To assess the extraction methods of egg yolk oil in ShiZhenKang (SZK) oil, which is used to treat eczema, a mice model of eczema was established by using 2,4-dinitrochlorobenzene (DNCB). The therapeutic effects of egg yolk oil extracted by different methods from SZK oil on the model of acute eczema in mice were evaluated. The oil yield rate of ethanol extraction is 42.06%. Its egg yolk oil is orange and has a rich, sweet, egg smell. Moreover, the SZK oil prepared from it has a very good therapeutic effect on the model of acute eczema in mice. The alcohol extraction method is the preferable method according to a comprehensive evaluation of each index of seven kinds of methods to extract the egg yolk oil. Eczema is a common skin disease in infants, mainly due to overnutrition, dyspepsia, food allergies, external stimulus and genetic factors ,2,3. TheCurrently, there are many kinds of treatments for infant eczema, For example, Chinese herbs, which belong to Chinese Traditional Medicine, have been successfully developed to treat infant eczema with relatively few side effects . ShiZhen2 extraction method, sub-critical propane extraction method and enzymatic methods [2 extraction methods are expensive and require expensive equipment [At present, there are many extracting methods for egg yolk oil, such as the dry distillation method, baking method, reduced pressure distillation, solvent extraction method, supercritical CO methods ,13,14. Equipment . The phoquipment ,17,18. Bquipment . Currentquipment ,21. In tThe egg yolk oils extracted by dry distillation, baking, and reduced pressure distillation were brown and disgusting a–c. The The mouse eczema model was established by application of 2,4-dinitrochlorobenzene (DNCB) solution to the dorsal skin of mice before administration of the different egg yolk oils. According to the visual inspection of the mice’s backs, the mice had varying degrees of eczema, which indicated that the model was established successfully. The eczema symptoms of the reduced pressure distillation group worsened three days after the administration and aggravated obviously five days after the administration, while the eczema symptoms of the other groups began to improve. The curative effect was similar to that obtained with paeonol (control drug group) .It could been seen that obvious erythema, edema, oozing, and auricle swelling appeared on the right ear of mice after stimulating the Delayed Type Hypersensitivity (DTH) reaction. Mouse auricle swelling was obviously improved after treatment with SZK oil obtained by the dry distillation, baking, and ethanol extraction methods and in the paeonol control, but the mice degree of auricle swelling of the reduced pressure distillation method group grew worse. This confirmed that except for the reduced pressure distillation method, the SZK oils prepared from egg yolk oil extracted by different methods had a good effect on the degree of mice auricle swelling, which had significant differences compared with the model group .The influence of eczema on the thymus index of each group  was: redThe histological images of auricle tissue confirmed that the mice model of eczema was successfully established b. The etThe use of egg yolk oil for topical treatment  appeared in the “set proved recipe” of the Northern Zhou Dynasty for the first time, and there are documents about treating sores, fistulas, dysentery and other diseases by oral treatment with egg yolk oil. This has been documented in ancient monographs, such as “Rihuazi Bencao”, “Compendium of Materia Medica”, “Chinese Eaglewood” and “Bencao Pinhui Jingyao” ,22,23,24This study evaluated the extraction methods of egg yolk oil according to the effects and characteristics of the egg yolk oil in eczema rehabilitation prescription . At presThe oil yield rates by the chloroform extraction method, petroleum ether extraction method, baking method, and the ethanol extraction method were all above 40%. However, oil extracted by baking method is black brown and has a burnt smell, so it is not suitable for the preparation of eczema liniments. The ethanol extraction and petroleum ether extraction method product is orange in color. In particular, the ethanol extraction method resulted in a rich egg fragrance and therefore is expected to be more easily accepted by patients. Zhang  preparedAs for the pharmacodynamics, the oils extracted by the ether, ethanol and petroleum ether extraction methods showed good curative effect on eczema skin lesions in mice. The carbonization method, baking method and ethanol extraction method products showed a good effect on the auricle swelling of the mice eczema model, which was significantly different compared with the model groups. The ether, chloroform and ethanol extraction method oils can reduce the mice thymus index that is the comparable to that of the control drug paeonol. The pathological section of auricle tissue in the mice model of eczema also confirmed that curative effect and the repair effect on the epithelial defects. The pathology images of auricle tissue showed that except for the reduced pressure distillation method oil, the SZK oils prepared from egg yolk oil extracted by different methods could reduce edema and oozing, alleviate keratinization, lighten denaturalization and putrescence of epithelial keratinocytes and decrease infiltration of inflammation cells.For economics and safety, chloroform and ethyl ether are chemically dangerous reagents, so both of them should be carefully considered if used for production, and petroleum ether is expensive, while ethanol, compared with ethyl ether, chloroform and petroleum ether, is both economic and safe. Jia  studied Overall, the ethanol extraction method showed high oil yield rate, orange color, rich egg fragrance and good therapeutic effect on acute eczema. The ethanol extraction method is thus the optimal egg oil extraction method to prepare SZK thanks to its relative safety and economic feasibility. In further experiments, in order to achieve a better application to clinical studies, the unique ingredients and main components in egg yolk oil extracted by the ethanol extraction method should be studied, and the number and variety of experimental animals should be increased.n = 10 for each group) were obtained from East innovation experimental animal science and Technology Services Department . Animal qualified number was SCXK (Xiang) 2009-0013. They were allowed to acclimate for one week. All mice were maintained at 21 °C on a 12-h light/12-h dark cycle with free access to water and food.All procedures in this study were approved by the Hunan University of Chinese Medicine Animal Care and Use Committee and were in compliance with the Guide for the Care and Use of Laboratory Animals. One hundred Kuming mice . Petroleum ether was obtained from Tianjin Fu Yu Fine Chemical Co. Ltd. . Anhydrous ethanol was purchased from Tianjin HengXing Chemical Reagent Co. Ltd. . Chloroform was purchased from Kaixin Chemical Reagent Co. Ltd. . 2,4-Dinitrochlorobenzene was purchased from Tianjin Guangfu Fine Chemical Research Institute . Acetone was obtained from Shenyang Yuanhang Chemical Co. Ltd. . Kuh-seng exctracts and borneol were purchased from Cunrentang Co. Ltd. . Paeonol was obtained from Ningbo Tianzhen Pharmaceutical Co. Ltd. . Water-bath pot (HH-S), Circulating water vacuum pump (SHZ-DIII), Rotary evaporator (RE-2000) and constant temperature heating magnetic stirrer (III) were purchased from Gongyi City Yuhua Instrument Co. Ltd. . Far infrared fast constant temperature drying box was obtained from Shanghai Yuejin Medical Optical Instruments Factory . TDl0001 electronic balance was purchased from YuYao Jinnuo Balance Instrument Co. Ltd. . TG328B analytical balance was purchased from Shanghai Banlance Instrument Technical Co. Ltd. . Animal shaver was obtained from Ningbo Daxie Development Zone Heng Qin Electric Appliance Factory . Olympus electron microscope was purchased from Olympus Corporation .Dry distillation method: egg yolk powder (174 g) was gently heated in the crucible until the water evaporated, and then heated with high heat to 275 °C until the egg yolk oil precipitated out. The oil obtained was separated from the residue with an asepsis gauze and weighed with an electronic balance.Baking method: egg yolk powder (100 g) was put onto a Buchner funnel lined with 60 mesh barbed wire and covered with a dish. The bottom of the funnel was put into a beaker, and placed in a constant temperature drying box, which was heated slowly up to 240 °C for 30 min. The obtained egg yolk oil was collected and weighed in the beaker.Reduced pressure distillation method: egg yolk (100 g) was put into four round bottom flasks and distilled on the electric heating set, which was adjusted to 250 °C. The egg yolk was distilled under reduced pressure (0.05–0.06 MPa) for 4 h in. The oil was collected and weighed.Ether extraction method: egg yolk powder (70 g) was added to ether (560 mL) and the oil was extracted under water bath heating at room temperature for 1.5 h. The remaining egg yolk powder was extracted again by ether (420 mL) for 1 h. After the removal of ether, the oil was collected and weighed.Ethanol extraction method: egg yolk powder (100 g) was added to 95% ethanol (800 mL) and extracted under water bath heating at 85 °C for 1.5 h, followed by addition of 95% ethanol (600 mL) to the remaining powder for 1 h. After the removal of ethanol, the oil was collected and weighed.Petroleum ether extraction method: egg yolk powder (100 g) was added to petroleum ether (800 mL) and extracted under water bath heating at 65 °C for 1.5 h, and then more petroleum ether (600 mL) was added to the remaining powder for extraction for 1 h more. After removal of petroleum ether, the oil was collected and weighed.Chloroform extraction method: egg yolk powder (100 g) was placed in a Soxhlet extractor filled with chloroform (200 mL) and heated in a 65 °C water bath for 1 h. The extraction was repeated four times until the