Prevalence, geographic distribution and risk factors of Eimeria species on commercial broiler farms in Guangdong, China.

BACKGROUND: Coccidiosis is one of the most frequently reported diseases in chickens, causing a significant economic impact on the poultry industry. However, there have been no previous studies evaluating the prevalence of this disease in broiler farms in Guangdong province. Therefore, this study aims to conduct an epidemiological investigation into the occurrence of Eimeria species and associated risk factors in intensive management conditions across four regions in Guangdong province, China. A total of 394 fecal samples were collected from 89 broiler farms in Guangdong province. The prevalence of Eimeria species infection was determined using PCR, and the occurrence of Clostridium perfringens type A was assessed using quantitative real-time PCR. RESULTS: The results showed an overall prevalence of 98.88% (88/89) at the farm level and 87.06% (343/394) at the flock level. All seven Eimeria species were identified, with E. acervulina (72.53%; 64/89), E. tenella (68.54%; 61/89), and E. mitis (66.29%; 59/89) at the farm level, and E. acervulina (36.55%; 144/394), E. mitis (35.28%; 139/394), and E. tenella (34.01%; 134/394) at the flock level. The predominant species combination observed was a co-infection of all seven Eimeria species (6.74%; 6/89), followed by a combination of E. acervulina, E. tenella, E. mitis, E. necatrix, E. brunetti, and E. maxima (5.62%, 5/89). A combination of E. acervulina, E. tenella, E. mitis, E. necatrix, E. brunetti, and E. praecox (4.49%; 4/89) was also observed at the farm level. Furthermore, the study identified several potential risk factors associated with the prevalence of Eimeria species, including farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were identified as potential risk factors associated with prevalence of Eimeria species. Univariate and multivariate analyses revealed a significant association between E. necatrix infection and both grower chickens (OR = 10.86; 95% CI: 1.92-61.36; p < 0.05) and adult chickens (OR = 24.97; 95% CI: 4.29-145.15; p < 0.001) compared to starter chickens at the farm level. Additionally, farms that used groundwater (OR = 0.27; 95% CI: 0.08-0.94; p < 0.05) were less likely to have E. maxima compared to those that used running water. At the flock level, the prevalence of E. tenella was significantly higher in the Pearl River Delta (OR = 2.48; 95% CI: 1.0-6.15; p = 0.05) compared to eastern Guangdong. Interestingly, flocks with indigenous birds were less likely to have E. brunetti (OR = 0.48; 95% CI: 0.26-0.89; p < 0.05) compared to flocks with indigenous crossbred birds. Furthermore, flocks that used anticoccidial drugs (OR = 0.09; 95% CI: 0.03-0.31; p < 0.001) or a combination of vaccines and anticoccidial drugs (OR = 0.06; 95% CI: 0.01-0.25; p < 0.001) were less likely to be positive for E. tenella compared to flocks that only used vaccines. Finally, flocks with C. perfringens type A infection were significantly more likely to have E. necatrix (OR = 3.26; 95% CI: 1.96-5.43; p < 0.001), E. tenella (OR = 2.14; 95% CI: 1.36-3.36; p < 0.001), E. brunetti (OR = 2.48; 95% CI: 1.45-4.23; p < 0.001), and E. acervulina (OR = 2.62; 95% CI: 1.69-4.06; p < 0.001) compared to flocks without C. perfringens type A. CONCLUSIONS: This study conducted an investigation on the prevalence, distribution, and risk factors associated with Eimeria species infection in broiler chickens in Guangdong. The farm-level prevalence of Eimeria species was higher than the previous prevalence figures for other areas and countries. E. brunetti was identified at higher prevalence in Guangdong than previously survived prevalence in different regions in China. Farm location, chicken age, drinking water source, control strategy, and the presence of C. perfringens type A were considered as potential risk factors associated with prevalence of Eimeria species. It is imperative to underscore the necessity for further surveys to delve deeper into the occurrence of Eimeria species under intensive management conditions for different flock purposes.

Seroprevalence of Epizootic Hemorrhagic Disease Virus in Guangdong Cattle Farms during 2013-2017, China.

