy Caused by Lawsonia intracellularis in a Foal in Brazil

An 8-month-old foal in Brazil presented with fever, colic, diarrhea, hypoproteinemia (especially hypoalbuminemia), leukocytosis, and hyperfibrinogenemia and became lethargic and anorexic with a poor body score. A diagnosis of Lawsonia intracellularis proliferative enteropathy was confirmed by fecal polymerase chain reaction and serologic testing, and the foal was successfully treated with oxytetracycline followed by azithromycin.     

Comparison of a commercial ELISA with an indirect fluorescent antibody test to detect antibodies to Lawsonia intracellularis in experimentally challenged pigs

Objective The ability of a new commercial ELISA to detect pigs with subclinical proliferative enteropathy (PE) was compared with the traditional indirect fluorescent antibody test (IFAT). Methods Serum samples were selected from pigs with known Lawsonia intracellularis infection status and clinical signs of PE, but the sample population consisted predominantly of pigs subclinically affected by PE. Results Significant association and agreement were shown between the ELISA and IFAT assays. ELISA results correlated well with the duration of L. intracellularis shedding as detected by polymerase chain reaction. Conclusion ELISA can be successfully used to monitor L. intracellularis infection in pigs.     

Transmission of Lawsonia intracellularis to weanling foals using feces from experimentally infected rabbits

The aim of this study was to investigate whether feces from rabbits experimentally infected with Lawsonia intracellularis were infectious to foals. Two rabbits were infected with L. intracellularis, while two rabbits served as controls. Eight foals received daily feces from either the infected or the control rabbits. All rabbits and foals were monitored daily for clinical signs for the entire study period (21 days for rabbits, 42 days for foals). Feces and blood were collected for the PCR detection of L. intracellularis and serologic analysis, respectively. None of the infected rabbits or foals developed clinical signs compatible with proliferative enteropathy. All infected rabbits and foals shed L. intracellularis in their feces and all seroconverted. The results support the role of rabbits as asymptomatic amplifiers of L. intracellularis and their role as sources of infection for susceptible foals.     

Oral Infection of Weanling Foals with an Equine Isolate of Lawsonia intracellularis,Agent of Equine Proliferative Enteropathy

Background: Equine proliferative enteropathy (EPE) is an emerging disease of weanling foals. Objectives: Describe clinical, hematologic, biochemical, serologic, molecular, and ultrasonographic findings in foals experimentally infected with Lawsonia intracellularis. Animals: Eight foals. Methods: Recently weaned foals were assigned to either the challenge (n = 3), the sentinel (n = 3), or the control (n = 2) group. Foals were experimentally challenged via intragastric inoculation of 3 × 10¹⁰L. intracellularis organisms grown in culture. Each experimentally infected foal was housed with a sentinel foal in order to assess feco‐oral transmission. All foals were monitored daily for the development of clinical abnormalities and were weighed once weekly for the duration of the study (90 days). Abdominal ultrasound examination was performed weekly. Feces were collected every other day for 60 days, then weekly for an additional 30 days for the quantitative molecular detection of L. intracellularis. Blood was collected weekly for hematologic, biochemical, and serologic analysis. Results: Only challenged foals developed transient clinical signs of EPE consisting of anorexia, lethargy, fever, loose feces, and peripheral edema. Two challenged foals developed transient hypoalbuminemia. Fecal shedding of L. intracellularis was first detected in the challenged foals between days 12 and 18 postinoculation and lasted for 7–21 days. Seroconversion was documented in all challenged foals and in 1 sentinel foal. The remaining sentinel and control foals remained unaffected. Conclusions and Clinical Importance: Clinical EPE of variable severity was induced in all foals infected with L. intracellularis. Furthermore, L. intracellularis can be transmitted via the feco‐oral route to susceptible herdmates.     

Faecal shedding and serological cross‐sectional study of Lawsonia intracellularis in horses in the state of Minas Gerais,Brazil

Reason for performing the study: Proliferative enteropathy, caused by the intracellular bacterium Lawsonia intracellularis, has been described in horses in Australia, the USA, Canada and European countries but has not been reported in Latin America. The prevalence of the disease in horses worldwide is unknown. Objective: To evaluate the presence of subclinical L. intracellularis infection in horses in the state of Minas Gerais, Brazil. Methods: A longitudinal study using serology and PCR for detecting antibodies (IgG) and shedding of L. intracellularis in faecal samples, respectively, was conducted using a total of 223 horses from 14 different horse farms in Minas Gerais, and from the Veterinary School of UFMG equine herds in Minas Gerais. The immunoperoxidase technique in glass slides was used as the serological test. Results: Twenty‐one horse sera had immunoglobulin G titres of 1:60 and were considered positive. The PCR technique in faeces for L. intracellularis DNA identified 7 horses as faecal shedders. Horses shedding the organism appeared healthy, indicating that subclinical infection of L. intracellularis occurred in the horses. Conclusion: Seropositivity and detection of faecal shedding of L. intracellularis indicates the presence of the agent in the equine population in Minas Gerais. Potential relevance: Results of this study should alert clinicians in countries where proliferative enteropthy in horses has not been reported to consider this disease as a possible cause of enteric disease.     

