High and low virulence Staphylococcus aureus strains in a rabbit skin infection model

In the present study, an in vivo rabbit skin infection model was developed to reproduce the lesions caused by high and low virulence Staphylococcus aureus strains from rabbits. "O"-shaped dermal skin lesions were induced on the shaved flanks of anaesthetised rabbits using a tattoo pin and pincers. The induced lesions on the flanks of four groups of 10 rabbits were then inoculated by topical application of 0.1 ml of 10(8)cfu S. aureus bacteria. One group was inoculated with a typical high virulence (HV) S. aureus strain from rabbits, one group received an atypical HV strain and two groups were inoculated with low virulence (LV) strains. Five animals were kept as negative controls. The development, appearance and size of abscesses were scored daily for a period of 2 weeks. The infection model showed reproducible results for the different S. aureus inoculation groups. Inoculation of the skin with the typical HV strain resulted in significantly larger abscesses than those caused by the LV strains. The atypical HV strain caused abscesses of a size intermediate to that obtained with the HV and LV strains. In rabbits infected with LV strains, most of the lesions had healed by day 14 post-inoculation. The devised infection model is able to reliably reproduce the virulence properties of HV and LV S. aureus strains.     

Genotypic and phenotypic screening of high and low virulence Staphylococcus aureus isolates from rabbits for biofilm formation and MSCRAMMs

At rabbit flock level, two types of Staphylococcus aureus infections can be distinguished. In the first type, caused by low virulence strains, the infection remains limited to a small number of animals. The second type of infection is caused by the high virulence strains, which spread throughout the rabbitry. The pathogenetic capacity of a particular S. aureus strain is attributed to a combination of extracellular factors and invasive properties such as adherence and biofilm formation. Twenty eight high virulence and 34 low virulence S. aureus isolates recovered from rabbits between 1998 and 2003 were used to study slime production on Congo red Agar (CRA) and prevalence of bap, icaA and icaD associated with biofilm formation. Furthermore these strains were screened for the presence of bbp, clfA, clfB, cna, ebpS, eno, fnbA, fnbB and fib encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The presence of icaA and icaD was not correlated with slime production on CRA. Bap was absent in all strains. All rabbit S. aureus strains harboured clfA and clfB. The prevalences of ebpS, eno, fnbA and fib did not reveal striking differences between high and low virulence strains. FnbB prevalence in high virulence isolates was lower than in low virulence isolates and cna was absent in high virulence strains. It was remarkable that only high virulence strains were positive for bbp. Further research is necessary to elucidate the significance of bbp in the pathogenesis of high virulence strains.     

International dissemination of a high virulence rabbit Staphylococcus aureus clone

High virulence rabbit Staphylococcus aureus strains cause chronic and spreading problems of mastitis, pododermatitis and subcutaneous abscesses on rabbit flock level, whereas infections with low virulence strains are limited to individual rabbits. In the present report, 13 high virulence rabbit S. aureus strains, selected out of a large collection of strains isolated in five European countries between 1983 and 2004, were genotyped using pulsed-field gel electrophoresis, spa typing, multilocus sequence typing (MLST) and accessory gene regulator (agr) group typing. Two low virulence rabbit S. aureus strains were also included in the study. The results indicate the clonal origin of high virulence rabbit S. aureus strains present in Europe. Furthermore, the results of MLST and spa typing form a basis for international epidemiology of rabbit S. aureus strains, as these DNA sequence-based typing techniques can easily be used for intercentre comparisons.     

Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.     

