Epidemiology of intramammary infections with Staphylococcus aureus and mastitis streptococci in a dairy cattle herd with a history of recurrent clinical mastitis.

The aim of the present work was to examine a dairy herd with an anamnesis of recurrent clinical mastitis and decreased milk production. A total of 239 individual cow milk samples originating from asymptomatic cows were collected at four-month intervals and examined mainly for the presence of Staphylococcus aureus and mastitis streptococci using standard cultivation methods. In total, 29.7% and 9.2% samples were positive for S. aureus and mastitis streptococci, respectively. Unlike for mastitis streptococci, the prevalence of animals positive for S. aureus had an increasing trend (p<0.05; Chi-squared test for trend) with rising parity. Despite in vitro susceptibility of S. aureus to potentiated penicillins and cephalosporins, the persistence of S. aureus was observed in cows undergoing intramammary treatment with amoxicillin/clavulanic acid (a potentiated penicillin antibiotic). All isolates of S. aureus were biofilm-positive and had the same macrorestriction pattern. Furthermore, no dependence was observed between the occurrence of S. aureus in milk and previous cases of clinical mastitis, reproductive and periparturient disorders and administration of antibiotics. In contrast to S. aureus, the occurrence of mastitis streptococci in milk was linked with previous cases of clinical mastitis and intramammary administration of antibiotics.

Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, 'Cattle' or Type II and 'Sheep' or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA-positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR-REA revealed that sheep and goat populations on the island are infected primarily by 'Sheep' strains. Only three Map isolates from goats originated from one farm were characterized as 'Cattle' strains.

Culture- and quantitative IS900 real-time PCR-based analysis of the persistence of Mycobacterium avium subsp. paratuberculosis in a controlled dairy cow farm environment.

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.

[Nocardia farcinica as the causative agent of a brain abscess in a patient with interstitial lung disease]. / Nocardia farcinica jako puvodce abscesu mozku u pacienta s intersticiálním onemocnením plic.

This case report describes a two-step protocol for the identification of the causative agent of nocardiosis in a patient with brain abscess, antibiotic susceptibility testing and etiological treatment after neurosurgery. The patient treated with corticosteroids for pulmonary fibrosis and presenting with multiple neurological manifestations was admitted to a neurosurgery clinic. CT and contrast MRI revealed an expansive multilocular lesion 45 x 35 mm in size in the left parietal lobe, differentially diagnosed as malignant glioma. The lesion was biopsied and the histology showed a brain abscess containing white blood cells and dead tissue. The aspirated pus culture yielded bacteria of the genus Nocardia that were further identified, in the first step, by phenotypic methods (Gram positivity, partial acidoresistance, airborne mycelium detection, growth at 45 degrees C, lysozyme resistance and antibiotic resistance phenotype) as belonging to resistance phenotype V., v.s. N. farcinica (resistance to aminoglycosides except amikacin and to third-generation cephalosporins). In the second step of the polyphasic identification, rDNA was isolated and a 1000 bp part of the 16S rRNA gene was sequenced. Sequence comparison with the GenBank database using BLAST software identified the agent as N. farcinica (100%). The isolate was tested for susceptibility by the NCCLS /CLSI dilution method and showed good susceptibility to co-trimoxazole, amikacin and imipenem. The patient was treated with long-term intravenous cotrimoxazole acid in combination with amikacin and his clinical condition and laboratory parameters of inflammation improved. N. farcinica is among the three most frequently isolated Nocardia species in Europe as well as in the Czech Republic where it was repeatedly recovered from the lungs and respiratory tract of immunocompromised patients with systemic nocardiosis.

Comparison of the conventional culture, the manual fluorescent MGIT system and the automated fluorescent MGIT 960 culture system for the detection of Mycobacterium avium ssp. avium in tissues of naturally infected hens.

Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold's and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).

Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR.

The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.

Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues.

