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An electrochemical biosensor for the rapid genetic identification of Musang King durian

More than 200 different cultivars of durian exist worldwide but Durio zibethinus or Musang King (MK) is the most premium and prized durian fruit among the recommended varieties. Early identification of this premium variety is critical to protect from non-authentic MK durian cultivars. However, the MK variety's morphological traits are nearly identical to other varieties. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape and this requires trained personnel for the morphology observation. To enable the rapid identification of the MK variety, PCR amplification of ten durian varieties using six gene candidates from the chloroplast genome was first performed to obtain DNA probes that were specific to the MK durian variety. PCR amplification of ten durian varieties using primers designed confirmed that the nadhA gene sequence showed an obvious difference in the MK variety from other durian varieties. The unique sequence of MK was used as a DNA probe to develop an electrochemical biosensor for the direct identification of the MK durian variety. The electrochemical biosensor was based on the hybridization response of the immobilized DNA probe with the target DNA from the MK variety and was monitored via differential pulse voltammetry technique. Under optimal conditions, the DNA electrochemical biosensor showed a low detection limit at 10% of MK genomic DNA concentration with a wide linear calibration range of 0.05–1.5 µM (R2 = 0.9891) and RSD value of 3.77% (n = 3). The results of the developed DNA biosensor provide high promise for the development of portable sensors employed in the determination of MK variety in the field.

www.nature.com/scientificreports/ to obtain new cultivars from one breeding generation 7 . To shorten the period of selection and to make it costeffective for durian breeding, it is essential to have a durian varieties identification tool for progeny selection. However, identification of MK variety is challenging due to some varieties showing similar morphological characteristics, which are difficult to distinguish and not applicable to opened durian fruits without shells in the markets. The distinctive odours of different durian cultivars, including MK have also been biochemically studied and characterized as a complex suite of odor-active compounds including sulfur volatiles, esters, alcohols, and acids 8,9 . This, therefore, results in a greater demand for quality planting materials of specified variety. The determination of the durian identity can be ideally conducted by the integration of morphological and molecular characterizations. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape 7, 10 and could only be by trained personnel. In 2018, a draft whole-genome assembly of the MK cultivar was reported in Singapore, providing a piece of useful information for durian agronomy 11 . The complete reported genome of MK may aid in the identification of cultivar-specific sequences, particularly Single nucleotide polymorphism (SNPs) associated with crucial cultivar-specific features (such as flavor, texture, and odor), and it may enable the molecular barcoding of distinct durian cultivars for rapid quality control and identification.
However, SNP marker validation entails labor-intensive and costly 12 . Furthermore, SNP genotyping analysis of one sample at a time is extremely expensive and time-consuming 13 . Moreover, SNPs are less polymorphic than other molecular markers because of their biallelic nature 13 . The other existing DNA markers technology, such as simple sequence repeats (SSR) [14][15][16] , inter-simple sequence repeat (ISSR) 17,18 , and random amplified polymorphic DNA (RAPD) markers 19,20 have been deployed in the identification of selected durian varieties. Molecular markers are nucleotide sequences that represent variation in nucleotide sequences across individuals of a species and are located at a known position on the chromosome. DNA markers are widely utilized in practical agriculture monitoring, mostly due to their reliability, stability, reproducibility, efficiency, and cost-effectiveness. In 2018, the simple sequence repeats (SSR) marker was employed as an alternative technique to study genetic variation among Durian varieties in Malaysia owing to its co-dominant inheritance, multi-allelic nature, and high reproducibility compared to other markers 13,16 . However, the development process of SSR marker is quite lengthy and expensive, and throughput is low due to automation and output data management limitations 21 .Though RAPD and ISSR markers are a suitable alternative to provide useful information for the identification of plant variety and cultivar, general genetic diversity assessment, population genetic structure and plant protection, however, the dominant character of these markers considers them inappropriate for DNA fingerprinting and genetic variation analysis 22 . Notably, the information derived from dominant genetic markers is less informative than that derived from co-dominant genetic markers, as codominant inheritance of markers can differentiate between homozygous and heterozygous states based on the generated alleles but not dominant inheritance of markers. Furthermore, some dominant genetic markers are known to suffer from poor reproducibility 13,23 , raising concerns about the feasibility and reliability of using dominant genetic markers for downstream applications.
With the advent of DNA biosensor technology, the gap in durian variety identification can be bridged. Therefore, in this work, we attempted to develop an electrochemical DNA biosensor, which can be useful to differentiate and to distinguish the identities among the durian cultivars. This technique is highly sensitive, simple, and low cost for MK durian variety determination with good accuracy. Electrochemical DNA biosensors have been widely adopted in agriculture, particularly in disease diagnosis, due to their potential to give higher sensitivity, faster analysis, portability, and lower cost than traditional technologies 24 . The selection of the right immobilization method and the suitability of matrix type for DNA probe immobilization are imperative owing to producing a high-performance DNA biosensor. The selection of DNA probe is based on DNA barcoding for plants technique, wherein genes encoding a mitochondrial cytochrome oxidase (COI, coxI) subunit were extensively employed 25,26 . The development of the DNA barcoding technique allows for high rapididentification of plant varieties and cultivars efficiently 13 . The chloroplast genome in plants is considered a potential candidate for barcoding genes due to conserved gene order, high copy number and simple amplification by PCR 13,27 .
In this research, the six genes candidate from DNA barcoding for plants were deployed by PCR amplification of ten durian varieties to identify DNA sequences that are unique to the MK variety but not found in other durian varieties and to develop as DNA probes. The DNA probe specific to the MK durian variety was used further to develop an electrochemical biosensor for the direct identification of the MK durian variety. The biosensor was constructed from the carbon-paste screen-printed electrode (SPE), which was modified by depositing with gold nanoparticles (AuNPs) and acrylic microspheres (AcMPs) containing succinimide functional groups as the matrix for DNA probe immobilization. The succinimide functional group of the acrylic microspheres become a linker to immobilize aminated DNA probe via covalent bonds 28,29 . The acrylic microspheres provide the advantages of small size and serve a large surface area to volume ratio for DNA probe immobilization on the surface, thus avoiding any barriers to the diffusion of reactants and products 30 . The overall electrode design provides a wide surface area for chemical reactions and excellent surface immobilization properties, which can significantly increase the effectiveness of the electrochemistry detection assay [31][32][33] . Thus, acrylic microspheres (AcMPs) based DNA biosensors have the potential to serve as a promising platform for the development of rapid, sensitive, specific, and portable diagnostic tools for DNA detection [34][35][36] .

