Clinical and haematological characterisation of Mycoplasma suis infections in splenectomised and non-splenectomised pigs.

Mycoplasma suis causes infectious anaemia in pigs (IAP), which can manifest in various degrees of severity depending on the virulence and the host's susceptibility. As M. suis cannot be cultured in vitro experimental infections of splenectomised animals play an essential role for pathogenesis research. The aim of the present study was to characterise the course of experimental infection using the highly virulent and red blood cell (RBC-) invasive M. suis strain KI3806, to compare the experimental course in splenectomised and non-splenectomised pigs and to correlate clinical and haematological parameters with M. suis blood loads. All infected splenectomised pigs (n=7) were PCR-positive 2 days post infection (DPI) with maximum mean bacterial loads of 1.61 × 10(10)M. suis/mL on 8 DPI. They developed severe anaemia and massive hypoglycaemia by 8 DPI and had to be euthanised preterm (until 8 DPI) without seroconversion. The non-splenectomised pigs (n=7) became PCR-positive within 23 DPI and reached a maximum mean M. suis load of 1.64 × 10(5)M. suis/mL on 8 DPI. They developed mild anaemia, massive skin alterations with petechiae and haemorrhagic diathesis and seroconverted within 35 DPI. The study demonstrated that experimental infection of splenectomised pigs with the highly virulent M. suis strain KI3806 induces a fulminant course of infection. In contrast, M. suis strain KI3806 induces a mild course of disease in non-splenectomised pigs, which resembles the situation in naturally infected pigs. Therefore, these infection models are valuable for future pathogenesis studies on acute and chronic M. suis infections.

Porcine ear necrosis syndrome: a preliminary investigation of putative infectious agents in piglets and mycotoxins in feed.

The aim of this study was to identify the causative factors of porcine ear necrosis syndrome (PENS) in 72 pigs, 5.5-10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all piglets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At the same time, serum samples were taken for serological analysis and viral PCR, and feed was sampled for mycotoxin analysis. The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44 and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Seven piglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasma suis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15 samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxynivalenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multifactorial in origin and that although infectious agents can be involved in the development of the syndrome they are not the exclusive triggering factor.

[Porcine eperythrozoonosis: from Eperythrozoon suis to Mycoplasma suis]. / Porzine Eperythrozoonose: von Eperythrozoon suis zu Mycoplasma suis.

Mycoplasma suis (formerly known as Eperythrozoon suis ) is the most prevalent agent causing haemolytic anaemia in swine. The disease is also known as porcine eperythrozoonosis. M.suis is a small, pleomorphic bacteria parasitizing porcine erythrocytes. To date, no in vitro cultivation system for M.suis has been established and, therefore, our knowledge about the characteristics of M.suis and the pathogenesis of porcine eperythrozoonosis is rather limited. M.suis can cause acute disease, but the major significance of M.suis infections lies in the fact that M.suis can establish chronic and persistent infections leading to a higher susceptibility to other infections, especially of the respiratory and digestive tracts. The present article summarizes the current knowledge of the pathogen, the clinical signs and pathogenesis, diagnostic as well as therapy and prophylaxis.

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples.

AIMS: In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag. METHODS AND RESULTS: Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB, as well as the selective 16S rDNA sequence regions of B. anthracis, of the Bacillus cereus group and of Bacillus subtilis. Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected. CONCLUSIONS: This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal (rpoB) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.

Mycoplasma suis invades porcine erythrocytes.

Mycoplasma suis belongs to the hemotrophic mycoplasma group and causes infectious anemia in pigs. According to the present state of knowledge, this organism adheres to the surface of erythrocytes but does not invade them. We found a novel M. suis isolate that caused severe anemia in pigs with a fatal disease course. Interestingly, only marginal numbers of the bacteria were visible on and between the erythrocytes in acridine orange-stained blood smears for acutely diseased pigs, whereas very high loads of M. suis were detected in the same blood samples by quantitative PCR. These findings indicated that M. suis is capable of invading erythrocytes. By use of fluorescent labeling of M. suis and examination by confocal laser scanning microscopy, as well as scanning and transmission electron microscopy, we proved that the localization of M. suis was intracellular. This organism invades erythrocytes in an endocytosis-like process and is initially surrounded by two membranes, and it was also found floating freely in the cytoplasm. In conclusion, we were able to prove for the first time that a member of the hemotrophic mycoplasma group is able to invade the erythrocytes of its host. Such colonization should protect the bacterial cells from the host's immune response and hamper antibiotic treatment. In addition, an intracellular life cycle may explain the chronic nature of hemotrophic mycoplasma infections and should serve as the foundation for novel strategies in hemotrophic mycoplasma research (e.g., treatment or prophylaxis).

Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks.

