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.

[Subclinical alveolar echinococcosis in a dog initially showing clinical features of leptospirosis]. / Subklinische alveoläre Echinokokkose bei einem Hund mit anfänglicher Symptomatik einer Leptospireninfektion.

A 3.5-year-old male Labrador retriever dog showed a short history of illness characterized by vomiting, apathy, and fever. Ultrasonographically, large nodular liver masses of high echogenicity were noted in both left and right liver lobes. Cytological and bacteriological examinations revealed a neutrophilic hepatitis without detectable agents. During treatment with doxycycline a four-fold decrease of serum titers to Leptospira (L.) icterohaemorrhagiae and L. sejroe was detected in paired serum samples by use of the complement-fixation test. The dog remained without clinical signs and no significant biochemical changes were recorded. However, ultrasonsographic examinations showed a progression of the hepatic lesions, presenting now as nodular parts with high echogenicity and cavernous parts with lower echogenicity. Diagnostic laparotomy was performed and the dog was euthanized due to severity of hepatic lesions. Histopathologically, a severe chronic granulomatous hepatitis with numerous parasitic structures was diagnosed. Morphology of the parasitic structures was comparable to the metacestode stage of Echinococcus multilocularis.

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.

Rare human skin infection with Corynebacterium ulcerans: transmission by a domestic cat.

Corynebacterium ulcerans is mainly known for its ability to cause animal infections. Some strains of C. ulcerans produce diphtheria toxin, which can cause life-threatening cardiopathies and neuropathies in humans. Human cutaneous C. ulcerans infection is a very rare disease that mimics classical cutaneous diphtheria. We present a very rare case of a C. ulcerans skin infection caused by a non-diphtheria toxin-producing strain of C. ulcerans that resolved after 3 weeks of therapy with amoxicillin-clavulanate. A pet cat was the probable source of infection. The presence of C. ulcerans in the mouth of the cat was confirmed by 16S rRNA gene analysis and the API Coryne system. In cases of human infection with potentially toxigenic corynebacteria, it is important to determine the species and examine the isolate for diphtheria toxin production. If toxigenicity is present, diphtheria antitoxin should be administered immediately. Carriers and potential infectious sources of C. ulcerans include not only domestic livestock but also pet animals. For the primary prevention of disease caused by diphtheria toxin-producing corynebacteria, vaccination with diphtheria toxoid is recommended.

Fermentation of six different forages in the semi-continuous fermentation technique Caesitec.

The aim of the present study was to compare carbohydrate degradation of forages which store carbohydrates either predominantly as fructan or starch, in horses' hindgut. The effects of an abrupt change from hay-based feeding to green fodder-based feeding on the caecal flora were tested with the in vitro hindgut simulation technique 'Caesitec'. Six trials with different forages (English ryegrass, tall fescue, grass mixture-horses, grass mixture-cows, lucerne, white clover) were conducted. During a 4-day stabilisation period, samples were taken once a day before loading the fermenters with hay. After diet-change to forage-based feeding, samples were taken four times a day. Ammonia and pH-value were measured before and 1, 2 and 6 h after loading the 'Caesitec'. Gas formation was measured daily. Bacterial numbers, lactate and short chain fatty acids were detected at four time-points of each trial. The grass mixtures contained the highest amounts of fructan. The pH-values were in the physiological range from pH 6 up to 7 (6.58-6.83) by feeding all forages. Gas formation, anaerobic and aerobic bacterial numbers increased after diet change from hay to any forage. The maximum amount of fructan (3.75 g/kg) in swiss pasture did not cause a permanent pathological change in the hindgut-flora.

[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.

Sonographic findings in an alpaca with Mycobacterium kansasii infection.

This case report describes the clinical, ultrasonographic and postmortem findings in an alpaca with Mycobacterium kansasii infection. The alpaca was referred because of chronic weight loss and weakness. The results of clinical examination, haematology and serum biochemistry were not diagnostic. Ultrasonography of the liver revealed multiple, hyperechogenic lesions with a diameter of 1 to 3 cm. Histological evaluation of a liver biopsy sample showed acute, multifocal, suppurative, necrotising hepatitis. Despite treatment with antibiotics, the alpaca died. Postmortem examination revealed nodular to coalescing lesions in the liver, lungs, mediastinum, pleura and greater omentum, which could not be differentiated macroscopically or histologically from lesions caused by tuberculosis. Ziehl-Neelsen staining showed massive numbers of rods within epithelioid macrophages, which were identified as Mycobacterium kansasii by polymerase chain reaction analysis.

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).

Haemotrophic mycoplasmas: recent advances in Mycoplasma suis.

Haemotrophic mycoplasmas (haemoplasmas) are uncultivable, small epicellular, cell wall less, tetracycline-sensitive bacteria that attach to the surface of host erythrocytes. Today, haemotrophic mycoplasmas are found in a large number of animals, with Mycoplasma suis being the porcine pathogen. Haemoplasmas can cause infections which are clinically marked, either by an overt life-threatening haemolytic anaemia or a mild chronic anaemia, by illthrift, infertility, and immune suppression. The life cycle of haemoplasmas on the surface of nucleus-less red blood cells is unique for mycoplasma and therefore, it is evident that these haemotrophic pathogens must have features that allow them to colonise and replicate on red blood cells. However, the mechanisms of adhesion and replication of M. suis on erythrocytes, for instance, as well as the significance of metabolic interchanges between the agent and the target cells, are completely unknown to date. Far from having gained clear insight into the clinical significance of the haemoplasmas, our knowledge about the physiology, genetics, and host-pathogen interaction of this novel group of bacteria within the Mollicutes order is rather limited. This can be explained primarily by the unculturability of these bacteria. The enormous advances in molecular biology witnessed in recent years have had a major impact on several areas of biological sciences, i.e. the fields of modern medical bacteriology and infectious diseases. This review describes progress made in research of the pathobiology of M. suis these past few years.

