High Virulence Isolates Research Articles

Prevalence of adhesin and toxin genes among isolates of Staphylococcus aureus obtained from mastitic cattle

Staphylococcus aureus is recognized as a major etiological agent of the most important udder disease; mastitis. The virulence potential of S. aureus isolates is attributed to a combination of extracellular factors and invasive properties that are controlled at the genomic level. In this study molecular variations were studied among the 128 S. aureus isolates obtained from the mastitic crossbred cattle. The polymorphic patterns of protein A, coagulase, and IG genes were studied in the isolates using the PCR and PCR–RFLP methods. In addition, presence of other virulence genes responsible for the expression of adhesins and toxins was also screened. A considerable genetic variation was observed in amplified fragment of SPA, IG and CLF genes. The PCR–RFLP of COA gene yielded five different patterns. Occurrence of EBP, ENO and FNBA was found to be common in both high and low virulence isolates. However, the prevalence of FNBB and toxin genes was higher in clinical than subclinical isolates. The distribution proportions of adhesin genes EBP, FIB, FNBB, CNA, BBP, MAP CAP5, and CAP8 were 60.2, 59.4, 21.9, 6.3, 5.5, 80.5, 56.3, and 22.7%; for AGR I, II, III, and IV were 44.5, 32.8, 12.5, and 10.2%; and for toxins genes HLB, SEB, SEC, SED, SEE, SEG, and SEI were 82.0, 3.1, 5.5, 3.9, 0.8, 16.4, and 55.5%, respectively. The proportions of FNBB, EBP, FIB, HLB, BBP, SEG, and SEI genes were observed more in clinical cases. The significant genetic variations in SPA, COA, IG, and CLF genes were useful to differentiate the isolates that might be valuable for mastitis reduction programme.

Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus

Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon ( Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser 32 to Ala 32 amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser 32 amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzyme's catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure–function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

Uropathogenic Escherichia coli with usp gene induces the rapid early stage apoptosis of HeLa cells

Objective To understand the distribution of pathogenic genes in uropathogenic Esche-richia coli (UPEC) from urinary specimen and to analyze the pathogenicity of UPEC and their mechanism of apoptosis to HeLa cells. Methods We have analyzed 6 pathogenic genes among the 28 strains of the clini-cally isolated E. coli from urinary tract infected patients. The 6 pathogenic genes were surveyed by using the PCR amplification of the target genes. The adhesion experiments and tryphan-blue staining was used to screen the phenotype of the pathogenic strains, while Annexin V/PI method was applied to study the strains to cause apoptosis of the HeLa cells, which was further confirmed with electronic microscopy. Results We have detected 6 strains that carried the usp gene. Phenotype screening identified two high virulent isolates (strain 6N and 27N) from the 6 strains. Strain 27N and 6N contained very similar virulence gene profile ex-cept that strain 27N did not contain usp gene. Both strains can destroy HeLa cell within 3 hours causing cell death. Results of apoptosis detected by flow cytometry revealed that strain 6N induced 20.75% of HeLa cells to an early stage apoptosis within 1.5 hours. On the other hand, strain 27N induced only 1.55% of HeLa cells to apeptosis. Conclusion High virulent UPEC strain carrying usp gene can induce HeLa cell rapid early apoptosis. Key words: Uropathogenic Escherichia coli; usp gene; HeLa cell; Rapid early stage apoptosis

ISSR for comparison of cross-inoculation potential of Colletotrichum capsici causing chilli anthracnose

