Tracking fungi in soil with monoclonal antibodies

Species of the genus Trichoderma are ubiquitous soil-borne fungi that exhibit antagonism towards a number of economically important plant-pathogenic fungi and oomycetes. This review discusses recent developments in the use of monoclonal antibodies to detect these fungi in their natural soil environments and to quantify their population dynamics during antagonistic interactions with saprotrophic competitors in soil-based systems. Immunological approaches to detection and quantification are examined in relation to conventional plate enrichment techniques and to nucleic acid-based procedures. An example of recent research using a mAb-based assay to quantify the effects of saprotrophic competition on the growth of Trichoderma isolates in mixed species, soil-based, microcosms is presented. Future technological developments in immunoassays for tracking Trichoderma populations in soil are discussed and results presented showing the accurate detection and visualization of a plant growth-promoting isolate of T. hamatum in the rhizosphere of lettuce using mAb-based immunodiagnostic assays.     

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.     

Outbreak of clinical listeriosis in sheep: evaluation from possible contamination routes from feed to raw produce and humans

We report the results of clinical and microbiological investigations on Listeria monocytogenes infections in a flock of 55 sheep and describe the implications for the safety of the raw milk and raw-milk cheeses produced in the on-farm dairy. The outbreak was caused by feeding grass silage, which was contaminated with 5 log10 CFU L. monocytogenes/g. Clinically, although having been fed from the same batch of silage, abortive (nine ewes), encephalitic (one ewe) and septicaemic (four ewes) forms of listeriosis were observed during the outbreak phase. As the starting point of feeding the contaminated silage was known we could calculate an incubation period of 18+/-2 and 26 days for the abortive and the encephalitic form of listeriosis, respectively. Pathologically, the septicaemic cases suffered from Listeria accumulation at comparable numbers in visceral organs but not in the brain. Only a single ewe developed central nervous symptoms and a rhomb-encephalitis was immunohistologically confirmed. In this case the infection proceeded from the nasal mucosa into the brain, with no infections of the liver, spleen and other visceral organs. Sampling of the cheese production chain, the farm environment and the persons living at the farm revealed the exposure of a farm-worker to an isolate genetically indistinguishable from the outbreak clone, obviously through the consumption of faecally contaminated bovine raw milk. The cheese under processing was free of Listeria because, as a result of intensive consultations, the farmer ensured a proper acidification of the cheese. The epidemiological findings suggest that food safety matters should be assessed in any case where infection of food-producing animals with potential human pathogens is observed.     

Field detection of resistance to isometamidium chloride and diminazene aceturate in Trypanosoma vivax from the region of the Boucle du Mouhoun in Burkina Faso

A longitudinal study assessed the chemoresistance to isometamidium chloride (ISM) and diminazene aceturate (DA) in the region of the Boucle du Mouhoun in Burkina Faso. A preliminary cross-sectional survey allowed the identification of the 10 villages with the highest parasitological prevalences (from 2.1% to 16.1%). In each of these 10 villages, two herds of approximately 50 bovines were selected, one being treated with ISM (1mg/kg b.w.) and the other remaining untreated as control group. All animals (treated and untreated herds) becoming infected were treated with DA (3.5mg/kg b.w.). In total, 978 head of cattle were followed up. Fortnightly controls of the parasitaemia and PCV were carried out during 8 weeks. The main trypanosome species was Trypanosoma vivax (83.6%) followed by Trypanosoma congolense (16.4%). In two villages, less than 25% of the control untreated cattle became positive indicating no need to use prophylactic treatment. These two villages were not further studied. Resistance to ISM was observed in 5 of the remaining 8 villages (Débé, Bendougou, Kangotenga, Mou and Laro) where the relative risk (control/treated hazard ratios) of becoming infected was lower than 2 i.e. between 0.89 (95% CI: 0.43-2.74) and 1.75 (95% CI: 0.57-5.37). In contrast, this study did not show evidence of resistance to DA in the surveyed villages with only 8.6% (n=93) of the cattle relapsing after treatment. Our results suggest that because of the low prevalence of multiple resistances in the area a meticulous use of the sanative pair system would constitute the best option to delay as much as possible the spread of chemoresistance till complete eradication of the disease by vector control operations.     