Prevalence of antibodies to Lawsonia intracellularis in pig herds in Australia

Objective Proliferative enteropathy (PE) of pigs is caused by Lawsonia intracellularis. Clinical severity appears to depend, at least partly, on the infective dose and strain of L. intracellularis. Serological tests are able to detect subclinical disease. The Bioscreen ELISA for detecting L. intracellularis-specific antibodies is widely used to monitor the circulating antibody status of pigs in Australia, but its sensitivity and specificity have not been reported. The aim of the present study was to measure the seroprevalence of antibodies to L. intracellularis in growing pigs in Australia. Methods Test sera were sourced from 1817 serum samples collected from finisher pigs from 63 herds across Australia in 2001, selected from a larger sample of 180 herds to represent the contribution that each herd size makes to the number of pigs produced. The test sera were the most recent collection of pig sera from all states and samples had been stored at −80°C from 2001 until testing was conducted in 2008. Sera were tested using the BioScreen ELISA. Results All herds tested positive for L. intracellularis-specific antibodies. The mean percentage of positive samples within each herd was 84.2% (range 31.3–100%). Conclusions Lawsonia intracellularis is endemic in pig herds in Australia and cost-effective strategies to reduce reliance on antibiotics, such as vaccination and/or all-in/all-out pig flow coupled with cleaning and disinfection of pens, are warranted.     

The critical threshold of Lawsonia intracellularis in pig faeces that causes reduced average daily weight gains in experimentally challenged pigs

Serology indicates that Lawsonia intracellularis infection is widespread in many countries, with most pigs seroconverting before 22 weeks of age. However, the majority of animals appear to be sub-clinically affected, demonstrated by the low reported prevalence of diarrhoea. Production losses caused by sub-clinical proliferative enteropathy (PE) are more difficult to diagnose, indicating the need for a quantitative L. intracellularis assay that correlates well with disease severity. In previous studies, increasing numbers of L. intracellularis in pig faeces, quantified with a real time polymerase chain reaction (qPCR), showed a strong negative correlation with average daily gain (ADG).In this study, the association between faecal L. intracellularis numbers and PE severity was examined in two L. intracellularis experimental challenge trials (n₁ = 32 and n₂ = 95). The number of L. intracellularis shed in individual faeces was determined by qPCR on days 0, 7, 14, 17 and 21 days post challenge, and average daily gain was recorded over the same period. The severity of histopathological lesions of PE was scored at 21 days post challenge. L. intracellularis numbers correlated well with histopathology severity and faecal consistency scores (r = 0.72 and 0.68, respectively), and negatively with ADG (r = ?0.44). Large reductions in ADG (131 g/day) occurred when the number of L. intracellularis shed by experimentally challenged pigs increased from 10⁷ to 10⁸ L. intracellularis, although smaller ADG reductions were also observed (15 g/day) when the number of L. intracellularis increased from 10⁶ to 10⁷ L. intracellularis.     

Reduced use of antimicrobials after vaccination of pigs against porcine proliferative enteropathy in a Danish SPF herd

The present study explored whether the use of group medication with antibiotics in a Danish pig herd was reduced after vaccination of the pigs against proliferative enteropathy (PE) caused by Lawsonia intracellularis. 7900 pigs originating from a single commercial sow herd were vaccinated against L. intracellularis, whereas 7756 pigs were kept as non-vaccinated controls. The pigs were included batch-wise in the study with every second batch being vaccinated. In the vaccinated batches, the consumption of oxytetracykline to treat PE was reduced by 79%, with a significantly lower number of pigs being treated (P < 0.0001). Vaccination also resulted in a highly significant improvement of average daily weight gain (+ 46 g/day; P = 9.55 × 10^-31) and carcase weight (+ 1.25 kg; P = 4.54 × 10^-05) as well as a shortened fattening period (-8 days; P = 2.01 × 10^-45).