Thirty-four isolates of Colletotrichum spp. including 2 species, Colletotrichumgloeosporioides and Colletotrichum capsici, from anthracnose on Bell pepper, Long cayenne pepper and Bird’s eye chilli were isolated and their pathogenicity was proven via fruit inoculation. Pathogenicity tests divided pathogenic potential into low, medium and high virulence groups. It is clearly revealed that C. capsici from the three tested hosts expressed the highest virulent isolates. Cross-inoculation of three high virulent isolates of C. capsici in accordance with three chilli varieties showed that all isolates could produce anthracnose symptom in the same lesions. All tested isolates developed lesions after co-inoculation of all hosts. Inter simple sequence repeat (ISSR) analysis indicated that there are two distinct groups of C. gloeosporioides andC. capsici. Furthermore, genetic diversity was correlated with geographic distribution, while there was no clear relationship between genetic diversity and pathogenic variability. But it is clearly demonstrated that whereas C. gloeosporioides appears in the same geographic area as C. capsici, it causes lower disease incidence. Key words: Anthracnose, chilli, Colletotrichum capsici, cross-inoculation.

High virulent clinical isolates of Mycobacterium abscessus from patients with the upper lobe fibrocavitary form of pulmonary disease

Pulmonary disease caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, can be classified into two distinct types of clinical disease; the upper lobe fibrocavitary (UC) form and nodular bronchiectatic (NB) form. However, the relationship between mycobacterial strain virulence and disease type in the pulmonary M. abscessus diseases has not been reported. To determine the differential virulence between strains causing two forms of disease, we obtained clinical isolates from patients with the UC and NB form of pulmonary disease caused by M. abscessus. In present study, we investigated the intracellular growth of clinical isolates in macrophages and their pathogenicity in C57BL/6 mice. For the isolates from the UC form, intracellular macrophage growth was faster and higher levels of cytokines were induced in macrophages than for those from NB form. Moreover, severe lung inflammation was only observed in mice intranasally infected with the isolate from the UC form with the increase of bacterial load. These findings suggest that M. abscessus isolates from the UC form of pulmonary disease are more virulent than those from NB form. This differential virulence of clinical strains may be one of the important factors involved in the determination of the disease form of pulmonary M. abscessus disease.

Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.

Positive correlation between low adhesion of group AStreptococcusto mammalian cells and virulence in a mouse model

We previously reported that a mouse model reflected, at least in part, the pathogenic mechanism of bacteremia observed during streptococcal toxic shock syndrome caused by group A Streptococcus (GAS). We have extended this study by assaying the in vitro adhesion of these same isolates to mammalian cells. Unexpectedly, we found that high-virulence GAS isolates in the mouse model showed low adhesion to the host cells. Similarly, the rate of recovery from the peritoneal cavity and cardiac blood of mice after intraperitonial injection was higher for high- than for low-virulence strains. Levels of expression of molecules that affect the adhesion of GAS to host cells were not significantly correlated with GAS virulence. Taken together, these results indicate that the invasiveness of GAS, reflected as higher virulence, is correlated directly with lower adhesion to host cells.

Selection of Beauveria bassiana isolates to control Alphitobius diaperinus

This paper presents results on isolates of the entomopathogenic fungus Beauveria bassiana for Alphitobius diaperinus (Coleoptera: Tenebrionidae) control. Of the 30 isolates tested, four (CG 71, CG 152, UNIOESTE 4, and UNIOESTE 40) resulted in mortality rates ⩾40% within 10 days. These mortality rates could be considered high because of the resistance of these species to B. bassiana. Tests were conducted using these isolates to estimate LC 50, mortality rate over time, vegetative growth, and conidial production on artificial medium and on insects. Isolates CG 71, CG 152, and UNIOESTE 4 showed the best performance and great potential to be used in an integrated management program in poultry farms to control A. diaperinus. Also, the molecular profiles of 12 isolates were analyzed using the RAPD technique. The high-virulence isolates presented a more homogeneous RAPD pattern than the others. Genetic sequencing of the ITS region was performed for one of the virulent isolates (UNIOESTE 4) and compared with sequences deposited at the NCBI database, confirming its taxonomical position as belonging to B. bassiana Clade A.

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.

Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation

Equine herpesvirus-1 (EHV-1) is responsible for respiratory disease and abortion in pregnant mares. Some high virulence isolates of EHV-1 also cause neurological disease. The pathogenesis of both abortion and neurological disease relates in part, to thrombus formation occurring in the pregnant uterus and central nervous system. The differences in disease outcome may relate to differing abilities of high and low virulence EHV-1 isolates to cause cell-associated viraemia, infect endothelial cells and cause thrombosis at sites distant from the respiratory tract. This study attempted to identify in vitro assays, which could be used to characterise the interaction between these isolates, equine endothelial cells and clotting factors. No significant difference was found between the growth kinetics of high and low virulence isolates of EHV-1 in polarised endothelial cells. For both isolates, virus was released preferentially from the apical surface of the polarised cells. The functional effects of viral infection on endothelial cells, with reference to virally-induced thrombosis were then investigated. Endothelial cells were grown on microcarrier beads, infected with EHV-1 and assayed for procoagulant activity. No significant difference in clotting time was observed between mock and EHV-1 infected endothelial cells in microcarrier cultures. Thus the degree of thrombosis may reflect a more complex interaction between endothelial cells, circulating leucocytes and other factors in the microenvironment.

Sequential Pathology after Experimental Infection with Marine Viral Hemorrhagic Septicemia Virus Isolates of Low and High Virulence in Turbot (Scophthalmus maximus L.)

Three marine viral hemorrhagic septicemia virus isolates were used to bath challenge turbot with the purpose of studying mortality and the pathology and antigen distribution over time. Two high-virulence isolates, 860/94, 4p168 and a low-virulence isolate 1p3 from a Baltic Sea herring were used. Organ samples were collected sequentially at 2, 4, 7, 10, 15, 20, 25, and 45 days postinfection. Specimens were processed for virology, histopathology, and immunohistochemistry. Organs during the early stages of infection (from 2 to 7 days) had virus isolation from all three groups only on day 7. Virus titer in kidney and heart sampled at day 25 was higher for the two virulent isolates compared with the low-virulence isolate. The viral distribution in situ of the two more virulent isolates from turbot (860/94) and herring (4p168) resembled viral hemorrhagic septicemia in rainbow trout with regard to the target organs. Early infection of endothelial cells in both kidney and heart was observed. Accumulated mean mortality was 41.5% for the turbot isolate 860/94, 48% for the herring isolate 4p168, and 3.5% for the herring isolate 1p3. This study revealed that the isolates from turbot (860/94) and herring (4p168) induced significantly higher mortality compared with the virus-free control and the herring isolate (1p3). The onset of mortality is markedly later in turbot compared with what is seen in rainbow trout.

In vitro and in vivo translational efficiencies of the 5′ untranslated region from eight genotype 2 bovine viral diarrhea virus field isolates

We determined the in vitro and in vivo translational efficiency mediated by the internal ribosomal entry site (IRES) from eight BVDV2 field isolates varying in virulence using a bicistronic reporter vector in rabbit reticulocyte lysates (RRL), and in primate and bovine cell lines. Using a T7-promoter system, the high virulence isolates had greater translational efficiencies in bovine lymphocytes (BL-3 cells), than did the low virulence isolates. The low virulence isolates translated with greater efficiencies than the high virulence isolates in RRL, African green monkey kidney (CV-1) and bovine turbinate (BT) cells. Our results demonstrate that despite a high degree of sequence identity in the 5′ untranslated region (UTR), subtle differences in the primary and secondary structures, as well as differences in cell lines, influence translational efficiencies.

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.

Characterization of Iranian Isolates of Fusarium oxysporum on the Basis of RAPD Analysis, Virulence and Vegetative Compatibility

