Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

Histopathological, immunohistochemical and ultraestructural evaluation of inflammatory response in Arius genus fish under experimental inoculation of BCG

The aim of this study was to evaluate the inflammatory response kinetics after experimental inoculation with BCG in the primitive Arius sp. fish. The BCG was applied through the intramuscular injection in the caudal peduncular region, and the samples were collected for the analyses at days 1, 3, 7, 14, 21, and 33 post-injection. Acute phase inflammatory infiltrate was characterized by the predominant mononuclear cells, intersticial edema, and muscular tissue necrosis. As the inflammatory response evolved, a large number of multinuclear giant cells were formed containing the BCG. These giant cells were positive for the S100 protein at the histochemical analysis, which demonstrate the macrofage activity, confirmed by the ultra-structural analysis showing the lack of the cytoplasmic membrane enveloping the many nuclei within the giant cell. These results led to the conclusion that Arius sp. fish injected with the BCG showed a difuse inflammatory response characterized by a large number of mononuclear cells, absence of granuloma formation, and predominant giant cells.

Mycoplasma pneumoniae and/or Chlamydophila pneumoniae inoculation causing different aggravations in cholesterol-induced atherosclerosis in apoE KO male mice

Background: Chamydophila pneumoniae (CP) and/or Mycoplasma pneumoniae ( MP) are two bacteria detected in vulnerable atheromas. In this study we aimed to analyze whether CP and/or MP aggravates atherosclerosis induced by cholesterol-enriched diet in C57BL/6 apoE KO male mice. Thirty male apoE KO mice aged eight weeks fed by a diet containing 1% cholesterol until 32 weeks of age were divided into four groups: the first was inoculated with CP (n = 7), the second with MP (n = 12), the third with both CP + MP ( n = 5), and the fourth with saline (sham n = 6). The animals were re-inoculated at 36 weeks of age, and sacrificed at 40 weeks of age. Two ascending aorta and one aortic arch segments were sampled. In the most severely obstructed segment, vessel diameter, plaque height, percentage of luminal obstruction and the degree of adventitial inflammation were analyzed. The plaque area/intimal surface ratio was obtained by measuring all three segments. The adventitial inflammation was semiquantified (0 absent, 1 mild, 2 moderate, and 3 diffuse). Results: The mean and standard deviation of plaque height, % luminal obstruction, external diameter, the plaque area/intimal surface ratio and the adventitial inflammation values are the following for each group: MP (0.20 +/- 12 mm, 69 +/- 26%, 0.38 +/- 0.11 mm, 0.04 +/- 0.04 and 0.22 +/- 0.67), CP (0.23 +/- 0.08 mm, 90 +/- 26%, 0.37 +/- 0.08 mm, 0.04 +/- 0.03, and 0.44 +/- 0.53), MP + CP ( 18 +/- 0.08 mm, 84 +/- 4.0%, 0.35 +/- 0.25 mm, 0.03 +/- 0.03 and 1.33 +/- 0.82) and sham (0.08 +/- 0.09 mm, 42 +/- 46%, 0.30 +/- 0.10 mm, 0.02 +/- 0.03 and 0.71 +/- 0.76). A wider area of plaque/intimal surface was observed in MP + CP inoculated groups (p = 0.07 and 0.06) as well as an increased plaque height in CP (p = 0.01) in comparison with sham group. There was also an increased luminal obstruction (p = 0.047) in CP inoculated group in comparison to sham group. Adventitial inflammation in MP + CP inoculated group was higher than MP, CP and the sham groups (p = 0.02). Conclusion: Inoculation of CP, MP or both agents in C57BL/6 apoE KO male mice caused aggravation of experimental atherosclerosis induced by cholesterol-enriched diet, with distinct characteristics. CP inoculation increased the plaque height with positive vessel remodeling and co-inoculation of MP + CP caused the highest adventitial inflammation measures.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Effect of bacterial inoculation, plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (Solanum tuberosum)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.

Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Evaluation of the Experimental Inoculation of Cryptococcus albidus and Cryptococcus laurentii in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37A degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Effect of Mn deficiency and legume inoculation on rhizosphere pH in highly alkaline soils