对FC株猪源性肠毒素型大肠杆菌致病因子的研究

FC菌株是一株从腹泻仔猪粪便中分离的肠毒素型大肠杆菌(Enterotoxigenic E.coli,ETEC)。在MRHA反应中,本菌能凝集人O型、豚鼠、马、绵羊、牛、鸡和兔的红细胞,对人O型和豚鼠红细胞有很高的血凝性,血抗K88和K99血清不能抑制其对豚鼠和绵羊红细胞的血凝。在体外小肠上皮细胞吸附试验中,本菌对仔猪小肠上皮细胞具有强烈的吸附作用;透射电镜和扫描电镜观察证实了FC株菌除表面具有一种纤毛样结构外,还能定居在仔猪小肠段。血清学试验结果表明,本菌的O抗原属于O101。K88和987P两种抗血清均不能凝集本菌,而K99和F41抗血清均可凝集。对纯化的FC株菌粘着素抗原作等电聚焦和聚丙烯酰胺凝胶电泳分析,结果表明,该菌的粘着素是由等电点分别为4.61和9.78,分子量分别为29500和17500的两种蛋白质抗原所组成。此外,用乳鼠胃内投服试验和兔肠结扎试验证明,该菌只产生热稳定肠毒素。总之,本菌是一株能产生ST的K99,F41的肠毒素型大肠杆菌。     

黑麦日粮中添加不同酶制剂对肉仔鸡生产性能及肠道食糜粘度的影响

本文用１８０只肉仔鸡,进行饲养试验,研究黑麦日粮添加４种不同酶制剂,ＮＱ、ＲＭ１、ＲＭ２和ＲＭ３,对肉仔鸡的生产性能以及肠道食糜及泄殖腔粪样粘度的影响。酶制剂ＮＱ和ＲＭ１含有高的木聚糖酶活（７７００ＩＵ／ｇ,３４５０ＩＵ／ｇ）能极显著改进（Ｐ＜０．０１）增重（８．４％,１６．０％）和饲料转化率（１５．４％,１４．４％）,ＲＭ２,含有相对低的木聚糖酶（６０ＩＵ／ｇ）和相对高的阿拉伯呋喃糖苷酶活（５．３ＩＵ／ｇ）,产生类似的效果（Ｐ＜０．０１）：增重提高１０．５％,饲料转化率提高１１．８％,然而ＲＭ３,由于含木聚糖酶活较低（５ＩＵ／ｇ）和较高的蛋白酶活（８０ＩＵ／ｇ）,则对增重无效,但亦可改善饲料转化率（６．４％,Ｐ＜０．０５）。上述酶制剂在降低肠道食糜和泄殖腔粪样粘度方面与增重和饲料转化率有相同的趋势。一般说来,日粮中添加的酶量与食糜相应的粘度成倒数关系。这些结果证明：具有较高木聚糖酶活的酶制剂能明显降低肠道食糜和泄殖腔粪样的粘度,从而改善饲喂黑麦日粮肉仔鸡的生产性能。     

Symbiotic N<Subscript>2</Subscript> fixation and nitrate utilisation in irrigated lucerne (<Emphasis Type="Italic">Medicago sativa</Emphasis>) systems

Symbiotic N₂ fixation by lucerne (Medicago sativa) has capacity to provide significant inputs of N to agro-ecosystems, and the species has also been shown to scavenge soil mineral N and thus act as a sink for excess reactive N. The balance between these two N cycle processes was investigated in an extensive irrigated lucerne growing region where nitrate contamination of groundwater has been reported. We sampled 18 permanent pure lucerne stands under irrigation for standing dry matter, total shoot N, and N₂ fixation using ¹⁵N natural abundance along with activity of the inducible enzyme nitrate reductase as indicators of use of soil NO₃⁻ by lucerne. On average 65% of lucerne N was obtained from symbiotic N₂ fixation. Converting standing dry matter estimates to annual N₂ fixation amounts we calculated average N₂ fixation of 311 kg N/ha, including N in roots and nodules. Uptake of N from soil by lucerne was calculated to be 181 kg N/ha/year. We were not able to identify the source of this soil mineral N, although nitrate reductase activity of lucerne was higher than that of non-N₂ fixing species examined.     