Multiplex PCR assay for the detection of high virulence rabbit Staphylococcus aureus strains

High virulence rabbit Staphylococcus aureus strains, which are clonal in origin, are responsible for the spread of chronic staphylococcosis at the rabbit flock level. The aim of the present study was to develop a multiplex PCR assay that can be used for the identification of these high virulence strains. Two targets of the assay were the bbp and the selm genes, which have recently been shown to occur specifically in high virulence isolates. A third target was a sequence designated "flank", which was derived from a previously generated high virulence specific RAPD pattern. Furthermore, the femA gene, which is specific for S. aureus, was incorporated in order to avoid false negative results due to insufficient DNA preparation. The multiplex PCR was successful at differentiating the 26 typical high virulence and 50 low virulence rabbit S. aureus strains incorporated in the present study. Therefore it is useful for the initial screening of newly acquired breeding stock, in order to prevent the intake of high virulence strains in rabbitries.     

Secreted antigens as virulence-associated markers in Staphylococcus aureus strains from rabbits

Western blot analysis was performed from the culture supernatant of 59 rabbit Staphylococcus aureus strains, classified as high and low virulence strains according to their epidemiological behaviour in commercial rabbitries, bio-, phage- and RAPD-type. Fourteen extracellular antigen bands (A-N) were recognised using sera of rabbits immunised with washed, viable high virulence S. aureus bacteria. Eleven of these bands were found in high virulence as well as in low virulence strains. The band A, approximately 78 kDa, was not seen in any of the 27 high virulence strains, except for one strain which was also typical in other aspects, was detected in all, but one of the low virulence strains. The M and N bands with molecular masses of approximately 29 and 27 kDa, respectively, were recognised in all high virulence strains except for the atypical strain, but in none of the low virulence strains. This indicates that the latter two antigens may be virulence-associated markers for S. aureus strains from rabbits.     

Microarray-based genotyping of Staphylococcus aureus isolates from camels

Staphylococcus aureus is a common cause of mastitis and other diseases in camels. In order to obtain data on population structure as well as on the carriage of toxin genes and resistance markers, a collection of 45 isolates from dromedaries of Dubai, United Arab Emirates, were genotyped. These isolates belonged to clonal complexes CC6 (twenty isolates; 44.44%), CC30 (sixteen isolates; 35.56%), CC188 (five isolates; 11.11%), CC152 (1 isolate, 2.2%) and to a previously un-described sequence type (ST1755: arcc-18, aroe-115, glpf-6, gmk-2 pta-109, tpi-50 and yqil-2; three isolates; 6.67%). Resistance genes proved to be rare. Only three out of 45 isolates (6.67%) carried the beta-lactamase operon. The tetracycline resistance gene tetK was also detected in three isolates (6.67%). Neither the mecA gene, defining MRSA, nor other resistance genes were found. Common virulence markers included leukocidin genes lukD+lukE (in twenty-five isolates; 55.56%), the staphylokinase gene sak (twenty-two isolates; 48.89%), the enterotoxin gene cluster egc (fifteen isolates; 33.33%), and a distinct variant of the enterotoxin A gene (sea-320E, GenBank AY196686.1; thirteen isolates; 28.89%). One CC152 isolate was positive for genes encoding the Panton-Valentine leukocidin (lukF-PV+lukS-PV). This study provides first genotyping data on the population structure and the presence of toxin genes and resistance markers of S. aureus strains in Middle Eastern camels.     

Rabbit staphylococcosis: difficult solutions for serious problems

Staphylococcus aureus infections are a major problem in rabbitries. The main manifestations are subcutaneous abscesses, mastitis, pododermatitis and septicaemia. Two patterns of infection can be distinguished. In the first type, clinical signs remain limited to a small number of rabbits in a flock. This type has little economic importance and is caused by low-virulence S. aureus strains. In the second type, the disease shows an epidemic spread. Consequences are poor production results, infertility and death. This leads to chronic problems and a subsequent decline in production. The latter type is caused by high-virulence strains. Biotyping, phage typing and RAPD typing contribute to the characterisation of high-virulence S. aureus strains. Administration of antibiotics, disinfection of the environment and vaccination are not able to solve the problems. Therefore, the only effective measure is to cull the entire flock and to restart with a new rabbit population after thorough disinfection. Limiting the introduction of new rabbits in existing rabbitries and reducing contacts between rabbitries to an absolute minimum are currently the only way to face this most difficult problem.     