Results and discussion
DNA probe determination of 'Musang King'. The sequencing analysis of ten durian varieties showed that there are some unique regions found in loci nadhA gene sequence and had obvious comparison among ten durian varieties in which D99, D145, MDUR88, D175, MDUR78, D24, D168, D200, and MDUR79 durian varieties sequences containing sequence lesion of 17 bases but not in MK DNA sequence (Fig. 1). This comparison implies that the unique sequence in the MK DNA sequence can be used as a DNA probe to differentiate between www.nature.com/scientificreports/ MK with the other durian varieties via electrochemical biosensor. Whereas the other five gene candidates did not show any significant differences among the ten durian varieties sequence (Fig. S1) DNA barcode candidates are based on chloroplast genome that provides some advantages such as simple and stable genetic structure, it is haploid, undergoes no (or very rare) recombination, it is generally uniparentally transmitted, and universal primers that are suitable to use for amplifying target sequences 37 . Additionally, chloroplast genomes are high conservation over nuclear and mitochondrial genomes. Therefore, partial chloroplast genome sequences are preferred and suitable to use for phylogenetic studies and species/varieties identification and discrimination with similar morphology characteristics [38][39][40][41][42][43] . DNA barcoding has been deployed in prior studies owing to its effectiveness in plant variety and cultivar identification. The chloroplast genome is considered a promising candidate for barcoding genes in various land plants due to its advantages, such as conserved gene order, high copy number, and ease of amplification by PCR 13 . The vast of studies on plant barcoding employs one or a few plastid areas, including the protein-coding rbcL and matK regions, the noncoding spacer trnH-psbA, and ITS regions. For instance, the three chloroplast genomes (ITS2 and cp gene matK and rpl32-trnL (UAG)) were successfully identified and discriminated 12 commercial varieties of kiwi fruit 44 . Likewise, Emirati date palm cultivars were able to discriminate using the chloroplast intergenic spacer psbK-psbI 45 . In Xinjiang, China, Artemisia L. varieties were identified using the chloroplast genome of ITS region which revealed the highest identification efficacy 46 .
The response of the biosensor towards target DNA concentrations. Under the optimal conditions, the analytical performance of the DNA biosensor was examined using the immobilized DNA probe by the response of the AcMP-AuNP-modified carbon SPEs electrode towards an increasing concentration of synthetic target DNA (1 µM, 1.5 µM, 0.1 µM, 0.05 µM, and 0.01 µM), non-complementary (NC) DNA and no target (NT) at a scan rate of 0.04 V s −1 , the results were illustrated in Fig. 2. The current response was increased steadily with increasing target DNA concentration at the electrode in experiment (a). The highest peak was 1.5 µM followed by 1.0 µM, 0.1 µM, 0.05 µM, 0.01 µM, and NC. The DPV peak reading of all concentrations was in the range of − 0.55 V to − 0.45 V. This indicates more DNA duplex was formed on the electrode through DNA hybridization reactions followed by anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) intercalation. Additionally, significant current differences were observed in the DPV peak from all concentrations, implying that the DNA probes were successfully immobilized onto the AcMP via covalent bonds between succinimide group of AcMP and amine functional group of the DNA probe 28,29 . The concentration of 1.5 µM was the highest peak, indicated hybridization where intercalation of AQMS had occurred in double-stranded DNA (dsDNA) formed in the microsphere surface with the current peak value of 3.34 µA. However, the DPV peak of NT was the lowest of other concentrations due to no DNA hybridization reaction occurring, indicating there was no specific adsorption of AQMS redox indicator onto AcMP-AuNP-based DNA modified carbon SPEs electrode 47 .
The DNA biosensor responses with five different concentrations of synthetic target DNA and no target that has been hybridized on DNA probe immobilized on AcMPs are shown in Fig. 2b. DNA biosensor response increased proportionally with increasing concentrations of synthetic DNA target immobilised. This suggests that the capacity of immobilized DNA probe to hybridize with complementary of five different synthetic target DNA concentrations has increased with an increase in DNA target attached to the AcMP-AuNP. The linear response range of the DNA biosensor from different concentrations of synthetic target DNA demonstrates that the oxidation peak current of DNA increased linearly with increasing synthetic target DNA concentration and revealed a satisfactory correlation coefficient value of R 2 = 0.9891, and the linear equation was expressed y = 2.1006x − 0.193 with a low detection limit of 0.01 µM (Fig. 2c). The large binding surface area of the AcMPs enabled a large number of DNA molecules to bind covalently to the AcMPs via succinimide functional groups, ultimately enhance the analytical performance of the DNA biosensor in terms of dynamic linear range and detection limit 30 .
The specificity of the biosensor response. Figure 3a shows the DPV response of DNA biosensors with different Durian varieties (MK, D24, MDUR88, MDUR78, MDUR79, D168, D200, D145, D99, D175) and blank (without DNA) under optimum conditions. The DPV peak near − 0.55 V indicates the oxidation of AQMS that has been intercalated into the dsDNA formed from MK DNA probe hybridized with MK complementary DNA. This result revealed that the designed DNA probe from MK is highly specific to MK. MK variety yielded the highest current response as expected and demonstrated that the immobilized MK DNA probe was selective  The sensitivity of the biosensor. To investigate the sensitivity of the biosensor, increasing amounts of MK genomic DNA were added to triplicate reactions containing a decreasing amount of MDUR88 genomic DNA. The genomic DNA mixture containing 0%, 10%, 20%, %, 40%, 60%, 80%, and 100% of MK genomic DNA. This is for the purpose of determining the biosensor response towards MK genomic DNA purity as to whether it is derived solely from clone or hybrid. Figure 4a represents the DPV of different genomic DNA concentrations of MK (0%, 10%, 20%, 40%, 60%, 80%, and 100%) in the present of MDUR88 genomic DNA. The current response of this proposed biosensor clearly increased with the increase of the target concentrations from 0 to 100%, followed by intercalation of AQMS in between the dsDNA ( Fig. 4a and b). The DNA biosensor showed a good linear relationship with the percentage of MK gDNA (Fig. 4c), and the linear equation was expressed y = 3x − 0.1 (R 2 = 0.9712) with a detection limit down to 10% of MK gDNA for sensitivity analysis. The reproducibility of the DNA biosensor was performed with 100% of MK gDNA. DNA biosensors gave satisfactory reproducibility results with 3.77%. The DPV peak reading of all concentrations was at − 0.55 V. This suggests an increase in hybridization between the immobilized DNA probes with the target DNA when the DNA concentrations increased.
Biosensor response validation with real durian DNA samples. In order to prove the effectiveness of the biosensor in determining and distinguishing the Durian MK variety from other Durian varieties, a total of 27 different durian field samples were analysed with the electrochemical DNA biosensor. Figure 5 illustrates the effect of different durian DNA samples on the biosensor response. Based on the DPV diagram in Fig. 5a-d, all field samples were identified as MK variety except for DS2 and DS18 from Sintok, Kedah, which yielded current as low as the non-MK control sample MDUR88. As shown in Fig. 5e, the mean of 24 MK samples is 0.93, and the electrochemical response of the biosensor for all samples was comparable with the MK standard, which revealed the current of 0.93 ± 0.25 µA except for MKS2 and MKS18. These results from the electrochemical DNA biosensor have been validated with the standard PCR-based method and followed by sequencing to determine the 27 durian field samples. PCR amplification of 27 durian samples produced 300 bp amplicon except for DS2 and DS18 samples (Fig. S2). With the results tabulated in Table 1, both methods provided the same result for

Conclusion
In this study, an electrochemical DNA biosensor was successfully developed with high specificity, good sensitivity, wide linear response ranges, and low detection limit in the determination of MK durian. Furthermore, the electrochemical DNA biosensor showed a good response to the MK DNA target, which implies that the biosensor is sensitive and has high selectivity to determine MK variety. Moreover, the developed MK DNA biosensor can assist farmers for early identification of MK durian variety in order to avoid confusion in the propagation system, which is economically advantageous in agriculture sectors. Furthermore, farmers can benefit from the use of biosensors for the identification of MK durian variety since it can help them to ensure the authenticity of MK, which is crucial for export activities to foreign nations. Early determination and confirmation of MK's authenticity at the seedling stage is also critical to ensuring that farmers do not suffer significant losses due to their large investment in Mk durian trees. Employing DNA biosensors to identify MK durian variety also has the advantage of allowing farmers or dealers to obtain proof of certification for export permission to an international market. The identification of MK is also not only beneficial to farmers, but it can also protect the rights of consumers in meeting the high demand for trees or fruit as MK is a durian variety that has a high global market.   ATT GAC GGC TCA AGNACA ACC GTA TAG AGT TTT GGC TAT CCT TTG TAT AGA TTCC CAT ACA TGA  TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG GAT GGT  TAG GAA CAC CAA AAA   Yes  Yes   2  DZ4   TCA TTT TGA CGG GCT CAA GAT CAA CCG TAT AGA GTT TTG GCT ATC CTT TGT ATA GAT TCC CAT  ACA TGA TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG  GAT GGT TAG GAA CAC  The DNA sequences of PCR products from ten durian varieties were aligned using Clustal Omega (https:// www. ebi. ac. uk/ Tools/ msa/ clust alo/) and unique DNA sequences that can differentiate MK from the other nine varieties were identified and used as a DNA probe in DNA biosensor development for MK determination.
Design of DNA probe for biosensor. DNA probe, target DNA and non-complementary DNA (Table 3) were designed based on the bioinformatics study of nadhA gene (based on unique DNA sequence revealed from sequence alignment of 10 durian varieties).  NTGA CGG CTC AAG ATC AAC CGT ATA GAG TTT TGG CTA TCC TTT GTA TAG ATTCC CAT ACA TGA  TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG GAT GGT  TAG GAA CAC CAA AA   Yes  Yes   26  DS39   TTG ACG GCT CAA ANACA ACC GTA TAG AGT TTT GGC TAT CCT TTG TAT AGA TTCC CAT ACA TGA  TAG ATT CCC ATA CAT GTA TTG CCA AAC CAA ACG GGG GAT TGA ACA AAA AAA TGA GTG GAT GGT  TAG GAA CAC     Fabrication of DNA biosensor. Two mg of acrylic microspheres were weighed in a microcentrifuge tube and 300 µL of 1 µM animated DNA probes was added to immobilize the DNA onto the microspheres for 24 h at 4 °C. The DNA /AcMPs were collected after 24 h by centrifuging at 1000 rpm for 8 min. The supernatant (which contains DNA probes) was kept for future use in DNA immobilization. The microspheres with K-phosphate were washed several times, and the wash solution was discarded. Fresh 0.05 M K-phosphate (pH 7.0) buffer was added to re-suspense the DNA microspheres. One mg of gold nanoparticles (AuNPs) was suspended in 300 µL ethanol and 10 µL of AuNPs suspension were drop-coated onto C-SPE. The electrode AuNPs/SPE is then left to dry at room temperature for 1-2 h. AcMPs immobilized with DNA probes were pipetted (8 µL) and dropped it onto AuNPs/CSPE and stored at 4 °C until dry for 24 h. 10 µL of solution containing DNA target, 0.05 M of Na-phosphate buffer and 1.0 mM AQMS was dropped onto the electrode surface and incubated for 40 min for hybridization of the probe with the target. The electrode was then rinsed several times with 0.05 M K-phosphate buffer. An amount of 100 µL fresh 0.05 M K-phosphate was dropped onto the electrode surface, and the DPV was scanned at the potential range of (− 0.8 V to − 0.2 V) of the electrode using CSPE as a working electrode, platinum as the counter electrode and Ag as a reference electrode. The AQMS peak should appear at approximately − 0.55 V. All the experiments were performed in triplicate. The SPE construction and design mechanism of the electrochemical DNA biosensor is illustrated in Fig. 6. Sensitivity of biosensor system. The biosensor experiment was performed on different concentration of MK percentage (100%, 80%, 60%, 40%, 20%, 10%, and 0%), where genomic DNA of MK variety was mixed with genomic DNA from MDUR88 variety. In 1 µL of DNA sample was containing total of 10 ng MK and MDUR88. The combination of MK and MDUR88 gDNA used in this study were shown in the Table 4.

