Search

Percutaneous needle electrolysis (PNE) is a physiotherapy technique that has been shown to be effective in different pathologies such as tendinopathies or mammary fistula. For many years, theoretical bactericidal and germicidal effects have been attributed to this type of galvanic currents, partly explained by the changes in pH that it generates. However, these effects have not yet been demonstrated. The aim of this study was to evaluate the bactericidal effect and the changes in pH caused by PNE. S. aureus were prepared in two different solutions (TSB and saline solution) and in different concentrations (from 9 to 6 Log10 CFU/mL). Bacteria were treated with three experimental PNE doses to assess bacterial death levels and the changes caused to the pH of the medium. The viable cell count showed that all experimental PNE doses had a bactericidal effect against a high concentration (9 Log10 CFU/mL) of S. aureus in saline solution (p S. aureus and the level of this effect was mainly modulated by the solution, the bacterial concentration and the dose. Changes affecting pH were modulated by the type of solution and there was no relationship between this and bacterial death.

The obligate intracellular bacterium Chlamydia abortus causes abortion and constitutes a worldwide threat for livestock. Plant-derived flavonoids have antimicrobial effects against veterinary and human pathogens and may be of help in the fight against C. abortus. The anti-infective efficacy against C. abortus of the flavonoids apigenin and naringenin, and of four of their derived metabolites was explored using an in vitro model of ovine trophoblast cells. Overall, the anti-infective effectiveness was apigenin > naringenin > benzoic acid > 3-(4-hydroxyphenyl) propionic acid ~ 4-hydroxyphenyl acetic acid ~ 4-hydroxybenzoic. At the lowest concentration tested (10 μM), apigenin, naringenin and benzoic acid inhibited the formation of C. abortus inclusions by 80%, 67%, and 39%, respectively. The cytotoxicity and anti-proliferative effects on the trophoblast host cells also differed greatly between the tested compounds. Our findings suggest that flavonoids may be of therapeutic value against C. abortus infection but metabolic conversion has a substantial and variable effect on their anti-chlamydial activity. Our results also support the notion that the mechanisms of anti-infective action may involve combined effects of the compounds against the host cells and the bacteria.

• The ability of the nematode-trapping fungus Arthrobotrys oligospora and the nematode egg parasite Verticillium chlamydosporium to colonize barley (Hordeum vulgare) and tomato (Lycopersicum esculentum) roots was examined, together with capability of the fungi to induce cell wall modifications in root cells. • Chemotropism was studied using an agar plate technique. Root colonization was investigated with light microscopy and scanning electron microscopy, while compounds involved in fungus–plant interactions were studied histochemically. • Only A. oligospora responded chemotropically to roots. Colonization of barley and tomato by both fungi involved appressoria to facilitate epidermis penetration. V. chlamydosporium colonized tomato root epidermis and produced chlamydospores. Papillae, appositions and lignitubers ensheathing hyphae on tomato were also found. Phenolics (including lignin), protein deposits and callose were present in papillae in both hosts. Both fungi were still present in epidermal cells 3 months after inoculation. • Nematophagous fungi colonized endophytically monocotyledon and dicotyledon plant roots. Arthrobotrys oligospora seemed to be more aggressive than V. chlamydosporium on barley roots. Both fungi induced cell wall modifications, but these did not prevent growth. The response of root cells to colonization by nematophagous fungi may have profound implications in the performance of these organisms as biocontrol agents of plant parasitic nematodes. (Less)

Ruminant livestock is the main reservoir of Coxiella burnetii (Cb), but little is known about the role of wildlife and ticks in its epidemiology. The Iberian ibex (Capra pyrenaica, Schinz 1838) population of "Ports de Tortosa i Beseit" (NE Spain) suffers intense tick infestations and low reproduction rates. This study aims to (1) assess the relationship between infection in ibexes (detection of serum antibodies and/or of Cb DNA in tissues) and Cb DNA presence in ticks hosted by the same ibexes; and (2) identify Cb associated risk factors. Between 2011 and 2015, serum (n = 130), spleen (n = 72), lymph node (n = 89) and tick (n = 669) samples from 134 hunter-harvested ibexes were collected. Antibody detection was performed by ELISA and Cb DNA presence was assessed by PCR. Potential risk factors were assessed with regression tree models. Although 30% of the ibexes (39/130; 95%CI, [10%-29.8%]) had antibodies, Cb DNA was detected in only 9.8% of the ibexes (11/112; 95%CI [7.6%-27.25%]). The prevalence of Cb-carrier ticks averaged 10% and exceeded 20% for the genus Haemaphysalis. However, lacking correlation between infection in ibexes and their ticks does not support tick-to-ibex transmission or vice versa. Tree modelling points to host, population and environmental factors as drivers of Cb infection in ticks and suggests connections with the domestic cycle. The percentage of Cb-carrier ticks detected is noteworthy. Along with heavy tick infestations, it suggests vector potential for these tick species, especially for the genera Rhipicephalus and Haemaphysalis. Since vector competence has not been assessed in these tick species, a classic vector role cannot be proposed nor discarded, but promoter factors of vector capacity occur. In addition, the risk of tick-borne infection through tick excreta should not be neglected. While the airborne route is the preeminent route for Cb infection, ticks' contribution to Cb epidemiology deserves further attention.

Chlamydia abortus, like other members of the family Chlamydiaceae, have a unique intracellular developmental cycle that is characterized by its chronic nature. Infection of a flock can remain undetected for months, until abortion occurs the following reproductive season but, to date, neither the location nor the mechanisms that maintain this latent phase are fully understood. Studies have shown that IL-10 produced as a response to certain micro-organisms sustains the intracellular survival of pathogens and increases host susceptibility to chlamydial infections. In order to induce a sustained infection C. abortus, transgenic mice that constitutively express IL-10 were infected and the immunological mechanisms that maintain infection in these mice were compared with the mechanisms of a resistant wild-type mouse strain. Viable bacteria could be detected in different tissues of transgenic mice up to 28 days after infection, as analysed by bacterial isolation and immunohistochemistry. Chronic infection in these mice was associated with an impaired recruitment of macrophages, decreased iNOS activity at the site of infection and a more diffuse distribution of inflammatory cells in the liver. This murine model can be of great help for understanding the immunological and bacterial mechanisms that lead to chronic chlamydial infections.

The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis.