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.

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.

Development and evaluation of a Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR assay.

There are specificity questions inherent in most of the current PCR systems that amplify the MAP IS900 sequence as an indicator for Mycobacterium avium subspecies paratuberculosis (MAP) presence due to false positives associated with IS900-like sequences that exists in other Mycobacterium species besides MAP. We developed a multiplex PCR system designed to enhance specificity for MAP detection in a single PCR reaction. This PCR assay co-amplifies the mycobacterium species 16S rRNA gene, MAP IS900 and f57 sequences. The assay incorporates an internal PCR amplification control (IC) template system enabling PCR amplification conditions to be assessed in each reaction. The assay's specificity was confirmed by testing 10 isolates of MAP and 59 isolates of other bacterial species. In parallel we also applied on the same samples, one of the established nested PCR systems that targets only the IS900 sequence. The multiplex PCR assay was highly specific for MAP, the nested PCR system was also positive with several other mycobacterium species. In this context, we report for the first time false positive IS900 PCR signals in Mycobacterium chelonae, Mycobacterium terrae and Mycobacterium xenopi strains associated with one of the established PCR systems widely used for MAP detection. Finally, the potential application of this multiplex PCR system in milk analysis is demonstrated through analysis of raw milk samples artificially contaminated with MAP.

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by culture and serology.

Faecal samples from 186 dairy cows representing ten commercial dairy herds with sporadic clinical paratuberculosis (group A), and from 100 dairy cows from herds without a history of paratuberculosis (group B) were cultured for Mycobacterium avium subspecies paratuberculosis (MAP). Two different decontamination methods, a NaOH/oxalic acid method and treatment with 0.75% hexadecylpyridinium chloride (HPC) were performed prior to inoculation of Loewenstein-Jensen agar slants with and without mycobactin. Cultures were incubated for 16 weeks. Acid-fast staining bacteria were identified as MAP on the basis of mycobactin dependency and by PCR-RFLP analysis of the IS 1311-insertion element of M. avium. MAP was grown from 15 out of 186 group A animals (8.1%) whereas faecal culture for MAP was consistently negative in group B. The growth rate of MAP was significantly higher (8.1% vs. 1.6%) and the contamination rate of cultures was significantly lower (17.6% vs. 21.5%) in faecal samples decontaminated with NaOH/oxalic acid than with HPC-treated faecal samples (p<0.01, McNemar's test). Atypical mycobacteria which were grown from 46.8% of NaOH/oxalic acid treated specimens were not obtained from any of the HPC-treated samples. A commercial ELISA with MAP-lipoarabinomannan as the antigen was used to detect MAP-antibodies in unabsorbed sera from all animals. The percentage of ELISA-positive cows was 16.8%. Overall agreement between antibody detection and MAP-positive faecal culture was 15.4%.

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.

[Humoral immune response in breeding sows after vaccination with a herd-specific Chlamydophila abortus vaccine]. / Humorale Immunantwort von Zuchtsauen nach Impfung mit einer stallspezifischen Chlamydophila abortus-Vakzine.

A chlamydial vaccine efficacy trial with assessment of the clinical acceptability and serum antibody responses was performed in breeding sows. A BGM cell culture derived vaccine containing 10(8)/ml formalin-inactivated purified elementary bodies (Eb.) in sterile 0.15 M saline was prepared from Chlamydophila (Ch.) abortus strain OCHL03/99 which has been isolated in the herd from a sample of vaginal discharge. Vaccination was performed as a randomised trial with parallel treatment of a vaccinated group (25 sows) and non-vaccinated control group (20 sows). Sows received two 2.0-ml doses of vaccine intramuscularly at a three week interval. Control sows were dosed with sterile 0.15 M saline, accordingly. Serological response to vaccination was measured by ELISA with a total of 204 blood serum samples (114 from the vaccine group; 90 from the control group) using crude chlamydial LPS as the antigen. Compared to the control group, vaccinated sows showed a marked primary and secondary IgG serum antibody response following the two vaccinations. Antibody levels peaked between week 7 and 14 after priming vaccination, declined incrementally until week 27 but remained significantly higher than the corresponding sham-immune control levels and the prevaccination values of the vaccine group (p < 0.05). Western blot analysis of solubilized whole Eb. of Ch. abortus, Ch. pecorum, and Chlamydia (C.) suis with pre- and postvaccination sera confirmed that vaccination induced an antibody response preferentially against a range of 13 chlamydial antigens including the 40 kDa MOMP of Ch. abortus. Clinical side effects consisting of a transient mild local inflammatory reaction at the site of injection were observed in approx. 30% of vaccinated sows. These results provide the basis for further clinical evaluation of the Ch. abortus vaccine to protect sows from chlamydia-induced reproductive disorders.

Herpes simplex encephalitis in a domestic rabbit (Oryctolagus cuniculus).

An adult domestic rabbit showing neurological signs was subjected to euthanasia. At necropsy, macroscopical lesions were absent. Histopathologically, extensive lesions were seen, particularly in the cerebral cortex. Non-suppurative meningitis was present and there was lymphocytic and plasmacytic perivascular cuffing in the neuropil. The cerebral cortex showed extensive segmental neuronal and glial necrosis. Within the necrotic areas, large amphophilic intranuclear inclusion bodies were present in neurons and glial cells. Immunohistochemically, neurons and glial cells in the affected areas were labelled by polyclonal antibodies against both herpes simplex virus-1 (HSV-1) and HSV-2. The agent was classified as HSV-1 by polymerase chain reaction analysis. This is only the second reported natural case of herpes simplex infection in a rabbit.