Sequence analysis of a RAPD band differentiating high and low virulence Staphylococcus aureus strains from rabbits.

RAPD typing revealed the presence of a nucleotide band in typical high virulence rabbit Staphylococcus aureus strains which was absent in low virulence strains and in an atypical high virulence strain. The nucleotide sequence of this band was determined. Primers within this sequence were developed and PCR products of eight typical high virulence, one atypical high virulence and nine low virulence rabbit S. aureus strains were sequenced. All low virulence strains and the atypical high virulence strain revealed a constant difference with the typical high virulence strains for nucleotide 377 of the 1055bp sequence. The eight typical high virulence strains possessed a guanine base on this site, while the other strains tested showed an adenine base. These findings support the hypothesis on the clonal origin of typical high virulence rabbit S. aureus strains. After comparison with databases, two open reading frames (ORF) were identified within the sequence, which appeared to encode two structural ribosomal proteins. The single nucleotide mutation does not affect the amino acid sequence of the protein it encodes for.     

Genotypic characterization of Staphylococcus aureus strains isolated from rabbit lesions

Since staphylococcal infections are the main pathological problem in rabbit does, the objective of this study was to characterize epidemiologically Staphylococcus aureus isolates from different lesion types in rabbits. Using 3 genetic markers (coagulase, staphylococcal protein A and clumping factor B genes), 22 different genotypes were identified among 301 isolates recovered from 259 rabbit does with 10 different kinds of chronic purulent lesions. These infected rabbits were obtained from 30 herds located in the Valencia province on the Spanish Mediterranean coast. The most frequent genotype was designated A1/II1/delta (coa/spa/clfB combination genotype) and represented 70.76% of the isolates. Although most genotypes were previously identified in other countries, novel types were also documented. No specificity between genotypes and nature of the pathologic process could be identified. After genetic comparison between strains from different origins, the results may suggest that rabbit, bovine and human S. aureus isolates are not clonally related, suggesting that specific host-dependent pathogenic factors may have evolved independently in these species. These differences indicate that a rational and effective strategy to control infections caused by rabbit-specific isolates may be advantageous.     

Effect on hematopoietic tissue of experimental infection of calves with noncytopathic type 2 bovine viral diarrhea virus

To investigate the hematologic abnormalities observed with noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2), calves 6 to 8 mo old were inoculated with an isolate of either high virulence (HV24515) or low virulence (LV11Q); control animals received the same volume of uninfected cell-culture supernatant. Peripheral blood neutrophil, lymphocyte, and platelet counts decreased in all the virus-inoculated calves but were significantly lower and remained decreased longer in the calves given HV24515. For each isolate, a decrease in the number of mature myeloid cells in the bone marrow coincided with the development of neutropenia, but the depletion persisted significantly longer (4 to 6 d) in the calves given HV24515. In the bone marrow of calves given LV11Q, the number of proliferating myeloid cells increased in proportion to the decrease in the number of mature myeloid cells. In the calves inoculated with HV24515, BVDV antigen was observed in bone marrow cells when the peripheral blood counts were lowest. Megakaryocytes were the predominant cell type exhibiting positive BVDV staining; myeloid cells rarely stained positively. Viral antigen was not observed in the bone marrow of calves given LV11Q. These experiments demonstrated that ncpBVDV-2 isolates of both high and low virulence caused decreased leukocyte and platelet counts, but only the high-virulence HV24515 isolate caused a delay in the production of myeloid proliferating cells. The delay may contribute to the ability of certain ncpBVDV-2 isolates to induce severe disease.     