egg yolk was white. After the removal of chloroform, the oil was collected and weighed.Fresh eggs were bought from a local Chinese market, and then they were cooked by bringing tap water to a boil and then kept simmering for 20 min. After being cooled in tap water for 15 min, eggs were shelled and egg yolk and egg white were separated. Wet pellets from egg were produced, through 14 mesh sieves, and then placed into the constant temperature drying box at 37 °C for 12–20 h until the yolk granules dried. The experimental samples were kept in sealed plastic bags in refrigerator at a temperature of +4 °C until experiment. The egg yolk oil was extracted by different methods as follows:All these extraction experiments were repeated five times according to the above extraction methodx. Oil yield rate % = (mass of egg yolk oil/mass of egg yolk powder) × 100%.SZK oil was obtained by mixing kuh-seng extracts (1.5 g), and borneol (0.5 g) with egg yolk oil (20 g) extracted according to the different extraction methods.Grouping and experimental preparation: after one week of adaptive feeding, the 100 mice were randomly divided into 10 groups, including blank control group, model control group, dry distillation method group, baking method group, reduced pressure distillation method group, ether extraction group, ethanol extraction group, petroleum ether extraction group, chloroform extraction group and paeonol (as positive drug) control drug group. Each group included five male mice and five female mice. All mice were sheared with an area of 3 cm × 3 cm on the abdomen and 1 cm × 2 cm on the left back.Sensitization: Except for the blank control group, 50 μL 5% DNCB acetone solution was evenly smeared onto the mice abdomen sheared zone to sensitize on the 1st day, and repeated on the 2nd day.DTH reaction stimulation: 10 μL and 15 μL of 1% DNCB were evenly smeared on the right ear in mice (both sides) and left back sheared zone on the 7th day, respectively. The ear and back skin of the blank control group were smeared similarly with DNCB All mice were stimulated again with a double dose on the 9th and 10th day.After 24 h of stimulation, each group of mice was treated with the corresponding drugs on the right ear and back sheared zone. The inside of the right ear and back sheared zone were treated with 0.05 mL and 0.1 mL SZK oil, respectively, twice per day for 5 days. The mice were killed 2 h after the last treatment.The skin changes, such as erythema and edema, on the back of experimental mice were observed daily since the start of drug treatment. The mice were scored two times per week according to the severity of erythema .The auricular tissue samples were immediately removed using a 7 mm hole puncher when the mice were executed and then weighed. The weight difference between the left and right ear was defined as swelling degree, and the ratio (%) of the weight difference of the left ear and right ear was defined as swelling rate:The thymus and spleen of each group of mice were weighed after the mice were executed and the thymus index and spleen index were calculated, respectively:The auricle tissues from the right ear of mice were cut into slices, dyed by hematoxylin and eosin (HE) staining, and observed under microscope.t test was used for statistical comparisons between any two specific experimental groups. Multiple group comparisons were done by ANOVA using a least significant difference post hoc analysis . p < 0.05 was considered statistically significant.Results are expressed as means ± standard deviation of the mean (etc. The results indicated that ethanol extraction was the optimal extraction method. This method preserves the main functions of the eczema rehabilitation prescription, is suitable for preparation production and has a good pharmacodynamic performance.Using the egg yolk powder as raw material, egg yolk oil was prepared by the distillation, baking, reduced pressure distillation, ether extraction, ethanol extraction, petroleum ether extraction and chloroform extraction methods, respectively. SZK oil was then made by using the obtained egg yolk oils to evaluate the egg yolk oil extraction methods by their pharmacodynamic indexes combined with oil yield rate, characteristics, color, smell,


Transport is a stressful procedure that can affect adversely the welfare of pigs and pork quality. It is well known that response to the journey is influenced by the genetic type. However, very little is known on the response of local breeds to the transport procedures despite the increasing interest in the welfare of animals during transport. The objective of this study was to investigate the effect of short distance transport on behaviour, blood parameters and meat quality traits of Apulo-Calabrese  and crossbreed [Duroc × (Landrace × Large White)] pigs. Statistical analyses were done using univariate and multivariate approaches. Both approaches showed that glucose, albumin/globulin, urea, and aspartate aminotransferase concentrations were influenced by the genetic type. Despite at loading Apulo-Calabrese pigs were less reluctant to move and showed a lower vocalization, physiological response to the short distance transport was more intense in this breed when compared with crossbreeds. With regards to meat quality, higher a* and lower L* coordinates were found in Apulo-Calabrese which indicates darker and more reddish meat than crossbreeds. The results from this study may provide insight into the response of local breeds to the transport procedures.longissimus thoracis samples. Univariate and multivariate analyses showed that glucose, albumin/globulin, urea, and AST at exsanguination were influenced by the genetic type. Apulo-Calabrese showed the highest increase in blood values of lactate, creatinine, sodium and potassium after the short distance transport. Behavioural occurrences were similar in both genetic types during unloading and lairage. Small differences were observed for meat quality although significantly higher a* and lower L* were found in Apulo-Calabrese pigs, showing meat with a deeper red colour than crossbreeds.Despite the increasing interest in the welfare of animals during transport, very little is known on the response of local pig breeds to the transport procedures. This study aims to compare the effect of short journey on behaviour, blood parameters, and meat quality traits in 51 Apulo-Calabrese and 52 crossbreed [Duroc × (Landrace × Large White)] pigs. All the animals were blood sampled five days before delivery  and at exsanguination for the analysis of creatine kinase, cortisol, glucose, lactate, albumin, albumin/globulin, total protein, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase, alkaline phosphate, sodium, and potassium. Post mortem pH, color, drip loss, cooking loss, and Warner-Bratzler shear force were measured at different times in  Stress associated with transport has been documented in pigs by a large number of studies ,2,3. AccIn pigs, extensive studies on blood parameters after transport have been carried out in conventional commercial breeds and their crossbreeds, whilst little information exists on local pig breeds such as Erhualian  and BasqAmong the indigenous Italian pig breeds is Apulo-Calabrese which is included in the list of endangered breeds by the United Nations’ Food and Agriculture Organization  and regiThe aim of this study was therefore to investigate the effect of short distance transportation on behavioural response, blood parameters, and meat quality traits of Apulo-Calabrese with respect to crossbreeds.Blood collection at the farm was carried out by a veterinarian in conjunction with routine sampling for sanitary controls. Transportation and slaughter of all pigs were carried out in compliance with EC regulation No 1/2005 and EC regulation No 1099/2013, respectively.Fifty-one Apulo-Calabrese pigs registered in the herd book of ANAS and 52 crossbreeds [Duroc × (Landrace × Large White)] were used. Apulo-Calabrese pigs were born in this farm from the mating of 13 sows by seven boars of Apulo-Calabrese whilst crossbreeds were bought at about 30 kg live weight from another piggery in the same region. All the pigs were fattened in the same finish facility in separate pens (7–10 pigs per pen) according to their genetic type and were fed the same commercial diet  in a liquid feeding system with dry feed and water mixed in a 1:4 ratio. All the pigs were identified by a numbered plastic ear tags. Apulo-Calabrese pigs were slaughtered when they reached 135 kg live weight (364 ± 58 days old) due to their slow growth whilst crossbreeds were slaughtered at approximately 155 kg live weight (300–330 days old).Approximately 12 h prior to transport, feed was withdrawn. Loading was carried out at 7 a.m. using a mobile ramp  available at the farm. Pigs were delivered through three consignments to the slaughterhouse. At each delivery, pigs from the four pens were herded by electric prods and walked the same distance (25 m) to reach the ramp which was positioned in correspondence with the facility door. The lorry was a hydraulic three tier equipped with internal partition and mechanical ventilation on the left side. Pigs were transported for approximately 1 h to a local processing plant  on two decks with a space allowance of about 0.50 m²/100 kg live weight. Unloading was done using the ramp of the lorry and pigs were driven for 10 m to the lairage pens where they rested for 30 min. Outdoor temperature and relative humidity were recorded for each journey by a thermo-hygrometer  during loading and unloading at the entrance of the ramp and at the entrance of the resting pen, respectively . During The behaviour recordings during loading at the farm and unloading at the abattoir included slipping, falling, reluctance to move, turning back, overlapping, and vocalization as previously described ,23. Lyin−), and potassium (K+). All biochemical parameters except for glucose, were measured in serum obtained from blood collected in serum separator tubes