Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.     

Isozyme characterization of genetic diversity in Minnesota populations of purple loosestrife,Lythrum salicaria (lythraceae)

Starch gel electrophoresis of plant proteins was used to identify purple loosestrife (Lythrum spp.) cultivars and weedy populations. Preliminary determinations were made as to what degree weedy loosestrife populations were related (or genetically similar) to populations of L. alatum, L. virgatum, and horticultural cultivars. Cluster analysis of the data indicated that native L. alatum was genetically different from all populations of purple loosestrife and cultivars examined. The L. salicaria and L. virgatum cultivars, as groups, were not genetically distinguishable from the weedy populations analyzed. Seven cultivars of L. salicaria origin analyzed as a group were not distinguishable from the eight cultivars of L. virgatum origin, indicating that separation by cultivar origin may not be feasible. While the two “groups” were not distinguishable, most individual cultivars could be distinguished from one another by isozyme phenotype. Genetic variation was high within populations of weedy purple loosestrife but low among populations, which is characteristic of polyploid, perennial plant species that are widely distributed. Geographic location did not consistently correlate with genetic similarity.     

Identification of Russian wheat aphid (Homoptera: Aphididae) populations virulent to the Dn4 resistance gene

The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious worldwide pest of wheat and barley. Russian wheat aphid populations from Hungary, Russia, and Syria have previously been identified as virulent to D. noxia (Dn) 4, the gene in all Russian wheat aphid-resistant cultivars produced in Colorado. However, the virulence of Russian wheat aphid populations from central Europe, North Africa, and South America to existing Dn genes has not been assessed. Experiments with plants containing several different Dn genes demonstrated that populations from Chile, the Czech Republic, and Ethiopia are also virulent to Dn4. The Czech population was also virulent to plants containing the Dnx gene in wheat plant introduction PI220127. The Ethiopian population was also virulent to plants containing the Dny gene in the Russian wheat aphid-resistant 'Stanton' produced in Kansas. The Chilean and Ethiopian populations were unaffected by the antibiosis resistance in Dn4 plants. There were significantly more nymphs of the Chilean population on plants of Dn4 than on Dn6 plants at both 18 and 23 d postinfestation, and the Ethiopian population attained a significantly greater weight on Dn4 plants than on plants containing Dn5 or Dn6. These newly characterized virulent Russian wheat aphid populations pose a distinct threat to existing or proposed wheat cultivars possessing Dn4.     

Ribotyping to differentiate Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme isolated from bovine ruminal contents and liver abscesses.

Differences in biological activities (hemagglutination, hemolytic, leukotoxic, and virulence) and ribotypes between the two subspecies of Fusobacterium necrophorum of bovine ruminal and liver abscess origins were investigated. Hemagglutination activity was present in all hepatic, but only some ruminal, strains of Fusobacterium necrophorum subsp. necrophorum. Ruminal F. necrophorum subsp. necrophorum had low leukotoxin titers yet was virulent in mice. Fusobacterium necrophorum subsp. funduliforme of hepatic or ruminal origin had no hemagglutination activity, had low hemolytic and leukotoxic activities, and was less virulent to mice. For ribotyping, chromosomal DNAs of 10 F. necrophorum subsp. necrophorum and 11 F. necrophorum subsp. funduliforme isolates were digested with restriction endonucleases (EcoRI, EcoRV, SalI, PstI, and HaeIII) and examined by restriction fragment length polymorphisms after hybridizing with a digoxigenin-labeled cDNA probe transcribed from a mixture of 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzyme for ribotyping was EcoRI. The presence or absence of two distinct bands of 2.6 and 4.3 kb differentiated the two subspecies. Regardless of the origin, only F. necrophorum subsp. necrophorum, a virulent subspecies, had a ca. 2.6-kb band, whereas F. necrophorum subsp. funduliforme, a less virulent subspecies, had a ca. 4.3-kb band. Ribotyping appears to be a useful technique to genetically differentiate the two subspecies of F. necrophorum.     

Neurovirulence of type 1 polioviruses isolated from sewage in Japan.