Climate change is an obvious threat to agriculture, food security and conservation of plant genetic resources. Potato is a globally important food. In Peru, there is high variability and diversity of wild and cultivated species, such that they are considered as one of the region’s most vulnerable to the impact of climate change. With these considerations, we report the results of an investigation in which a preliminary screening of the reaction to drought of 67 native potato morphotypes was conducted to verify for differences in morphological indicators of growth, development, and production of tubers in response to continuous irrigation (CI) and restricted irrigation (RI) treatments. In the course of this study, 21 irrigations were applied to plants by CI (every 1-2 days) and, in the same period, 7 irrigations were applied to plants by RI (every 5-6 days). Comparison of characteristics average in all the morphotypes with plants under CI and RI, indicated that RI did not show differences in height of plants, length of internodes, or leaf and terminal leaflet indexes, and that RI reduced the stem thickness and dry weight of foliage. In this study, we highlight the morphotypes whose plant characteristics present higher averages with respect to their clonal counterparts that were treated with CI. The positive relationship between the number and weight of tubers harvested, as well as the significant reduction of both components of low RI yield is confirmed. In addition, we also highlight the response of some morphotypes that present higher tuber yield under RI. The native morphotypes that were identified as tolerant (according their responses in the characteristics of their plants and yield of tubers under RI) are as follows: BGR 19 (“Rayhuana”); BGR 99 (“Yuracc ñahui hualash”); BGR 170 (“Yana utcush”); and BGR 238 (“Muru huayro”).

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. The evolution of its genome has exhibited differences in virulence among strains of the same species. Listeria monocytogenes LIPI-3 (Listeria Pathogenicity Island 3) and LIPI-1 (Listeria Pathogenicity Island 1) are considered responsible for the increased virulence in some strains. The aim of this study was to detect LIPI-1 genes and the llsX gene belonging to LIPI-3 in invasive strains of L. monocytogenes and to establish whether there is a relationship among the invasiveness, presence of the llsX and LIPI-1 genes, and the source of the strains. The results showed that 70% of the strains were invasive, and all these strains except one possessed LIPI-1, which suggests that although there is a correlation between LIPI-1 and invasiveness, the independent mechanisms of LIPI-1 may contribute to invasiveness. In contrast, 35% of the total strains were positive for llsX and were invasive; thus, the results revealed that there is a strong association between llsX and the invasiveness of L. monocytogenes in HEp-2 cells (HeLa contaminant/epithelial in origin). In addition, there is no other association with any other variable in this study. Moreover, the authors found that LIPI-1 and llsX are more frequently found in fresh than in frozen vegetables. Together, the findings provide an approximation for the better understanding of Listeriolysin S (LLS) and its role in the pathogenesis of L. monocytogenes, and a possible relation between virulence factors and food-storage temperature.

Between 2010 and 2013, an outbreak of caseous lymphadenitis (CLA) occurred in a captive stock of Iberian ibexes (Capra pyrenaica, Schinz 1838) maintained for conservation purposes in the National Game Reserve ‘Ports de Tortosa i Beseit’ (PTB), northeastern Spain. The aim of this study was to assess the CLA status in the free-ranging donor population of ibexes. An ELISA test to detect antibodies to CLA was performed in 360 hunter-harvested ibexes between 2007 and 2013. A spatial analysis and recursive partitioning approaches with regression tree models were used to explore CLA-associated risk factors. Nineteen per cent of ibexes had antibodies to CLA. Significant differences in seroprevalence were observed among game management areas but no clear trends of CLA occurrence were observed over the study period. Ibexes from areas of higher population density and living close to aggregation points displayed a higher probability of testing positive to CLA. These results suggest that CLA is endemic in the Iberian ibex population inhabiting PTB and density-dependent risk factors. To the best of our knowledge, this is the first record of CLA maintenance in a free-ranging wild Caprinae population.

Intragastric infection mimics the natural route of infection of Chlamydia abortus (etiological agent of ovine enzootic abortion). In the mouse model, intragastric experimental infection induces very mild signs of infection followed by late term abortions, as it is shown by the natural ovine host. In order to evaluate the immune mechanisms associated to the dissemination of the pathogen from the gastrointestinal tract, we have administered an intragastric dose of C. abortus to pregnant mice. Systemic and local expression of cytokines, tissue colonization and excretion of bacteria after parturition were monitored during pregnancy. Susceptible CBA/J mice showed a higher bacterial colonization of the placenta and excretion of live bacteria after parturition that were related to a higher local IL-10 expression. By contrast, resistant C57BL/6 mouse strain had higher local IFN-γ mRNA expression in the placenta just before parturition and a transient bacterial colonization of the reproductive tract, with no excretion of C. abortus after parturition. In summary, intragastric infection not only mimics the natural route of infection of C. abortus, but can also be useful in order to understand the immunopathogenesis of chlamydial abortion in the mouse.

Background Identifying the aetiological agent of atypical pneumonia in human can sometimes be a tedious process, especially in cases where Mycoplasma pneumoniae, Legionella species and Chlamydia pneumoniae are ruled out. In such cases, a correct anamnesis of the patient is basic to clarify which pathogens might have produced the infection. For this reason, health professionals including veterinarians and laboratory personnel working with zoonotic pathogens should keep their doctors informed. Case presentation A human case of atypical pneumonia linked to Chlamydia abortus is reported. A 47-year-old male, a veterinarian researcher into chlamydiae, developed respiratory symptoms, breathing problems and high fever. Serological analyses ruled out the involvement of several respiratory pathogens, such as M. pneumoniae, Legionella pneumophila, Rickettsia conorii and C. pneumoniae, and Chlamydia abortus was identified as the possible aetiological agent of the infection. The isolation of C. abortus from the patient’s sputum and subsequent molecular analysis confirmed the presence of this microorganism. Conclusion As far as we know, although C. abortus has not been previously described as capable of causing pneumonia in humans, this is the first reported case of atypical pneumonia in which C. abortus is thought to have played an aetiological role.

Caprine tuberculosis is caused by bacteria of the Mycobacterium tuberculosis complex (Mycobacterium bovis and Mycobacterium caprae). Although typical tuberculoid granulomata are usually observed in the lungs and lymph nodes of infected goats, the presence of cavitary lesions with exuberant mycobacterial growth is also a common feature in this species. The aim of this study was to characterize the immunological mechanisms that lead to liquefaction and cavity formation by comparing granulomata and cavitary lesions. Samples from animals positive by skin testing were collected for microscopical and immunohistochemical examination. Samples were also collected for analysis of cytokine gene expression in the lesions by real time polymerase chain reaction. There were marked differences between granulomata and cavitary lesions. In cavitary lesions there was a substantial population of neutrophils and a significant decrease in the number of CD4(+) T cells, with concomitant increases in other T-cell populations (CD8(+) and cells expressing the γδ form of the T-cell receptor). The enzyme iNOS was strongly expressed by macrophages in the cavitary lesions. There was no difference in the balance of pro- and anti-inflammatory cytokine gene expression in the lesions. These findings suggest that cavitary lesions are reactivation sites, where conditions are optimal for Mycobacterium proliferation and that immunological mechanisms may underlie the severe destruction of lung tissue that characterizes the cavitary pathology.