with gel separator and clot activator , let to clot and centrifuged at 1300× g at 20 °C  for 10 min. Plasma for the measurement of glucose was harvested from anticoagulated blood collected in Na2EDTA test tubes containing the glycolysis inhibitor potassium fluoride , centrifuged at 1300× g at 20 °C for 10 min. Parameters were measured by colorimetric assays on automated analyser  with the exception of cortisol. Serum cortisol was determined using the Kit Immulite One Cortisol .The animals were blood sampled five days before delivery as reference for basal blood parameter level (T0) and at exsanguination (T1) for the analysis of creatine kinase (CK), cortisol, glucose, lactate, albumin, albumin/globulin ratio, total protein, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), sodium  based on the scale developed by the Danish Meat Research Institute (DMRI) . The DMRlongissimus thoracis muscle (LT) at the level of six/seven thoracic vertebra were recorded at 1, 3, and 6 h post mortem on the left side, with a pH-meter  equipped with Crison electrode  and an automatic temperature compensation probe. At 6 h post mortem the left side was sectioned in the primal cuts and a sample of LT muscle between 6 and 10 thoracic vertebra (10 cm thickness) was collected. After 30 min of blooming, L*, a*, and b* coordinates were determined using Minolta chroma-meter  with D65 light source and 0 °C viewing geometry. Samples were transferred (by air) to a laboratory of the Department of Agricultural and Food Sciences . Measurements of pH were repeated at 24 and 72 h post mortem and colour measurements were repeated at 24, 72, and 144 h after slaughter. At the same time interval, cooking loss was determined on a slice of the LT muscle according to Honikel [Measurements of pH on the  Honikel  and Warn Honikel .RYR1) and Rendement Napole , major genes that influence the reactivity of pigs to stress and pork quality, genomic DNA was isolated from blood collected in tubes containing EDTA as anticoagulant. Genotyping of the c.599G > A single nucleotide polymorphism (SNP) PRKAG3 gene and the g.1843C > T SNP of the RYR1 gene were done by PCR-RFLP analyses [In order to determine the genotype of the Halothane .+ results. All transformed estimates were back-transformed for presentation to their original scale. Blood parameters were analysed using the mixed model (PROC MIXED of SAS) including the genetic type (two levels), sampling time (two levels: T0 and T1) and their interaction as fixed effects and subject within the day of slaughter as random effect. Sex as a fixed effect and hot carcass weight as a covariate were initially included in the model but they never reached statistical significance (p > 0.05) and were removed. Differences between means were tested by the Tukey-Kramer test (p < 0.05).Data from blood parameters were transformed to meet assumptions of homogeneity of variance and normality of residuals. Concentrations of CK, lactate, albumin, albumin/globulin ratio, creatinine, and AST were log10 transformed. A square root transformation was used to normalize cortisol and ALT results, while an inverse transformation was used to normalize glucose and Kp > 0.05) and were removed from the model. The data of drip loss was analysed using the same model without measuring time.Data of pH, colour, cooking loss and shear force were analysed using PROC MIXED of SAS for repeated measures. The same model for blood parameters was used replacing sampling time factor with measuring time . Sex and hot carcass weight did not reach the significant level  and basal blood parameters (T0) has been used to perform an unsupervised multivariate principal component analysis (PCA). All the new variables resulting from the difference between T1 and T0 blood parameters were normally distributed except for cortisol difference, which was root squared transformed in order to meet normal distribution criteria. Furthermore, a PCA has also been used to test the presence of differences in meat quality traits between the two studied genetic types. Unsupervised PCAs have been performed using on 3.4.4 . The datPROC GLIMMIX was used to analyse the effects of genetic type on skin damage scores recorded on each quarter separately as well as on the whole carcass. Because these data approximated a Poisson distribution, the GLIMMIX procedure’s POISSON option was used. The differences in least squares means (L.S.M.) were evaluated using Tukey–Kramer’s test.RYR1 gene [PRKAG3 gene [Both genetic types did not carry the recessive allele (c.1843T) of the YR1 gene  and the AG3 gene  that infAG3 gene ,30.Few research studies have been focused on the effect of transport on local breeds ,17 althop < 0.05) of reluctance to move and vocalization (p > 0.05) on skin damage score .Behavioural occurrences on both genetic types were collected at loading, unloading and lairage. The two genetic types showed no differences in the behavioural occurrences during unloading and lairage, while at loading Apulo-Calabrese pigs showed significantly lower percentages (lization . During p < 0.05) all blood parameters, except ALP and sodium whilst the effect of genetic type was significant (p < 0.05) for glucose, albumin/globulin ratio, urea, creatinine, AST and potassium. Of particular interest was the interaction between the genetic type and the sampling time since it lays emphasis on the possibility that variation of plasma components between basal and exsanguination is influenced by the genetic type. This interaction was statistically significant (p < 0.05) for lactate, albumin/globulin, urea, creatinine, AST, ALT, ALP, sodium, and potassium.Sampling time statistically influenced (p < 0.05) were found in both genetic types. Higher levels of lactate in the blood have been associated with physical stress [At exsanguination, significantly higher levels of lactate  of albumin/globulin were found at exsanguination in Apulo-Calabrese than the crossbreeds, the values obtained were however within the normal physiological range for pigs [Significantly lower levels  were found in Apulo Calabrese at the basal condition when compared with the crossbreeds whereas serum urea did not differ between the two genetic types. At exsanguination Apulo-Calabrese showed significantly higher levels of urea and lower levels of creatinine when compared with crossbreeds. The values of urea and creatinine obtained in this study were within the normal physiological range for pigs and were in agreement with the results found by Dikic et al. [Higher levels of urea and creatinine in the blood have been associated with food deprivation stress and an increase in physical activity as a result of the transport procedures . In thisc et al.  in localp < 0.05) of AST were found in Apulo-Calabrese at exsanguination when compared with crossbreeds. It is interesting to note that the concentration of AST increased slightly in Apulo-Calabrese from T0 to T1, unlike in the crossbreeds which demonstrated a remarkable increase at slaughter compared with the values obtained at the basal level. According to Pugliese and Sirtori [AST, ALT, and ALP are chemical indicators of tissue function: elevated levels of these enzymes occur when liver and pancreas are damaged. Significantly higher levels  decrease was reported in the first 6 h after slaughter, which stabilized during subsequent measurements. A similar pH trend was observed by Shen et al. [Post mortem pH decline a was simn et al.  when comp < 0.05) than those recorded in the meat of crossbreeds in all measuring times, with the exception of those recorded at 144 h after slaughter. The L* coordinate in the meat of crossbreeds stabilized after 24 h whilst that of Apulo-Calabrese maintained an almost constant increase. According to Scheffler and Gerrard [p < 0.05) values of a* (L* (lightness) measured at 6, 24, 72, and 144 h after slaughter increased progressively as post mortem time increased b. The L* Gerrard  post mores of a* c were foHigher values of cooking losses were reported for both genetic types at 24 h post mortem a compareWarner-Bratzler shear force measured after drip and cooking loss b decreaslongissimus thoracis pH, the estimated L.S.M. for pH at 24 h were the most divergent between the two genetic types  when compared with the pH measured at the other times. This result suggests that using a combined statistical approach may allow to highlight the main differences that would have not been appreciable with the use of univariate statistics alone.The variables that weighted most in PC2 were colour coordinates a* at 24 h (−0.409), a* at 6 h (−0.394), a* at 72 h (−0.368), a* at 144 h (−0.363), L* at 72 h (−0.257), and pH measured at 24 h (0.276). The high weights observed for a* colour coordinate are in agreement with the significant differences obtained from univariate analysis reported in Overall, the results obtained in this study broaden the knowledge on the Apulo-Calabrese pig breed, which showed higher levels of lactate, urea and AST after short distance transport indicating a more intense physiological response when compared with crossbreeds. With regards to meat quality, similar trends for pH, drip loss and cooking loss were found for both genetic types. The higher a* coordinate found in Apulo-Calabrese pig indicates that meat from this breed has a deeper red colour and can be used for the production of typical cured meat which on the basis of the gathered evidence could be produced without the use of additives intended to improve colour. The results in this preliminary study may provide insight into the response of local breeds to the transport process. However, due to practical restraints, in this pilot research it was not possible to investigate the effects of different pre-slaughter treatments on the behavioural and physiological responses of the two genetic types, further research is needed to evaluate the effect of different transport durations and handling practices on welfare and meat quality traits when transporting local pig breeds.


