Prevalence study of Staphylococcus aureus in quarter milk samples of dairy cows in the Canton of Bern, Switzerland.

In the present study, the prevalence of S. aureus in mammary gland quarters of dairy cows in Switzerland was estimated and a risk factor analysis was carried out. Dairy cows were selected by one-step-cluster sampling with stratification by herd size. Forty-seven of 50 randomly chosen farms participated in the study, resulting in 603 cows and 2388 quarter samples. Milk samples were collected in all herds on two occasions two weeks apart. In 6% of cows (95% CI: 2.7-9.3%) at least one milk sample was positive for S. aureus and from 2% (0.8-3.2%) of all quarters, S. aureus was cultured at least once. In four quarters a latent S. aureus infection (agent detected and somatic cell count (SCC) <100,000cell/ml) was diagnosed. Multivariable hierarchic logistical regression analysis yielded five significant risk factors for observing S. aureus in a milk sample: high SCC, a S. aureus-positive neighbouring quarter, a palpable induration in the quarter, and a wound, scar tissue or crush injury affecting the teat. The type of housing (P=0.1596) was also a factor that remained in the model. The mentioned risk factors must be considered during the evaluation of herds with S. aureus problems. The occurrence of latent S. aureus infections emphasises that not only quarters with a high SCC but all quarters of all cows must be cultured for control measures to be effective.

[Occurrence of Clostridium perfringens type A and type C in piglets of the Swiss swine population]. / Vorkommen von Clostridium perfringens Typ A und Typ C bei Saugferkeln in der schweizerischen Schweinepopulation.

Necrotizing enteritis (NE) of newborn piglets still represents an economical problem in Swiss pig breeding and production. The aim of our study was to identify risk factors for NE and evaluate the prevalence of C. perfringens with the toxingenes cpb and cpb2 in Swiss pig breeding farms. The prevalence of theses C. perfringens was investigated using fecal swabs followed by bacteriological culturing and genotyping. Close proximity to other breeding farms and large herd sizes were shown to predispose to NE. C. perfringens type C, carrying the genes cpa, cpb and cpb2 were frequently identified in herds with acute outbreaks of NE. Farms not affected by NE or those using prophylactic vaccination against NE were predominantly positive for C. perfringens type A strains with cpb2 and showed much lower prevalence of C. perfringens type C, compared to acutely affected herds. Our results demonstrate that C. perfringens type A strains with cpb2 are not associated with NE. Besides typical necropsy finding, only the identification of cpb can be used for the diagnosis of NE in affected herds.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan) multiplex PCRs for the detection of C. perfringens alpha-, beta-, beta2-, epsilon-, entero- and iota-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne alpha-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located alpha-toxin gene.

Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome.

Based on our clinical experience on bovine mastitis, we hypothesized that subtypes of Staphylococcus aureus (S. aureus) exist which differ in their contagious and pathogenic properties. In order to investigate this hypothesis, we analyzed strains of S. aureus isolated from spontaneous intramammary infection (IMI) with their virulence gene patterns and genotypes obtained by PCR amplification of the 16S-23S rRNA intergenic spacer (RS-PCR). The genotypes were then associated with epidemiological and clinical data including 26 herds. The results demonstrated a high association between genotypes and virulence gene patterns as well as between epidemiological and pathogenic properties of S. aureus. In particular, genotype B was related to high contagiosity and increased pathogenicity whereas the other types (C, OG) were found with infection of single cows. Because of the high clinical relevance, our results indicate the need to subtype the IMI-associated strains of S. aureus in the future.

[The seroprevalence of Actinobacillus pleuropneumoniae in Swiss pig breeding herds--a study with the ApxIV ELISA]. / Zur Seroprävalenz von Actinobacillus pleuropneumoniae in schweizerischen Schweinezuchtbetrieben--eine Studie mit dem ApxLV ELISA.