Chlamydiae infect male genital organs of ruminants. However, little is known about their prevalence. Hence, we investigated fresh and cryopreserved semen (bulls: n=304; rams: n=78; bucks: n=44) by polymerase chain reaction (PCR), as well as genital organs (bulls: n=13; rams: n=10; bucks: n=6) by immunohistochemistry (IHC) and PCR. Sera from bulls (n=104) and small ruminants (n=61) were tested by LPS and rMOMP (recombinant major outer membrane protein) ELISA and competitive ELISA (cELISA), respectively. Three PCR assays were compared in this study for detection of chlamydial DNA in semen: 16S rRNA, IGS-S (intergenic spacer 16S/23S-short), and IGS-L (intergenic spacer 16S/23S-long) PCRs. PCR sensitivity and inhibitory effects were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. In bull semen, detection limits of the 16S, IGS-S and IGS-L PCRs were 10, 10, 100 templates, respectively. However, PCR sensitivity was reduced in ram and buck semen suggesting the presence of potential PCR inhibitors. Of 304 bull semen samples, the 16S PCR revealed DNA of chlamydiae in 20 samples (6.6%), including Cp. abortus (n=2), Cp. psittaci (n=1), Chlamydia suis (n=2), and Chlamydia-like organisms (n=15). In rams, one semen sample was positive for Chlamydia-like organism. All investigated male genital organs were negative for Chlamydia. Serology revealed 47.1% (49/104) positive bulls by LPS ELISA. Of these, 30 samples were positive by rMOMP ELISA, predominantly for Cp. pecorum. In small ruminants, cELISA displayed 34.8% (16/46) and 60% (9/15) positivity for Cp. abortus in rams and bucks, respectively. There was no correlation between serology and PCR of semen. The presence of chlamydiae in semen suggests the possibility of venereal transmission, although risk may be low in Switzerland.

Detection of chlamydiae in boar semen and genital tracts.

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.

Mycoplasma suis antigens recognized during humoral immune response in experimentally infected pigs.

Today, serodiagnostic tests for Mycoplasma suis infections in pigs have low accuracies. The development of novel serodiagnostic strategies requires a detailed analysis of the humoral immune response elicited by M. suis and, in particular, the identification of antigenic proteins of the agent. For this study, indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses were performed using pre- and sequential postinoculation sera from M. suis-infected and mock-infected control pigs. M. suis purified from porcine blood served as the antigen. Eight M. suis-specific antigens (p33, p40, p45, p57, p61, p70, p73, and p83) were identified as targets of the immunoglobulin G (IgG) antibody response during experimental infection, with p40, p45, and p70 being the preferentially recognized M. suis antigens. Besides the M. suis-specific antigens, porcine immunoglobulins were identified in blood-derived M. suis preparations. By immunoglobulin depletion, the specificity of the M. suis antigen for use in indirect ELISA was significantly improved. M. suis-specific Western blot and ELISA reactions were observed in all infected pigs by 14 days postinfection at the latest and until week 14, the end of the experiments. During acute clinical attacks of eperythrozoonosis, a derailment of the antibody response, determined by decreases in both the M. suis net ELISA values and the numbers of M. suis-specific immunoblot bands, was accompanied by peaking levels of autoreactive IgG antibodies. In conclusion, the M. suis-specific antigens found to stimulate specific IgG antibodies are potentially useful for the development of novel serodiagnostic tests.

Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

Diagnosis of porcine proliferative enteropathy: detection of Lawsonia intracellularis by pathological examinations, polymerase chain reaction and cell culture inoculation.

A total of 21 pigs aged 7-17 weeks with clinical symptoms suggestive for Porcine Proliferative Enteropathy were examined for Lawsonia intracellularis by analysing the following parameters: (i) intestinal gross and histological lesions, (ii) presence of comma-shaped bacteria in enterocytes by Warthin-Starry and a modified Ziehl-Neelsen stain, (iii) PCR amplification of L. intracellularis DNA from intestinal mucosa by using two oligonucleotide primer pairs targeting a 255-bp DNA fragment of the 16S rDNA-gene and a 319-bp DNA fragment of the L. intracellularis chromosome. Specificity of PCR reactions was confirmed by using DNA extracted from the L. intracellularis reference strain N343 (ATCC 55672) as well as by DNA sequence comparisons of PCR amplification products with data bank entries. Intestinal gross lesion indicative for PPE were observed in 20 pigs (95.2%). For all 21 pigs, the L. intracellularis aetiology was confirmed by histological as well as bacterioscopical examinations. Specific PCR amplification products were obtained from 20 pigs (95.2%). Taking PCR positivity as the definite criterion, L. intracellularis was diagnosed in 20 pigs from 11 herds in seven Swiss cantons (Argovia, Berne, Fribourg, Grisons, Lucerne, Schwyz, Thurgovia). To grow L. intracellularisin vitro, the cell culture method of Lawson et al. (J. Clin. Microbiol. 1993: 31, 1136-1142) was adopted. Inocula prepared from heavily infected fresh and frozen ileal mucosa of 15 pigs were cultured in rat enterocytic IEC-18 cells (ATCC CRL 1589). Six cell culture passages of 10 days each were completed. The reference strain N343 was examined for cultivability, accordingly. Except for occasional specific PCR amplifications from cell cultures up to the second passage, any indications for growth of L. intracellularis in IEC-18 cells were not found.