Few studies have investigated the role of raptors as natural reservoirs of Chlamydiaceae spp. and the preferred anatomical sites where these bacteria can be detected in non-symptomatic wild birds. We investigated the occurrence of Chlamydiaceae in 54 non-symptomatic adult free-living birds belonging to 14 species sampled upon reception in a raptor rehabilitation centre in Spain, and ten juvenile birds from five species born and reared in the centre for subsequent release into the wild. Swabs from conjunctivae, choanae and cloacae were taken to detect Chlamydiaceae DNA by a family-specific polymerase chain reaction (PCR) and a nested genus- and species-specific PCR. Chlamydiaceae DNA was detected in adult raptors belonging to 12 species (85.7%), mainly in conjunctival (40.6%) and, to a lesser extent, choanal (17.2%) swabs, but never in cloacal samples. Neither the genus nor the species of Chlamydiaceae could be confirmed by the nested PCR assay. Our results suggest that most of the raptor species investigated, especially the Common Kestrel (Falco tinnunculus) and Eurasian Griffon Vulture (Gyps fulvus), can be natural reservoirs of Chlamydiaceae spp. in the wild. Failure to identify the organisms at genus and species level might have been due to the poor quality and low concentration of DNA in the samples or to the presence of hitherto unclassified Chlamydiaceae species.

The entomopathogenic fungi Beauveria bassiana, Lecanicillium dimorphum and L. cf. psalliotae can survive and colonize living palm tissue as endophytes. The molecular interaction between these biocontrol agent fungi and the date palm Phoenix dactylifera L. was investigated using proteomic techniques. Field date palms inoculated with these fungi were analyzed 15 and 30 days after inoculation in two independent bioassays. In vitro date palms were also inoculated with B. bassiana or L. cf. psalliotae. Qualitative and quantitative differences in protein accumulation between controls (not inoculated) and inoculated palms were found using 2-DE analysis, and some of these responsive proteins could be identified using MALDI/TOF-TOF. Proteins involved in plant defence or stress response were induced in P. dactylifera leaves as a response to endophytic colonization by entomopathogenic fungi in field date palms. Proteins related with photosynthesis and energy metabolism were also affected by entomopathogenic fungi colonization. A myosin heavy chain-like protein was accumulated in in vitro palms inoculated with these fungi. This suggests that endophytic colonization by these entomopathogenic fungi modulates plant defence responses and energy metabolism in field date palms and possibly modulates the expression of cell division-related proteins in in vitro palms at proteomic level.

Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.

Chlamydophila (C.) abortus is an obligate intracellular bacterium able to colonize the placenta of several species of mammals, which may induce abortion in the last third of pregnancy. The infection affects mainly small ruminants resulting in major economic losses in farming industries worldwide. Furthermore, its zoonotic risk has been reported in pregnant farmers or abattoir workers. Mouse models have been widely used to study both the pathology of the disease and the role of immune cells in controlling infection. Moreover, this animal experimental model has been considered a useful tool to evaluate new vaccine candidates and adjuvants that could prevent abortion and reduce fetal death. Future studies using these models will provide and reveal information about the precise mechanisms in the immune response against C. abortus and will increase the knowledge about poorly understood issues such as chlamydial persistence.

Surveys (in 2002 and 2003) were performed for fungal endophytes in roots of 24 plant species growing at 12 sites (coastal and inland soils, both sandy soils and salt marshes) under either water or salt stress in the Alicante province (Southeast Spain). All plant species examined were colonized by endophytic fungi. A total of 1830 fungal isolates were obtained and identified by morphological and molecular [internal transcribed spacer (ITS) and translation elongation factor-1α gene region (TEF-1α) sequencing] techniques. One hundred and forty-two fungal species were identified, belonging to 57 genera. Sterile mycelia were assigned to 177 morphospecies. Fusarium and Phoma species were the most frequent genera, followed by Aspergillus, Alternaria and Acremonium. Fungal root endophytic communities were influenced by the soil type where their respective host plants grew, but not by location (coastal or inland sites). Fusarium oxysporum, Aspergillus fumigatus and Alternaria chlamydospora contributed most to the differences found between endophytic communities from sandy and saline soils. Host preference was found for three Fusarium species studied. Fusarium oxysporum and Fusarium solani were especially isolated from plants of the family Leguminosae, while Fusarium equiseti showed a preference for Lygeum spartum (Gramineae). In some cases, specificity could be related to intra-specific variability as shown by sequencing of the TEF-1α in the genus Fusarium.

Ovine enzootic abortion (OEA) is caused by Chlamydophila abortus, an intracellular bacterium which acts by infecting the placenta, causing abortion in the last term of gestation. The main prevention strategy against OEA is the vaccination of flocks. An effective vaccine against C. abortus must induce a Th1-like specific immune response, which is characterized by the early production of IFN-gamma and the activation of CD8(+)T cells. Moreover, vaccine effectiveness could be modulated by the functioning of the innate immunity. The purpose of this study was to ascertain how polymorphonuclear neutrophils (PMNs) and NK cells might influence vaccine-induced protection. The live attenuated 1B vaccine and two inactivated experimental vaccines, adjuvated with aluminium hydroxide (AH) or QS-21 (QS), were used in PMN-depleted or NK cell-depleted mice. For PMN depletion, RB6-8C5 monoclonal antibody, which recognizes GR1(+) receptors (Robben, P.M., LaRegina, M., Kuziel, W.A., Sibley, L.D. 2005. Recruitment of Gr-1(+) monocytes is essential for control of acute toxoplasmosis. The Journal of Experimental Medicine 201, 1761-1769.) was used, while for NK cell-depletion the anti-asialo GM1 polyclonal antibody was used. The depletion of PMNs caused 100% mortality in non-vaccinated mice (NV) and 60% mortality in the AH-vaccinated mice by day 10 p.i., while both groups showed a significant increase in their bacterial burden in the liver by day 4 p.i. The depletion of NK cells caused mortality only in the NV group (50% by day 10 p.i.), although this group and the 1B vaccinated mice showed an increased bacterial burden in the liver at day 4 p.i. Our results suggest that the importance of PMNs in inactivated vaccines depends on the adjuvant chosen. The results also demonstrated that the importance of NK cells is greater in live vaccines than in inactivated vaccines.