At the end of the national eradication program for Enzootic Pneumonia (EP) and Porcine Actinobacillosis (APP) in Switzerland (2003), A. pleuropneumoniae serotype 2 is considered to have been eradicated. There is no current information about the distribution of the other serotypes available. The ApxIV ELISA detects antibodies against all serotypes of A. pleuropneumoniae, without cross-reaction with other bacterial species. The aim of this study was to achieve actual data concerning the seroprevalence of A. pleuropneumoniae in breeding-herds and to validate the ApxIV ELISA under field conditions, especially for the diagnosis of latently infected breeding-herds without clinical signs, and to achieve more information about the role of herd book farms for the spread of the infectious agent. A total of 2068 serum samples from 96 pig herds in Switzerland were examinated. Over half of the examinated herd book farms showed positive results in this ELISA. 93% of the breeding herds were positive. On single animal level sensitivity was 96% and specifity 100%. Herd sensitivity ranged between 67% and 99%. Herd specifity was 100%. The results show that the ApxIV ELISA is a valuable tool for the detection of latently infected herds.

Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation.

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis (CFV) and C. fetus subsp. fetus (CFF). CFV is the causative agent of bovine genital campylobacteriosis, a highly contagious venereal disease that may lead to serious reproductive problems, including sterility and abortion. In contrast, CFF can be isolated from the gastrointestinal tract of a wide range of host species, is associated with abortion in sheep and cattle, and can also be isolated from local and systemic infections in humans. Despite differences in host and niche preferences, microbiological differentiation of the two subspecies of C. fetus is extremely difficult. This study describes the identification of a new insertion element, ISCfe1, which is present exclusively in CFV strains, with highly conserved specific ISCfe1 insertion sites. The results are useful for identification and differentiation of the two C. fetus subspecies and will help in understanding the evolution and pathogenesis of C. fetus.

A successful national control programme for enzootic respiratory diseases in pigs in Switzerland.

Before the start of systematic disease control, respiratory diseases in swine in Switzerland caused estimated losses of several million euros per year. In 1993, a national programme to control enzootic respiratory diseases in pigs was proposed, with the aim of reducing the incidence of clinical cases to less than 1%. Enzootic pneumonia (EP) caused by Mycoplasma hyopneumoniae and clinical cases of pleuropneumonia caused by any serotype of Actinobacillus pleuropneumoniae (APP) would be targeted, in addition to any cases with serological evidence of APP serotype 2. This control programme was initiated in 1996, region by region, and fully implemented by 2004. Clinical, epidemiological and laboratory test results were used to identify the appropriate disease control measures. Partial depopulation was used to control EP on breeding and breeding-finishing farms. Total depopulation was implemented on all farms affected with APP and finishing farms affected with EP Animal trade was strictly regulated during the programme and all suspected cases of respiratory disease in pigs were made notifiable. Continued monitoring is based on clinical suspicion of infection and/or the detection of gross pathological lesions at slaughter, followed by laboratory confirmation. In 2005, the incidence of clinical cases was less than 1%. Regulations have been introduced to control the international trade in live pigs and prevent the re-introduction of respiratory diseases into Switzerland.

Prevalence of classical swine fever, Aujeszky's disease and brucellosis in a population of wild boar in Switzerland.

During two survey rounds of a national surveillance system for infectious diseases in wild boar in Switzerland, each lasting four months from November to February, between 2001 and 2003, 1949 blood samples and 62 tissue samples from the spleen and 50 from the reproductive organs were collected from hunted wild boar. The survey was designed so that freedom from infection could be detected with a probability of 95 per cent at a threshold prevalence of less than 1 per cent for classical swine fever and Aujeszky's disease and less than 1.5 per cent for brucellosis. There was no serological evidence of classical swine fever or Aujeszky's disease, but brucellosis due to Brucella suis biovar 2 was confirmed serologically and by bacterial isolation.

[Cutaneous atypical mycobacteriosis due to Mycobacterium massiliense in a cat]. / Kutane atypische Mykobakteriose durch Mycobacterium massiliense bei einer Katze.

Fast growing mycobacteria are saprophytic bacteria that prevail in water and soil. They are opportunistic pathogens and may cause various infections if gaining entry into the body through a trauma. We herein describe the clinical presentation, pathology and diagnosis of the first case of cutaneous atypical mycobacteriosis due to Mycobacterium massiliense in a cat.