Sixteen type 1 poliovirus strains were isolated from a sewage disposal plant located downstream of the Oyabe River in Japan between October 1993 and September 1995. The isolates were intratypically differentiated as vaccine-derived strains. Neutralizing antigenicity analysis with monoclonal antibodies and estimation of neurovirulence by mutant analysis by PCR and restriction enzyme cleavage (MAPREC) were performed for 13 type 1 strains of these isolates. The isolates were classified into three groups. Group I (five strains) had a variant type of antigenicity and neurovirulent phenotype. Group II (four strains) had the vaccine type of antigenicity and neurovirulent phenotype. Group III (four strains) had the vaccine type of antigenicity and an attenuated phenotype. Furthermore, it was demonstrated that the virulent isolates were neutralized by human sera obtained after oral poliomyelitis vaccine (OPV) administration, and the sera of rats immunized with inactivated poliovirus vaccine. Although vaccination was effective against virulent polioviruses, virulent viruses will continue to exist in the environment as long as OPV is in use.     

Studies on resistance of pea to pea seed borne mosaic virus and new pathotypes

Studies were carried out to search for virulent pathotypes of pea seed borne mosaic virus (PSbMV) on pea and to explore new sources of resistance in French and Indian pea collections. A virulent pathotype, PSbMV-Pi, capable of partially overcoming the recessively resistant gene sbm 1, was identified for the first time in an Indian pea line. PSbMV-Pi did not produce visible symptoms on sbm 1 lines, on which it had a reduced multiplication and could no longer be detected by ELISA seven wk after inoculation. However, it multiplied normally on the susceptible cultivars and could be differentiated from other strains on a set of strain differentials. Also, another strain, PSbMV-Pv, of the virulent pathotype, that appeared to multiply slightly better than PSbMV-Pi on sbm 1 lines, was recovered from the local strain of PSbMV.
Five new sources of resistance to PSbMV were screened from the Indian pea collection by a rapid screening based on the assumption that, in germplasm collections, the virus is generalised to all susceptible lines by aphid vectors. The initial step of testing a few testas of each germplasm line in ELISA and the rejection of positive lines eliminated 85% of the germplasm before further testing in the field. The inheritance studies on four of the five resistant lines lead to reidentification of the sbm 1 gene. The sbm 1 lines behaved fairly well under heavy inoculum pressure in the pea fields in 1989.     

The origin of Russian cultivars of red clover ( Trifolium pratense L.) and their genetic relationships to wild populations in the Urals

Propagation and breeding of red clover ( Trifolium pratense L.) in Russia was initiated about 200 years ago but the origin of present-day cultivars is disputed. Some authors argue that most Russian cultivars were derived from western European ones, whereas others support a Russian origin of the cultivars from local wild populations. In the present study we assessed the genetic variation at 17 allozyme loci in seven Russian cultivars, bearing the names of localities of the Urals, two American ones that have been used in Russia for scientific experiments and seven wild populations of the Urals and Western Siberia. Variation at the 17 protein loci supports the western European origin of the cultivars and also indicates that gene flow between cultivars and wild populations was limited or has not acted sufficiently long to affect the genetic composition of the red clover wild populations of the Urals.     

Origin and genetic structure of feral rye in the western United States

Feral rye (Secale cereale) is a serious, introduced weed of dry land agricultural regions of the western United States. It closely resembles cultivated cereal rye (Secale cereale cereale L.) with the exception of having a shattering seed head. Feral rye may have originated from hybridization of cultivated rye with mountain rye, Secale strictum, as past studies of northern Californian populations suggest, or directly from volunteer cultivated rye. We characterized the genetic structure of feral rye populations across a broad geographical range and reexamined evidence for hybrid origin versus direct evolution from domesticated cultivars. Eighteen feral populations were examined from three climatically distinct regions in the western United States. Seven cultivars, four mountain rye accessions, and one wild annual relative (Secale cereale ancestrale) were included in our analysis as possible progenitors of feral rye. Individual plants were scored for 14 allozyme and three microsatellite loci. Estimates of genetic diversity in feral populations were relatively high compared to those of the possible progenitors, suggesting that the weed had not undergone a genetic bottleneck. Weed populations had no geographical structure at either a broad or a local scale, suggesting idiosyncratic colonization and gene-flow histories at each site. Feral rye populations were no more closely related to mountain rye than cultivars were. They were, however, weakly clustered as a distinct lineage relative to cultivars. Our results do not support an interspecific hybrid origin for feral rye, but do suggest that the sampled populations of feral rye share a common ancestry that may explain its weedy nature.     