Mouse models have been widely used to test candidate vaccines against Chlamydophila abortus infection in mice. Although the induction of a systemic infection by endogenous or intraperitoneal inoculation is a useful tool for understanding the immune mechanism involved in the protection conferred by the vaccination, a different approach is necessary to understand other factors of the infection, such as mucosal immunity or the colonization of target organs. To test whether C. abortus intranasal model of infection in mice is a useful tool for testing vaccines in a first group of experiments mice, were infected intranasally with C. abortus to characterize the model of infection. When this model was used to test vaccines, two inactivated experimental vaccines, one of them adjuvated with QS-21 and another with aluminium hydroxide, and a live attenuated vaccine (strain 1B) were used. Non-vaccinated control mice died within the first 8 days, after displaying substantial loss of weight. Histologically, the mice showed lobar fibrinopurulent bronchointerstitial pneumonia. Prior immunization with QS-21 adjuvated vaccine or 1B vaccine presented mortality and the recipients showed a greater number of T cells in the lesions, especially CD8+ T cells, than the control mice and mice immunized with vaccine adjuvated with aluminium hydroxide. The results confirm that the C. abortus intranasal model of infection in mice is a useful tool for testing vaccines

Light and scanning electron microscopy together with fungal isolation techniques were used to detect entomopathogenic fungi within young and adult date palm (Phoenix dactylifera) petioles and to assess fungal survival in leaf tissues. The entomopathogenic fungi Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium c.f. psalliotae survived inside leaf tissues at least 30 days after inoculation. Entomopathogenic fungi colonized inoculated petioles endophytically and were recovered up to 3 cm from the inoculation site. Fungi were detected inside the parenchyma and sparsely within vascular tissue using microscopy techniques. Our results show that the entomopathogenic fungi used in this study survived and colonized date palm tissues in bioassays both under laboratory and field experimental conditions with no evidence of significant damage.

Low molecular weight chitosan (4.6 kDa) and N-/2(3)-(dodec-2-enyl)succinoyl/-derivatives of different degrees of substitution were tested for their antimicrobial activity against Escherichia coli, Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, Candida kruisei and Fusarium oxysporum f. sp. radicis lycopersici. The results indicated that the chitosans show high activities against all bacteria, yeast and filamentous fungus. In fact, they suppressed fungal colony growth and inhibited fungal spore germination at 0.01% (w/v) concentration. A higher activity of chitosan and its derivatives was obtained with a substitution degree of 3 mol% for E. coli and B. subtilis, while a substitution degree of 9 mol% was usually preferable for increased activity against P. aureofaciens, E. agglomerans and C. kruesei.

Fungal inflorescence rot of date palms, caused by Mauginiella scaettae, was detected at all four sites surveyed in Elx, SE Spain. There was a higher incidence of the disease in male than in female inflorescences. The pathogen responsible for the disease was isolated and identified as M. scaettae. Sequencing of the internal transcribed spacer (ITS) region of this fungus demonstrated that it is close to Phaeosphaeria.

This study provides evidence that egg-parasitic nematophagous fungi, Pochonia chlamydosporia, Pochonia rubescens and Lecanicillium lecanii, can also reduce root colonisation and root damage by a fungal pathogen. Interactions of nematophagous fungi with the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt), and their influence on severity of the root disease it causes were studied in laboratory and pot experiments. In Petri dish experiments the three nematophagous fungi reduced colonisation of barley roots by Ggt and also reduced necrotic symptoms. On the contrary, root colonisation by nematophagous fungi was unaffected by Ggt. In growth tube experiments, the three nematophagous fungi again reduced Ggt root colonisation and increased effective root length of barley seedlings. This was true for both simultaneous and sequential inoculation of nematophagous fungi versus Ggt. In the pot experiments the inoculum of the tested fungi in soil was applied in the same pot, as a mixture or in layers, or in coupled pots used for wheat grown with a split-root system. The nematophagous fungi P. chlamydosporia (isolate 4624) and L. lecanii (isolate 4629), mixed with Ggt or in split root systems with the pathogen, promoted growth of wheat (i.e. increased shoot weight), although no disease reduction was found. In split root systems, lower levels of peroxidase activity were found in seedlings inoculated with Ggt in combination with the nematophagous isolates 4624 and 4629 than when the take-all fungus was applied alone. Our results show that nematophagous fungi reduce root colonisation by Ggt, root damage and stress induced senescence in Ggt-inoculated plants.

Chlamydophila abortus is the aetiological agent of enzootic abortion in small ruminants in which it infects the placenta to cause abortion during the last trimester of gestation. In a mouse model, a Th1 immune response involving IFN-gamma production and CD8+ T cells is necessary for the infection to be resolved. The authors previously demonstrated that infection with Nippostrongylus brasiliensis, a rodent gastrointestinal nematode extensively used in experimental models to induce Th2 responses, alters the specific immune response against C. abortus infection, increasing bacterial multiplication in liver and reducing specific IFN-gamma production. The aim of the present work was to clarify whether a Th2 immune response has any influence on the success of vaccination using both inactivated and attenuated vaccines. The results showed that the Th2 response established prior to vaccination did not influence the induction of protection offered by the vaccines. However, the effectiveness of this protective response can be altered, depending on the adjuvant employed in the inactivated vaccines, when the Th2 response is established after vaccination, just before challenge with C. abortus.

Wildlife and notably deer species - due to the increasing relevance of deer farming worldwide - may contribute to the maintenance of Coxiella burnetii, the causal agent of Q fever. Currently, there are no precedents linking exposure to deer species with human Q fever cases. However, a human case of Q fever was recently diagnosed in a red deer (Cervus elaphus) farm, which led us to investigate whether deer could be a source for environmental contamination with C. burnetii and ascertain the implication of C. burnetii in reproductive failure in the farm. Blood serum and vaginal swabs were collected from hinds either experiencing or not reproductive failure and tested to detect the presence of antibodies and DNA, respectively, of C. burnetii, Chlamydia abortus, Neospora caninum and Toxoplasma gondii. Serology and PCR results suggest C. burnetii was the primary cause of the reproductive failure. We identified vaginal shedding of C. burnetii in hinds, confirming red deer as a source of Q fever zoonotic infection., EU FP7: ANTIGONE. Grant Number: 278976. CDTI (Centro para el Desarrollo Tecnológico Industrial, Spanish Ministry for Economy and Competitiveness).