Macoma balthica, Macoma phenax, Mulinia sp., Rangiacuneata, Myaarenaria, Guekensia demissa, and an undetermined mussel species) from five collection sites was analyzed for the presence of type A influenza virus by qPCR targeting the matrix gene. Of the 300 tissue samples analyzed, 13 samples (4.3%) tested positive for presence of influenza virus A matrix gene. To our knowledge, this is the first report of detection of IAV in the tissue of any bivalve mollusk from a natural water body.We evaluated the prevalence of influenza A virus (IAV) in different species of bivalves inhabiting natural water bodies in waterfowl habitat along the Delmarva Peninsula and Chesapeake Bay in eastern Maryland. Bivalve tissue from clam and mussel specimens ( Crassostrea virginica) alone is substantial; typical filtration rates are approximated as 5 L water/h/g dry tissue during peak filtration periods in summer [Rangia cuneata has been reported to range from 7.7 to 29 g C m−2 throughout fresh and brackish water up to approximately 10 ppt salinity in Chesapeake Bay and some of its major tributaries, and even the small Baltic clam  has comparable biomass levels in the region [Cryptosporidium parvum and Giardia sp., bacteria such as Vibrio sp. and fecal coliforms, and enteric viral agents such as Hepatitis A virus [Dreissena polymorpha) from the Shannon River drainage in Ireland were found to carry C. parvum oocysts, Giardia lamblia cysts, Encephalitozoon intestinalis spores, and Enterocytozoan beineusi spores [Giardia sp. cysts have also been recovered from Macoma spp. clams in the Rhode River subestuary [Bivalve mollusks provide invaluable benefits to their aquatic environments. As suspension feeders, bivalves such as clams, mussels, and oysters filter particulate matter from bay water and are widely regarded as a water quality control mechanism in the Chesapeake Bay ecosystem ,2. Filtrn summer , translae region . Chesape A virus ,7. Otheri spores . In the D. polymorpha) and Asian clams (Corbicula fluminea) under laboratory conditions [Type A influenza viruses (IAV) are infectious to many species of wild and domestic birds as well as many mammals. With multiple viral subtypes that vary considerably in pathogenicity among hosts, the environmental distribution and longevity of IAV is an important consideration for wildlife, domestic animal, and human health interests. Persistence of IAV in pond sediment from Alaskan waterfowl habitat has been demonstrated with a virus detection rate greater than 50% among samples . In watenditions ,14.Building upon this previously demonstrated premise that filter-feeding bivalves may bioconcentrate IAV in a controlled setting, the purpose of this study was to evaluate the capacity of native bivalves to accumulate detectable levels of IAV within a natural water body in waterfowl habitat in a region where large numbers of waterfowl concentrate during migration and overwinter. The coastal Delmarva Peninsula in Maryland is part of the Chesapeake Bay catchment and serves as an ideal location for this study, as it provides habitat for both a variety of bivalves in fresh, brackish, and saltwater and a high density of overwintering migratory waterfowl with a proven capacity to carry IAV.G. demissa mussels (25%) from Reed Creek, four of the 40 (10%) M. phenax clams sampled from Goldsborough Creek, and two of the 59 (3.4%) M. balthica clam specimens from Trippe Creek  were positive for the IAV matrix gene. Positive samples represented 12 individual mollusks (12/264 or 4.5%) including six of the 24 pe Creek . As prevMacoma spp. clams and mussels. The Macoma species are widespread and abundant in sediment of shallow waters throughout the Chesapeake Bay and its tributaries, acting as both suspension feeders and deposit feeders [Guekensia demissa, the positive mussel specimen, is a suspension feeder in the intertidal zone in Chesapeake Bay and its tributaries [These bivalve specimens testing positive for IAV represented three different species including  feeders ,15. Guekbutaries . The posbutaries . Water tbutaries .Avian influenza viruses (AIV) are carried subclinically by a variety of waterfowl inhabiting the Chesapeake Bay region . Low patLow pathogenic AIV is generally of little consequence to infected waterfowl. Still, it is important to monitor, recognize, and characterize these viruses present in a region such as this one that supports a diversity and high density of susceptible waterfowl and shorebirds as well as a major poultry production industry. Monitoring efforts that utilize sentinel species for the detection of IAV in the environment could enhance biosurveillance programs for these pathogens, and the potential value of bivalve molluscan species as sentinel biosurveillance tools for IAV in the aquatic ecosystem merits further examination. Moreover, the role of filter feeding invertebrate species in the disease ecology of avian influenza in this region remains mostly undescribed and warrants exploration. It is plausible that with further investigation, seasonal variability may be noted in potential IAV recovery from bivalves related to changes in water quality parameters like temperature and salinity, variation in bivalve feeding behavior, and the abundance of waterfowl as primary reservoirs of the virus.M. balthica, Macoma phenax, Mulinia sp., R. cuneata, Mya arenaria) and mussels (Guekensia demissa and an undetermined species) were collected from each of four sampling locations in tidal riverine habitat of the Delmarva Peninsula and one location in the Chesapeake Bay in November 2015  and two samples per bivalve specimen were collected, processed and analyzed from the larger species (Rangia sp. and G. demissa). Tissue samples were handled aseptically and treated individually throughout the entire process as follows using flame-sterilized dissecting instruments. Whole bivalves were thawed slowly on ice, and external surfaces were disinfected with 70% ethanol. Approximately 30 mg of composite tissue  was collected for each sample and placed in sterile bead ruptor tubes (2mm ceramic beads) with 600 mL TRIzol® Reagent . Samples were subjected to bead homogenization on an Omni Bead Ruptor 24 bead mill homogenizer  for three cycles of 40 sec (6 m/s) with intermittent 3 min intervals of samples cooled on ice. Supernatants were collected from the homogenized specimens for RNA extraction via the E.Z.N.A. Viral RNA Kit and Protocol . All extracted nucleic acid samples were stored at −20 °C until analyzed. Sample RNA was transcribed to cDNA using the Applied Biosystems high-capacity cDNA reverse transcription kit , using random hexamers, according to the manufacturer’s protocol.Three hundred tissue samples from bivalve specimens were processed for analysis. One tissue sample per bivalve specimen was collected, processed, and analyzed from the smaller species  for use as a positive control standard. Primers and the synthetic control sequence are listed in ® Green PCR Master Mix  and 10 pM of each forward and reverse primer. Cycling parameters were 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 58 °C for 15 s and 72 °C for 20 s. An appropriately sized amplification product was confirmed for each reaction by electrophoresis of 5 µL of the reaction product through a 1.2% I.D.NA™ agarose gel  at 100 V for 45 min.All samples were screened for the presence of IAV via quantitative PCR targeting the influenza A matrix gene . A parti


Treponema and Filifactor) and in the oral microbiota (e.g. Moraxella and Capnocytophaga) of healthy cats. Phylogenetic beta diversity analysis showed that the microbiota of periodontally healthy cats were distinguishable from diseased cats. Even though most of the genera known to be associated with periodontal disease were also identified in healthy cats, they were present at significantly lower relative abundance. Remarkably, alpha diversity was found to be higher in the disease groups compared to healthy animals. These results suggest a pathological mechanism involving opportunistic behavior. Our findings corroborate those in the current literature regarding the complexity of the subgingival microbiota of the domestic cat and reveal both differences and similarities among periodontally healthy and diseased cats.Periodontitis is a common and important health problem in domestic cats. The subgingival microbiota of cats diagnosed with chronic periodontitis (CP), aggressive periodontitis (AP), and feline chronic gingivostomatitis (FCGS) are not well characterized. Thus, the aim of the present study was to characterize and compare the periodontal microbiota of periodontally healthy cats versus cats diagnosed with CP, AP, and FCGS by using next-generation sequencing. In total, 44 domestic cats were enrolled, and 139 subgingival samples were subjected to 16S rRNA gene sequencing to investigate the microbiota composition of each periodontal group evaluated. Our results identified several key genera previously described in periodontal disease (e.g.  It is characterized by loss of periodontal attachment, tooth mobility and eventual tooth loss3. It causes pain, gingival bleeding, reduced food intake, and may impact overall health5. Given its prevalence, as well as its local and systemic effects, periodontitis is a serious animal welfare issue that can impact quality of life. The pathogenesis of periodontitis involves the presence of subgingival bacterial plaque that initially leads to gingivitis and marginal tissue inflammation; if not treated it may progress to loss of periodontal attachment6.Periodontitis is an inflammatory disease that is highly prevalent in domestic cats8. Chronic periodontitis (CP) is the most common form and is characterized by slow progression1; the prevalence of CP increases with age2. In contrast, aggressive periodontitis (AP) has a higher rate of progression and typically begins during early stages of life9. Although subgingival bacteria play a crucial role in the disease, determination of the causal pathogen(s) has been inconclusive despite advances in the microbiology of subgingival biofilms in health and in different forms of periodontal disease9. In humans, it has been suggested that AP is associated with a mixed and complex microbial flora, with heterogeneity in the types and proportions of microorganisms recovered from individuals with the disease9. On the other hand, the supposed association of AP with an increased load of specific periodontal bacteria underscores the importance of bacteria to the disease process, and consequently the importance of microbiologic tests10.Periodontitis can be classified as chronic or aggressive11. FCGS may affect cats of any age, and treatment usually involves lifelong medical therapy or extensive surgery. Many cats fail to respond to treatment, and some are euthanized due to a poor quality of life13. Even though the etiology of FCGS is unknown, bacteria are thought to play a role in the disease pathogenesis16. Additionally, cats with FCGS have been shown to develop more severe and extensive forms of periodontitis compared to cats without the disease17. However, it is unknown whether the latter is due to underlying differences in the subgingival microbiome compared to cats without FGCS.Another important disease is feline chronic gingivostomatitis (FCGS), which causes severe pain and distress and is characterized