The members of Mycobacterium avium species, comprising M. avium subsp. paratuberculosis, M. a. hominissuis, M. a. avium, M. a. silvaticum, are currently the most prevalent opportunistic pathogenic mycobacteria causing mycobacterial infection in animals and humans. The ability to distinguish between these subspecies is of relevance for proper diagnosis and control programmes of the diseases. The aim of this study was to design a fast and specific PCR strategy for the detection and differentiation of M. avium subspecies from the solid plate cultures for use in routine veterinary diagnosis. We have developed a multiplex PCR based on IS900, IS901, IS1245 and the dnaJ gene. This method allows the detection of M. a. paratuberculosis, M. a. hominissuis and M. a. avium/M. a. silvaticum in one PCR reaction and theoretically enables mixed infections of M. a. paratuberculosis and M. a. avium or M. a. paratuberculosis and M. a. hominissuis to be revealed. The sensitivity of this multiplex PCR is 10(3)CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep.

Mycobacterium avium subsp. avium distribution studied in a naturally infected hen flock and in the environment by culture, serotyping and IS901 RFLP methods.

Mycobacterium avium subsp. avium (MAA) of serotype 2 and genotype IS901+ and IS1245+ was cultured from 21 naturally infected hens (Gallus domesticus) from one smallholder aviary. From a total of 330 samples taken from hens, 124 mycobacteria were detected. Out of which MAA was detected in 103 (35.7%) of 288 tissues, in 4 (19.0%) of 21 swabs of cloacae and in 9 (42.9%) of 21 faeces samples, 8 other conditionally pathogenic mycobacterial species were also isolated. Tuberculous (TB) lesions were found in the liver, spleen and intestinal organs of seven hens. The isolates of MAA (n=58) from 16 infected hens (7 with TB lesions and 9 without TB lesions) were found to be of 3 IS901 RFLP types AE (n=48), AD (n=4) and E (n=6), where these MAA isolates are highly virulent to hens. Mixed infections with IS901 RFLP types (AE and AD) and (AE and E) were also evident in seven hens. From a total of 35 examined environmental samples, 23 mycobacterial isolates were detected. Out of which four (17.4%) MAA isolates of IS901 RFLP type AE and 19 (82.6%) other isolates of conditionally pathogenic mycobacteria were detected. The finding of identical IS901 RFLP types from both tissues and faecal isolates confirms that infected domestic hens are the principal source of infection for other susceptible hosts and lead to the contamination of the surrounding environment. The presence of different IS901 RFLP types in tissue isolates may indicate the repeated incidence of MAA infection and the occurrence of polyclonal infection.

[Invasive streptococcal infections]. / Invazivní streptokokové infekce.

The severity of streptococcal infections depends upon different virulence of individual strains of its causative agent. The most important species are beta-haemolytic group A streptococci (GAS). Clinical manifestations include skin affections, respiratory tract infections and, in particular, serious systemic invasive infections. The pathogenicity of GAS is derived from cell wall components and extracellular products, especially toxins with properties of the so-called superantigens. Less invasive forms of the disease are include necrotizing fasciitis, myositis, pneumonia, sepsis without focus, arthritis, meningitis, puerperal sepsis, streptococcal toxic shock syndrome (STSS) and severe course of erysipelas and cellulitis with blood culture positive for GAS. In most cases, soft tissue infections dominate, often accompanied by chronic diseases of lower extremities in elderly patients. The other clinical forms are rather rare. In children, the condition is clearly frequently related to chickenpox. The generally accepted therapeutic management comprises comprehensive intensive care, early administration of penicillin in combination with clindamycin, and surgical intervention. The use of intravenous immunoglobulins (IVIG), elimination methods and hyperbaric oxygen are under discussion. The slight increase in cases and ineffective prevention require rapid assessment of diagnosis and adequate treatment as a protracted course of the condition is connected with a high mortality rate.

Avian tuberculosis in naturally infected captive water birds of the Ardeideae and Threskiornithidae families studied by serotyping, IS901 RFLP typing, and virulence for poultry.