Chlamydiaceae are intracellular bacteria responsible for a variety of infections, ranging from asymptomatic to very severe, in humans and animals. We have investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in Chlamydophila abortus infection using PARP-1−/− and their littermates PARP-1+/+ mice. Infection was resolved more efficiently by PARP-1−/− than PARP-1+/+ mice. However, the inflammatory response was similar in both strains, suggesting a potential role for PARP-1 in the cross-talk between this microorganism and the host cells. PARP-1−/− fibroblasts showed a 10-fold lower rate of chlamydiae production than PARP-1+/+. Moreover, a strong inhibition of bacterial production was also observed after pharmacological inhibition of PARP-1 activity in McCoy cells. Likewise, PARP-1 inhibition induced a higher level of cell death of infected cells, interfering in this way with the normal bacterial cell cycle. Overall, we identify PARP-1 as a new molecule involved in chlamydial developmental cycle, although the intrinsic mechanisms deserve further studies.

A Chlamydophila abortus-induced abortion model was carried out on the basis of the experimental infection of ewes at day 75 of gestation. The infection induced abortions and the birth of weak lambs during the last 3 weeks of pregnancy. To study the kinetics of the infection in the placenta and in other organs, infected ewes were killed at 105, 120, and 130 days of gestation and also several days after abortion or parturition. Infected ewes developed a systemic infection that caused a mild and transient pneumonia and focal hepatitis. Pathologic changes were observed in placentas at 120 day of gestation, although the lesions varied between animals and even between placentomes of the same placenta. The first placental area infected was the maternal stroma and epithelium next to the intercaruncular areas, where neutrophilic response seemed to control the infection. A substantial degree of multiplication of C. abortus was then observed in the trophoblast cells of the placentome, periplacentomal choriallantoic membranes, and hilius, with an inflammatory exudate composed mainly of neutrophils, some macrophages, and very scarce lymphocytes. After abortion, the lesions affected the intercotyledonary areas of the aborted placentas, whereas in the uterus significant lymphocyte infiltration was observed, together with a rapid decrease of the C. abortus antigen in the degenerated caruncular tissues.

The protective efficacy of two inactivated commercial (A, B) and two new inactivated vaccines (M7, QS) against ovine enzootic abortion was determined in two separate experiments in sheep. Vaccine A contained chlamydiae propagated in chicken embryos, adjuvated with Marcol 82, and vaccine B contained chlamydiae cultured in cell monolayers, adjuvated with aluminium hydroxide. For the preparation of the experimental vaccines, Chlamydophila abortus AB7 strain was cultured in McCoy cells and adjuvated with QS-21 (QS) or Montanide ISA 773 (M7). The ewes were vaccinated twice subcutaneously and challenged at 90 days of gestation. Protection was evaluated by clinical, bacteriological and serological examinations, and compared to two control groups: one of infected but not vaccinated ewes, and another of vaccinated but not infected ewes. The experimental vaccines induced considerably better protection than the two commercial ones. The new vaccine M7 especially showed no abortions, a good antibody response, the highest newborn lamb weights and the lowest level of C. abortus shedding at lambing.

Chlamydophila abortus is a gram-negative obligate intracellular bacterium and the etiological agent of ovine enzootic abortion (OEA), an economically important disease in many countries. Inactivated vaccines have been reported to induce immunity in ewes and they have been used for many years. However, some outbreaks have been reported in correctly vaccinated flocks, so it is clear that new vaccines are necessary to address adequate protection and to avoid the shedding of the microorganism. This idea lead us to design inactivated vaccines, in a previously established mouse model, evaluating different inactivation procedures and new adjuvants. To assess the protection conferred, the results were analyzed on the basis of clinical signs and the isolation of C. abortus from spleen. These findings were correlated with the immune response induced by the vaccines, as determined by the production of C. abortus -specific IFN-γ and IL-4 from splenocyte cultures and the detection of IgG isotypes in serum. BEI was found to be the best C. abortus -inactivation procedure. The inactivated vaccines adjuvated with QS-21 (QS) or Montanide 773 (M7) induced the best protection both against homologous and heterologous challenge, with an adequate (Th1-like) immune response. Finally, these selected vaccines were evaluated in a pregnant mouse model, in which they were seen to confer good protection and to avoid the C. abortus persistence in uterus after delivery. With these results, this mouse model could be considered as an adequate tool for selecting and optimizing effective vaccines against OEA.

Chlamydophila abortus (Chlamydia psittaci serotype 1) is an obligate intracellular bacterium and the etiological agent of enzootic abortion in small ruminants. The disease is serious because of the economic losses it causes and the potential zoonotic risk for pregnant women (36). Mouse models have been widely used to study the immune response to this bacterium (5, 7, 10, 30) and the pathogenesis of chlamydial abortion. It has been observed that the inoculation of pregnant mice with C. abortus causes late-term abortions similar to those observed in cases of natural or experimentally induced abortion in ruminants (6). In experimental infection, the establishment of an effective immune response after the systemic spread of C. abortus is able to eliminate the infection in every organ except the placenta of pregnant animals, where the multiplication of the bacterium induces abortion (6). The effective immune response observed in the spleen and liver involves neutrophils, which act as a first line of defense (3) and participate in the recruitment of other leukocyte subpopulations (32). Also, a strong Th1-like specific immune response is rapidly induced, involving the production of proinflammatory cytokines, especially gamma interferon (IFN-γ) (30), and activation of T cells, with CD8+ T cells playing an important role in the resolution of the infection (7, 10). However, it has been reported recently that an interleukin 12 (IL-12)-dependent overproduction of IFN-γ may result in an increase in morbidity and pathology (9). The role of B cells in the immune response against C. abortus has been poorly studied, although it has been demonstrated that the passive transfer of specific antibodies protects pregnant animals against chlamydial abortion (11). Thus, B cells not only play a pivotal role in the production and amplification of humoral immune responses but also act as antigen-presenting cells (APCs) in the generation of T-cell-mediated immune responses (22). With the development of B-cell-deficient mice (μMT) through disruption of the transmembrane portion of the μ chain gene (21), the functions of B cells other than antibody production can readily be studied in infections in which the cellular immune response is important. Recent studies have identified several of these functions in the response against parasites, bacteria, and viruses. B cells play an immunomodulatory role in Schistosoma mansoni infection (19) and are involved in protective immunity against Salmonella enterica serovar Typhimurium (31). The lack of B cells leads to a suboptimal vaccinal response to Toxoplasma gondii (38) and to a delay in dissemination of and development of lung pathology in response to Mycobacterium tuberculosis (1). B cells have a role in early protective immunity against Francisella tularensis infection (8), play a nonantibody role during primary (26) and secondary (23) respiratory infection with Bordetella pertussis, and mediate innate and CD4+-T-cell-specific immune responses in herpes simplex virus infections (12). Several studies have been performed with another species of the family Chlamydiaceae, Chlamydia trachomatis, producing contradictory results (20, 33, 40, 44). Thus, in order to deepen our knowledge of the immune mechanisms involved in C. abortus clearance, it is necessary to study the potential functions of B cells in primary and secondary infection. In this study, we placed special emphasis on the relationship between the lack of B cells and neutrophilia, which has been described in the immune response to some intracellular pathogens (2, 39), and on the incidence of this correlation in the early proinflammatory cytokine response and subsequent inflammation-associated pathology.