Colonisation of rabbits with Staphylococcus aureus after experimental infection with high and low virulence strains

Four groups of 12 rabbits each were inoculated in the nose with strains with suspected differences in virulence. In the two groups infected with strains from severe outbreaks, belonging to a rabbit-pathogenic biotype-phage type combination, 6-12 rabbits were found positive at the successive bacteriological samplings over a period of 28 days. In the two other groups, infected with strains obtained from rabbitries without a history of staphylococcosis, the number of Staphylococcus aureus positive animals quickly became negative but increased again after 1 week to 1-5 positive animals until the end of the experiment. Two rabbits in each group inoculated with a high virulence strain developed purulent skin lesions, while in the groups inoculated with low virulence strains, all animals remained clinically healthy. Results indicate that colonisation capacity is an important virulence determinant in rabbit staphylococcosis.     

Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully.     

Differentiation between high and low virulence Staphylococcus aureus strains from rabbits by randomly amplified polymorphic DNA (RAPD) analysis

Randomly Amplified Polymorphic DNA (RAPD) typing was performed on 53 rabbit Staphylococcus aureus strains. Twenty-three strains isolated in 13 different rabbitries with chronic problems of staphylococcosis, showed the same RAPD banding pattern. Twenty of these strains belonged to the 'mixed CV-C' biotype and to the phage-type 3A/3C/55/71, previously described to be highly virulent in rabbits, and three strains belonged to other biotypes or phage-types. None of the strains isolated from rabbitries without chronic problems of staphylococcosis showed this specific RAPD pattern. RAPD analysis can be used as a rapid and reliable test method to differentiate between the characteristic genotype corresponding to high virulence and other S. aureus strains from rabbits. This is useful for the diagnosis and prevention of the introduction of these highly virulent strains in industrial rabbitries.     

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from birds

The present study was designed to comparatively investigate 19 Staphylococcus aureus strains isolated from specimens of 19 different birds during routine microbiological diagnostics. The S. aureus strains were characterized genotypically by polymerase chain reaction (PCR) amplification using 62 different oligonucleotide primers amplifying genes encoding staphylococcal cell surface proteins, exoproteins and two classes of the accessory gene regulator agr. All 19 investigated S. aureus were positive for the gene segment encoding a S. aureus-specific part of the 23S rRNA, the genes encoding thermostable nuclease (nuc), clumping factor (clfA) and coagulase (coa) and the gene segments encoding the Xr-repetitive region and the immunoglobulin G (IgG)-binding region of protein A (spa). In addition, all tested strains were positive for the genes hla and fnbA and negative for the genes seb, sec, sed, see, sej, tst, eta and etb. The remaining genes, including sbi, hlb, fnbB, ebpS, cna (domains A and B), cap5, cap8, set1, agr class I, agr class II, sea, seg, seh and sei were detected in a variable number of isolates. The presented data give an overview on the distribution of virulence determinants of S. aureus strains isolated from birds. This might be useful to understand the role of these virulence determinants in bird infections.     

奶牛乳房炎金黄色葡萄球菌毒力因子及荚膜基因的PCR检测

为研究金黄色葡萄球菌(S.aureus)的因型特征,本实验利用聚合酶链式反(PCR)方法,对S.aureus的3个标准株和30个临床分离株的凝集因子A、凝固酶、溶血素等33种致病因子进行了检测.结果表明各分离株在毒力因子方面存在一定差异.这些差异的检测将为进一步研究S.aureus的致病性和有效防治提供参考.     

Association of avian reovirus M and S genes with viral behavior in vivo. I. Viral persistence

Persistent infections were initiated in chickens with four different avian reovirus strains of varying virulence. Chickens 1 day old, 1 week old, or 2 weeks old were inoculated with each. Eight weeks later, isolates from all four parent strains were obtained; all isolates but one were from the tendons, and that was from the pancreas. Biochemical characterization of the isolates showed their genomes to be similar to those of the parent strains, although the proteins of the persistent isolates occasionally appeared to migrate differently from those of the original strains. Hybridization studies of the genes of the isolates indicated that at least two genes, S2 and S4, consistently seemed to undergo the greatest degree of mutation in the most virulent strain. These data suggest that the S2 and S4 genes may be associated with initiation and maintenance of persistent infection in vivo, and that changes in these genes may be noted by 56 days postinfection.     