Copper‐Induced Oxidative Stress and Responses of the Antioxidant System in Roots of Medicago sativa

The effects of various copper (Cu) concentrations on the antioxidative system in the roots of Medicago sativa were explored. The results indicated that the Cu content of the roots reached a value of 854 μg g^?1 DW at 10 μm Cu and a value of 4415 μg g^?1 DW at 100 μm Cu, suggesting that M. sativa has better ability to tolerate and accumulate Cu than other Cu‐bioaccumulators, and is a potential plant for phytoremediation. Treatment with Cu resulted in a significant increment in the levels of H₂O₂, O₂˙^? and OH˙. The reduced form of ascorbate and glutathione reached a peak at 30 μm Cu, and was followed by a sharp depletion to a lower level than that of the control. In contrast, the levels of the oxidised forms of ascorbate and glutathione showed a progressive increment with increasing Cu concentrations, suggesting that the antioxidant system was unable to cope with Cu stress at higher Cu levels. Under the Cu concentrations tested, the activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) increased at lower Cu concentrations, and then decreased, reaching a maximum at 30 μm of Cu for APX and GR, at 10 μm for CAT, whereas the activities of guaiacol peroxidase (POD, EC 1.11.1.7) were gradually increased with increasing Cu concentrations. PAGE analysis of superoxide dismutase (SOD, EC 1.1.5.1.1) revealed that one band is a Mn‐SOD and five bands are identified as Cu, Zn‐SOD, whereas Fe‐SOD isoforms were not found in the roots of alfalfa. Cu at 10–100 μm increased the intensity of constitutive isozymes of CAT, APX and POD, whereas it decreased the intensity of isozymes of glucose‐6‐phosphate dehydrogenase (G6PDH, EC 1.1.1.49) significantly. The activities of lipoxygenases (LOX, EC 1.13.11.12) were gradually augmented with increasing Cu concentrations, demonstrating that LOXs are probably involved in production of lipid hydroperoxides and superoxide anion. There was a continuous and pronounced enhancement in the activity of esterase (EST, EC 3.1.1.1) in roots treated with 10–30 Cu μm , whereas EST activity in roots exposed to above 30 μm Cu declined, suggesting that EST plays a protective role under lower Cu concentrations stress.     

5‐Aminolevulinic Acid Activates Antioxidative Defence System and Seedling Growth in Brassica napus L. under Water‐Deficit Stress

The present study assesses the effects of 5‐aminolevulinic acid (ALA, 0, 0.1, 1 and 10 mg l^?1) on the growth of oilseed rape (Brassica napus L. cv. ZS758) seedlings under water‐deficit stress induced by polyethylene glycol (PEG 6000, 0 and ?0.3 MPa). Water‐deficit stress imposed negative effects on seedling growth by reducing shoot biomass, cotyledon water potential, chlorophyll content and non‐enzymatic antioxidants (glutathione and ascorbic acid) levels. On the other hand, water‐deficit stress enhanced the malondialdehyde (MDA) content, reactive oxygen species (ROS) production, enzymatic antioxidants activities, reduced/oxidized glutathione ratio (GSH/GSSG) and reduced/oxidized ascorbic acid (ASA/DHA) ratio in seedlings. Application of ALA at lower dosages (0.1 and 1 mg l^?1) improved shoot weight and chlorophyll contents, and decreased MDA in rape seedlings, whereas moderately higher dosage of ALA (10 mg l^?1) hampered the growth. The study also indicated that 1 mg l^?1 ALA improved chlorophyll content, but reduced MDA content and ROS production significantly under water‐deficit stress. Lower dosages of ALA (0.1 and 1 mg l^?1) also enhanced GSH/GSSG and ASA/DHA as compared to the seedlings under water‐deficit stress. The antioxidant enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase and superoxide dismutase) enhanced their activities remarkably with 1 mg l^?1 ALA treatment under water‐deficit stress. It was also revealed that 1 mg l^?1 ALA treatment alone induced the expression of APX, CAT and GR substantially and under water‐deficit stress conditions ALA treatment could induce the expression of POD, CAT and GR to a certain degree. These results indicated that 0.1–1 mg l^?1 ALA could enhance the water‐deficit stress tolerance of oilseed seedlings through improving the biomass accumulation, maintaining a relative high ratio of GSH/GSSG and ASA/DHA, enhancing the activities of the specific antioxidant enzymes and inducing the expression of the specific antioxidant enzyme genes.