An experimental mouse model to analyse the interaction between the immune responses elicited following infection with Nippostrongylus brasiliensis and Chlamydophila abortus has been established. Mice infected withC. abortus 7 days after N. brasiliensis showed an increased bacterial multiplication in spleen and liver compared to bacteria-alone infected mice. However the morbidity of these mice, expressed as weight loss, was significantly lower. Analysis of the immune responses elicited showed that spleen from co-infected mice had reduced IFN-gamma production in response to C. abortus antigen. The bias towards a type 2 response in co-infected mice was confirmed by an increase in the production of IL-4 and in the lower ratio IgG2a/IgG1. In pregnant mice co-infection caused a delay in the time of abortion and an increased systemic susceptibility to C. abortus infection.

The present investigation deals with the main factors involved in early infection of nematode eggs by fungal parasites. We studied the effect of hydrophobicity on appressorium formation by germlings of Pochonia rubescens (syn. Verticillium suchlasporium), P. chlamydosporia (syn. V. chlamydosporium) and Lecanicillium lecanii (syn. V. lecanii). Appressoria were frequently formed on hydrophobic surfaces such as polyvinyl chloride or polystyrene and were infrequently formed on hydrophilic materials such as glass or aluminium. Infected eggs probed with the FITC-labelled lectin Concanavalin-A showed intense labelling corresponding to appressoria formed by fungal parasites on the eggshell surface. Proteolytic activity was found in extracts from conidia and germlings of fungal parasites (especially P. chlamydosporia) in the absence of nematode eggs. Addition of the serine proteinase inhibitors phenylmetylsulphonyl fluoride (PMSF) or diisopropyl fluorophosphonate (DFP) to the extracts reduced their proteolytic activity. PMSF was the most effective inhibitor. Zymography also revealed proteolytic activity in extracts from the three fungi tested. This activity mostly corresponded to bands of Rf's of substrate degradation similar to that of purified main protease (P32) from P. rubescens. Other bands with molecular weight higher than P32 (low Rf) were found especially for P. chlamydosporia extracts. For L. lecanii only bands of low Rf were found. Serum anti-P32 partially inhibited proteolytic activity of extracts from conidia and germlings. Application of PMSF and DFP to the inoculum, reduced egg penetration for the three species studied. PMSF caused the highest reduction in eggs infected by L. lecanii, while DFP significantly reduced egg infection by both P. chlamydosporia and L. lecanii. Our results therefore show hydrophobicity, appresorium formation and protease production as factors involved in early parasitism of nematode eggs.

Background: In 2010, an Iberian ibex (Capra pyrenaica hispanica) stock reservoir was established for conservation purposes in north-eastern Spain. Eighteen ibexes were captured in the wild and housed in a 17 hectare enclosure. Once in captivity, a caseous lymphadenitis (CLA) outbreak occurred and ibex handlings were carried out at six-month intervals between 2010 and 2013 to perform health examinations and sampling. Treatment with a bacterin-based autovaccine and penicillin G benzatine was added during the third and subsequent handlings, when infection by Corynebacterium pseudotuberculosis was confirmed. Changes in lesion score, serum anti-C. pseudotuberculosis antibodies and haematological parameters were analyzed to assess captivity effects, disease emergence and treatment efficacy. Serum acute phase proteins (APP) Haptoglobin (Hp), Amyloid A (SAA) and Acid Soluble Glycoprotein (ASG) concentrations were also determined to evaluate their usefulness as indicators of clinical status.Once in captivity, 12 out of 14 ibexes (85.7%) seroconverted, preceding the emergence of clinical signs; moreover, TP, WBC, eosinophil and platelet cell counts increased while monocyte and basophil cell counts decreased. After treatment, casualties and fistulas disappeared and both packed cell volume (PCV) and haemoglobin concentration significantly increased. Hp, SAA and ASG values were under the limit of detection or showed no significant differences. Conclusions: A role for captivity in contagion rate is suggested by the increase in antibody levels against C. pseudotuberculosis and the emergence of clinical signs. Although boosted by captivity, this is the first report of an outbreak of caseous lymphadenitis displaying high morbidity and mortality in wild ungulates. Treatment consisting of both vaccination and antibiotic therapy seemed to prevent mortality and alleviate disease severity, but was not reflected in the humoural response. Haematology and APP were not useful indicators in our study, perhaps due to the sampling frequency. Presumably endemic and irrelevant in the wild, this common disease of domestic small ruminants is complicating conservation efforts for the Iberian ibex in north-eastern Spain.

Chlamydophila abortus (Chlamydia psittaci serotype 1) is a gram-negative intracellular bacterium that replicates in cell phagosomes, thus preventing their fusion with lysosomes (12). This bacterium, which has been recorded worldwide, is the etiologic agent of enzootic abortion in small ruminants, infecting the placenta and causing abortion during the last trimester of gestation (35). In addition to the economic losses it causes, this pathogen also represents a potential zoonotic risk for pregnant women (7). Mouse models have been widely used to study the pathogenesis and immune response to chlamydial infections. Mice experimentally infected with the bacteria show a fever syndrome followed by abortion. This syndrome is similar to that presented in the natural infection of chlamydial abortion in humans and small ruminants. The host response is immediately activated in organs such as the liver and the spleen. Meanwhile, the bacteria replicate in the placenta, which is an immunocompromised organ (6). Although innate immune mechanisms, especially neutrophils, play an important role (3), chlamydial infection is ultimately controlled by a specific Th1 immune response characterized by the production of high levels of gamma interferon (IFN-γ) and the presence of T cells, particularly CD8+ T cells (5, 9, 11, 25, 26). However, the mechanisms involved in the development of this cellular immune response have been poorly studied in C. abortus infection. In murine experimental infections with other species of the family Chlamydiaceae, such as Chlamydia trachomatis and Chlamydophila pneumoniae, this response has been defined, at least in part, as being interleukin-12 (IL-12)-dependent (15, 21). We have recently reported that IL-12 is produced early during C. abortus infection (5), and it has been reported that treatment with exogenous IL-12 confers immediate and long-term protection in susceptible BALB/c mice intranasally infected with C. abortus (17). IL-12 is a heterodimeric cytokine, which is produced primarily by phagocytic cells and dendritic cells in response to infections caused by intracellular pathogens such as bacteria (19, 39), fungi (10, 23), protozoa (1, 14), and viruses (24, 28). It has been reported that IL-12 increases NK cell and T-lymphocyte cytotoxic activity, favors Th1 differentiation, and triggers the production of IFN-γ and other proinflammatory cytokines (22, 41). However, some recent findings point to a more complicated picture, the role of IL-12 in the immune response to certain intracellular pathogens seeming to be ambiguous. Indeed, this cytokine has both protective and pathogenic functions in infections with species such as Plasmodium (43, 44) and Leishmania (37). Furthermore, in the host response to some virus infections, a Th1 response was established in the absence of IL-12. This response was characterized by both T-cell IFN-γ release and immunoglobulin G2a (IgG2a) production (29, 42). The aim of this study, therefore, was to further define the role of endogenous IL-12 in host resistance against C. abortus infection. For this purpose, we used wild-type (WT) C57BL/6 mice, a strain that is relatively resistant to chlamydial infection (8), and IL-12 p40-deficient mice (IL-12−/−) to assess the IL-12-mediated mechanisms that control the primary replication of the bacteria in the immune response to C. abortus infection.