by focal or diffuse chronic inflammation of the gingiva and oral mucosa18. Recent studies have made use of bacterial 16S rDNA sequencing to describe the feline oral microbial composition20. Sturgeon20 and colleagues characterized the oral microbiota of healthy cats using next-generation sequencing and found it to be highly diverse, rich, and even. Also, comparisons of the feline subgingival microbiota among healthy, gingivitis, and mild periodontitis cases were recently described based on pyrosequencing6. Porphyromonas was the most abundant genus in healthy cats, along with Moraxella and Fusobacterium, whereas Peptostreptococcaceae was the most abundant family in gingivitis and mild periodontitis6. Dewhirst et al.19, using bacterial culture and DNA sequencing, showed that for periodontitis as well as for other oral diseases, members of the phyla Spirochaetes and Bacteroidetes are key pathogens19, e.g. Treponema denticola and Porphyromonas gingivalis21, respectively. Pasteurella multocida subsp. multocida was found to be significantly more prevalent in affected cats compared to controls when using culture-dependent and independent methods in one study14.Techniques based on DNA sequencing and advanced bioinformatics tools can help unravel the complexity of the subgingival microbiota in periodontal health and diseaseIn order to better understand the possible role of the subgingival microbiota in periodontal health and disease in cats, we have applied next-generation sequencing techniques to assess and compare the periodontal microbiota from healthy cats and cats with CP, AP, and FCGS.The intrinsic characteristics of enrolled animals were analyzed. There was no statistically significant difference among disease groups  regarding sex  or breed distribution (domestic vs purebred) using a chi-square test. A Kruskal-Wallis test was used to analyze differences among groups regarding age and body weight. A statistically significant difference was identified among disease groups regarding age . Comparisons for all pairs using the Dunn method showed that cats with AP were significantly younger compared to cats with CP ; significant differences were not found between the other disease groups. No statistically significant differences were found among diseases groups in terms of body weight. Median, minimum and maximum age and body weight are presented in Table DNA amplification using barcoded primers and next-generation sequencing of the V4 region of the 16 rRNA gene were completed for all samples collected. The sequencing run was performed using a MiSeq sequencer and the reagent kit v2 (300-cycles) . Quality filtered reads were de-multiplexed, and the total number of reads was 5,372,557; the average coverage was 38,651 reads per sample, with a standard deviation of 11,454; the number of reads per sample ranged from 8,476 to 82,373 ; and the median length per read was 301 bases.The relative distributions of bacteria at the phylum level identified by 16S rDNA gene sequencing are depicted in Fig. Treponema was the most abundant genus in the disease groups. However, the relative abundance of Treponema was significantly higher only in the CP group compared to the healthy group (P = 0.0423). Snowella was more abundant in the AP group when compared to the two other disease groups. In addition, the relative abundance of Filifactor differed significantly between the healthy and AP groups , and between CP and FCGS (P = 0.0127). Candidatus Tammella differed significantly only between the healthy group and AP (P = 0.0332).Each bacterial taxon selected by the screening model was subjected to the Dunn method to examine potential differences across groups. Figure  P = 0.043. SnowelEnhydrobacter, Moraxella, and Capnocytophaga were the most abundant genera in healthy cats. When compared to the AP group, the genera Enhydrobacter (P = 0.0459), Moraxella (P = 0.0134), Bergeyella (P = 0.0151), Corynebacterium (P = 0.0288), and Comamonas (P = 0.0063) were found to be significantly more abundant in healthy cats. Furthermore, Moraxella (P = 0.0066), Capnocytophaga (P = 0.0002), Bergeyella (P = 0.0395), Dichelobacter (P = 0.0096), Bibersteinia (P = 0.0074), Actinobacillus (P = 0.0032), Comamonas (P = 0.0032), and Myroides (P = 0.0394) were less abundant in FCGS than in the healthy group. When comparisons were performed with the CP group, significant lower relative abundance of Corynebacterium (P = 0.0488) and Capnocytophaga (P = 0.0488) were identified only when compared to healthy cats.Bacterial genera identified as being significantly less abundant in the disease groups compared to the healthy group are reported in Fig. The alpha diversity was determined by Chao1 and Shannon indices and is depicted in Fig. To assess microbial community similarities among the different groups, beta diversity was calculated by using UniFrac distance metrics. Regardless of the UniFrac metric used  microbial communities significantly clustered by periodontal status  were detected as significant in the disease groups, as were genera known to be constituents of the oral microbiota of healthy cats (e.g. Moraxella and Capnocytophaga). However, we also identified significant genera which, to our knowledge, have not yet been associated with periodontal disease in cats, thus demanding further in-depth studies. Additionally, we found higher alpha diversity in the disease groups compared to the healthy cats. Notably, regardless of the method used to evaluate the beta diversity, healthy cats were found to harbor a microbial community that differed from that detected in diseased cats. Therefore, this study corroborated previous data and revealed new insights into feline periodontal disease and its different clinical forms and presentations.To better understand the subgingival microbiota of CP, AP, FCGS and periodontally healthy domestic cats, we used a 16S target sequencing approach. Significantly different relative abundances were identified among groups, both between healthy and diseased cats and among diseases. As was expected, genera with pathogenic species  were distinguishable. When comparisons were used to evaluate differences between groups, significant Unifrac distances were identified between healthy and disease groups. Accordingly, Chao1 and Shannon indices were higher in diseased cats. A previous study on periodontitis in humans using 16S pyrosequencing revealed that community diversity was higher in disease sites than in healthy sitesIn summary, our study corroborates findings in the literature regarding the complexity of the subgingival microbiome of the domestic cat and demonstrates differences and/or similarities based on periodontal status. We found higher bacterial diversity in the microbiomes of diseased sites compared to healthy sites, highlighting the important role played by bacterial biofilms in ecologically unstable tissue environments. Also, a high prevalence and significance of bacteria previously described as pathogens was demonstrated in disease groups. Additionally, beta diversity showed dissimilar bacterial composition between the groups studied. Further studies should be conducted with advanced DNA sequencing tools to improve the understanding of these diseases in cats based on genes and microorganisms from subgingival samples.Experimental protocols using cats were reviewed and approved by Cornell University Institutional Animal Care and Use Committee (protocol number 2015-0117). Informed consent for study participation was obtained from all cat owners prior to experimental sampling. The methods were carried out in accordance with the approved guidelines.36. Briefly, a gingival index of 0 was assigned when there was no visible gingival inflammation or bleeding upon periodontal probing; a gingival index of 1 was assigned when there was gingival erythema/edema but no bleeding upon probing; a gingival index of 2 was assigned when there was gingival erythema/edema and mild gingival bleeding upon probing; a gingival index of 3 was assigned when there was gingival erythema/edema and spontaneous bleeding without probing. Cats with periodontal probing depths <1 mm, no radiographic evidence of alveolar bone loss, and a gingival index of 1 or less on every tooth, were considered healthy controls. For the purposes of this study, CP was diagnosed when the cat had clinical and/or radiographic evidence of clinical attachment loss in 1 or more teeth regardless of the severity, had a gingival index of 2 or less in at least 70% of the teeth present, and had no signs of inflammation or ulceration beyond the mucogingival junction. AP was diagnosed when the cat had a gingival index of 3 affecting 30% or more of the teeth present; had more than 50% of clinical attachment loss affecting 30% or more of the teeth present; and had no signs of inflammation or ulceration beyond the mucogingival junction or at the caudal oral mucosa. A diagnosis of FCGS was made based on clinical examination findings including ulcerative or ulcero-proliferative lesions of the caudal oral mucosa, as previously described37.Periodontal status was assessed by a board-certified veterinary dentist based on periodontal probing and full-mouth radiographic findings. The Silness-Löe index was used to determine the gingival index during periodontal examinationFor the purposes of this study, when applicable, the stage of progression of periodontitis of sampled teeth was determined based on the following criteria: mild periodontitis, if there was clinical or radiographic evidence of less than 25% of attachment loss; moderate, if there was clinical or radiographic evidence of 25–50% of attachment loss; or severe if there was clinical or radiographic evidence of more than 50% of attachment loss.Animals that had received systemic antibiotics during the previous 4 weeks were excluded. Only cases in which full-mouth radiographs and periodontal probing and charting were performed were included in this study.The sampled population consisted of client-owned domestic cats originally presented to the Cornell University Hospital for Animals (CUHA) Dentistry and Oral Surgery Service for diagnosis and treatment of periodontal disease and/or FCGS. In total, 139 subgingival samples, up to 7 samples per animal, were obtained from 44 cats, each individual was classified in only one category. The sex distribution of sampled animals was 23 males (52.3%) and 21 (47.7%) females. The breed distribution was 39 domestic and 5 purebred cats . Out of the 139 samples, 27 samples came from 6 healthy control animals, 50 samples from 20 cats with CP, 39 samples from 11 cats with AP, and 23 samples from 7 cats with FCGS. Of the samples collected from the CP group, 31 (62%) were from sites previously diagnosed with mild periodontitis, 15 (30%) were from sites diagnosed with moderate periodontitis, and 4 (8%) were from sites with severe periodontitis. In the AP group, 21 (53.8%) samples were collected from sites diagnosed with mild periodontitis, 16 (41%) samples were from sites with moderate periodontitis, and 2 (5.1%) samples were collected from sites diagnosed with severe periodontitis. For the cats with FCGS, 10 (43.5%) samples were collected from sites diagnosed with mild periodontitis, and 9 (39.1%) and 4 (17.4%) samples were collected from sites diagnosed with moderate and severe periodontitis, respectively.Only canine