Number of Colonies Produced by 22 Isolates of Powdery Mildew on Primary Leaves of 10 Barley Cultivars

Primary leaves of 10 barley cultivars were uniformly inoculated with 22 virulent powdery mildew isolates using a jet spore trap. The number of colonies per unit area leaf was counted. Significant differences in number of colonies among cultivars were found, indicating different degrees of susceptibility among cultivars. While cultivars were rather consistent in their rankings for number of colonies produced by most isolates, interactions between some cultivars and isolates were found. In spite of great differences in geographical origin of cultivars and isolates, the interactions were not associated with origin.     

Diversity of Sinorhizobium strains nodulating Medicago sativa from different Iranian regions

Alfalfa is believed to have originated in north-western Iran and has a long history of coexistence with its bacterial symbiont Sinorhizobium in soils of Iran. However, little is known about the diversity of Sinorhizobium strains nodulating Iranian alfalfa genotypes. In this study, Sinorhizobium populations were sampled from eight different Iranian sites using three cultivars of Medicago sativa as trap host plants. A total of 982 rhizobial strains were isolated and species were identified showing a large prevalence of Sinorhizobium meliloti over Sinorhizobium medicae. Analysis of salt tolerance demonstrated a great phenotypic diversity. The genetic diversity of the Sinorhizobium isolates was analysed using BOX-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Patterns ofBOX-PCR fingerprinting were statistically analysed with AMOVA to evaluate the role of plant variety and site of origin in the genetic variance observed. Results indicated that most of the total molecular variance was attributable to divergence among strains isolated from different sites and cultivars (intrapopulation, strain-by-strain variance). Moreover, the analysis showed the presence of two geographic populations (west and northwest), indicating that the effect of the site of origin could be more relevant in shaping population genetic diversity than the effect of cultivar or individual plant.     

Flowering in cultivars of sesame (Sesamum indicum) Differing in photoperiodic sensitivity

Studies of flowering in sesame (Sesamum indicum L.) under different photoperiods revealed that cultivars Glauca, Venezuela-52 and Oro were short day strains while a fourth cultivar, Aceitera, proved to be day length neutral. Sensitivity to varying photoperiods, then, does not seem to be a universal characteristic of all sesame cultivars. Also, no correlation was found between photoperiodic class and latitude of origin in the cultivars studied. Inheritance studies involved crosses between the day neutral cultivar and two of the short day cultivars. In all cases, the F₂ populations demonstrated continuous variation. Most of the F₂ variability was of genetic origin and heritability values ranged from 86 to 96%. Additionally, transgressive segregation was observed for both early and late flowering. It is proposed that a minimum of three loci are involved in the inheritance of photoperiodic response in the sesame cultivars studied.     

Fluorescent Pseudomonads Associated with Diseases of Wheat in South Africa1

A survey of fluorescent pseudomonads associated with diseased wheat was conducted in South Africa during 1987 and 1988. Phenotypic features of 87 local strains were compared with those of 10 reference strains. Five groups were distinguished. Group 1 (nine reference and 16 local strains) was classified as Pseudomonas syringae pv. syringae. Group 2 (four local strains) was similar to group 1 but did not produce levan on nutrient sucrose agar. Group 3 (one reference and 33 local strains) also resembled P. s. pv. syringae, but did not elicit a hypersensitive reaction on tobacco. Group 4 (20 local strains) was mostly isolated from plants with atypical symptoms (total melanism) found in a single geographical region (Villiers) within South Africa. These strains had uniform characteristics, but failed to induce melanism on inoculated test plants. Group 5 (14 local strains) was not uniform. Twenty-eight representative local strains, selected from each of the five groups, and the 10 reference strains were used in pathogenicity and virulence tests. The four most virulent local strains were used to screen 14 wheat cultivars grown commercially in South Africa. Five of the cultivars were susceptible to these strains. Symptoms on leaves of naturally-infected plants corresponded with those already described, but the typical ear symptom (basal glume rot) was absent.     

