Low Virulent Isolates Research Articles

Virulence variation among isolates of western equine encephalitis virus in an outbred mouse model

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed < or =2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 10(3) p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 10(3) p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

Genetic diversity and pathogenicity of Fusarium oxysporum isolated from wilted Welsh onion in Japan

Thirty isolates of Fusarium oxysporum from wilted Welsh onion plants were examined for their diversity in nucleotide sequences of the ribosomal DNA (rDNA) intergenic spacer (IGS) region and for pathogenicity with regard to five Welsh onion cultivars. Phylogenetic analysis based on the IGS sequences revealed polyphyletic origins of the isolates and a relationship between phylogeny and pathogenicity; low virulence isolates differed genetically from those with high and moderate virulence. Mating type analysis revealed that all F. oxysporum isolates were MAT1-1 idiomorphs, suggesting that the pathogens may be clonal in the fields examined.

Cysteine Peptidase Expression in Trichomonas vaginalis Isolates Displaying High- and Low-Virulence Phenotypes

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.

Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate

The study aimed to evaluate the effect of an infection with low virulent isolates of M. hyopneumoniae (LV1 and LV2) on the subsequent infection with a highly virulent isolate (HV). Fifty-five, 3-week-old piglets free of M. hyopneumoniae were randomly allocated to 6 different groups. At 4 weeks of age (D0), groups LV1–HV and LV1 were intratracheally inoculated with LV1, groups LV2–HV and LV2 with LV2, and group HV with sterile culture medium. Four weeks later (D28), the pigs of these different groups were either intratracheally inoculated with the highly virulent isolate (groups LV1–HV, LV2–HV, HV) or with sterile culture medium (groups LV1 and LV2). A negative control group consisted of pigs inoculated with sterile culture medium on D0 and D28. All animals were necropsied at 28 days after the second inoculation (D56). Clinical symptoms were evaluated daily using a respiratory disease score (RDS). After necropsy, macroscopic and histopathological lung lesions were quantified and immunofluorescence (IF) testing on lung tissue and nested PCR on BAL fluid were performed for the detection of M. hyopneumoniae. Disease signs and lung lesions were not observed in pigs of the negative control group. In the other groups, there were no or only very mild clinical symptoms from D0 until D28. A significant increase in the average RDS values was, however, observed during D28–D56, especially in groups LV1–HV (1.48) and LV2–HV (1.49), in group HV (0.79), and to a lesser extent in groups LV1 (0.50) and LV2 (0.65) ( P < 0.05). The clinical symptoms during D28–D56, the lung lesions and intensity of IF staining were more pronounced in groups LV1–HV, LV2–HV and HV compared to groups LV1 and LV2. All pigs, except those from the negative control group, were positive on IF testing and PCR at D56. The present study demonstrates that pigs inoculated with low virulent isolates of M. hyopneumoniae are not protected against a subsequent infection with a highly virulent isolate 4 weeks later and may even develop more severe disease signs. This indicates that subsequent infections with different M. hyopneumoniae isolates may lead to more severe clinical disease in a pig herd.

Identification of high- and low-virulent strains ofChlamydia pneumoniaeby their characterization in a mouse pneumonia model

Contradicting reports exist about the pathogenicity of Chlamydia pneumoniae and the severity of the respiratory disease they cause. This study aimed to clarify, in mice, our hypothesis that marked differences in virulence of well-defined C. pneumoniae strains might exist for lung infections. C57BL/6J mice were intranasally infected with equal amounts of five different, identically prepared laboratory strains of C. pneumoniae. Based on the clinical score, weight, histopathological score, the granulocyte marker-enzyme myeloperoxidase, and the amount of Chlamydiae in the lung tissue, the C. pneumoniae isolates exhibited clear differences in overall growth characteristics or clearance, and pathological potential. Thus, we could identify chlamydial strains (Kajaani-K6 and CWL-029), where mice became seriously ill, as well as a relatively low-virulent isolate (TWAR-183). Cytokine profiles also varied drastically between the five strains in extent and kinetic. Our results indicate that C. pneumoniae isolates differ markedly with regard to their interaction with the host and their pathological potential. This might also be true for the infection in humans. Because the genomic diversity of C. pneumoniae is rather small, more subtle genomic deviations account most likely for the apparent functional differences. Our results will be useful to identify additional virulence factors in the future.

Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells

T-cell activation is essential for protection against Burkholderia pseudomallei infection. Using bone marrow-derived dendritic cells (BMDC) isolated from partially resistant C57BL/6 and susceptible BALB/c mice, the degree of BMDC activation in the presence of B. pseudomallei was investigated. Maturation, cytokine production and internalization of B. pseudomallei by BMDC was assessed in response to infection with a highly virulent and a low-virulent clinical isolate. Maturation was determined by identifying the up-regulation of cell-surface markers CD11c and CD86. IL-1beta and IL-12p40 expression were assessed by reverse-transcriptase PCR. The uptake of B. pseudomallei by BMDC was measured using an internalization assay. This study demonstrated that B. pseudomallei isolates stimulate the maturation of BMDC to the same degree regardless of virulence. However, maturation of BMDC was significantly increased in BALB/c mice compared with C57BL/6 mice. Additionally, the uptake of B. pseudomallei by BMDC was significantly greater with the highly virulent isolate compared with the low-virulent isolate. Expression of IL-12 and IL-1beta following infection with B. pseudomallei was up-regulated. The differences observed may have implications in the development of an effective immune response to B. pseudomallei.

Isolation and characterization of a bovine isolate of Neospora caninum with low virulence

Neospora caninum tachyzoites were isolated from the brain of an asymptomatic naturally infected calf with precolostral-specific antibodies. The new isolate, named Nc-Spain 1H, was identified as a member of the N. caninum species based on its internal transcribed spacer 1 (ITS-1) sequence and was genetically characterized using microsatellite markers. Multilocus analysis showed that Nc-Spain 1H was genetically different from other N. caninum isolates. We compared the in vitro tachyzoite yield and viability rate of the Nc-Spain 1H and Nc-1 isolates in a plaque assay. The lower tachyzoite yields displayed by Nc-Spain 1H were complemented with a significantly lower viability rate. Moreover, in an in vitro tachyzoite–bradyzoite stage conversion assay, the percentage of Nc-Spain 1H bradyzoite conversion was similar to that of the cystogenic isolate Nc-Liv, with the exception that Nc-Spain 1H produced only intermediate bradyzoites. The pathogenicity of Nc-Spain 1H was examined in BALB/c mice, and the results demonstrated that Nc-Spain 1H failed to induce clinical signs or mortality and that no parasite DNA was detected in the brain during the chronic stage of infection. In a pregnant mouse model, Nc-1 infection resulted in high transplacental transmission, leading to a high neonatal mortality rate over time. In contrast, the offspring survival rate from Nc-Spain 1H-infected dams was almost 100%, and N. caninum DNA was detected in only one pup. These data show that Nc-Spain 1H appears to be a low virulence isolate and may be a suitable candidate for live vaccine development.

Evaluation of the Sensitivity of Reverse-Transcription Polymerase Chain Reaction to Detect Porcine Reproductive and Respiratory Syndrome Virus on Individual and Pooled Samples from Boars