teeth and carnassial teeth (maxillary fourth premolar and/or mandibular first molar teeth) were sampled. The sample side (i.e. right vs. left) was selected randomly. In the disease groups , only samples from teeth with periodontitis were included for analysis. Of the 27 samples collected from healthy cats, 13 (48.1%) were obtained from canine teeth and 14 (51.9%) from carnassial teeth. Of the 50 samples obtained from cats with CP, 24 (48%) were obtained from canine teeth and 26 from carnassial teeth. Of the 39 samples obtained from cats with AP, 17 (43.6%) were obtained from canine teeth and 22 (56.4%) from carnassial teeth. Of the 23 samples obtained from cats with FCGS, 6 (26.1%) were obtained from canine teeth and 17 (73.9%) from carnassial teeth, mainly because canine teeth in cats with FCGS often did not have clinical or radiographic signs of periodontitis.38 was used for collection of subgingival samples. Briefly, cats were clinically evaluated and sampled under general anesthesia using standard-of-care protocols supervised by a board-certified veterinary anesthesiologist. Each sterile endodontic paper point  was gently inserted into the sulcular area and gently rubbed along at least half of the circumference of individual teeth. Samples were aseptically collected, labeled, and stored individually in 1.5 ml sterile polypropylene microcentrifuge tubes, placed on ice, transported to the laboratory within 4 h, and then frozen at –80 °C.Subgingival samples were obtained prior to any instrumentation or disinfection, or systemic antibiotic administration. In the present study, the established technique of using a sterile endodontic paper pointTM distilled water  into a 1.5 mL microcentrifuge tube containing the paper point, which was placed in a vortex mixer  and vortexed/washed for 10 minutes. Paper points were removed from the microcentrifuge tubes and the remaining liquid was submitted to centrifugation for 5 min at 13,000 rpm at room temperature. The pellet obtained was used for DNA extraction, which was performed using a DNeasy® PowerFood® Microbial Kit  following the manufacturer’s instructions.DNA was extracted from paper points by adding 1.0 mL of UltraPure39, all DNA samples were amplified using different 12-bp error-correcting Golay barcodes for 16S rRNA gene PCR (http://www.earthmicrobiome.org)40. Amplifications were performed using 10 µM of each primer, EconoTaq Plus Green 1x Master Mix , 10 ng–100 ng of individual metagenomic DNA and UltraPureTM distilled water  to bring the final reaction volume to 25 µL; each DNA sample was amplified in triplicate and in all PCR plates a blank (no DNA added) was included. Also, a negative control  and a positive control  were included. PCR conditions were: initial denaturing at 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 50 °C for 1 min, and 72 °C for 90 s; and final elongation at 72 °C for 10 min. Pooled replicates were loaded in agarose gels  stained with 0.5 mg/ml ethidium bromide; electrophoresis was completed and the presence of amplicons was verified.Amplification of the V4 hypervariable region of the bacterial 16S rDNA gene for each sample was carried out by polymerase chain reaction (PCR) using primers 515F and 806R according to a previously optimized method for the Illumina MiSeq platform® Inc., San Diego, CA). Final equimolar libraries were sequenced using the MiSeq Reagent Kit V2–300 cycles .Amplified DNA was purified using a Gel/PCR Fragments Extraction Kit  and the concentration of each purified DNA sample was determined using a NanoDrop ND-1000 spectrophotometer . Aliquots of all samples were standardized to the same concentration and pooled for sequencing on the Illumina MiSeq platform  based on 97% identity against the Greengenes reference database43 (May 2013 release). Using USEARCH42, low abundance clusters were filtered, and chimeric sequences removed. Representative sequences for each OTU were compared against the Greengenes database for taxonomy assignment, and only full-length, high-quality reads (−r = 0) were used for data analysis. Phylogenetic trees were generated from the filtered alignment using FastTree44. Shannon diversity index and Chao1 richness index output were generated by the QIIME pipeline; prior to index estimation, the sample library was rarefied to an equal depth of 10,000 sequences using QIIME. To determine how taxa were related within and between subgingival microbiota of healthy cats and cats diagnosed with CP, AP, FCGS, both unweighted and weighted UniFrac distance metrics were generated in QIIME45. To account for uneven sequencing depth across samples, all sample libraries were rarefied to an equal depth of 10,000 sequences before estimating the unweighted and weighted UniFrac. Also, phylum- and genus-level OTU tables were generated using the MiSeq Reporter Metagenomics Workflow, which is based on the Greengenes database (http://greengenes.lbl.gov/). The output from this workflow is a classification of reads at multiple taxonomic levels .All 16S rRNA gene sequences generated were processed through the open-source pipeline Quantitative Insights into Microbial Ecology (QIIME) version 1.7.0-devDescriptive statistical analysis was completed in JMP Pro 11  using the chi-square test of independence to evaluate whether cat gender and breed differed significantly between groups. The Kruskal-Wallis test and the Dunn method were used to determine whether there were statistically significant differences between groups regarding age and body weight; both tests were completed using JMP Pro 11.The OTU tables obtained from bioinformatics analyses were used to describe the relative abundances of bacterial phyla and genera within the healthy and diseased cats. Each value obtained indicates the percentage relative frequency of reads with 16S rRNA genes annotated to the indicated taxonomic level. The arithmetic mean of the relative abundance of each OTU was calculated for each individual cat, a single parameter.The distribution of bacterial phyla across the groups studied was presented according to the average of relative abundance calculated by animal enrolled as described. The microbial profile of healthy and disease groups for the most abundant phyla was fitted using GraphPad Prism 7 .Response screening analysis was performed in JMP Pro 11 to determine which bacterial taxa are most associated or most important to the different periodontal status groups. The relative abundances of each genus found to have a False Discovery Rate (FDR) of <0.1 were illustrated by box-whiskers plots. A nonparametric comparison for all pairs using the Dunn method were performed using JMP Pro 11 to evaluate statistical differences among the different groups evaluated.Chao 1 richness and Shannon diversity indexes were calculated using QIIME. These diversity indexes were compared within all periodontal statuses using ANOVA in JMP Pro 11, and the Tukey-Kramer test (P < 0.05) was used to adjust for multiple comparisons. Box-whiskers plots were built using GraphPad Prism 7 .46 by QIIME (http://qiime.org/scripts/compare_categories.html). Principal coordinates were computed from the calculated UniFrac distance matrixes to compress dimensionality into three-dimensional principal coordinate analysis (PCoA) plots created by the “beta_diversity_through_plots.py” script in QIIME and visualized by EMPeror47. For calculation of pairwise ecological distances, the Mann-Whitney test followed by Holm–Bonferroni multiple comparison correction were used.Differences between microbial communities (beta-diversity) based on phylogenetic information visualized on the PCoA plots were calculated by permutational multivariate analysis of variance (PERMANOVA) with 999 permutations


Among the tested stress conditions, AmAChE1 expression was induced under the brood rearing-suppressed, crowding and heat shock conditions. The hsps, particularly hsp70 and hsp90, responded to seven of nine stress conditions tested, confirming that hsp expression profiles can serve as a general stress marker. Taken together, AmAChE1 expression is not suitable for using as a stress marker due to its limited response. Nevertheless, AmAChE1 expression appears to be connected, at least in part, to heat shock response and other pathways. Considering that AmAChE1 likely regulates the ACh titer particularly in non-neuronal tissues, thereby modulating the signal cascades mediated by mAChR, the AmAChE1 expression profile under different conditions likely provides important information on its physiological roles in honey bees.The honey bee acetylcholinesterase 1 (AmAChE1) has been suggested to be related to stress response as judged from its elevated expression level under brood rearing-suppressed conditions. To further investigate the involvement of AmAChE1 expression in the stress response and its physiological functions, we analyzed altered expression profiles of AmAChE1 induced by diverse stress factors. In addition, transcription profiles of several heat shock protein (Hsp) genes ( Apis mellifera, plays a major role in the pollination of commercial crops and native plants1. In addition to the pollination value, honey bees produce various beneficial products, such as honey, wax, pollen, propolis, royal jelly and venom. The total economic value of honey bees for agriculture in the United States was estimated to be $21.22 to $54.75 billion per year in 20152. Since first reported in the United States in late 2006, colony collapse disorder (CCD), which refers to the phenomenon of sharp declines of honey bee colonies, has been reported in most parts of the world3, causing serious damage to the agricultural industry4. Although the exact mechanisms of CCD are poorly understood, chronic accumulation of several stressors, including pesticides, parasites, malnutrition and pathogens, is known to cause CCD7. Therefore, it is imperative to understand the stress response systems of honey bees, and any markers for prior detection of stress in honey bee colonies would be useful for proactive management.The western honey bee, 10. In particular, some cellular stress responses such as the expression levels of heat shock proteins (Hsps) and cortico-releasing hormone-binding protein (CRH-BP) have been more recently used to evaluate stress in honey bees under a variety of stressors, including capture, transport, harnessing, cold, heat, and UV light11. As various stresses are known to trigger the heat shock response (HSR), Hsps involved in the HSR can serve as stress markers for detecting and quantifying cellular stress levels12. Vitellogenin (Vg) has also been suggested as a plausible candidate for a stress marker in that it provides a protective role against oxidative stress and is regulated by the juvenile hormone, which is also considered to be related with stress responses14. Biogenic amines, such as dopamine, serotonin and octopamine, have also been studied in connection with stress responses16.To assess honey bee stress levels, several indicators for stress responses, including physiological, behavioral and cellular stress responses, have been used under various stress conditions17. Based on the assumption that the suppression of brood rearing can cause a stressful condition in honey bee colonies, AmAChE1 was suggested to be involved in the stress response or stress management in honey bees17. The readthrough