Avian tuberculosis was detected in one flock of 38 water birds of the families Ardeideae (n = 20) and Threskiornithidae (n = 18). Mycobacterium avium subsp. avium (MAA, serotype 1, genotype IS901+ and IS1245+) was more often (p = 0.01) detected in tissue and/or faecal samples in 18 (90.0%) birds form the Ardeideae family: little egret (Egretta garzetta), buff-backed heron (Bubulcus ibis), great white egret (Egretta alba), and bittern (Botaurus stellaris) in comparison to two (11.1%) birds from the Threskiornithidae family: sacred ibis (Threskiornis aethiopicus). Avian tuberculosis was not diagnosed in spoonbills (Platalea leucorodia). Tuberculous lesions were found in nine birds. MAA isolates of IS901 RFLP type F-C3 were present in all of the 20 infected birds and in all environmental isolates. A mixed infection with the MAA isolates of three RFLP types F-C3 (tissue isolate), G-C3, and T-C3 (faecal isolates) was found in one sacred ibis. All 20 tissue isolates of IS901 RFLP type F-C3 from 20 birds and 8 environmental MAA isolates were fully virulent in pullets, whilst the isolates of RFLP types G-C3 and T-C3 were non-virulent in pullets. All of the tested MAA isolates had the same IS1245 RFLP "bird profile". In 12 of 20 infected birds with MAA M.a. hominissuis isolates of serotypes 4, 8, 9 and genotype IS901- and IS1245+ were detected and in 8 other birds mycobacteria not belonging to the M. avium complex were found. The presence of MAA in the environment may be a source for further spread of the causal agent of avian tuberculosis among other groups of animals in zoological gardens, farm animals, and also among their keepers.

[Rapidly progressing streptococcal sepsis in a patient after transplantation of a cadaver kidney]. / Rychle progredující streptokoková sepse u nemocného po transplantaci kadaverózní ledviny.

The authors submit the case-history of an adverse rapidly progressing phlegmon and septicaemia in a patient after transplantation of a cadaverous kidney with a functional graft. The patient was admitted repeatedly to the Transplantation Centre of the Urological Clinic, Faculty Hospital Hradec Králové with acute cellular rejection for corticosteroid treatment.

[Cooperation between the urologist and microbiologist in the diagnosis and treatment of sepsis. I. Predisposing factors and sources of sepsis in patients with urologic diseases]. / Spolupráce urologa a mikrobiologa v diagnostice a lécení sepse. I--predisponující faktory a zdroje sepse u urologických nemocných.

The most important source of septicemia caused by Gram-negative microorganisms, which has an ever rising trend, is the urogenital tract. The majority of these sepsis is of nosocomial origin (74%). At the Urological Clinic of the Faculty Hospital in Hradec Králové during 1984-1987 sepsis, based on a positive haemoculture, was diagnosed in 52 patients. During the investigation period the number of sepsis almost trebled. The most frequent predisposing factor was obstruction of the urinary pathways concomitant infection (41X) and instrumental procedures (25X). In the majority several factors were involved simultaneously. Septic disease occurred in particular in patients with the basic diagnosis of a malignant disease (36%) and in obstructive pyelonephritis (23%). Its incidence was significantly more frequent in more advanced age groups. In 63% it was urosepsis, in 8% the source was a cannula in the subclavian vein and in the remainder it did not prove possible to reveal the source of septicaemia.

[Cooperation between the urologist and microbiologist in the diagnosis and treatment of sepsis. II. The most common microbial species and their sensitivity to antimicrobial drugs in sepsis at the Urologic Clinic of the Medical School Hospital]. / Spolupráce urologa a mikrobiologa v diagnostice a lécení sepse. II--Nejcastejsí mikrobiální druhy a jejich citlivost na antimikrobiální látky u sepsí na urologické klinice FN.

The authors investigated the spectrum of aetiological agents and their sensitivity to antimicrobial substances in 52 patients with septicaemia at the Urological Clinic in Hradec Králové during 1984-1987. They found a predominating number of Gram-negative bacteria (82%). The most frequent agent producing sepsis was Klebsiella pneumoniae (19X), its source were in 52% the urinary pathways and 16% the cannula in the subclavian vein. In nine instances the aetiological agent was Escherichia coli and 5 times Proteus mirabilis. The above microorganisms are the most important pathogenic organisms of the urinary pathways. In all cases of bacteriaemia urosepsis was involved. The source of sepsis caused by Pseudomonas aeruginosa (7X) was in 43% the urinary tract, in particular in conjunction with instrumental procedures and in 14% a cannula in the subclavian vein. The most effective antimicrobial substance in Gram-negative bacteraemia in the investigated group was amicacin (100%); high sensitivity was recorded also in colistin. In Gram-positive bacteriaemia, where most frequently enterococci were isolated (6X), ampicillin and vancomycin were most effective.