AbstractFusarium yellow disease of chickpea (Cicer arietinum), caused by the vascular wilt pathogen Fusarium oxysporum, is a major production constraint in chickpea‐growing regions of Iran. Fifteen isolates of F. oxysporum were classified on the basis of vegetative compatibility (the ability to form heterokaryons). Vegetative compatibility was assessed by pairing nit mutants. The isolates were assigned to three vegetative compatibility groups (VCGs): VCG1, VCG2, and VCG3 with 5, 3, and 7 members, respectively. These isolates were also characterized by virulence assay. On the basis of disease severity on chickpea seedlings, they were divided into three groups: highly virulent (HV), moderately virulent (MV) and weakly virulent (WV). Twelve arbitrary decamer primers were tested on the fungal isolates by polymerase chain reaction. Random amplified polymorphic DNA (RAPD) analysis indicated considerable genetic variation among these isolates. Comparison of the results of VCG tests and the virulence assay indicated that all seven HV isolates belong to a single vegetative compatibility group (VCG3), and all three WV isolates are placed in VCG1. There was no relationship between RAPD groups and the other two characteristics of Iranian F. oxysporum isolates.

Verbesserung der Krankheitsresistenz von Kohlgem�se: 1. Turnip mosaic virus(TuMV)

Das Turnip mosaic virus (TuMV) ist weltweit verbreitet und gehort zur Gruppe der Potyviren. Es hat einen auserordentlich grosen Wirtspflanzenkreis von uber 300 Arten und wird nicht persistent durch 89 Aphidenarten ubertragen. Bisher sind beim TuMV zwolf Pathotypen bekannt. Im Kohlgemuse verursacht das TuMV die so genannte Kohlschwarzringfleckigkeit. Dabei handelt es sich um eine wirtschaftlich wichtige Virose, die beim Weiskohl bis zu 25 % Ertragsausfall und Gewebenekrotisierungen, insbesondere wahrend der Kuhllagerung, bewirken kann. Zur Sicherung eines hohen Ertrags- und Qualitatsniveaus bei Kohlgemuse wurde begonnen, die Resistenz gegen diese Virose zu verbessern. Mit aktuellen, weitgehend charakterisierten Erregerherkunften des TuMV wurden Resistenzprufmethoden weiterentwickelt und neue Resistenzquellen in der Familie der Brassicaceae erschlossen. Resistenzen gegen mehrere TuMV-Pathotypen liesen sich in B. oleracea-Primitivformen, in Chinakohl (B. rapa ssp. pekinensis), in Meerrettich (Armoracia ru-sticana), in Radies bzw. Rettich (Raphanus sativus) sowie in sexuell erzeugten Raphanobrassica-Hybriden nachweisen. Die evaluierten TuMV-Resistenzen wurden entsprechend der phylogenetischen Distanz zum Kohl durch Kreuzung bzw. durch Protoplastenfusion in Kulturformen des Kohls ubertragen.

In vitro characterisation of high and low virulence isolates of equine herpesvirus-1 and -4

Basic in vitro characteristics of high and low virulence isolates of equine herpesviruses-1 and -4 were investigated with particular reference made to the Ab4 and V592 isolates of EHV-1 as both have distinct endotheliotropism and clinical outcomes in pony challenge studies. Additionally, some EHV-4 isolates that showed variations in clinical outcome were included in some experiments. The aim of the study was to identify an in vitro characteristic that would differentiate strains of known virulence. Such a system could then be applied to vaccine and virulence studies as an effective screening tool. Viral growth kinetics in a variety of cell culture systems, plaque size, ability to replicate in fetal endothelium in organ culture, and sensitivity to acyclovir were compared. No reliable marker system that differentiated between higher and lower virulence isolates of EHV-1 and EHV-4 was identified.

Use of RAPD, enzyme activity staining, and colony size to differentiate phytopathogenic Fuzarium Oxysporum isolates from Iran