AChE (AChE-R), which is a soluble variant of mammalian AChE and likely a molecular homolog of AmAChE1, is induced by various stressors and thus functions as a stress signal molecule20. In addition, it was reported that the pinewood nematode (Bursaphelenchus xylophilus) and fruit fly (Drosophila melanogaster) generate soluble AChE upon chemical stress22. Taken together, these support the notion that AmAChE1 is likely an element of the stress regulation pathway in honey bees.In recent studies, the expression of honey bee acetylcholinesterase 1 (AmAChE1) was determined to be highly correlated with the state of brood rearing by increasing when brood rearing was suppressedVarroa mite infestation, bacterial challenge (Escherichia coli and Staphylococcus aureus) and brood-rearing suppression. In addition, we determined the transcription profiles of Hsps  and Vg and evaluated the connection between AmAChE1 expression and the Hsp-mediated stress response pathway. The putative roles of AmAChE1 in stress regulation and other physiologies were further discussed.In this study, to further investigate the involvement of AmAChE1 expression in the stress response and its physiological functions, we analyzed altered expression profiles of AmAChE1 induced by diverse factors, including chemicals , temperature (heat and cold shock), UV-B irradiation, crowding, dehydration, starvation, Amace1) and several stress marker genes. However, the expression level of AmAChE1 as judged by Western blotting was not well correlated with the mRNA level of Amace1. When this discrepancy occurred, the protein expression level of AmAChE1 was primarily used for data interpretation. The results are summarized in Table Worker bees were treated with each stress factor and then the protein expression levels of AmAChE1 were primarily evaluated and compared with those of control bees, along with transcription levels of AmAChE1 gene  results, the stress marker genes showed different expression patterns depending on tissues and age , 90 (p = 0.001) and vg (p = 0.009), but this was not observed in the abdomens of forager bees. When forager heads were compared, however, expression levels of hsp10 (p = 0.049), 60 (p = 0.051) and 70 (p = 0.027) were higher in the control groups compared to the treated groups, whereas no significant differences in abdomens were observed. In forager bees, the overall transcription levels of the hsp and vg genes were higher in the control compared to nurse bees.AmAChE1 protein expression levels in the worker bees collected on day 0, 7 (nurse) and 21 (forager) from the brood rearing-suppressed and control groups were investigated. On day 7 and 21, significant induction of AmAChE1 expression was observed in both the head and abdomen of brood rearing-suppressed samples whereas no induction was observed in control samples Fig. , which iage Fig. . When abvg in the head on day 2 (p < 0.001) and in the abdomen on day 4 (p < 0.0001) were lower in treated groups than those of the control.In the crowding treatment, honey bee workers in the crowded hive showed an increase in AmAChE1 expression, which was more distinct in nurse bees than in forager bees Fig. . Howeverhsp and vg marker genes was clearly upregulated  in the head upon heat shock  and hsp60 (p = 0.015) only increased in heat-shocked samples compared to the control  induced the expression of AmAChE1  and choline acetyltransferase (ChAT) distributed in non-neuronal tissues29 support the presence of a functional and non-neuronal cholinergic system regulated by AmAChE1 in honey bees. As AmAChE1 possesses an extremely low catalytic activity toward ACh compared to the neuronal AmAChE2, it appears to function mainly as an ACh-sequestration protein27. With this in mind, the functions of AmAChE1 seem to be associated with the non-neuronal cholinergic system mediating chronic stress response via ACh sequestration rather than with the neuronal cholinergic system modulating acute stress response.AmAChE1 is soluble like the mammalian AChE-R and distributed in both neuronal and non-neuronal tissues, including fat body and hemolymph17, expressed AmAChE1 is likely secreted from the fat body and circulates via hemolymph. Detection of soluble AChE with weak enzyme activity in honey bee hemolymph supports this notion28. Thus, the heat-induced expression of AmAChE1 appears to exert systemic effects on the whole body. Based on the facts that the toxicity of organophosphorus (OP) and carbamate (CB) insecticides increases in proportion to temperature increase30 and that AmAChE1 provides chemical defense against OP and CB insecticides27, the augmented AmAChE1 level likely provides extra protection against toxic chemicals particularly under heat-stressed conditions. Another possible scenario is that AmAChE1 circulating the body reduces the overall amount of ACh through sequestration or degradation, thereby regulating the cholinergic signal pathways. Thus, ACh concentration reduction in hemolymph by AmAChE1 would selectively influence the physiology of different tissues. In humans, plasma ACh can promote macrophage phagocytosis via stimulation of nAChR, resulting in enhancement of the cellular immune response31. Although the same ACh-related immune pathway has not been reported in honey bees, the recent identification of major cholinergic components such as ChAT and nAChR in honey bee hemocytes suggests the existence of functional ACh-mediated immune regulation pathways, especially in non-neuronal tissues29, and further indicates the role of AmAChE1 in the immune system of honey bees. If this is the case, AmAChE1 overexpression following heat shock is expected to reduce the ACh concentration, thus resulting in the downregulation of the immune response of individual bees.In heat shock treatment, the abdomen sample showed a higher AmAChE1 expression than the head sample. Considering that AmAChE1 expressed in the abdomen is mostly derived from the fat bodyhsp genes was induced in bees that received heat shock, suggesting the activation of the HSR pathway , where heat shock suppressed the immune gene transcription in the abdomen whereas the immune challenge to the abdominal cuticle resulted in decreased expression of multiple HSR genes32. Such a mutually antagonistic relationship between the HSR and immune response was explained by the trade-offs theory of ecological immunology33. In the eusocial honey bees, as both the HSR and immune response require large energy resources, trade-offs between these two responses at the individual level would be necessary to allocate limited resources in a manner that eventually benefits the colony32.Interestingly, transcription of all tested ay Table . Thus, t17. Taken together, AmAChE1 expression is induced in both nurse and forager bees by brood rearing suppression regardless of forager bee activity.Under the brood rearing suppression condition, which was generated by confining the queen bee in a plastic queen cage, AmAChE1 expression was induced and maintained from at least day 7 to day 21. This result is consistent with the previous finding when brood rearing suppression was induced by confining the entire hive inside a screen tent, which completely blocked the free foraging activityhsp and vg genes, was more apparent in nurse bees compared to forager bees  of both nurse and forager bees under the brood rearing-suppressed condition can be speculated to cause the alteration of energy metabolism. In rats, one of the mechanisms regulating secretion of insulin, the main component of energy metabolism, is mediated by the binding of ACh to the M3 mAChR subtype of pancreatic β cells36. All of these may suggest some possibility of metabolic regulation mediated by the AmAChE1-ACh-mAChR circuit, perhaps via the insulin/insulin-like growth factor signaling (IIS) pathway in honey bees.Considering that the ACh-mAChR pathway is involved in the regulation of energy metabolismhsp and vg transcription under the crowding condition supports this notion. If this is the case, the crowding condition  may not act as a stress factor to honey bees at least under the experimental conditions used in this study. If assuming that colony-level immunity can be boosted by increased colony density, the putative downregulation of the immune response by overexpressed AmAChE1 in individual worker bees would be compensated.Crowding treatment induced the expression of AmAChE1, which was more apparent in nurse bees than in forager bees. This differential expression of AmAChE1 between nurse and forager bees appears to be due to the fact that nurse bees stay inside the hives, thus being constantly exposed to the crowding condition, whereas forager bees spend most of their time outside for foraging during the daytime and are less exposed to the crowding condition. Considering that a bee colony is a superorganism consisted of numerous worker bees with different functions, the increase in colony density may alleviate an individual worker bee’s workload, which likely reduces the cellular stress level in individual bees. Overall downregulation of the 37. If assuming that a sudden increase of population could work as a signal for extrinsic invasion to the colony, it can be speculated that the AmAChE1-mediated reduction of the ACh titer leads to a decrease in the sensitivity for nestmate recognition, thereby temporarily enhancing a colony’s defense capability.ACh also might be related with nestmate recognition. Treatment of a muscarinic agonist to young honey bees improves recognition of nestmates whereas muscarinic antagonist treatment increases attacks on nestmatesAmace1 transcription levels. This inconsistency between mRNA and protein levels suggests that certain post-transcriptional control, such as pumilio-mediated translational regulation, likely plays an important role in the expression of AmAChE1. As the Amace1 transcript has six pumilio-binding domains in the 3’ UTR and one in the 5’ UTR , suggesting their potential as general stress markers  and assist the folding of signaling-related proteins such as kinases, steroid hormone receptors and transcription factors39. The overtranscription of hsp70 and hsp90 particularly in brood rearing-suppressed nurse bees likely suggests an increased rate of protein translation, which is in good agreement with the over-represented protein biosynthesis pathway under the brood rearing-suppressed condition, as observed in the gene-set enrichment analysis of worker bee transcriptomes34. Likewise, both hsp70 and hsp90 were overtranscribed upon UV-B irradiation stress, indicating that Hsp70 and Hsp90 function as chaperones to protect or rescue UV-damaged proteins in nurse bees. Hsp60, the mitochondrial protein, is induced by diverse stress factors such as water-immersion40, metals, heavy metals and chemicals43. Hsp10, as a co-chaperonin of Hsp60, makes a complex with Hsp6044, which explains the similar transcription patterns of hsp10 and hsp60. Although both Hsp10 and Hsp60 were not induced by chemical treatment unlike previous reports43, high induction by heat shock suggests their important role for protecting cells from stress.Among the rs Table . Increasvg . Since an increase of the JH titer and decrease of Vg are the two major factors for nurse-forager transition13 and pre-exposure to sunlight increases the JH level of young bees54, it can be speculated that the