[Field validation of a real-time PCR test for the detection of Mycoplasma hyopneumoniae in nasal swabs of live pigs]. / Feldvalidierung einer real-time PCR zum Nachweis von Mycoplasma hyopneumoniae in Nasentupfermaterial von lebenden Schweinen.

Enzootic pneumonia (EP) of pigs, caused by Mycoplasma hyopneumoniae has been a notifiable disease in Switzerland since May 2003. The diagnosis of EP has been based on multiple methods, including clinical, bacteriological and epidemiological findings as well as pathological examination of lungs (mosaic diagnosis). With the recent development of a real-time PCR (rtPCR) assay with 2 target sequences a new detection method for M. hyopneumoniae became available. This assay was tested for its applicability to nasal swab material from live animals. Pigs from 74 herds (average 10 pigs per herd) were tested. Using the mosaic diagnosis, 22 herds were classified as EP positive and 52 as EP negative. From the 730 collected swab samples we were able to demonstrate that the rtPCR test was 100% specific. In cases of cough the sensitivity on herd level of the rtPCR is 100%. On single animal level and in herds without cough the sensitivity was lower. In such cases, only a positive result would be proof for an infection with M. hyopneumoniae. Our study shows that the rtPCR on nasal swabs from live pigs allows a fast and accurate diagnosis in cases of suspected EP.

Detection of the beta2 toxin gene of Clostridium perfringens in diarrhoeic piglets in The Netherlands and Switzerland.

The two studies presented here were done to determine the prevalence of the alpha, beta, epsilon and enterotoxin genes and the novel beta2 toxin gene of Clostridium perfringens in neonatal or pre-weaned piglets with diarrhoea or necrotic enteritis. All C. perfringens isolates were positive for the alpha and negative for the epsilon and enterotoxin gene, implying that only non-enterotoxigenic type A and C strains were detected. The most important findings were the relatively high prevalence of the beta2 toxin gene in isolates from diarrhoeic piglets in both studies, and, in one of the two studies, absence of strains with only the alpha and beta toxin gene. These data are supportive for the suggestion of a causal relationship of beta2 toxin-producing strains with digestive tract diseases in piglets.

LPS-induced, but not interferon-gamma-induced procoagulant activity of suspended human macrophages is followed by a refractory state of low procoagulant expression.

Monocyte-derived macrophages cultured under a variety of conditions were assessed for expression of procoagulant activity (PCA) upon induction by various triggers, using a semiautomated turbidimetric recalcification time assay in a kinetic ELISA reader. Macrophages cultured in a nonadherent (teflon) culture system and seeded in microtiter plates responded with PCA expression to lipopolysaccharide (LPS), to toxic shock-syndrom toxin-1 (TSST-1) and to surface-bound IgG, but not to surface-bound albumin, nor to interferon-gamma (IFN-gamma). In contrast, macrophages stimulated in teflon containers by IFN-gamma showed a strong PCA response peaking around 24 hr after stimulation, but they failed to secrete tumour necrosis factor (TNF). Suspended IFN-gamma-stimulated cells showed a similar response upon 2nd stimulation by LPS or IgG after adherence to microtiter plates as did nonprimed counterparts. In contrast, cells primed in suspension, then cultured in adherence secreted dramatically enhanced amounts of TNF when compared with nonprimed cells. Macrophages stimulated in suspension with LPS showed a PCA response of similar magnitude, which was accompanied by TNF secretion. PCA of both IFN-gamma-primed and LPS-exposed suspension culture cells was largely due to the surface expression of tissue factor, and to a lesser extent of a prothrombinase-like activity, as evidenced by PCA testing with factor-X-deficient plasma. The kinetics of LPS-induced PCA differed from IFN-gamma-induced PCA, in that PCA peaked at 6 hr and fell to insignificant levels after 24 hr. When transferred to microtiter plates at this time, they could be restimulated neither with LPS, nor with surface-adherent IgG nor with IFN-gamma. Evidence was obtained that the failure to express PCA was due to a refractory state of the cells rather than to the generation of cell-bound or secreted inhibitors of coagulation. The loss of PCA expression could be prevented by pre-exposure to IFN-gamma. Thus, PCA expression may be dissociated from other functional and/or activation parameters (e.g. TNF secretion). For the first time, a state in which cells are completely unresponsive to PCA induction has been identified. Should lower LPS concentrations also be found to induce such a refractory state, our results may be of pathophysiological significance.