Characterization of highly virulent Escherichia coli strains by ribosomal DNA restriction fragment length polymorphism

Patterns of ribosomal DNA polymorphism were examined to compare carboxylesterase B type B1 strains and B2 strains of Escherichia coli isolated from extra-intestinal infections. DNA from 14 type B2 strains showing the presence of alpha-haemolysin and mannose-resistant haemagglutinin and lethality to mice and 14 type B1 strains lacking these characteristics, was digested with HindIII, EcoRI, BamHI or BglII restriction enzymes and analysed by Southern blotting. The obtained ribotypes clearly differentiated the B2 strains from the B1 strains. These results indicate that genotypes of the highly virulent B2 strains are different from that of the less virulent B1 strains.     

I. Pathogenic variation in fungi and bacteria and mycorrhizal compatibility: Pathogenic variation in Verticillium albo-atrum isolates from hop (Humulus lupulus)

Non-lethal (M strain) ‘fluctuating’ wilt was first identified in hops in 1924. Lethal (V strain) ‘progressive’ wilt appeared in the 1930s and spread rapidly through Kent, destroying all commercial varieties. By 1965 the disease was largely controlled by use of new resistant cultivars, accompanied by restricted nitrogen fertilisers, non-cultivation (with weed control by overall herbicides), and high standards of field hygiene. Between 1968 and 1971 severe wilt developed in previously highly resistant clones, used as ‘controls’ in routine field resistance tests. Verticillium isolates from the two farms supplying the natural inoculum for these tests during 1968–71 were more virulent than previously encountered V strains, some causing severe wilt in all resistant cultivars. Subsequent surveys supported by pathogenicity tests against resistant cultivars indicated the existence of two ‘super virulent’ strains on eight farms in Kent. The appearance of mild (M) strains in the 1920s, virulent (VI) strains in the 1930s, and ‘super-virulent’ (V2 and V3) strains in the 1960s follow 400 years of apparent freedom from wilt. This suggests that the initial breakage of resistance barriers was critical and subsequent variations in an apparently complex, polygenically controlled resistance system are rapidly being matched by the pathogen.     

Phylogenetic background of Orange lily (Lilium bulbiferum s.l.) cultivars from a genetically isolated environment

Domesticated Orange lily ( LILIUM BULBIFERUM s.l.) cultivars do not typically produce seeds, and Orange lily is not native to Finland. Therefore, back crossing of the cultivars with wild species has not been possible. Genetic variability and genuineness of eight Finnish traditionally-grown Orange lily cultivars was studied. RAPD patterns were compared between the cultivars and genuine Orange lily ( LILIUM BULBIFERUM L.), and a related Dauricum group. The results showed partition of tested genotypes into four groups, L. CANADENSE as the outgroup. The cultivars were divided into two subgroups where the trait to form bulbils was characteristic to subgroup I. The cultivated strains differed from each other as much as from the seedling strains, but were genetically closer to genuine Orange lily than the Dauricum group. This indicates that the cultivars are genuine forms of Orange lily species. The special morphological features of the cultivars have likely been formed during centuries-long genetic isolation from natural populations.     

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.     

The identification and fitness of virulent potato cystnematode populations (Globodera pallida) selected on resistant Solarium vernei hybrids for up to eleven generations

Selection of Globodera pallida populations on resistant Solanum vernei-hybrids resulted in distinct virulent strains after eleven generations. Some of these virulent populations were assessed on their environmental fitness under field-type conditions. All reproduced less well in unsterilised soil, but virulent populations were less affected by environmental variation than their avirulent counterparts. Evaluation of their reproductive ability could not equate virulence to overall enhanced or reduced genotypic fitness compared with their avirulent counterparts. These populations were shown to be genetically distinct from their unselected counterparts using isoelectric focusing and specific enzyme staining. The control and management of virulent G. pallida populations is discussed.     