UV irradiation in fact accelerated the nurse-forager transition. The overall reduction in vg transcription in nurse bees following UV exposure can be explained if it is assumed that the necessary Vg reduction for the labor-transition following UV irradiation masks the demand for a Vg increase for the UV-induced oxidative stress.As reported in the midge hsp genes following heat shock. Although not as complete as the heat stress, brood rearing suppression also resulted in both AmAChE1 expression and the transcription of two hsps (hsp70 and hsp90). This simultaneous upregulation of AmAChE1 and hsps under the stress conditions suggests that there is crosstalk between the expression of AmAChE1 and Hsps. Transcription of hsps is controlled by heat shock factor 1 (HSF1), of which activation is modulated via various protein kinase pathways by heat shock55. Considering that HSF1 is specific for hsp transcription, activation of Amace1 may not be directly regulated by HSR but rather by the upstream kinase pathways. Considering that Hsp expression is regulated by mAChR activation in the rat hippocampus56 and AmAChE1 regulates the ACh titer, perhaps by sequestration, AmAChE1 likely exerts its functions via the mAChR-mediated pathway in the honey bee as well. If this is the case, as the cAMP-dependent protein kinase (PKA) pathway is known to activate HSF155, AmAChE1 appears to exert its function via the PKA pathway mediated by mAChR, thereby modulating the stress response. In contrast, the mutually antagonistic relationship between the HSR and the immune response, putatively mediated by AmAChE1, also suggests the presence of antagonistic crosstalk between the HSF1 activation and immune regulationThe AmAChE1 expression was accompanied by the transcription of all tested hsp genes exhibited the opposite tendency. If assuming that overexpression of Hsps reflects various cellular stresses, this finding supports the notion that the crowding condition in fact did not induce cellular stress. The lack of correlation between AmAChE1 expression and hsp transcription further suggests that AmAChE1 expression does not necessarily indicate the presence of cellular stress in honey bees.Under the crowding condition that induced AmAChE1 expression, however, transcription of all hsp genes, particularly hsp70 and hsp90 that responded to six of nine stress conditions tested, AmAChE1 expression was only induced under three out of 12 stress conditions. Thus, these results suggest that AmAChE1 is inappropriate as a general stress marker. However, AmAChE1 expression appears to be connected, at least in part, to the HSR. Considering that AmAChE1 regulates the ACh titer particularly in non-neuronal tissues, thereby modulating the signal cascades mediated by mAChR, the AmAChE1 expression profile in honey bees likely provides important information on physiological changes in honey bees.By determining the AmAChE1 expression profile following diverse stress factors, we tried to evaluate the possibility of AmAChE1 as a general stress marker in honey bees. Unlike 2 gas (30 s). Anesthetized bees were placed in a 474-ml transparent plastic disposable cup (30 workers/cup) and provided with a piece of pollen bread (1 cm3) and 50% sucrose solution in a 5-ml plastic syringe. These bees were maintained at 32 °C in 40–60% relative humidity under a dark condition, which was the standard test condition in this study. The 50% sucrose solution was replaced every day. When exact age synchronization was needed, newly emerged worker bees were marked with paint  and collected later at appropriate dates. After each treatment, honey bees were flash-frozen with liquid nitrogen and stored in a −80 °C deep freezer until used. All stress experiments along with appropriate controls were conducted with three biological replicates.Colonies of western honey bees  have been kept at the Gwanak campus of Seoul National University . Young worker bees located in the center of brood rearing cells were considered as nurse bees and old worker bees that enter the hive from foraging and are mostly found around the honey/pollen cells were considered as forager bees. Nurse or forager bees were collected from combs with a brush, placed in a plastic cage and briefly anesthetized with COE, coli and S. aureus were cultured in LB broth with gentle shaking until the OD600 value became 0.6 and the resulting cultures  were centrifuged at 13,000 rpm for 5 min. Each pellet was mixed with 4 ml of 10% sucrose solution to obtain the final working solution . Nurse bees were oral-challenged by providing with 10% sucrose solution containing bacteria under the standard condition. Control nurse bees were provided with a 10% sucrose solution without bacteria. Both bacteria-challenged and control honey bees were collected after 24 h.Bacterial challenge (Escherichia coli and Staphylococcus aureus): Varroa mite infestation: Live Varroa mites were collected from a Varroa mite-infested beehive using sugar powder57. Test nurse bees were collected from a Varroa mite-uninfested hive. Two or three Varroa mites were manually attached to each of the healthy nurse bees. Varroa mite-attached and unattached control nurse bees were incubated respectively under the standard condition for 24 h and collected. Nurse bees naturally infested with Varroa mites in the infected hive were also collected as a reference.Brood rearing suppression stress: For the collection of honey bee workers under the normal brood rearing condition, newly emerged workers were paint-marked from hives actively engaged in brood rearing. The paint-marked bees were then collected on day 0, 7 and 21, flash-frozen and immediately stored at −80 °C until use. To create the brood rearing-suppressed condition, honeycomb containing eggs, brood and pupa were removed from the hive and the queen was confined inside a plastic queen cage with a few nurse bees. On the same day, newly emerged workers were marked with paint and then collected in the same way on day 0, 7 and 21. Collected bees were flash-frozen and immediately stored at −80 °C until use.58.Starvation/Dehydration: For starvation stress, nurse bees were provided with only water instead of a 50% sucrose solution, with other conditions being equal to the standard condition. For dehydration stress, nurse bees were supplemented with solid sucrose instead of a 50% sucrose solution under the same standard condition. Control bees were maintained under the standard condition. Treated and control bees were collected after 24 and 48 hCrowding stress: Similar colony densities  were maintained in two different hives, each with a super . For the crowding treatment, bees in a super (eight combs) of one hive were transferred into the bottom hive, thereby roughly doubling the density. The other hive was maintained without any change in colony density and used as the control. Nurse bees and forager bees from each hive were collected on day 0, 2, 4, 6 and 8.59. In contrast, nurse bees are less often exposed to low temperatures compared to forager bees, thus being more likely to be vulnerable to cold shock. Nurse bees were collected from a hive and immediately placed in separate incubators with different temperature settings . Treated bees were incubated under the standard condition except for the temperature and collected at 24 h post-treatment.Thermal stress (heat and cold shock): For both heat shock and cold shock treatments, nurse bees were used considering that forager bees are normally exposed to a diverse range of temperatures and adapt more efficiently to heat shock through the expression of HspChemical stress : For the treatment of imidacloprid, of which most natural exposure to honey bees is via the oral route, forager bees were provided with a 50% sucrose solution containing 0, 4, 16, 64, and 256 ppb imidacloprid, with other conditions being equal to the standard. Treated bees were collected after seven days of incubation.As for the treatment of fluvalinate, of which most plausible exposure is via contact, forager bees were placed in a Mason jar precoated with an appropriate amount of fluvalinate and then maintained for 14 days with the provision of pollen bread and a 50% sucrose solution. The Mason jar was coated with 5 ml fluvalinate-acetone solution  using a rolling machine under a fume hood. Bees were collected 14-days post-treatment.UV irradiation (oxidative stress): Under the assumption that nurse bees are more sensitive to UV irradiation, paint-marked seven-day-old nurse bees were used for the UV irradiation. Nurse bees were collected from a hive without direct exposure to sunlight. Half of the collected bees were then exposed to UV (peak at 305–310 nm wavelength) generated by a 20 W UV-B lamp  at a distance of 15 cm away for 4 h under the standard condition and collected immediately. The other half of the collected bees were placed in the dark for 4 h under the standard condition and collected as the control.Honey bee samples were separated into the head and abdomen. The abdomen was further dissected to remove the gut and the gut-removed abdomen carcass was used as the ‘abdomen’ sample in this study. For protein extraction, 10 heads or abdomens were homogenized with 0.1 M Tris-Hcl buffer (pH 7.8) containing 0.5% Triton X-100 and a protease inhibitor cocktail  using a bullet blender . The homogenized samples were centrifuged at 12,000 g at 4 °C for 10 min. The supernatants were filtered through column type D  to remove lipids. The BCA assay was used to determine protein concentrations of the extracted protein samples.60. An aliquot (10 μg) of a protein sample was separated on a 7.5% native-polyacrylamide gel in duplicate at 120 V for 90 min. After the native-PAGE, Western blotting was performed with anti-insect AChE1 antibody as previously described17.To determine whether AmAChE1 expression is involved in responses to different stress, protein expression levels of AmAChE1 following treatment of various stressors were primarily evaluated by Western blotting and compared with those of the control beesTo measure mRNA levels of genes, qPCR was conducted. Total RNA was extracted from 10 heads or gut-removed abdomens with TRI reagent  following the instructions of the manufacturer. The extracted RNA was treated with DNaseI  to remove genomic DNA. After DNaseI treatment, the first strand cDNA was synthesized from 2.5 μg of total RNA with Superscript IV reverse transcriptase  at 55 °C for 10 min.hsp 10, 60, 70 and 90 as representatives of four major insect Hsp families, small Hsps, Hsp60, 70 and 90 families61, and the Vg gene (vg) were used as reference genes for stress along with the (Amace1). PCR primers for the stress reference genes and the ribosomal protein S5 (RPS5) gene, which was used as a qPCR reference gene, are listed in Table. Amace1 along with those of the reference stress genes was evaluated following various stress treatments.The *(p < 0.05), **(p < 0.01) and ***(p < 0.001). For comparison of multiple sample data, one-way or two-way ANOVA test followed by Tukey’s multiple comparison test was performed, and significant differences in mean values (p < 0.05) were marked with different alphabets.A statistical analysis of the qPCR data was performed using Prism 6.0 . For a comparison of two-sample data, Student’s t-test was performed and a significant difference was marked with Supplementary file