Antimicrobial resistance and resistance genes in Staphylococcus aureus strains from rabbits

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.     

Clinical, microbiological, and molecular characterization of methicillin-resistant Staphylococcus aureus infections of cats

OBJECTIVE: To compare clinical information obtained from medical records of cats with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S aureus (MSSA) infections, evaluate antibiograms of MRSA and MSSA for multiple-drug resistance (MDR), and characterize the strain type and staphylococcal chromosome cassette (SCC)mec type of each MRSA. SAMPLE POPULATION: 70 S aureus isolates obtained from 46 cats. PROCEDURES: Clinical information obtained from medical records, including signalment, clinical signs, histologic examination of affected tissues, and outcomes, was compared between the 2 groups. Composite antibiograms of MRSA and MSSA were compared statistically. The MRSA strains were characterized by use of pulsed-field gel electrophoresis and SCCmec typing. RESULTS: No statistical differences in signalment or subjective differences in clinical signs or outcomes were detected between groups with MRSA or MSSA infection. Significant differences in antimicrobial resistance were detected, with MRSA having complete resistance to fluoroquinolone and macrolide antimicrobials, whereas MSSA maintained a high frequency of susceptibility. Seven pulsed-field patterns were observed in 15 MRSA strains; all but 1 were highly related. All MRSA isolates contained a type II SCCmec element. CONCLUSIONS AND CLINICAL RELEVANCE: Because MDR cannot be predicted in staphylococcal infections in cats on the basis of clinical signalment, culture and susceptibility testing are recommended whenever initial empirical treatment is unsuccessful. Molecular characterization of MRSA strains suggests that there has been reverse-zoonotic transmission from humans. IMPACT FOR HUMAN MEDICINE: The SCCmec type II element is typically associated with nosocomial MRSA infections of people. Cats may serve as reservoirs for MRSA infections in humans.     

Effects of noncytopathic type 2 Bovine viral diarrhea virus on the proliferation of bone marrow progenitor cells

The purpose of this study was to investigate the effects of isolates of noncytopathic type 2 Bovine viral diarrhea virus (ncpBVDV-2) of high and low virulence on the proliferation of bone marrow progenitor cells. Holstein calves 6 to 7 mo old and BVDV-na?ve were inoculated intranasally with a BVDV isolate of high virulence (HV24515), a BVDV isolate of low virulence (LV11Q), or uninfected cell culture medium. Serial bone marrow and peripheral blood samples were collected before and after inoculation. Bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 d, and the mean number of colony-forming unit-granulocyte-macrophage (CFU-GM) colonies was determined. Tritiated (3H)-thymidine uptake by BMMCs was determined to indicate overall proliferative capacity. Virus isolation was done on concurrent samples of BMMCs and peripheral blood. Virus was isolated from BMMCs and peripheral blood buffy-coat cells as early as day 2 or 3 after inoculation. Neutropenia developed in both groups inoculated with a BVDV isolate. However, in the calves given LV11Q, neutrophil counts rebounded earlier in response to increased proliferation of BMMCs, whereas the response was delayed in calves given HV24515. Thymidine uptake was significantly increased (P = 0.0047) in BMMCs after inoculation compared with before inoculation in the calves given LV11Q but not in those given HV24515 or in the control calves. The median number of CFU-GM colonies was significantly decreased (P = 0.0164) after inoculation compared with before inoculation in the calves given HV24515, whereas there was no significant difference in the calves given LV11Q or in the control calves. The data support the hypothesis that the prolonged neutropenia observed in calves given HV24515 results at least in part from decreased proliferative capacity of bone marrow progenitor cells.