Epizootic hemorrhagic disease (EHD) is an infectious viral disease caused by epizootic hemorrhagic disease virus (EHDV) and EHDV frequently circulates in wild and domestic ruminants. Sporadic outbreaks of EHD have caused thousands of deaths and stillbirths on cattle farms. However, not much is known about the circulating status of EHDV in Guangdong, southern China. To estimate the seroprevalence of EHDV in Guangdong province, 2886 cattle serum samples were collected from 2013 to 2017 and tested for antibodies against EHDV using a competitive ELISA. The overall seroprevalence of EHDV reached 57.87% and was highest in autumn (75.34%). A subset of positive samples were serotyped by a serum neutralization test, showing that EHDV serotypes 1 and 5-8 were circulating in Guangdong. In addition, EHDV prevalence always peaked in autumn, while eastern Guangdong had the highest EHDV seropositivity over the five-year period, displaying apparent temporal-spatial distribution of EHDV prevalence. A binary logistic model analysis indicated a significant association between cattle with BTV infections and seroprevalence of EHDV (OR = 1.70, p < 0.001). The co-infection of different serotypes of EHDV and BTV raises a high risk of potential genomic reassortment and is likely to pose a significant threat to cattle, thus urging more surveillance to monitor their circulating dynamics in China.

Ancylostoma ceylanicum Infection in a Miniature Schnauzer Dog Breed.

PURPOSE: Canine hookworm disease is a global zoonotic parasitic disease caused by a variety of nematodes in families Ancylostomatidae, including Ancylostoma spp., Necator spp., and Uncinaria spp., in the small intestine (mainly the duodenum) of dogs. The disease is widely distributed in China. The purpose of this study is to systematically diagnose and treat canine hookworm disease through the case of miniaturization Schnauzer dog feed infected with A. ceylanicum, so as to provide experimental basis for subsequent prevention and control of canine hookworm disease. METHODS: In the current study, we isolated hookworm eggs from a diseased miniature schnauzer, then the polymerase chain reaction (PCR) was used to amplify the ITS1-5.8S-ITS2 gene sequence from genomic DNA extracted from hookworms. Phylogenetic analysis based on ITS1-5.8S-ITS2 gene sequence sequences was inferred using MEGA-X. After phylogenetic analysis, etiologic and symptomatic therapies were used to treat the canine hookworm disease. RESULTS: The sequencing results showed that the length of the ITS1-5.8S-ITS2 gene sequence was approximately 960 bp, and ITS1 and ITS2 were extracted to analyze similarity with other hookworms to build a phylogenetic tree. After phylogenetic analysis, the results showed that the diseased miniature schnauzer was infected by A. ceylanicum. Using etiologic and symptomatic therapies, the sick dog with an A. ceylanicum infection was also treated for 5 days. CONCLUSIONS: To our knowledge, this is the first report of diagnosis and treatment for canine hookworm disease in Guangzhou city. In addition, with the improvement of economic level, the scale of pet dog breeding is also increasing. The diagnostic methods and treatment schemes adopted in this report will help to standardize the prevention and control of canine hookworm disease.

Hexamethyldisilazane Lithium (LiHMDS)-Promoted Hydroboration of Alkynes and Alkenes with Pinacolborane.

Lithium-promoted hydroboration of alkynes and alkenes using commercially available hexamethyldisilazane lithium as a precatalyst and HBpin as a hydride source has been developed. This method will be appealing for organic synthesis because of its remarkable substrate tolerance and good yields. Mechanistic studies revealed that the hydroboration proceeds through the in situ-formed BH3 species, which acts to drive the turnover of the hydroboration of alkynes and alkenes.

Experimental induction of necrotic enteritis with or without predisposing factors using netB positive Clostridium perfringens strains.