Fusarium oxysporum is a common soilborn plant pathogen with a worldwide distribution. Fusarium yellows disease of chickpea (Cicer arientinum) caused by F.oxysporum is one of the most destructive soilborn disease which is a major production constraint in chickpea-growing regions of Iran. Three laboratory methods of genomic DNA using random primers, enzyme activity staining, and colony size determination have been used to discriminate between highly virulent (HV) and weakly virulent (WV) isolates of F. oxysporum. On the basis of colony size (a traditional morphological method) and the ability of isolates to produce pectic enzymes, five HV isolates were differentiated from three WV isolates. The HV isolates formed large colony (ranging from 10.1 to 12.6 mm in diameter) and showed the same enzyme pattern,while the WV isolates produced small colony (ranging from 5.8 to 7.8 mm in diameter) and had not detectable enzyme activity in the stained overlaying gel. Twelve arbitrary 10-mer primers were tested on these 8 isolates of F. oxysporum by Polymerase Chain Reaction (PCR). Cluster analysis of the data from the DNA amplification by Random Amplified Polymorphic DNA (RAPD), differentiated HV from WV isolates. The results obtained from RAPD test confirmed the classification of these eight isolates based on pathogenicity test, colony size, and enzyme activity staining into two groups (HV and WV).

Variation in In Vitro α-Amylase and Protease Activity Is Related to the Virulence of Aspergillus flavus Isolates

Variation in the ability of Aspergillus flavus isolates to spread between cotton boll locules was previously shown to be at least partially related to variation in the production of a specific polygalacturonase (pectinase P2C). To determine if non-pectolytic hydrolase differences between low- and high-virulence isolates exist and, thus, could also potentially contribute to virulence differences, the present investigation was undertaken. Two A. flavus isolates, AF12 with low virulence and lacking pectinase P2C and AF13 with high virulence and producing pectinase P2C, were compared for production of nonpectolytic hydrolases after growth in 10% potato dextrose broth. Activity of amylases, cellulases, xylanases, and proteases was quantified using the radial diffusion/cup plate technique followed by differential staining. Culture filtrates also were subjected to native polyacrylamide gel electrophoresis. Both isolates produced amylases, proteases, and xylanases, whereas cellulases were not detected for either. AF13 produced more amylase than AF12, and this difference was supported by amylase isoform differences between isolates on native polyacrylamide gel electrophoresis gels. AF13 also produced more protease than AF12; however, isoform differences between isolates were inconclusive. These variations in other hydrolytic activities (besides pectinases)may contribute to virulence differences in cotton bolls between AF12 and AF13.

Virulence Markers in the 5′ Untranslated Region of Genotype 2 Bovine Viral Diarrhea Virus Isolates

Virulence markers to distinguish high from low virulence bovine viral diarrhea virus genotype 2 isolates have not been previously reported. The objective of this study was to identify virulence markers by evaluating the primary and secondary structures of the 5′-untranslated region of low and high virulence bovine viral diarrhea virus genotype 2 isolates. The nucleotide sequences of the entire 5′-untranslated region mRNA of eight bovine viral diarrhea virus genotype 2 isolates, four of high virulence and four of low virulence, and two genotype 1 reference isolates were determined using a polymerase chain reaction and a 5′ Rapid Amplification of cDNA Ends System. Two nucleotide substitutions were identified in the internal ribosomal entry site that distinguished the high virulence from the low virulence genotype 2 isolates. The low virulence isolates had a cytosine at position 219, whereas the high virulence isolates had a uracil. At position 278, a uracil or cytosine was found in the low and high virulence groups, respectively. The substituted bases are virulence markers that were used to identify bovine viral diarrhea virus genotype 2 isolates of high virulence.

Possible relationship between aggressiveness of two <em>Eutypa lata</em> isolates and their polyamines and abscisic acid content

<p style="text-align: justify;">By HPLC analysis, we showed that myceliums of two <em>Eutypa lata</em> isolates cultivated in Enikson and Petersson medium culture synthesized polyamines (Putrescine, Spermidine and Spermine) and abscisic acid (ABA).</p><p style="text-align: justify;">ln this medium, myceliums of high virulent isolate (BXI-IO) produced fewer polyamines than less virulent isolate (BX 1-5) while inverse phenomenon was observed for ABA content. The two growth regulators were diffused in medium culture in which their levels increase with age of mycelium. In the end, variations of PA and ABA in myceliums show antagonist effect of the two growth regulators in <em>Eutypa lata</em> fungi.</p>