Characterization of Newcastle Disease Viruses in Wild and Domestic Birds in Luxembourg from 2006 to 2008

Newcastle disease virus (NDV) is one of the most important viral diseases of birds. Wild birds constitute a natural reservoir of low-virulence viruses, while poultry are the main reservoir of virulent strains. Exchange of virus between these reservoirs represents a risk for both bird populations. Samples from wild and domestic birds collected between 2006 and 2010 in Luxembourg were analyzed for NDV. Three similar avirulent genotype I strains were found in ducks during consecutive years, suggesting that the virus may have survived and spread locally. However, separate introductions cannot be excluded, because no recent complete F gene sequences of genotype I from other European countries are available. Detection of vaccine-like strains in wild waterbirds suggested the spread of vaccine strains, despite the nonvaccination policy in Luxembourg. Among domestic birds, only one chicken was positive for a genotype II strain differing from the LaSota vaccine and exhibiting a so-far-unrecognized fusion protein cleavage site of predicted low virulence. Three genotype VI strains from pigeons were the only virulent strains found. The circulation of NDV in wild and free-ranging domestic birds warrants continuous surveillance because of increased concern that low-virulence wild-bird viruses could become more virulent in domestic populations.     

Theileria annulata: attenuation of a schizont-infected cell line by prolonged in vitro culture is not caused by the preferential growth of particular host cell types

Flow cytometry and monoclonal antibodies to bovine leucocyte surface antigens were used to identify the types of host cells that the sporozoites of Theileria annulata infect in cattle, to determine whether virulent schizont-infected cell lines (lines) differed phenotypically from avirulent lines, and to establish whether attenuation in vitro was accompanied by the preferential growth of particular host cell types. The surface antigens of four pairs of T. annulata (Ta) (Hisar) lines derived ex vivo and in vitro, including the virulent ex vivo-derived Ta Hisar S45 line, were consistent with a myeloid origin for all lines, irrespective of their derivation. The profiles of lines derived from cattle inoculated with a virulent line showed that the schizonts liberated from inoculated cells had transferred to myeloid cells. A number of other lines infected with different stocks of T. annulata expressed myeloid markers; a single line expressed CD21, a B cell marker. During prolonged in vitro culture, the parasites in the ex vivo (virulent)- and in vitro (avirulent)-derived Ta Hisar S45 myeloid lines became clonal, as defined by glucose phosphate isomerase (GPI) polymorphism, and the virulent line became attenuated. The two lines retained phenotypic profiles indicative of a myeloid origin but coexpressed some lymphoid antigens (CD2, CD4, CD8), although not CD3. Cloned schizont-infected lines, representing the three parasite GPI isotypes which constituted the virulent line, expressed similar patterns of myeloid and lymphoid markers to the virulent parent line. Some schizont-infected clones failed to establish as lines during the early weeks of culture because the cells died as the parasites differentiated into merozoites at 37 degrees C, the temperature at which schizont-infected cells normally grow exponentially. These results provided no evidence that prolonged culture induces preferential growth or loss of particular host cell types. However, a number of the alterations in host cell surface antigens induced by prolonged culture were shown to be linked to permanent changes in the parasite genome.     

Genetic diversity of Babesia bovis in virulent and attenuated strains

The aim of this study was to compare the genetic diversity of the single copy Bv80 gene sequences of Babesia bovis in populations of attenuated and virulent parasites. PCR/ RT-PCR followed by cloning and sequence analyses of 4 attenuated and 4 virulent strains were performed. Multiple fragments in the range of 420 to 744 bp were amplified by PCR or RT-PCR. Cloning of the PCR fragments and sequence analyses revealed the presence of mixed subpopulations in either virulent or attenuated parasites with a total of 19 variants with 12 different sequences that differed in number and type of tandem repeats. High levels of intra- and inter-strain diversity of the Bv80 gene, with the presence of mixed populations of parasites were found in both the virulent field isolates and the attenuated vaccine strains. In addition, during the attenuation process, sequence analyses showed changes in the pattern of the parasite subpopulations. Despite high polymorphism found by sequence analyses, the patterns observed and the number of repeats, order, or motifs found could not discriminate between virulent field isolates and attenuated vaccine strains of the parasite.