BACKGROUND: Poultry necrotic enteritis (NE) is an economically important disease caused by C. perfringens. The disease causing ability of this bacterium is linked with the production of a wide variety of toxins. Among them, necrotic enteritis B-like (NetB) toxin is reported to be involved in the pathogenesis of NE; in addition there is some circumstantial evidence that tpeL toxin may enhance virulence, but this is yet to be definitely shown. The situation becomes more complicated in the presence of a number of predisposing factors like co-infection with coccidia, type of diet and use of high protein diet. These co-factors alter the intestinal environment, thereby favoring the production of more toxins, leading to a more severe disease. The objective of this study was to develop a successful animal model that would induce clinical signs and lesions of NE using C. perfringens type G strains obtained from field outbreaks. A separate trial was simultaneously considered to establish the role of dietary factor with coccidial co-infection in NE. RESULTS: The results have shown that use of net-B positive C. perfringens without predisposing factors induce moderate to severe NE (Av. Lesion score 1.79 ± 1.50). In a separate trial, addition of fish meal to a feed of C. perfringens challenged birds produced higher number of NE cases (Av. Lesion score 2.17 ± 1.28). However, use of less virulent E. necatrix strain along with fish meal in conjunction with net-B positive strain did not alter the severity of NE lesions in specific pathogen free chicken (Av. Lesion score 2.21 ± 1.13). CONCLUSIONS: This study suggests that virulent C. perfringens type G strains can induce NE lesions in the absence of other predisposing factors. Birds in the clostridia challenged group showed moderate to severe NE lesions. Use of less virulent coccidia strain contributed to a lesser extent in increasing the severity of disease. Maize based diet along with fishmeal (1:1) increased the severity of lesions but statistically it was non-significant. The NE lesions in all experimental groups were found to be present more frequently in the duodenum. In this way, this study provided an effective model for in vivo production of NE in poultry birds.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy.

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Autophagy induced by monensin serves as a mechanism for programmed death in Eimeria tenella.

Monensin (Mon), the first ionophoric antibiotic has widely been used for the treatment and prevention of coccidiosis in poultry until recently, however, at present; its efficacy has been compromised with the emergence of many Mon-resistant strains. Knowledge of the mode of the action of anti-parasitic agents is as important as for other antimicrobials, especially for discovery and long term use of the existing drugs. However, little is known about anti-parasitic drug: monensin's, mechanism of action and physiological alteration in Eimeria tenella. In this study, we explored Mon effects on the viability of Mon-Sensitive GZ (MonS-GZ) and Mon-Resistant GZ (MonR-GZ) Eimeria tenella strains using trypan blue staining and investigated Mon-induced autophagy using Western blotting, indirect immunofluorescence assay, and transmission electron microscopy. The results showed that monensin leads to programmed death of E. tenella parasites by inducing autophagy as a mechanism of anticoccidial action. Mon-induced autophagy was indicated by the decreased sporozoites survival rate, ATG8 over expression and localization, and intracellular vacuolar structures and autophagosomes formation in MonS-GZ strain while in MonR-GZ strains autophagy pathway was not triggered. The autophagy inhibitor 3-methyladenine (3-MA) effectively blocked programmed cell death and saved the MonS-GZ sporozoites. These findings indicated that autophagy serves as a potentially important mechanism of E. tenella cell death in response to Mon and disruption of the autophagy pathway may lead to emergence of drug resistance against this anti-parasitic drug.

Occurrence and genotypes of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in household, shelter, breeding, and pet market dogs in Guangzhou, southern China.

Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. are common intestinal protozoans that infect humans and animals worldwide. A survey that assessed the prevalence, molecular characteristics, and zoonotic potential of these pathogens was conducted on a variety of dogs in Guangzhou, southern China. A total of 651 canine stool samples from household (n = 199), shelter (n = 149), breeding (n = 237), and pet market dogs (n = 66) were collected from eight districts in Guangzhou. Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. were detected by PCR amplification of the SSU rRNA gene. Giardia duodenalis-positive specimens were further assigned into assemblages using the glutamate dehydrogenase gene. Cryptosporidium spp., G. duodenalis, and Blastocystis sp. were found in 21 (3.2%), 20 (3.1%), and 35 (5.4%) samples, respectively. The overall prevalence of shelter dogs (40.28%, 60/149) was significantly higher than that of household (3.0%, 6/199), breeding (2.1%, 5/237), and pet market dogs (7.5%, 5/66) (χ2 = 154.72, df = 3, P < 0.001). Deworming in the past 12 months had a strong protective effect on the risk of contracting parasite infections (P < 0.001). No significant differences were detected between age or sex groups (P > 0.05). Dog-specific C. canis (n = 19) and zoonotic C. parvum (n = 2) were the only two Cryptosporidium species. Sequence analysis revealed the presence of three G. duodenalis assemblages: dog-specific assemblages D (n = 14) and C (n = 5), and cat-specific F (n = 1). Zoonotic Blastocystis ST3 (n = 28) was the dominant subtype, followed by ST1 (n = 6) and ST10 (n = 1). To our knowledge, this is the first large-scale investigation on the occurrence and molecular characteristics of Blastocystis sp. in dogs in China. Our results indicated that the dogs seemed to play a negligible role as reservoirs for Cryptosporidium spp. and G. duodenalis transmission to humans, but they are potential novel suitable hosts of Blastocystis sp. A strict sentinel surveillance system of dogs should be established to minimise the zoonotic risk of spreading blastocystosis among humans and dogs.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

Understanding the relationship between urban blue infrastructure and land surface temperature.

Urban heat island (UHI) effect has serious negative impacts on urban ecosystems and human well-being. Mitigation of UHI using nature-based solutions is highly desirable. It was well known that urban green infrastructure (UGI), i.e., urban vegetation, can effectively mitigate UHI effect. However, the potential of urban blue infrastructure (UBI), i.e., urban surface water, on UHI mitigation is not well understood, although its potential to lower UHI effect via evaporation is similar to the biophysical mechanism of evapotranspiration through vegetation. In this paper, we study the relationship between UBI and land surface temperature (LST) in Wuhan city in central China, using a normalized difference water index (NDWI), maximum local cool island intensity and the maximum cooling distance as indicators for the cooling effects of UBI, respectively. We found a significant negative linear relationship between mean LST and NDWI after NDWI passes a critical threshold value. NDWI is an effective biophysical parameter to delineate the spatial distribution of UBI. The cooling effects of UBI are influenced both by its size and shape. Water surface temperature decreased logarithmically with increasing UBI size, critical threshold values of UBI size corresponding to maximum cooling efficiency do exists. Maximum cooling distance and maximum local cool island intensity are also affected by the shape and size of UBI, and exhibit seasonal and spatial variations. These results provide insights for urban landscape planning regarding how to use UBI as a nature-based solution to improve urban thermal environment.

The effect of autophagy on the survival and invasive activity of Eimeria tenella sporozoites.

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.

ISEcp1-mediated transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid in Escherichia coli.

BACKGROUND: CMY-2 is the most prevalent pAmpC ß-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. METHODS: Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1-PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. RESULTS: The blaCMY-2-containing region (ISEcp1-blaCMY-2-∆blc-∆yggR-∆tnp1-orf7-orf8-orf9-∆tnp2-∆hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10-8-10-9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. CONCLUSIONS: Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure.

Characterization of cattle-origin ticks from Southern China.

To characterize ticks in cattle from Guangdong Province and Guangxi Zhuang Nationality Autonomous Region, Southern China, 783 cattle in four localities were examined. Among them, 232 (29.63%) cattle were positive for tick infection. A total of 503 ticks collected in these cattle were further investigated. Two Rhipicephalus species, namely R. microplus and R. sanguineus, were firstly identified by morphological features. Thereinto, R. microplus is the prevalent species in cattle in southern China, with high prevalent in summer and autumn annually. Mixed infection of R. microplus and R. sanguineus was just found in yellow cattle. To further confirm the morphological identification of these cattle-origin ticks, a phylogeographic analysis inferred from the sequences of the ribosomal internal transcribed spacer-2 (ITS-2) was performed, and R. microplus and R. sanguineus were identified. However, the morphological taxonomy of R. microplus has been challenged in recent years. The mitochondrial cytochrome c oxidase subunit 1 (cox1) marker was then used to provide higher resolution of R. mircoplus complex. The re-constructed cox1 phylogenetic tree further identified these R. mircoplus tick samples as R. microplus Clade A. These findings illustrated the prevalence and characterization of cattle-origin ticks in Southern China for the first time, and provided base-line information for further control of tick and tick-borne disease in these areas.