Boar studs are continuously monitored for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) by testing different biological samples by reverse-transcription polymerase chain reaction (RT-PCR). In most cases, samples are run in pools, even though the impact of pooling on the sensitivity of RT-PCR is unknown. The objective of this study was to evaluate the feasibility of using PCR on pooled samples through the estimation of the sensitivity of RT-PCR on different biological samples run individually, in pools of 3 and in pools of 5. Twenty-nine boars were inoculated with a low virulent PRRSV isolate. Serum, blood swab, and semen samples were obtained from each boar every 2 to 3 days for 2 weeks. Each sample was tested by RT-PCR undiluted or diluted 1:3 and 1:5 with negative samples. Eleven of the 29 boars did not appear to get infected from the inoculum, as evidenced by no seroconversion 15 days after inoculation. Data from the other 18 boars showed that serum was the best sample to detect PRRSV during acute infection, with the blood swab sample performing almost as well. Semen samples failed to detect PRRSV infection in most of the cases. Pooling samples at pool sizes of 3 and 5 resulted in a decrease in the sensitivity of RT-PCR. Sensitivity was reduced by 6% and 8%, respectively, when serum or blood swab samples were run in pools of 5. The impact of pooling on the sensitivity of PCR was higher in samples taken during the beginning of the viremic period.

Protein variability among Mycoplasma hyopneumoniae isolates

Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein variability of Mycoplasma hyopneumoniae isolates. Fifty-six M. hyopneumoniae isolates from 6 different countries and 37 different herds were used. From eight herds, more than one isolate was available. All SDS-PAGE patterns of isolates originating from different herds were clearly divergent. Intra-species protein variability was quantified using the reference strain J and seven field strains all obtained from different herds and classified according to virulence. Between the field strains, a variability of 25% was found, while the culture-adapted strain J was clearly divergent and showed 30% variability with the field strains. No clustering according to virulence was obtained, but a protein band of about 181 kDa was present in the two highly virulent isolates whereas this protein band was absent in the moderately and low virulent isolates. Protein patterns of isolates derived from different animals from the same herd, were identical or differed in only a few protein bands. This study clearly indicates that, in agreement with previous studies on genomic diversity of M. hyopneumoniae isolates, proteomic variability within the species is high. Our study did not find clear evidence that more than one M. hyopneumoniae isolate circulates within a herd at a specific time point. The minor differences found between M. hyopneumoniae isolates from the same herd might reflect the organism's ability to alter its proteomic expression profile under field conditions.

Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28 DPI compared to the other groups. At 10 and 15 DPI, animals in the highly virulent group had significantly higher concentrations of TNF-α in BAL fluid. IL-1β concentration in this group was higher at 5 and 28 DPI compared to the other groups. From 10 DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.

Differential screening reveals genes differentially expressed in low- and high-virulence near-isogenic Phytophthora sojae lines

To explore the molecular mechanisms involved in virulence variations in Phytophthora sojae, the low-virulence isolate PS2 was inoculated successively on a resistant soybean ( Glycine max) cultivar. After 14 successive inoculations, a high-virulence progeny, termed PS2-vir, was obtained and demonstrated to exhibit lower oospore production. DNA fingerprinting revealed no large-scale DNA differences in PS2 and PS2-vir. A suppression subtractive hybridization (SSH) approach was developed to investigate differences in gene expression between PS2 and PS2-vir in the early stages of soybean infection. Of the 323 sequences chosen for examination, 74 putative unigenes were identified that exhibit high expression in PS2-vir. These sequences are predicted to encode proteins involved in energy production, protein biosynthesis, cell signaling, cell-wall biogenesis, and transcription regulation. Ten clones were selected for temporal expression analysis using RT-PCR based on the results of the dot-blot screens. The possible genetic mechanisms involved in these phenomena are discussed.

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.