Humoral and bronchial immune responses in cattle experimentally infected with Mycoplasma mycoides subsp. mycoides small colony type.

The course of immune reactions of the manifold antigens of Mycoplasma mycoides subsp. mycoides small colony type (SC) was analysed in serum and bronchial lavage of cattle experimentally infected with the African strain Afadé and the European strain L2 using Western-blots and complement fixation. Western-blot analysis of total antigens of both strains with sera from animals infected with the homologous and heterologous strain revealed the common dominant immunogenic antigens with the molecular masses of 110, 95, 85, 80, 72, 62, 48 and 39 kDa. The sequential sampling of the blood and bronchial lavages before and after contact infections allowed us to identify the antigens of 85, 80, 72, 48 and 39 kDa as particularly early immunogens. The IgA Western blots of the bronchial lavages showed distinct, early and persistent reactions to the 110, 85, 80, 72, 48 and 45 kDa proteins. These proteins were the predominant lipoproteins as determined by [14C]palmitic acid labelling. Only relatively weak reactions of the bronchial lavages were detected with IgG. In general immune responses were significantly stronger in the animals infected with the African strain Afadé, which gave positive results two weeks after contact infection. In contrast, the animals infected with the European strain L2 induced much lower reactions with a delay of three months after contact infection. In one animal strain L2 caused no sero-conversion and no infection. The results indicate a difference in virulence between the African strain Afadé and the European strain L2.

Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA.

[Identification and diagnosis of Taylorella equigenitalis by a DNA amplification method (PCR)]. / Identifikation und Diagnostik von Taylorella equigenitalis mittels einer DNA-Amplifikationsmethode (PCR).

A polymerase chain reaction (PCR) for identification of Taylorella equigenitalis was developed. The oligonucleotide primers are based on the DNA sequence of the rrs gene of T. equigenitalis, encoding for the 16S ribosomal RNA. Analysis of 21 strains of T. equigenitalis from England, USA and Switzerland showed an amplification product of 410 bp with identical Sau3A restriction profile. The sensitivity of the PCR-Assay was estimated to detect 50 to 500 bacteria of T. equigenitalis in a mixture with frequently found contaminants. Further analysis of culture from 60 genital swabs, taken in the course of the control of the contagious equine metritis in horses and donkeys, of experimental assays as well as of two positive cases from the diagnostic showed that this PCR-assay can be used to identify and to detect strains of T. equigenitalis. In addition, preliminary results indicate that the method is also applicable for direct in vitro establishment of the presence of T. equigenitalis in clinical samples.

Antimicrobial resistance in bacteria from Swiss veal calves at slaughter.

Bacteria with antimicrobial resistance can be transferred from animals to humans and may compromise antimicrobial treatment in case of infection. To determine the antimicrobial resistance situation in bacteria from Swiss veal calves, faecal samples from 500 randomly selected calves originating from 129 farms were collected at four big slaughterhouses. Samples were cultured for Escherichia coli, Enterococcus sp. and Campylobacter sp. and isolated strains were tested for antimicrobial susceptibility to selected antimicrobial agents by the minimal inhibitory concentration technique using the broth microdilution method. From 100 farms, data on farm management, animal husbandry and antimicrobial treatments of the calves were collected by questionnaire. Risk factors associated with antimicrobial resistance were identified by logistic regression. In total, 467 E. coli, 413 Enterococcus sp. and 202 Campylobacter sp. were isolated. Of those, 68.7%, 98.7% and 67.8%, respectively, were resistant to at least one of the tested antimicrobial agents. Resistance was mainly observed to antimicrobials frequently used in farm animals. Prevalence of resistance to antimicrobials important for human treatment was generally low. However, a rather high number of quinupristin/dalfopristin-resistant Enterococcus faecium and ciprofloxacin-resistant Campylobacter sp. were detected. External calf purchase, large finishing groups, feeding of milk by-products and administration of antimicrobials through feed upon arrival of the animals on the farm significantly increased the risk of antimicrobial resistance at farm level. Participation in a quality assurance programme and injection of a macrolide upon arrival of the animals on the farm had a protective effect. The present study showed that veal calves may serve as a reservoir for resistant bacteria. To ensure food safety, veal calves should be included in the national monitoring programme for antimicrobial resistance in farm animals. By improving farm management and calf husbandry the prevalence of resistance may be reduced.