Although plant growth is often limited at high pH, little is known about root-induced changes in the rhizospheres of plants growing in alkaline soils. The effect of Mn deficiency in Rhodes grass (Chloris gayana cv. Pioneer) and of legume inoculation in lucerne (Medicago sativa L. cv. Hunter River), on the rhizosphere pH of plants grown in highly alkaline bauxite residue was investigated. Rhizosphere pH was measured quantitatively, with a micro pH electrode, and qualitatively, with an agar/pH indicator solution. Manganese deficiency in Rhodes grass increased root-induced acidification of the rhizosphere in a soil profile in which N was supplied entirely as NO3-. Rhizosphere pH in the Mn deficient plants was up to 1.22 pH units lower than that of the bulk soil, while only 0.90 to 0.62 pH units lower in plants supplied with adequate Mn. When soil N was supplied entirely as NO3-, rhizosphere acidification was more efficient in inoculated lucerne (1.75 pH unit decrease) than in non-inoculated lucerne (1.16 pH unit decrease). This difference in capacity to lower rhizosphere pH is attributable to the ability of the inoculated lucerne to fix atmospheric N2 rather than relying on the soil N (NO3 ) reserves as the non-inoculated plants. Rhizosphere acidification in both Rhodes grass and lucerne was greatest in the meristematic root zone and least in the maturation root zone.

Effects of habitat modification in mangroves on the structure of mollusc and crab assemblages

The abundance and species richness of mollusc and crab assemblages were examined in a subtropical mangrove forest in Moreton Bay, Queensland, Australia, which has been disturbed and damaged by the construction of a wooden boardwalk and a path. Sections of the forest immediately adjacent to the boardwalk and path were compared with reference areas to determine whether changes to the small-scale structural complexity within the forest affected the benthic fauna. The disturbed area was characterised by having 65-80% fewer pneumatophores, significantly fewer species and individuals of molluscs, but significantly more species and individuals of crabs than the reference areas. The abundance of mangrove pneumatophores and the attached epiphytic algae were manipulated at two sites to determine whether observed differences in these features could account for the differences in the assemblage of molluscs in the disturbed area of the forest compared with reference areas. Five experimental treatments were used: undisturbed controls, pneumatophore removals (abundance reduced by ca. 65%), epiphytic algal removals (algae removed from ca. 65% of pneumatophores), pneumatophore disturbance controls and algal disturbance controls. The experimental reduction of the abundance of mangrove pneumatophores and the associated epiphytic algae led to significant declines (by as much as 83%) in the number of molluscs utilising the substratum in the modified plots. There was no significant difference in the abundance of molluscs in the pneumatophore and algal removal plots suggesting any effect was primarily related to removal of the epiphytic algae from the surface of the pneumatophores. The responses by the biota to the changes in the physical environment demonstrate that even relatively small-scale modifications to the physical structure of subtropical mangrove forests can lead to significant effects on the diversity and abundance of macrobenthic organisms in these habitats. Such modifications have the potential to cause cascading effects at higher trophic levels with a deterioration in the value of these habitats as nursery and feeding grounds. Future efforts at conservation of these estuarine environments must focus on the prevention or reduction of modifications to the physical structure and integrity of the system, rather than just on the prevention of loss of entire patches of habitat. (C) 2000 Elsevier Science B.V. All rights reserved.

A Mox homeobox gene in the gastropod mollusc Haliotis rufescens is differentially expressed during larval morphogenesis and metamorphosis

We have isolated a homeobox-containing cDNA from the gastropod mollusc Haliotis rufescens that is most similar to members of the Mox homeobox gene class, The derived Haliotis homeodomain sequence is 85% identical to mouse and frog Mox-2 homeodomains and 88.9% identical to the partial cnidarian cnox5-Hm homeodomain. Quantitative reverse transcription-polymerase chain reaction analysis of mRNA accumulation reveals that this gene, called HruMox, is expressed in the larva, but not in the early embryo, Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g. Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution, (C) 1997 Federation of European Biochemical Societies.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 X 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. Al; the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Point source inoculation of Mesocyclops (Copepoda : Cyclopidae) gives widespread control of Ochlerotatus and Aedes (Diptera : Culicidae) immatures in service manholes and pits in North Queensland, Australia

This study details the novel application of predacious copepods, genus Mesocyclops, for control of Ochlerotatus tremulus (Theobald) group and Aedes aegypti (L.) mosquito larvae in subterranean habitats in north Queensland, Australia. During June 1997, 50 Mesocyclops sp. I were inoculated into one service manhole in South Townsville. Wet season rainfall and flooding in both 1998 and 2000 was responsible for the dispersal of copepods via the underground pipe system to 29 of 35 manholes over an area of 1.33 km(2). Significant reductions in Aedes and Ochlerotatus larvae ensued. In these habitats, Mesocyclops and Metacyclops were able to survive dry periods, when substrate moisture content ranged from 13.8 to 79.9%. At the semiarid inland towns of Hughenden and Richmond, cracking clay soil prevents drainage of water from shallow service pits where Oc. tremulus immatures numbered from 292-18,460 per pit. Introduction of Mesocyclops copepods into these sites during May 1999 resulted in 100% control of Oc. tremulus for 18 mo. One uninoculated pit subsequently became positive for Mesocyclops with resultant control of mosquito larvae.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.