Field samples test. A total of 27 durian samples were collected from five different regions in Peninsular
Malaysia as shown in Table 5. The optimised DNA biosensor was then used for the determination MK gDNA via DPV method.
Validation. The biosensor performance of 27 MK samples was validated via PCR based-method using designed primers from nadhA gene sequence as shown in Table 6. The reaction mixture and PCR program used in validation were similar as used in the DNA probe selection for MK biosensor development. Amplicons were confirmed using gel electrophoresis. The PCR products were then purified and outsourced for DNA sequencing at 1st BASE, Malaysia. From the DNA sequence of PCR product, unique sequence of MK was identified, thus validate the biosensor performance in identifying MK field samples.

Data availability
The datasets generated and analysed during the current study are available in the European Variation Archive (EVA) at EMBL-EBI under accession number PRJEB55857 (https:// www. ebi. ac. uk/ eva/? eva-study= PRJEB 55857).
Additional raw data will be available upon request.


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

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Research on Ecological Protection and Restoration of Lijiang River Landscape in Guilin, Guangxi

Guilin landscape is the best in the world, and Lijiang River Basin is one of the most important landscape axis in Guilin landscape, so the landscape planning of Lijiang River Basin and its coast is particularly important. However, in recent years, the ecological environment of Lijiang River Basin and its coastal areas has deteriorated, and the beautiful scenery of Lijiang River is no longer the same as before, which has already attracted the attention of Guangxi Province and even the whole country. In order to respond to the call of building Guilin into a world-class international tourist attraction, it is urgent for us to create a beautiful urban tourism environment and to recreate the grand scenery of the Lijiang River. Based on the existing resources, environment and regional advantages of Lijiang River, this study analyzes the current situation of landscape ecological problems of Lijiang River in Guilin, and puts forward the ecological protection and restoration strategies suitable for Lijiang river ecological construction objectives. Based on the concept of ecological restoration and urban restoration, we analyze and research on the current situation and problems of landscape planning of Lijiang River in Guilin. The research actively discusses and summarizes the governance measures. It is suggested that, in close combination with regional characteristics, we should establish a resource-saving and environment-friendly sustainable development landscape industrial structure scheme suitable for regional ecological carrying capacity. Building a suitable ecological compensation mechanism of the Lijiang river landscape, it provides suggestions and references for the ecological protection and restoration of Lijiang River Basin and the sustainable development of Lijiang River landscape in Guilin.

INTRODUCTION
Guilin landscape is one of the best landscapes in China, and the Lijiang River Basin is one of the most important landscape axes in Guilin landscape. The Lijiang River originates from Maoer Mountain, the first peak of South China in Guilin, with a total length of 214 kilometers and a total area of 12,159 square kilometers. It involves 5 counties, 1 city and 6 urban areas under the administrative jurisdiction of Guilin. There are 14 national and autonomous regional nature reserves and wetland parks in the basin [1]. Therefore, the landscape planning of Lijiang River Basin and its coast is particularly important. However, in recent years, the ecological environment of Lijiang River Basin and its coastal areas has deteriorated, and the beauty of Lijiang River is no longer the same as before.
The environmental deterioration of the Lijiang River in Guilin has already attracted the attention of Guangxi Province and even the whole country. The 19th National Congress of the Communist Party of China proposed to promote the construction of Guilin as a world-class international tourist destination. The Development and Reform Commission of Guangxi Zhuang Autonomous Region and the people' s government of Guilin have also introduced relevant policies for the ecological protection and restoration of the Lijiang River [2]. Therefore, it is urgent to reproduce the prosperity of the Lijiang River, create the most beautiful Lijiang River and create a beautiful urban tourism environment. The ecological protection and restoration of Lijiang landscape is to repair the urban space, function system and environmental carrying capacity of Guilin, further improve the public service quality of Guilin and improve the level of infrastructure construction. Based on the concept of ecological restoration and urban restoration, this study analyzes the current situation and problems of landscape planning of Lijiang River in Guilin, and summarizes the governance measures. On this basis, suggestions are put forward for the future landscape of Lijiang River in Guilin to combine regional characteristics, consider ecological carrying capacity, build sustainability and establish corresponding ecological compensation mechanism, so as to provide reference for the ecological protection and restoration of landscape in Lijiang River Basin in Guilin.

STATUS ANALYSIS
In recent years, with the increasing intensity of tourism development and the development of industry and agriculture in the city and other factors, the ecological environment of Lijiang River Basin is deteriorating.

The change of upstream forest vegetation structure
There are well-preserved primitive forests in the upper reaches of Lijiang River Basin, with good ecological status. However, since the continuous expansion of the scope of human activities and the increase of human intervention, the vegetation structure in this area has undergone great changes. The proportion of primary forests has been declining, while the proportion of artificial forests and secondary forests has been increasing. This situation is easy to lead to increased soil erosion, reduced biodiversity and decreased soil water conservation capacity.

Aggravated desertification
The middle and lower reaches of the Lijiang River are karst areas with serious desertification problems. In addition to natural and geographical factors, human activities have also caused rocky desertification and desertification. For example, areas that have become bare after artificial development of mines and quarries ( Fig. 1 ) have not been well repaired, resulting in deterioration of the ecological environment.

Reduction of water area
The middle reaches of the Lijiang River Basin have a flat terrain, which is a plain area, once distributed with a large area of water, and has a network water resources system. The water veins conserve water, regulate urban microclimate, and regulate flood and water storage to reduce the impact of disasters. However, with the acceleration of urbanization, urban construction space area continues to expand ( Fig. 2 ), the original water area continues to decrease, wetland degradation and its area rapidly shrink [3]. This leads to the weakening of self-purification function of water body, the degradation of ecological function of water system, the decline of groundwater and surface water quality, the reduction of water storage capacity, and the drying of rivers.

Disaster-prone
The rainfall in Lijiang River Basin is abundant, the temporal and spatial distribution of rainfall is uneven, and the suddenness is strong, resulting in a large fluctuation of water level, and the upstream flood occurs. However, since the upstream rainfall will be converted into surface runoff flow to the middle and lower reaches, but the lower channel is narrow, and the insufficient water storage capacity and poor connectivity in karst areas will cause water accumulation due to poor drainage. The middle and lower reaches of the river basin have less vegetation, thin soil layer, poor water retention capacity, and serious surface water infiltration, which forms a dry karst surface, that is, the typical karst drought zone.

Deterioration of quality
Domestic wastewater, production wastewater, industrial and agricultural wastewater discharge indiscriminately along the river and the river water self-purification capacity is very low, the water quality of Lijiang River is deteriorating ( Fig. 3 ). Chemical pollutants discharged at will and difficult to degrade, algae increased, water and wetland area and quality decline, weak self-purification capacity of these factors are poor Lijiang River water quality.

Lag of ecological protection infrastructure
Lijiang River Basin has a wide coverage, long history of environmental protection, flood control, water conservancy, transportation and other infrastructure construction, and many debts. Urban sewage and solid waste treatment capacity is insufficient. Most counties have no spare water sources, and there are certain ecological risks. Urban and industrial park sewage, garbage disposal capacity and operational economic security is insufficient, the vast majority of villages did not establish sewage treatment facilities, garbage disposal facilities are simple, Lijiang River Basin only 240 natural villages initially established centralized sewage treatment facilities. The limited number of centralized treatment sites for industrial waste, medical waste, hazardous materials and animal corpses died of diseases in the study site, resulting in these pollution sources cannot be timely and effectively treated ( Fig. 4 ). Second pollution of soil surface and surface runoff is due to long-term accumulation of pollutants on the ground. The black and odorous water in the tributaries of the Lijiang River and the raising of livestock by coastal villagers have not been comprehensively and effectively controlled. Agriculture is facing serious pollution risks. The construction of ecological protection and management capacity of Lijiang River is lagging behind, and there is a lack of systematic and perfect monitoring and management tools, and the investigation and evaluation of ecological environment and scientific research monitoring are lagging behind.