A new commercially available ELISA (ELISAr-Chlamydia) for detecting antibodies against Chlamydophila abortus has been evaluated using sheep field serum samples. The ELISA is based on a recombinant antigen which expresses part of a protein from the 80–90 kDa family that is specific to C. abortus . Sera (105) from six flocks with confirmed ovine chlamydial abortion (OEA) outbreaks were used in this study, as well as sera (258) from 18 flocks which had suffered no OEA in the last lambing. The ELISAr-Chlamydia was compared with the complement fixation test (CFT) and with an ELISA using purified C. abortus elementary bodies (ELISA-EB), employing as reference technique a comparative microimmunofluorescence test that differentiates C. abortus infection from Chlamydophila pecorum infection. The results showed that the sensitivity of ELISAr-Chlamydia was 90.9% with a specificity of 85.9%, the sensitivity of CFT was 71.0% with a specificity of 83.6%, while the sensitivity of ELISA-EB was 95.2% and the specificity was 54.2%. Furthermore, ELISAr-Chlamydia was the test with fewer false positives resulting from positive reactivity to C. pecorum , although 15% of the sera positive for C. pecorum but negative for C. abortus antibodies reacted positively. This study demonstrated with field material that ELISAr-Chlamydia provides the most balanced results between sensitivity and specificity, especially in flocks with no clinical OEA but reactivity to C. abortus .

The aim of this work was to study the role of polymorphonuclear neutrophils (PMNs) in the clearance of infection, and in the development of specific immunity against Chlamydophila abortus (Chlamydia psittaci serotype 1) secondary infection. A pregnant mouse model depleted of neutrophils by the RB6-8C5 monoclonal antibody was used. No clinical signs were observed in depleted or non-depleted mice after secondary infection and no significant differences were observed in the litter size between the infected and control groups. In PMN-depleted mice C. abortus was not detected in the materno-fetal unit but merely produced low, persistent levels of infection in spleen and liver. In the non-depleted mice the level of infection was significantly lower, being resolved during the first few days post-reinfection. In both infected mice groups the immune response in the liver was quickly established and was seen to be composed mainly of CD4(+)T lymphocytes and macrophages. A Th1 response characterized by the presence of IFN-gamma and TNF-alpha in serum was observed during early infection, with significantly higher levels in the non-depleted animals. Our results suggest that PMNs have little influence on the control of C. abortus secondary infection, although they are a first line of defense and may influence the early production of TNF-alpha and IFN-gamma.

Six plant waste substrates: palm leaves (Phoenix dactylifera, Phoenix canariensis, Washingtonia filifera and Chamaerops humilis), Phoenix dactylifera seed and almond mesocarp, have been used to produce entomopathogenic (Verticillium lecanii, Paecilomyces farinosus, Metarhizium anisopliae, Beauveria bassiana) and mycoparasitic (Trichoderma harzianum and Gliocladium virens) fungi. Uninoculated plant waste substrates had very little microbial colonization. In inoculated substrates, germinating conidia of T. harzianum were found on P. canariensis leaves. Widenings of hyphal apex, resembling appressoria, were found for T. harzianum and B. bassiana on palm leaves. Hyphae of G. virens produced adhesives on the surface of palm leaves. The development of antagonistic fungi on plant waste substrates depended on the combination fungus–substrate tested. Of the entomopathogens P. farinosus did not grow on almond mesocarp. This fungus grew well on both P. dactylifera seed and P. canariensis leaves. P. farinosus grew extensively on the edges of the leaf fragments and then started colonizing the leaf surface. V. lecanii colonized and sporulated on almond mesocarp. Of all the plant waste substrates tested M. anisopliae only did grow on almond mesocarp. C. humilis leaves were excellent substrates for the growth and sporulation of both V. lecanii and B. bassiana. B. bassiana grew best on P. dactilyfera seed. The mycoparasitic fungi were, in general terms, faster and better colonizers of plant waste substrates than the entomopathogens. T. harzianum and G. virens sporulated well on P. dactilyfera seed C. humilis leaves also supported abundant growth of T. harzianum. P. canariensis leaves were also readily colonized by the fungus. Our results show that plant waste has potential as substrate for production and perhaps formulation of important biocontrol fungi.

Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.

Monoclonal antibodies (mAbs) were produced to find strain markers essential to the epidemiological study of chlamydial abortion of ruminants. Their specificity was tested on 53 C. psittaci strains including 35 ruminant strains isolated mainly from abortion, belonging to serotype 1 and which are invasive in our mouse model (Rodolakis et al., 1989), and 14 ruminant strains mostly intestinal, belonging to serotype 2 and non-invasive for mouse. One strain specific mAb was obtained reacting only with the non-invasive strain iB1. Six sub serotype 2 mAbs were found. They reacted only with some non-invasive strains. They allowed the distinction of 9 patterns of response among the 14 non-invasive strains. No serotype 2 specific mAbs reacting with all non-invasive serotype 2 strains were selected. In return all the invasive strains reacted with all the 18 serotype 1 specific mAbs produced. No cross-reactivities between invasive and non-invasive strains were observed, whereas common epitopes were demonstrated between invasive strains and avian or feline strains.