Change in sensitivity to lipopolysaccharide during the differentiation of human monocytes to macrophages in vitro.

Mononuclear phagocytes in distinct differentiation stages and cultured under different conditions were tested for their sensitivity towards lipopolysaccharide (LPS), using procoagulant activity (PCA) expression and tumor necrosis factor (TNF) production as indices. The response of mature monocyte-derived macrophages differed from that of freshly isolated monocytes 1) by higher levels of constitutive PCA, 2) by responding to approximately 1,000-fold lower concentrations of LPS with PCA and TNF production, and 3) by a faster rise in PCA and TNF production. Due to the high constitutive level of PCA expression, the PCA stimulation index for LPS was low in macrophages when compared with that in monocytes. Thus, during differentiation to macrophages, human monocytes acquire increased sensitivity to LPS (2 orders of magnitude more sensitive than a sensitive turbidimetric Limulus amoebocyte lysate assay). This exquisite sensitivity to LPS is expressed regardless of whether LPS is offered in the presence or absence of lipopolysaccharide binding protein-containing serum. This points to as yet uncharacterized pathways of high affinity interaction between LPS and macrophages.

Nocardia nova causing pulmonary nocardiosis of black crakes (Limnocorax flavirostra).

Natural nocardial infection has been reported in many different species including mammals and fish, but reports in birds remain uncommon. Eight juvenile Black Crakes (Limnocoraxflavirostra) died unexpectedly at the Basle Zoo. Necropsy revealed disseminated white, firm nodules, 1-3 mm in diameter, throughout the lung parenchyma. Histologically, the lungs contained multiple, often confluent granulomas with central necrosis. Delicate, gram-positive, 0.5- to 1.0-microm-wide, branching, occasionally beaded, filamentous organisms were visible in necrotic centers. These organisms were acid fast when stained with Fite-Faraco. No histologic lesions were seen in other organs. Nocardia nova was isolated from liver, spleen, kidney, and lung. Granulomatous and necrotizing nocardial pneumonia with agonal septicemia was diagnosed, suggesting an aerogenous infection. To our knowledge, this is the first reported epizootic outbreak of nocardiosis in birds, which is additionally unusual because it was caused by N. nova.

Development of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides subsp. mycoides SC.

A specific and sensitive test for the detection of Mycoplasma mycoides subsp. mycoides small colony type (SC), the aetiological agent of contagious bovine pleuropneumonia (CBPP) was developed using two nested PCR reactions. The PCR reactions are based on the nucleotide sequence of lipoprotein P72 of M. mycoides subsp. mycoides SC. The two specific oligonucleotide primer pairs were chosen to match those sequence segments of the P72 gene which differ most from the gene of the closely related lipoprotein P67 of Mycoplasma sp. bovine group 7 (strain PG50). The nested PCR reacted with all of the 34 different strains of M. mycoides subsp. mycoides SC analysed, and gave no amplification product with any of the closely related mycoplasmas tested, showing its high specificity. In bronchial lavage fluid experimentally contaminated with M. mycoides subsp. mycoides SC, the assay was able to detect as few as two viable cells per ml using a simple lysis procedure prior to the amplification step. With clinical samples, the sensitivity of the nested PCR was about 10(4)-10(5) higher than that of single PCR amplifications performed under the same conditions. The assay was also successfully used to detect M. mycoides subsp. mycoides SC in bronchial lavage fluid of experimentally infected cattle and proved to be more sensitive than classical culture methods.