Low capacity of conservation and utilization of ecological resources
Some industrial and mining enterprises which produce pollutants still appear in the red line of ecological protection along the Lijiang River. Ecological park foundation is weak, pollution control, clean production, recycling and reuse technology is relatively backward. The utilization of ecological resources is relatively inefficient, the innovation of ecological tourism is insufficient, and the comprehensive benefit is low. Due to the special geographical environment and vulnerable ecosystem characteristics of Lijiang River, the implementation of ecological protection and restoration measures will be more difficult than other regions. There are still immature areas in the protection and restoration technology of karst landform natural ecosystem, which restrict the development of ecological protection and restoration of Lijiang River.

Contradiction between ecological protection measures and people ' s livelihood
Deforestation, grazing, hunting and large-scale livestock breeding are prohibited in some key ecological protection areas in Lijiang River Basin. These activities are the source of local rural residents ' life and economy, so people ' s livelihood along the river has received a great impact, production and life has been greatly affected, industrial structure adjustment is difficult, it is difficult to form the scale of ecological economy in the short term, farmers transfer to industry constraints, the contradiction between population and environmental carrying capacity has become increasingly prominent. Therefore, while carrying out the ecological protection and restoration work in the Lijiang River, it will also face how to better protect the lives of residents along the river as little as possible. This requires us to explore the coordinated development of ecological benefits, economic benefits, social benefits and people ' s livelihood benefits, and seek a win-win, balanced management system and effective and suitable ecological compensation mechanism.

STRATEGY OF ECOLOGICAL PROTECTION AND RESTORATION
Effectively restore the damaged rocks, rivers, lakes, vegetation communities and ecosystems in the Lijiang River Basin, and restore the self-regulating function of the Lijiang River ecosystem. Continuously improve the level of urban public service quality, strengthen the construction of municipal infrastructure, explore and protect the historical and cultural connotation of the Lijiang River Basin in Guilin, based on the concept of city double fix [4], the ecological protection and repair of the Lijiang River Basin will be the city of Guilin. The

Advances in Economics, Business and Management Research, volume 654
ecological carrying capacity and functional system have been comprehensively and effectively repaired. The ecological protection and restoration of Lijiang River Basin is to protect and restore the urban ecological space of Guilin City [5], and also to improve the urban function and landscape effect. In view of the above problems in the Lijiang River Basin, such as the degradation of ecological functions, the lag of infrastructure, the poor protection and utilization of ecological resources, and the contradiction between relevant measures and people 's lives, this case consults relevant literature, and conducts on-the-spot investigation and research along the Lijiang River. Combined with the case of landscape ecological remediation in the land-water ecotone at home and abroad [6], the following references are put forward for the ecological protection and restoration measures of the Lijiang River landscape.

Coastal rural environmental remediation
It is necessary to carry out the improvement of living environment of rural residents in coastal areas, lay sewage collection pipe network, build rural sewage ecological treatment facilities, establish garbage collection [7], transportation and harmless treatment system in coastal villages, and implement comprehensive treatment of sewage and garbage in villages along the Lijiang River. Efficient treatment and utilization of livestock manure resources, migration and management of livestock farms, treatment and relocation of key pollution sources in the Lijiang River Basin, and implementation of various pollution control projects. Emphasis is placed on strengthening the clean-up and renovation of the problems such as random construction, random excavation, random farming, random management and environmental sanitation along the Lijiang River, and prohibiting illegal construction, illegal sand excavation, residential boats, catering boats, cage fish farming and livestock farming. Build characteristic tourism villages along the Lijiang River [8], and build a beautiful countryside with good ecological environment, leisure, livable, tourism, architectural style and natural environment.

Management of water quality
Open all sewage treatment plants in Guilin and build sewage treatment infrastructure to expand the scope of sewage collection and treatment so that sewage treatment can reach full coverage. The black and odorous water is treated by a series of means such as source control, garbage cleaning, sludge cleaning and drainage. Combined with the upper reaches of the Lijiang River reservoir, strengthen the allocation of water resources, improve the Lijiang River dry season ecological and landscape environment.
According to the construction goal of Guilin, the sponge city is constructed. On the basis of ensuring the normal drainage and waterlog prevention of the city, combined with the natural water system and artificial measures, the flood control and waterlog prevention are realized to the greatest extent, the water storage and storage are realized, and the water quality is purified to achieve the efficient utilization of atmospheric precipitation resources, and the ecological protection and restoration of the Lijiang River Basin are realized.

Forestry ecological protection
Reducing the proportion of fast-growing forest and economic forest, improving the compensation standard of ecological public welfare forest, increasing the proportion of ecological public welfare forest, improving the structure of forest species, improving the water conservation capacity of the upper reaches of the Lijiang River, and reducing soil erosion ; it is strictly prohibited to carry out production and operation activities such as reclamation and burning wasteland in ecological public welfare forest areas, and to increase the ecological restoration of the reclaimed forest areas. To protect the ecological environment of Qingshitan reservoir and its surrounding areas as a whole, repair the system and comprehensively manage it; construction of ecological landscape forest belt along the mainstream of Lijiang River. Under the premise of protecting the original vegetation, the artificial forest and residual forest were reconstructed or the stand quality was improved, and the long-term management and protection mechanism was established. Strengthen the infrastructure and capacity-building of natural protected areas in Lijiang River Basin, accelerate the construction of mountain forests and biodiversity ecological function areas around Lijiang River, carry out biodiversity background survey, establish monitoring, evaluation and early warning system, and strengthen the construction of protection and management infrastructure.

Developing ecological agriculture
Carrying out the industrial transfer in the core area of Lijiang River, promoting the construction of ecological park, combining with the tourism situation of Lijiang River in Guilin, under the background of returning farmland to forest to protect ecology, using the reserved part of agricultural land to establish an industrial model with local characteristics of Lijiang River combining agriculture, ecology and tourism. Production of high-quality agricultural products, improve product efficiency, in-depth mining of agricultural products function.

CONCLUSION
Due to its unique geographical location and unique Advances in Economics, Business and Management Research, volume 654 natural conditions, the Lijiang River Basin in Guilin has become the most ecological development space in Guilin. Through the comparative study of successful cases of ecological development at home and abroad and the analysis of the current situation of landscape ecological problems of the Lijiang River in Guilin, the ecological protection and restoration of the Lijiang River landscape should be combined with its existing resources, environment and regional advantages. Therefore, the development strategy suitable for the ecological construction goal of Lijiang River is put forward. Based on the concept of ecological restoration and urban restoration, through the analysis and research on the current situation and problems of landscape planning of Lijiang River in Guilin, the governance measures are actively explored and summarized. It is proposed that under the close combination of regional characteristics, the industrial structure scheme of resource-saving and environment-friendly sustainable development landscape suitable for regional ecological carrying capacity should be established, and the ecological compensation mechanism suitable for Lijiang River landscape should be established, so as to provide suggestions and references for the ecological protection and restoration of Lijiang River Basin in Guilin and the sustainable development of Lijiang River landscape.


s2fieldsofstudy:
Agricultural And Food Sciences
Environmental Science

text:
Bioaerosol exposure by farm type in Korea

Introduction and Objective. Bioaerosols exist in almost every environment and are known to be risk factors for a variety of diseases. Agricultural work involves high exposure to bioaerosols and its workplace concentrations affect the surrounding areas. The study evaluates bioaerosol concentrations in agricultural workplaces and residential areas according to farm type. Materials and method. In 2013–2015, a total of 381 samples were collected for endotoxin and microbial testing from three farm types: open field, greenhouse, and livestock facilities. Endotoxins were measured using a 37-mm glass fire filter connected to an air pump calibrated to 2 LPM. Microorganisms were measured using a gelatin filter and impaction (singlestage Andersen sampler). Results. The concentration of endotoxins and microorganisms at the livestock facilities was significantly higher than in the open fields and greenhouses (p<0.05). Among the livestock farms, the concentrations of endotoxins and gram-negative bacteria were highest at hog farms, and the concentrations of total bacteria and fungi were the highest in poultry houses. In both greenhouses and livestock facilities, the concentrations of bioaerosols were higher in the workplace than in a nearby residential area, and the difference in concentration was significantly greater in the case of livestock facilities. Conclusions. The concentrations of bioaerosols in agricultural workplaces and nearby residential areas were higher than in the control and general areas. Therefore, measures should be taken to prevent exposure to bioaerosols at agricultural workplaces and their vicinities.