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan-resistant but not chitosan-sensitive fungi. The plasma membranes of chitosan-sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan-resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady-state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.

A histological study was carried out on 58 formalin-fixed and paraffin-embedded samples of placenta from sheep and goats that had aborted, and the placental lesions were graded. Sequential histological sections of each cotyledon were then immunostained with specific antibodies and used for PCR detection of Chlamydophila abortus, Coxiella burnetii, Salmonella Abortusovis, Brucella melitensis, Listeria monocytogenes and Toxoplasma gondii. Most of the cotyledons showed different degrees of placentitis. The proportional agreement between the two techniques was 0.879 (kappa value 0.746). C abortus was the most prevalent pathogen. Mixed infections were common.

The present study reports the frequent isolation of the two date palm pathogens Thielaviopsis paradoxa (de Seynes) Hohn and T. punctulata (Hennebert) Paulin, Harrington et McNew from soil of date palm plantations at Elx, south-east Spain, using dilu- tion plate, direct soil plating or by soil treatment either with acetic acid or phenol. The two species showed a high isolation rate. T. punctulata detected from all samples (100% isolation rate), whereas, T. paradoxa showed 52% isolation rate.Total fungal colony count, ranged from 1.1x10 5 -6 x10 5 , CFU/g dry soil. Out of these, T. punculata comprised between 0.2-3.2% and T. paradoxa, between 0.5-4.4%. Both species were characterized by development of thick-walled aleuroconidia either singly (T. punctulata) or in chains (T. paradoxa) in addition to the phialoconidia. The widespread occurrence of the two pathogens in soil may contribute to the possibility of infection of newly transplanted offshoots of date palms.

Aims: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). Methods and Results: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis-lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine-labelled chitosan enters rapidly into conidia of both fungi, in an energy-dependent process. Conclusions: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. Significance and impact of the study: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.

Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.

Pregnant goats were inoculated subcutaneosly with Coxiella burnetii and the course of infection was studied. Abortion in the last third of pregnancy occurred in all infected animals. Tissues from the placenta and other organs were studied before and after abortion by immunohistochemistry and PCR analysis. After infection, mild lesions were observed in several maternal organs, mainly the mammary gland but also the lung and the liver. The trophoblast cells of the choriallantoic membrane were the first target cells of the placenta; there was, however, a substantial delay between initial infection and placental colonization. In the last weeks of pregnancy, just before abortion, massive bacterial multiplication was detected in the placenta. In this stage of infection a necrotic and suppurative placentitis separated the fetal trophoblast cells from maternal syncytial epithelium. Vasculitis was observed in the fetal mesenchyme. A strong maternal T-cell response was detected in the inter-placentomal areas but not in the placentomes, where only neutrophils and smaller numbers of macrophages were associated with the lesions. Neither lesions nor C. burnetii DNA were found in maternal organs in animals maintained until day 120 post-abortion.

The role of the specific cellular immune response is well established in Chlamydiaceae infections, but the importance of each T-cell subset seems to be species-dependent. This study was designed to clarify the role of T-cell subsets in the response to Chlamydophila abortus primary infection. C57BL/6 mice were depleted of CD4+ or CD8+, or both, by monoclonal antibody injections and subsequently infected with C. abortus. Mice were killed at intervals and samples were collected for bacteriological and histopathological analysis. Also carried out were spleen cell culture, cytokine quantification, immunolabelling for C. abortus antigen, and a TUNEL assay for apoptosis. CD8+ T cell-depleted mice all died within 12 days of C. abortus infection, while no mortality was observed in the other groups; surprisingly, CD4+ T cell-depleted mice showed lower morbidity (expressed as weight loss) than did a non-depleted (control) group. CD8+ T cell-depleted mice also differed from the other groups in showing a significantly higher chlamydial burden in the liver. CD8+ T cell-depleted mice also had a higher number of apoptotic cells in hepatic inflammatory foci and showed exacerbated IFN-gamma production by spleen cells after specific stimulation. Simultaneous depletion of both T-cell subpopulations led to a chronic infection, but not to early mortality. It is concluded that CD8+ T cells may play a role in the regulatory control of the CD4+ T-cell response and may have a direct cytotoxic or IFN-gamma-mediated effect on infected cells.

Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection. Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection. The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody. The number of NK cells increased during the infection both in spleen and liver. In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells. NK-depleted mice showed a substantial increase in their susceptibility to C. abortus infection, with high mortality rates and an increased burden of bacteria in the liver. Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci. Serum concentrations of IFN-gamma, a key cytokine in the control of C. abortus infection, were substantially reduced in the NK-depleted mice. To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody. These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci.

Culture filtrates of the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium growing on colloidal chitin showed increasing chitinolytic activity and production of two (32- and 43-kDa) main proteins. Maximum activity was found 18-20 days after inoculation, but V. suchlasporium always displayed higher activity. Zymography of such filtrates on carboxymethyl-chitin-Remazol brilliant violet 5R/acrylamide gels showed five bands of substrate degradation for V. suchlasporium and three for V. chlamydosporium. Filtrates with maximum activity were chromatographed on macroporous cross-linked chitin affinity matrix, showing a peak of main (50-60%) activity, which only contained a 43-kDa protein for both fungi. Zymography and colloidal chitin degradation showed that it was a single endochitinase (CHI43) with optimum pH range of 5.2-5.7. The main isoforms had pIs of 7.6 for V. suchlasporium and 7.9 for V. chlamydosporium. Eggs of the nematode Globodera pallida treated with CHI43 and the serine protease P32 from V. suchlasporium alone or in combination showed surface damage in comparison with controls when examined by scanning electron microscopy.

Neutrophil migration to the site of infection is a critical early step in host immunity to microbial pathogens, in which chemokines and their receptors play an important role. In this work, mice deficient in expression of the chemokine receptor CXCR2 were infected with Toxoplasma gondii and the outcome was monitored. Gene-deleted animals displayed completely defective neutrophil recruitment, which was apparent at 4 h and sustained for at least 36 h. KitW/KitW-v animals also displayed defective polymorphonuclear leukocyte migration, suggesting mast cells as one source of chemokines driving the response. Tachyzoite infection and replication were accelerated in CXCR2−/− animals, resulting in establishment of higher cyst numbers in the brain relative to wild-type controls. Furthermore, serum and spleen cell IFN-γ levels in infected, gene-deleted mice were reduced 60–75% relative to infected normal animals, and spleen cell TNF-α was likewise reduced by ∼50%. These results highlight an important role for CXCR2 in neutrophil migration, which may be important for early control of infection and induction of immunity during Toxoplasma infection.