Molecular and biochemical characterization of Eimeria tenella hexokinase.

Hexokinase (HK) is one of the key enzymes in the glycolytic pathway that catalyzes the phosphorylation of glucose. In the present study, we cloned the HK gene from the coccidian Eimeria tenella (EtHK), expressed EtHK as a His-tagged fusion protein, and characterized its primary biochemical features. Mutagenesis confirmed that residues S159, N216, and D217 are essential or important to the EtHK catalytic activity. EtHK exhibited high affinity for D-glucose (Km = 0.67 to 0.79 mM), but was also able to utilize 2-deoxy-D-glucose (Km = 5.66 mM), D-fructose (Km = 13.76 mM), and D-mannose (Km = 25.41 mM). We also observed that quercetin and mangiferin could inhibit the EtHK enzyme activity (IC50 values = 6.52 and 85.82 µM, respectively). Among the two inhibitors, mangiferin also inhibited the growth of E. tenella in vitro (MIC50 = 0.12 µM). These observations suggest that EtHK may be explored as potential drug target, and mangiferin and its analogs may be explored for developing anti-coccidial therapeutics.

Molecular characterization of Cryptosporidium spp. in domestic pigeons (Columba livia domestica) in Guangdong Province, Southern China.

To investigate the prevalence and assess the zoonotic transmission burden of Cryptosporidium species in domestic pigeons in Guangdong Province, Southern China, 244 fecal samples were collected from four pigeon breeding farms between June 2012 and March 2013. Cryptosporidium oocysts were purified by Sheather's sugar flotation technique and characterized by DNA sequencing of small subunit ribosomal RNA (SSU rRNA) gene. Cryptosporidium species were determined by comparison of sequences with corresponding Cryptosporidium sequences in GenBank and phylogenetic analysis using neighbor-joining (NJ) in MEGA5.2. The overall prevalence of Cryptosporidium infection in domestic pigeons in Guangdong Province was 0.82% (2/244). Two Cryptosporidium species, namely Cryptosporidium baileyi and Cryptosporidium meleagridis, were identified in Huizhou and Chaozhou farm, respectively. These findings confirmed the existence of C. meleagridis infection in domestic pigeons in China for the first time and provided base-line information for further studies to evaluate the public health risk from pigeon to human.

Functional characterizations of malonyl-CoA:acyl carrier protein transacylase (MCAT) in Eimeria tenella.

Eimeria tenella, an apicomplexan parasite in chickens, possesses an apicoplast and its associated metabolic pathways including the Type II fatty acid synthesis (FAS II). Malonyl-CoA:acyl-carry protein transacylase (MCAT) encoded by the fabD gene is one of the essential enzymes in the FAS II system. In the present study, the entire E. tenella MCAT gene (EtfabD) was cloned and sequenced. Immunolabeling located this protein in the apicoplast organelle in coccidial sporozoites. Functional replacement of the fabD gene with amber mutation of E. coli temperature-sensitive LA2-89 strain by E. tenella EtMCAT demonstrated that EcFabD and EtMCAT perform the same biochemical function. The recombinant EtMCAT protein was expressed and its general biochemical features were also determined. An alkaloid natural product corytuberine (CAS: 517-56-6) could specifically inhibit the EtMCAT activity (IC(50)=16.47µM), but the inhibition of parasite growth in vitro by corytuberine was very weak (the predicted MIC(50)=0.65mM).

Activity-based identification of secreted serine proteases of the filamentous fungus, Ophiostoma.

A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease.

Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amy1) gene from Ophiostoma floccosum.

UV mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein production after repeated rounds of mutagenic treatment. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the increase in total secreted protein was about six times greater than the parental strain. Secreted proteinase and lipase activities of the parental strain and four key mutants were also investigated. N-terminal sequencing of the five dominant protein bands separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an alpha-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the alpha-amylase amino acid sequence. The amplified fragment contained the entire gene encoding alpha-amylase (amy1) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single alpha-amylase gene locus.