INTRODUCTION
Bioaerosols are airborne particles of microbial, plant and animal origin and constitute a substantial part of organic dust [1]. Specifically, bioaerosols include fungal conidia and hyphae, bacterial vegetative cells and spores, endotoxins, glucans, mycotoxins or high-molecular-weight allergens, and organic dusts in general, composed of or derived from biological matter [2]. They are known to be risk factors for a variety of diseases, including infectious diseases, hypersensitivity, asthma, allergy, and cancer [3], of which infectious and respiratory diseases are the most common [4]. Infectious diseases are caused by bacteria, fungi, and viruses through the transmission of infectious agents by direct or indirect contact, airborne and vector-borne transmission [5,6]. Impairment of lung function and respiratory symptoms are common mild conditions to severe chronic respiratory diseases [4]. Cancer can be caused by oncogenic viruses and non-viral biological carcinogens, such as mycotoxins (e.g. ochratoxin A) [4]. For those reasons, interest in bioaerosol exposure in both occupational and residential indoor environments has increased during the past few decades.
Farms are workplaces with particularly high exposure to bioaerosols and agricultural activities also therefore increase the likelihood of exposure to bioaerosols [7]. Farms typically hold a large number of animals, such as poultry, swine, and cattle, mainly in indoor settings and at high densities. The variety of possible sources associated with those conditions produces complex mixtures of microorganisms within the bioaerosols emitted from farms [8][9][10][11][12]. This dynamic microbial composition places farm workers and nearby residents at high risk for health issues, such as accelerated declines in lung function, changes in blood pressure, nasal inflammation, secretory immunity, infectious diseases, as well as dermatological and gastrointestinal problems [13,14].
Currently in Korean agriculture, a rapid increase in the use of concentrated animal feeding operations in the livestock industry to grow swine, cattle, poultry, etc., in confined buildings and greenhouses has increased the probability that farm workers will be exposed to organic dust. Furthermore, most farmers live adjacent to their workplaces, therefore organic dust generated in the workplace could also contaminate residential spaces.

OBJECTIVE
The aim of this study was to evaluate the concentration of organic dust, including endotoxins, in workplaces and

MATERIALS AND METHOD
Study subjects. In 201-2015, samples from 21 kinds of crops were tested for endotoxins (n=381), total bacteria (n=363), and fungi (n=346) (Tab. 1). Farms were divided into three types: open field, greenhouse, and livestock facilities, with the latter category subdivided by animal: poultry, swine, and cattle. The open field farms grew crops such as grapes, green beans, and rice in a natural environment without the aid of any kind of enclosed artificial facility. Greenhouse farms grew most of their crops, mainly leafy vegetables, leeks, Chinese cabbage, peppers, cucumbers, tomatoes, and watermelons, within an enclosed structure to maintain optimal growing conditions. Livestock facilities grew poultry, swine, and cattle, with many of them having places within the workspace, such as manure storage areas, that had a very high probability of breeding microorganisms. When a farming residential area was adjacent to a workspace, such as a barn, the same items and conditions measured in the workspace were also measured in the residential area (living room, bedroom, kitchen), to evaluate whether organic dust generated in the workplace also contaminated the residential area.
Sampling method. Measurement of area samples was conducted to confirm microbial and endotoxin levels in the workplaces, and personal samples collected in the farmer's breathing zone to assess the exposure levels faced by individual farmers while carrying out their work activities. For the personal measurements, bioaerosol was sampled on sterilized gelatin filters (Cat. No. 225-9551, SKC Inc., USA) with Button aerosol sampler (Cat. No. 225-360, SKC Inc., USA) at a flow rate of 4 LPM using pumps (AirChek XR5000, Cat. No. 210-5001, SKC Inc., USA). The area samples were measured using Biostage single-stage impactor (model QuickTake 30 Biostage, Cat. No. 228-9530K, SKC Inc., USA) at a flow rate of 28.3 LPM. The endotoxin was sampled on sterilized glass fibre filters at a flow rate of 2 LPM using pumps (model AirChek XR5000, Cat. No. 210-5001, SKC Inc., USA). Endotoxins were measured in a two-stage cassette through a glass fibre filter sterilized at 180 °C for two hours during the entire agricultural work period. Local and individual endotoxin samples were acquired in the same manner. The flow rate of all measuring instruments was calibrated before and after each measurement, and the average value was applied. To measure the concentration of microbial and endotoxin levels of the general residential environment, indoor pollution was evaluated by the same method in the living room, bedroom, and kitchen. To compare the concentrations of microorganisms and endotoxins in the air, a control group (area sampling) was selected at a distance of about 100m from the farm, and measured simultaneously with the exposure group performing the agricultural work. Temperature and humidity were measured at all locations using direct-acting equipment (7545, TSI Inc., USA).
The number of samples by farm type was as follows: from open field farms, 4 personal, 20 workplace, and 7 control samples were taken; from greenhouses, 32 personal, 159 workplace, 15 residential area, and 42 control samples were taken; from the livestock facilities, 21 workplace, 9 residential area, and 5 control samples were taken from poultry farms, 8 workplace, 5 residential area, and 1 control samples were taken; from swine farms; and 48 workplace and 5 control samples were collected; from cattle farms. The total number of samples was 907 from greenhouse farms, 390 from livestock facilities, and 117 from open field farms.
Analysis method. Each gelatin filter was placed in a sterile tube with a 10 ml extraction solution (0.1% Pepton+0.01% Tween 80) immediately after measurement and stored in a refrigerator until analysis. The extraction was performed by shaking the tube for five minutes in a vortex mixer. After extraction, a 500 uL aliquot from the suspension was spread on tryptic soy agar, MacConkey agar, and Sabouraud Dextrose + Chloramphenicol Agar to cultivate bacteria, gram-negative bacteria, and fungi, respectively. In addition, the extract solution was diluted as necessary (10 -2 , 10 -3 ). The spread agar plates for personal samples and impactor agar plates for area samples were incubated at 37 ± 0.5 °C for 24, 48, 72, and 96 hours, and microbial cultures were counted as colonies. The concentration of airborne bacteria and fungi (CFU/m 3 ) was calculated by dividing the counted colonies by the volume of sampled air.
For endotoxin extraction, a glass fibre filter was placed in a sterile pryogen-free with 10 ml LAL water. The extraction was performed by shaking the tube for 15 min in a vortex mixer and then subjected to ultrasonication for one hour. The extracts were centrifuged at 2,500 r.pm. for 25 min, and only the supernatant was analyzed. Endotoxin activity was quantitated using a Turbidimetric-Kinetic Limulus amoebocyte lysate (LAL) assay (BioWhittaker, Walkersville, MD, USA), and its concentration expressed as endotoxin units (EU) per cubic meter (EU/m 3 ).
Statistical analysis. Data were analyzed using SPSS 18.0. T-testing, Mann-Whitney U testing, and Kruskal-Wallis H testing were performed to compare the concentrations on each type of farm, and to compare concentrations between the individual and local samples. All tests were two-sided, and p-values < 0.05 were considered statistically significant.
Ethical Considerations. The study was approved by the Institutional Review Board (IRB) of Hanyang University (Approval No. HYI-13-068-1). All study participants were fully informed about the purpose and procedure of the study and provided written consent. Table 1 shows the number of samples for each type of farm. Each farm type had subgroup samples: personal, workplace, residential area, and control area, and all samples were tested for endotoxins, total bacteria, gram-negative bacteria, and fungi. Table 2 shows the bioaerosol concentration, temperature, and relative humidity for each type of farm. Endotoxins, total bacteria, gram-negative bacteria, and fungi were measured. The concentrations at livestock facilities were significantly higher than those at open field and greenhouse facilities (p<0.05). In particular, gram-negative bacteria, which are known to contribute directly to the production of endotoxins, had an average concentration at livestock facilities that was about three times higher than that in The concentrations of total bacteria and gram-negative bacteria, but not fungi, were higher than those at poultry houses and cowsheds. In both the greenhouse and livestock facilities, the concentration of endotoxins in the workplace and residential areas was the highest, followed by total bacteria, gram-negative bacteria and fungi, respectively. The concentrations of endotoxins and microorganisms in the residential areas near greenhouses were higher than in the control areas. The difference in concentrations of endotoxins, total bacteria, and fungi was not statistically significant. The concentration of gram-negative bacteria in greenhouse workplaces was significantly lower than that in residential areas. The concentrations of endotoxins, total bacteria, gram-negative bacteria, and fungi in the workplaces of livestock facilities were all significantly higher than those in residential areas. The temperature of the open field and greenhouse farms was about 27 °C, and relative humidity -46-56%. The temperature of the poultry houses was 13.8 °C, and relative humidity -54.6%. In the cowsheds, the temperature was 10.4 °C, and relative humidity -59.0%. At the hog farms, the temperature was 20.9 °C, and relative humidity -74.6%. Table 3 compares the concentrations in the personal area samples from greenhouses. The concentrations of endotoxins and microorganisms were measured in samples taken from each of the people working in the greenhouse, the workplaces and control areas. The geometric mean concentrations of endotoxins and gram-negative bacteria in the personal samples were significantly higher than those from the workplace samples. The average temperatures measured with each sample were between 26.6°C-32.0°C, and relative humidity between 41.8%-49.6%.