Recovery of Young Olive Trees from Verticillium dahliae

Natural recovery from wilt disease symptoms was evaluated in young olive trees root dip inoculated with Verticillium dahliae in a growth chamber over a 12 week period and, later on, when the trees were transplanted in a V. dahliae-free soil in a lathhouse during a period of 127 weeks. Recovery in an individual tree was considered when a plant showed symptom remission after having reached a maximum value of symptom severity. Recovery accounted for 53% of 464 trees that showed wilt symptoms during observations in the two environments. The remaining trees died. Recurrent wilt symptoms were not observed in recovered trees, and recovery was usually accompanied by the production of new green tissues. Recovery was clearly higher in trees inoculated with a non-defoliating (ND) isolate (86.4%) of the pathogen than in those inoculated with a defoliating (D) isolate (23.9%). The percentage of recovery and the level of resistance were significantly correlated. Recovery accounted for 92.1% of the cases in resistant and moderately susceptible cultivars, reaching 100% in plants inoculated with the ND isolate (Table 2); meanwhile it was three times lower (30.1% of the plants) in susceptible and extremely susceptible diseased trees. In the lathhouse, periodical tissue isolations for monitoring the progress of infections over a period of 127 weeks in recovered trees, showed that the pathogen could only be isolated from trees 19 weeks after inoculation. Pathogen isolation was significantly higher from susceptible and extremely susceptible cultivars (84.6%) than from resistant and moderately susceptible ones (33.3%). Results showed that if a tree overcomes infection by pathogen from a single inoculation, and it is able to begin a recovery process, it will not express wilt symptoms again in a pathogen-free environment. The pathogen remained inactive or dead over time in recovered trees. Thus, new infections from rootlets would be necessary for new symptom expression. Recovery from Verticillium wilt is an important natural mechanism that occurs in a high percentage of infected olive trees, and can complement the resistance of the cultivar, particularly in conditions of low inoculum densities of low virulence isolates of the pathogen in the soil.

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.

Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments

Mycoplasma hyopneumoniae ( M. hyopneumoniae) is present in almost all swine herds worldwide, but transmission of the pathogen through herds is not yet fully clarified. The aim of this study, performed in 2003, was to investigate and to quantify the transmission of M. hyopneumoniae under experimental conditions by means of an adjusted reproduction ratio ( R n). This R n-value, calculated according to the final size method, expresses the mean number of secondary infections due to one typical infectious piglet during the nursery period. The period lasted from 4 to 10 weeks of age, corresponding with the nursery period used in most European production systems. Additionally, a comparison was made between transmissions of highly virulent and low virulent isolates. Forty-eight weaned piglets, free of M. hyopneumoniae, were housed in six separate pens. During 6 weeks, two animals experimentally infected with M. hyopneumoniae were housed together with six susceptible piglets. At the end of the study, the number of contact-infected animals was determined by the use of nPCR on bronchoalveolar lavage fluid. The R n-values of the highly virulent and the low virulent isolates were estimated to be 1.47 (0.68–5.38) and 0.85 (0.33–3.39), respectively. No significant difference between the groups was found ( P = 0.53). The overall R n was estimated to be 1.16 (0.94–4.08). Under the present experimental conditions, the transmission of M. hyopneumoniae, assessed for the first time by a reproduction ratio, shows that one piglet infected before weaning will infect on average one penmate during the nursery phase.

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.

Binding of laminin to Paracoccidioides brasiliensis induces a less severe pulmonary paracoccidioidomycosis caused by virulent and low-virulence isolates

The pathogenic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM). This pulmonary mycosis, acquired by inhalation of airborne propagules, may disseminate to several internal organs and tissues, leading to severe disease. Adhesion to host cell components is the first step involved in dissemination of pathogens. Previous studies showed that laminin, the most abundant glycoprotein of the basement membrane, binds to P. brasiliensis yeast cells, enhancing their pathogenicity in the hamster testicle model. As PCM is primarily a pulmonary infection, we studied the influence of previous treatment of yeast cells with laminin on the course of the intratracheal infection of resistant and susceptible mice using high-virulence (Pb18) and low-virulence (Pb265) P. brasiliensis isolates. Laminin treatment did not alter fungal loads, delayed-type hypersensitivity reactions, levels of pulmonary cytokines and production of specific antibodies in any group of Pb18-infected mice. However, early in the infection, a less intense inflammatory reaction was detected in the lungs of the laminin-treated groups. In addition, laminin treatment of Pb265 resulted in a less severe infection as revealed by the lower fungal loads recovered from lungs. Antibody and cytokine levels, however, did not change after laminin treatment. Altogether, our results demonstrate that laminin binding to yeast cells diminishes P. brasiliensis pathogenicity. The lower inflammatory response observed with the virulent isolate and the decreased pulmonary fungal burden with the low-virulence isolate indicate an inhibitory effect of laminin treatment on P. brasiliensis infectivity and interaction with pulmonary host cells or extracellular matrix proteins.