RESULTS
There was a relationship between the concentration of bioaerosols and the surrounding environment (Tab. 4). Total bacteria showed a moderate correlation with fungi (r=0.73), while gram-negative bacteria showed a moderate correlation with endotoxins (r=0.72). In addition, the correlation coefficient between endotoxins and total bacteria and fungi was 0.46-0.47, indicating a moderate correlation. On the other hand, temperature showed a weak negative correlation with the levels of endotoxins and total bacteria, while relative humidity showed a weak positive correlation with endotoxins and gram-negative bacteria.

DISCUSSION
In rural or agricultural environments, health problems caused by bioaerosols of pollen, grains, animals, and animal wastes have been reported for decades [15,16,17]. Recently, health problems caused by endotoxins have emerged with the most commonly reported being respiratory problems (e.g., rhinitis, asthma, asthma-like syndrome, bronchitis, and COPD). It is known that when the level of endotoxins in the air exceeds 30 EU/m 3 , toxicity begins to appear. Endotoxin exposure at the level of 100 EU/m 3 can cause respiratory tract inflammation, and exposure to 2,000 EU/m 3 could lead to severe pneumonia symptoms [18,19,20].
In order to prevent endotoxin-induced illness, some countries, including The Netherlands, but not Korea, have established exposure limits in the workplace or the general environment. The Netherlands recommends an exposure limit for the general population of 30 EU/m 3 , based on applying a safety factor of three to the occupational limit of 90 EU/m 3 [8]. The Occupational Health and Safety (OHS) research centre, one of the leading research centres in Canada, and the Institut de recherche Robert-Sauvé en santé et en sécurité du travail (IRSST), on the other hand, provides action levels based on the respiratory symptoms of exposed workers that require improvement of the working environment. When respiratory symptoms are reported, the action level is 10 times the background level, and in the absence of respiratory symptoms, the action level is 30 times the background level.
In South Korea, in the working environments of most industries, including manufacturing and agriculture, there are no established exposure limits for microorganisms. Instead, the 'Indoor Air Quality Control Act' of the Ministry of the Environment and 'Office Air Management Guideline' of the Ministry of Employment and Labour, suggest a microbial exposure standard for offices and some vulnerable living environments. For example, in multi-use facilities, such as medical institutions, postpartum care centres, nursing homes, day care centres and all offices, the total bacteria in the indoor air should be less than 800 CFU/m 3 [21]. To determine the approximate exposure level it is necessary   [22], and in a study of 14 hog farms by Yoo et al. in 2003 [23], the endotoxin concentration was 190.55 EU/m 3 , results which are similar to those of the current study.
Previous studies have shown that airway inflammation may occur at endotoxin levels below 90 EU/m 3 and the positive significant reaction of the respiratory tract occurred when the geometric mean endotoxin concentration reached 15.4 EU/m 3 [24]. Endotoxin exposure as low as 10-200 EU/m 3 can also negatively affect the respiratory system [25]. Thus, the work environments of hog farms and poultry houses measured in the current study are harmful and could cause acute respiratory symptoms in exposed workers. Proper ventilation and the use of respiratory protective equipment is necessary to protect the workers.
Although the endotoxin concentrations on open field farms, greenhouse farms, and cowsheds were lower than those at hog farms and poultry houses, even those low concentrations might adversely affect health. In a study in New Zealand, plywood workers exposed to 23 EU/m 3 of endotoxins had significantly more attacks of shortness of breath with wheezing than unexposed controls [26]. In addition, another study showed that the mean FEV1 on spirometry decreased among potato processing workers exposed to 56 EU/m 3 of endotoxins [27], and a further study found a mean decrease in the FEV1 of textile yarn workers exposed to endotoxin levels of 0.64 EU/m 3 [28]. A systematic review also concluded that respiratory health effects from exposure to low levels of airborne endotoxin (<100 EU/m 3 ) seem plausible [29]. Therefore, it is necessary to manage endotoxin exposure in all agricultural workplaces and nearby residential areas. In addition, further studies should be conducted on the health effects of exposure to low levels of endotoxins.
The average concentration of total bacteria on all types of farms, although not the control areas, was higher than the exposure limits set by ACGIH [30] in Canada [31], and all microbial concentrations in the livestock facilities were higher than in the open field and greenhouse facilities, with hog farms again having the highest levels. Canada is proposing an additional 1,000 CFU/m 3 for gram-negative bacteria. The concentrations of gram-negative bacteria found in the current study in all the workplaces and control areas were lower than the proposed Canadian limits. In the livestock facilities, which had a higher concentration of gram-negative bacteria than the other areas, the highest concentration was on the hog farms (0.73x10 3 CFU/m 3 ), followed by the poultry houses (0.63x10 3 CFU/m 3 ) and cowsheds (0.20 × 10 3 CFU/m 3 ).
A previous study in Korea reported a bacterial concentrations of 2.7 × 10 5 CFU/m 3 and 5.6 × 10 7 CFU/m 3 from hog farms and poultry houses, respectively [32], and also reported fungal concentrations of 4.9 × 10 3 and 2.1 × 10 3 CFU/m 3 , respectively. The remarkable difference in microbial concentrations between studies at similar work sites is presumed to be the result of the influence of temperature, humidity, and workplace conditions at the time of measurement. For example, in livestock workplaces, airborne microbial concentrations have been reported to be high during certain tasks, such as weighing, animal movement, and faecal-cleaning tasks [33]. Therefore, the current study, which did not take samples during such tasks, reports relatively low concentrations of microorganisms and fungi. When comparing concentrations of microorganisms in a workplace, it is necessary to take into consideration the working conditions and the season, along with environmental conditions, such as temperature and humidity, at the time of measurement.
The concentrations of organic dust in the residential areas of farmers were generally lower than those in the workplaces. However, the endotoxin and microbial concentrations were still about three times higher than in the control areas. Furthermore, the bacterial and fungal exposure levels in those residential areas exceeded the criteria set by both the 'Indoor Air Quality Control Act' of the Ministry of Environment (800 CFU/m 3 for total bacteria, 500 CFU/m 3 for fungi) [21], and the levels found in residential and office spaces in typical urban areas in South Korea. In a study of 69 offices, , reported total bacterial and fungal concentrations of 426 CFU/m 3 and 234 CFU/m 3 , respectively [34].
The high levels of microorganisms found in the residential areas of farmers in the current study presumably occurred through propagation from the contaminated workplaces near the residential area. The workplaces of many farms in South Korea are located within 100 m of their associated residential area. Another possible reason is that most Korean farmers enter their residential areas wearing the same clothes they wore during work. For greenhouses and livestock facilities that have relatively high concentrations of microorganisms, rest areas should be located as far away as possible from the workplace. In addition, protective measures, such as the installation of ventilation equipment, will be to change the behaviour of the workers, e. g. taking off their work clothes and showering before entering residential areas.
There are some limitations to consider when interpreting the results of this study. Firstly, the samples were collected at different times of the year, therefore the effects of environmental conditions (temperature, humidity, wind speed, etc.,) and seasonal factors could not be controlled. Secondly, the microbiological and endotoxin levels of some farms, especially the livestock facilities, might be underestimated because the measurement protocols did not adequately cover certain tasks, such as weighing, animal movement, and faecal-cleaning tasks which are known to generate high concentrations of endotoxins.

CONCLUSIONS
The measured levels of endotoxins in the workplaces at hog farms and poultry houses exceeded exposure limits, indicating that the health of farmers working under those conditions could be threatened. The total bacterial concentration also exceeded the exposure limit at all sites where it was measured. In addition, the concentrations of personal samples, which more closely reflect worker exposure, were higher than those of the area samples, which imply that the actual exposure levels of farmers might differ, even within the same workplace, depending on the tasks performed and the degree of work activity. Interestingly, exposure levels in residential and rest areas were significantly higher than in the control areas, although they were lower than in the workplaces. This was presumably caused by contamination from bioaerosols generated in the workplace.
In order to protect farmers from health hazards caused by bioaerosols, appropriate measures should be taken to reduce them in the workplace; countermeasures against pollution in residential and rest areas should also be considered.