Binding of laminin to Paracoccidioides brasiliensis induces a less severe pulmonary paracoccidioidomycosis caused by virulent and low-virulence isolates

The pathogenic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM). This pulmonary mycosis, acquired by inhalation of airborne propagules, may disseminate to several internal organs and tissues, leading to severe disease. Adhesion to host cell components is the first step involved in dissemination of pathogens. Previous studies showed that laminin, the most abundant glycoprotein of the basement membrane, binds to P. brasiliensis yeast cells, enhancing their pathogenicity in the hamster testicle model. As PCM is primarily a pulmonary infection, we studied the influence of previous treatment of yeast cells with laminin on the course of the intratracheal infection of resistant and susceptible mice using high-virulence (Pb18) and low-virulence (Pb265) P. brasiliensis isolates. Laminin treatment did not alter fungal loads, delayed-type hypersensitivity reactions, levels of pulmonary cytokines and production of specific antibodies in any group of Pb18-infected mice. However, early in the infection, a less intense inflammatory reaction was detected in the lungs of the laminin-treated groups. In addition, laminin treatment of Pb265 resulted in a less severe infection as revealed by the lower fungal loads recovered from lungs. Antibody and cytokine levels, however, did not change after laminin treatment. Altogether, our results demonstrate that laminin binding to yeast cells diminishes P. brasiliensis pathogenicity. The lower inflammatory response observed with the virulent isolate and the decreased pulmonary fungal burden with the low-virulence isolate indicate an inhibitory effect of laminin treatment on P. brasiliensis infectivity and interaction with pulmonary host cells or extracellular matrix proteins.

Review of Newcastle disease virus with particular references to immunity and vaccination

Newcastle disease (ND) is a highly contagious disease. The present paper deals with classification of ND virus (NDV), clinical signs and pathology, virus strain classification and molecular backgrounds for the pathogenicity. Major emphasis is reviewing immunity and vaccination. Clinical forms of the disease vary depending on many factors, but mainly on the virulence of Newcastle disease virus (NDV) strains. Virulent strains are considered List A pathogens by the ‘Office International des Epizooties' (OIE). The virulence has been traditionally determined using in vivo pathogenicity tests to distinguish between highly, moderately and low virulent isolates. More recently, molecular biological techniques like polymerase chain reaction and sequencing have been described to differentiate virulent from non-virulent strains.The systemic and mucosal immune systems are considered to function more or less independently. Systemic antibodies are essential elements in protection against ND, whereas the local antibodies limit multiplication of NDV at the site of entry. Cytotoxic T lymphocytes specific against NDV have been detected in the spleen of vaccinated birds, however, their contribution to protection remains to be elucidated. An increase of the number of various leukocyte subsets was noticed in the respiratory tract and the Harderian gland (HG), which favours involvement of local cellular immunity in the defence against NDV infection. It is tempting to speculate that the local lymphoid infiltrates are involved in first defence and that cytolytic cells clear virus by directly lysing infected target cells at the site of NDV inoculation. Secondly, various cell types, mainly T-lymphocytes and macrophages, may be equipped to produce a range of cytokines with antiviral activity and cytokines that stimulate B-lymphocytes to proliferate and differentiate into antibody-forming cells responsible for the local antibody production against NDV.Vaccination using inactivated virus induces mainly systemic immunity and usually protects birds from disease caused by virulent NDV strains. Mucosal vaccination would be more attractive, as it induces in addition to systemic immunity a local IgM, IgA, and IgG, response as well as a mucosal cellular immune response. These local responses contribute to protection against NDV infection of the respiratory tract mucosa, which is the first target tissue of infection with this virus.