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Dietary Effects of Free Insulin-Like Growth Factor-1 Supplementation On the Production Performance, Serum Tumor Necrosis Factor-A and Nutrient Digestibility in 50-80 Day Old Pigs

 This study determined the effect of dietary supplementation of insulin-like growth factor-1 (IGF-1) on the production performance, nutrient digestibility, complete blood count (CBC) and serum concentration of tumor necrosis factor-α (TNF-α) on 50 pigs aged 50-80 days (Large white x Landrace; 9.48 + 3.59 kg body weight; 50.6 d of age) , assigned randomly to five treatments (n=10), including one basal (A=0), and four treatment diets with IGF-1 supplementation (B=0.25, C=0.5, D=1, and E=2 kg/ton of feed, respectively). Feeding lasted for 28 days, after which 5 pigs from each treatment were selected to undergo nutrient digestibility test. At day 14 and 28, blood samples were extracted for CBC and TNF-α analysis. Results yielded no significant differences among treatments for body weight , feed intake, average daily gain, and feed conversion ratio. However, the average fecal score for days 1-28, treatment C differed significantly (P<0.05) being the poorest. There were no significant differences among the treatments on crude protein, crude fat, digestible energy and metabolizable energy. Nursery pigs fed diets with free IGF-1 have lower WBC count (P<0.05) and tended to be higher in RBC count (P=0.0607) at day 15. Pigs supplemented with free IGF-1 tended to have lower (P=0.0569) TNF-α serum concentrations compared to Control at day 14. Concluding, the dietary supplementation of IGF-1 does not significantly affect production performance and nutrient digestibility. Collectively, results indicated that supplementation of free-IGF-1 in piglets decreased WBC and serum TNF-α levels while increasing RBC indices in piglet 2 weeks after weaning.

Introduction
IGF-1 or Insulin-like Growth Factor 1 is a protein molecule with similar structure to Insulin. It is produced primarily by the liver and has been shown to stimulate growth and development as well as immune modulation. Animals naturally produce IGF-1, however, diseases and other stress factors compromise its production leading to poor growth and immune suppression. Insulin and insulin-like growth factor (IGF) , an opportunity may exist to enhance gut growth and development through supplementation of the newly weaned pig with exogenous growth factors. In an attempt to maintain and enhance intestinal health in pigs and improve productivity in the absence of in-feed antibiotics, researchers have evaluated a wide range of feed additives.. Hence, this study seeks to determine the dietary effects of free IGF-1 supplementation on the production performance, nutrient digestibility, complete blood count (CBC) and serum concentration of tumor necrosis factor α on weaners. The study was conducted at the Institute of Animal Science swine research facility, University of the Philippines Los Baños, Laguna.

Animals and Experimental Design
A total of 50 crossbred weaner pigs (Large white x Landrace) with an average body weight of 9.48 + 3.59 kg and 50.6 days of age were used in this study. The population consisted of 26 male pigs with an average body weight of 9.73 kg and 50.1 days of age and 24 female pigs with an average body weight of 9.20 kg and 51.1 days of age. These pigs were weaned at 35 days of age, stayed at the farrowing house for another 10 days, and transferred to the nursery building. Pigs were allowed to acclimatize to the new environment and the dietary treatments for 5 days before commencing the feeding trial. At the day of allotment, each pig were weighed and the sex were recorded. The allocations were done by weight and balanced by sex. Ten blocks were made, each having 5 pigs. The pigs were fed with basal diet (control group, treatment A) and 4 diets added with free IGF-1 at the rate of 0.25 (treatment B), 0.5 (treatment C), 1.0 (treatment D) and 2.0 (treatment E) kg/ton. The basal diet was formulated to meet or exceed nutrient requirements recommended by the Philippine Society of Animal Nutritionists Feed Reference Standards (2010) for swine ( Table 1). The free IGF-1 used in this trial was provided by Philchema, Inc. Free IGF-1 (immuTEIN™) is a unique source of activated IGF-1. Its proprietary technology increases the effectiveness of biological proteins which also include growth factors, cytokines, immunoglobulins, transfer factors and other functional proteins that are bioactivated and enhanced in immuTEIN. After allotment, each weaner pig was placed on individual and concrete-oored pens of 2.0 x 4.2 meters in size. To allow ad libitum access to feed and water, individual feeder and drinker were installed. The environmental temperature was recorded daily.

Sampling and Measurements
Study 1: Production Performance, Mortality and Fecal Score During allotment, pigs were weighed and designated as day 1 initial weight. Daily feed allowance per pig were recorded including refusal. Thereafter, pig were weighed on days 7,14, 21 and 28. To obtain average fecal score for each treatment, fecal consistency per pig was assessed and visually scored by 4 independent evaluators twice daily (9:00H and 16:00H) from days 1 to 28, based on fecal scoring guide ( Figure 1).

Study 2. Nutrient Digestibility
After 28 days of feeding trial, a total of 15 pigs (3 pigs per treatment group) were selected to undergo a nutrient digestibility test, done by allocating 1 metabolic cage per treatment that was implemented in 3 batches. In selecting the rst batch of pigs that went into the metabolic cages, pigs from each treatment were weighed -the heaviest pig from each treatment represented the rst batch. The same diet was fed and extended for three days, after which, 0.2% or 2 grams of chromic oxide per kg of mixed feed was added to serve as an indigestible marker for 8 days. Fecal and urine samples excreted that contained the indigestible marker were collected on day 5, 6, 7 and 8. These samples were pooled and stored at -20°C.
The same process was done on the second and third batches. The third batch, in turn, represented the lightest of the pigs and it took 24 days to complete the three batches. All fecal samples were oven-dried at 90°C for three weeks, grinded, and submitted to the laboratory for crude protein (CP), crude fat (CF) and gross energy determination. Urine samples were processed to obtain gross energy.

Study 3. Blood Assay
On day 14 of the feeding trial, blood was extracted from 8 pigs per treatment selected randomly. It was repeated on day 28 on the same pigs. Serum were obtained and stored at -80°C. Serum tumor necrosis factor α (TNF-α) was determined using ELISA kits speci c for TNF-α. Similarly, blood samples (10 ml) were collected randomly from 6 pigs per treatment on day 15 and 30. Samples were submitted to a thirdparty laboratory complete blood count (CBC).

Statistical Methods
Wilk-Shapiro was employed to test the normality followed by the test of homogeneity. The test of homogeneity of variances was done using Sphericity Test. Tukey's Studentized Range (HSD) test was employed for the signi cance among treatments. When the matrix is symmetric, MANOVA was used, if not, UNIVARIATE was used. Across all studies, P<0.05 was applied.

Results
There were no signi cant differences among treatments for the body weight, cumulative feed intake (FI), average daily gain (ADG), feed conversion ratio (FCR), and weekly fecal score (Table 2). However, when average fecal score was calculated for the entire period of day 1-28, treatment C (P< 0.05) had the poorest fecal score among the treatments. As shown on Table 3, there were no signi cant differences among the various treatments for crude protein (CP), CF (Crude Fat), digestible energy (DE) and metabolizable energy (ME). Pigs fed diets with free IGF-1 have lower WBC count (P<0.05) and tended to be higher in RBC count (P=0.0607) at day 15 but showed no treatment differences (p>0.05) at day 30 (Table 4). Other CBC parameters are the same in all treatments (P>0.05). Similarly, pigs supplemented with free IGF-1 tended to have lower (P=0.0569) TNF-α serum concentrations compared to Control at day 14 and also, did not differ (P>0.05) at day 28 (Table 5).There was no signi cant difference on the serum concentration of TNF-α among the various treatments in both sampling days.

Discussion
The current study showed no signi cant differences on the performance parameters of pigs fed with different levels of supplementation with IGF-1. There was also no signi cant differences on digestibility across treatments. A direct contrast against growth performance parameters could not be made because, most of the IGF-1 supplementation studies directly measured the capacity and development of the gastro intestinal tract (GIT). In an attempt to explain the results of IGF-1 supplementation, a number of factors have been listed and supported by earlier literature. These factors are the age of the pig used, the in uence of IGF-1 Binding Protein (IGFBP) and receptors, the effect of digestion on oral IGF-1 administration, the feed as well the quality of supplement incorporation, the variability of the treatments, experimental error as well as the total number of pigs used on this study.
Age of the Piglet and IGF-1 The data of Dunshea and Walton in 1995, strongly suggested that the subcutaneous infusion of IGF-1 analogue on pigs aged 0-27 days expressed not only increased piglet growth rate (ADG) in late lactation, but also increased the growth of small intestine, spleen and pancreas. In this instance, the authors suggested that the spleen size seen may re ect an enhanced immune response. In 1996, Burin et al., conducted a study involving oral supplementation of human recombinant IGF-1. Their results demonstrated that oral administration of human recombinant IGF-I during the rst 4 days after birth signi cantly increased small intestinal mucosal growth in formula-fed neonatal pigs. Bene cial effects of orally administered IGF-I have been reported in neonatal piglets by Houle et al., (2000) and Burin et al., (2001). However, no effects of oral IGF-1 on serum concentration, overall body weight, or growth of tissues other than the intestine were reported. Most recently, Donovan and Monaco, in 2004 concluded that, in general, there is no effect of oral IGF-1 on serum concentration, overall body weight or growth of tissues other than the intestine in neonatal pigs. On the current study, overall average age of pigs used at the beginning of the experiment was 50.6 days, the youngest pig recorded was 44 days while the oldest was 52 days. The average weight was 9.48 kg, with the lightest recorded at 5.60 kg and heaviest at 12.78 kg. The age of the pig is a major difference on from the studies cited, which were conducted on neonatal pigs, 27 days being the oldest animal. Age has a linear effect towards intestinal development (E rd et al., 1982) thereby directly proportional or translated into growth performance of the pigs.

IGF-1 Binding Protein and Receptors
To be able to express its effect, IGF-1 needs binding protein. Insulin-like growth factor (IGF) action is in uenced by the insulin-like growth factor binding proteins (IGFBPs). Binding Proteins (BP) control the distribution, function and activity of IGF-1 in vivo and are produced and expressed in at least one form in almost all tissues. It is thought that mechanisms controlling the expression of BPs during various physiological states will determine whether IGFs, both IGF-1 and IGF-2, have effects on IGF-mediated ). On this current study, however, these binding proteins and receptors were not studied nor quanti ed, such that no direct conclusion can be made on the its role on the magnitude of effect during IGF-1 supplementation. It could only be said, based on the above publications that, IGF-1 binding proteins (IGFBP) and receptors mitigates the effects of IGF-1. On this study, however, it is impossible to say whether or not that there is su cient amount of serum binding proteins and receptors or even their total absence or presence in the circulation during the phase of production and age of the pig utilized. It could only be strongly suggested, based on the above literature, that, binding protein's availability as well as the receptors, will dictate the site and mechanism of action of IGF-1. Thereby understanding that binding proteins and receptors are paramount to IGF-1 to cause an effect, it will nonetheless assist on explaining the results of this study.

Effect on Digestion of Orally Administered IGF-1
Burrin et al., in 1992 concluded that orally administered IGF-1 is stable to digestion during neonatal stage of the piglet. According to Schoknecht et al. (1993), exogenous IGF-1 (or analogue) treatment given to pigs does not feedback in a negative manner to inhibit endogenous IGF-1 secretion, thereby allowing the growth-promoting properties of this hormone to be expressed, his conclusion formed the basis of dose dependent IGF-1 feeding trials. However IGF-1 is rapidly degraded within the adult gastrointestinal tract (Xian et al., 1995;Rao et al., 1998), but it appears to survive digestion and exert effects within the neonatal intestine. On the current study, we cannot entirely conclude whether or not the orally supplemented IGF-1 was affected by gastro intestinal secretions. We can only refer to the abovementioned studies that digestion will exert an effect on the availability of circulating IGF-1, in particular with older pigs.

Other Factors
The health and nutritional status of the animal may very well affect the magnitude of response. The absence of severe diarrhea is suggestive of a mature GIT, absence of pathogenic challenge or stable immune system, thus none or minimum effect of supplementation can be achieved on an apparently healthy animals. The average fecal score of Group C (IGF-1 at 0.5kg/ton) which is statistically signi cant has the value of 1.77 which leaned towards moist feces, thereby negating the ability of the treatment to reduce cases of diarrhea. The overall signi cance of the fecal score is with the time factor. When the analysis was done per week, no signi cant difference among treatments were observed. However, when day 1 to 28 was treated as one period, statistical difference was observed. Interestingly, the other treatments are statistically the same. However, this could be taken as an important factor for further studies for the optimum inclusion rate for the free IGF-1 on diets. Again, it must be noted that the experiment was conducted in weaned pigs that were apparently healthy and that pathogenic infection is low or none at all, with no other known disease challenge that manifested at the start of the feeding trial. The cause for these ndings were, the least to say, unclear, but may be attributed to the timing, the quantity of diet obtained and amount consumed, the quality of IGF-1 incorporation in the diet, dietary composition, and age at weaning, that constitute differences on absorptive function in the GIT. Some of these are clearly the limitations of this study. In general, it is well-accepted that response to feed additives is dependent on age of the pig, disease level, environmental factors, and type of diet. It may be possible that at this stage of production, the pig has been well acclimatized on the diet showing consistent feed intake and weight gain. Healthy and well-fed animals with appropriate management, favorable environment, and minimal stress are generally less responsive to a feed additive.

CBC and TNF-α Concentration
Piglets are known to be susceptible to enteric infections due to not fully developed immunity and by contact to pathogens naturally present in the environment. . The increased rate of RBC production leads to better oxygen transport in blood. In a study that involved cardiopulmonary bypass of piglets, intravenous infusion of IGF-1 improved oxygen transport by reducing oxygen consumption as well as increasing cardiac output and oxygen delivery during the rst 6 hours post-operation (Li et al., 2004). Better oxygen transport promotes anabolism such as muscle tissue growth. Improvement in RBC indices has potential to improve growth rate and health of pigs even during times of stress. Exposure to micro-organisms commonly elicit the production of cytokines. These soluble factors enhance several innate immune functions that aim to limit the spread of infection. The onset of production of these cytokines is rapid, and several of them may reach systemic levels during a short period after infection. Thus, cytokines can serve as markers for ongoing infections and be used to discriminate between infections of bacterial or viral origin. Several bacterial compounds (peptidoglycan, lipotechoic acid, polysaccharide, various toxins, heat shock proteins, and super antigens) can induce the production of pro-in ammatory cytokines (Degre, 1996) and consequently also infections with Gram-positive bacteria can result in measurable blood levels of TNF-α , IL-6, IL-8, and G-CSF (Kragsbjerg et al., 1996). One study on porcine intestinal ion transport shows that TNF-α may be involved in the stimulation of NaCl secretion in the ileum (Alexander et al., 2001, 2002and Kandil et al., 1994. Their data suggest that the increased expression of TNF-α may be implicated in the development of post-weaning diarrhea. Pro-in ammatory cytokines, such as TNFα, play a vital role in the normal host resistance to infection, serving as immunomodulators and as mediators of in ammatory signs (Kiarie et al., 2009) thus, identifying the changes in serum concentration of which could exhibit the effect of free IGF-1 supplementation on in ammatory response. The results illustrated that there is no statistical difference between the treated and non-treated groups with regards to the TNFα (pg/ml) serum concentration. This could imply that, since there are no severe clinical signs of diarrhea within the control and treatment groups, an immunomodulatory response through the pro-in ammatory cytokine, TNF-α, was demonstrated at lower values. A study reported by Nyachoti et al. (2012) reported that increased TNF-α increased from 111.4 to 162.1 pg/ml upon ETEC challenge which proves that during in ammatory encounter, TNFα serum concentration will be on upsurge. In a study done by Kruse et al.
(2008), TNF-α increased in all animals after inoculation of Brachyspira hyodysenteriae, with a peak at 300 pg/ml. It must be emphasized that the release of TNF-α, and other in ammatory cytokines, are rapid and induced early in a disease or injury process. However, it's not exclusive to viral and bacterial infections. A study by Pie et al. (2004) suggested that Cytokines play a central role in immune cell response, but they also participate in the maintenance of tissue integrity. Changes in the cytokine network of the pig gut may be expected at weaning, because abrupt changes in dietary and environmental factors lead to important morphological and functional adaptations in the gut. The current results, could suggest that the TNF-a levels present on the serum could indicate an in ammatory response or could be attributed to weaning. Stress brought by weaning commonly induce production of pro-in ammatory cytokines, such as TNF-α (Kiarie et al., 2009). Upon the presence of in ammation, nursery pigs supplemented with free IGF-1 could be able to immunomodulate or decrease the level of serum TNF-α , therefore reducing the signs of in ammation. However, attributing these values entirely to infection could not be determined due to the numerous factors affecting TNF-a release.
Concluding, the dietary supplementation of insulin growth factor-1 (IGF-1) had no signi cant effect on the production performance, serum concentration of tumor necrosis factor alpha and nutrient digestibility in weaned pigs. Collectively, results indicated that supplementation of free-IGF-1 in piglets decreased WBC and serum TNF-α levels while increasing RBC indices in piglets 2 weeks after weaning. We can also measure earlier (e.g. day 7) to see possible greater response. It can be concluded that supplementation of free-IGF-1 can lessen the negative impact of post-weaning stress particularly response to pathogen infection and in ammation. The study offered suggestive evidence for the role of dietary supplementation of IGF-1 on fecal consistency. No diarrhea was observed on the entire trial period, although pigs fed with control diet yielded the same fecal score. Further study should be focused on this potential. The study appears to support the argument that age of the pig is an important factor to consider. This would suggest that future research may done on neonatal pigs or at pre weaning period with a considerable population. It is relevant to relate levels of IGF-1 in the circulation, thus, blood pro ling can be done periodically during the trial period. Future research into IGF-1 supplementation can also be done by measuring the weight and length of the small intestine, furthermore a histopathological examination can be performed to characterized the villous height and crypt depth. Modi cation of the basal diet can also be suggested. Other in ammatory cytokines can also be included as a measure of in ammation.

Declarations Acknowledgement
This study will not be completed without the invaluable assistance from Philchema Incorporated, Department of Science and Technology -Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development (PCAARRD) and the Institute of Animal Science-University of the Philippine, Los Baños, much appreciation is given to the people behind these institutions.   Figure 1