Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.

Survey on gastrointestinal parasites and detection of Cryptosporidium spp. on cattle in West Java,Indonesia

Objective:To evaluate the presence of gastrointestinal parasites on cattle in Indonesia because the prevalence of parasites varies between counlries depending on the terrain surrounding livestock farms and investigations in Indonesia have never been performed.Methods:Fecal samples from cattle at 35 farms in 7 districts in West Java,Indonesia,has been examined using the floatation or sedimentation methods,and a immunofluorescence assay and experimentally inoculation to mice for Cryptosporidium or Giardia spp.Results:153 of 394 examined cattle(38.8%)were infected with gastrointestinal parasites.The prevalence of Eimeria spp.,Nematoda spp.(including Oesophagustomum and Bunostomum-like),Fasciola gigantica and Paramphistomum spp.was 22.4%,11.2%,12.5%and 3.8%,respectively.Cryptosporidium andersoni(C.andersoni)was also found in two samples.One isolate of this parasite was confirmed to be transmitted to mice,in contrast to the isolates from other countries.Conclusions:although this survey is preliminary,the results shows that the infection of gastrointestinal parasites in Indonesia was not high,but these infected cattle could be as a potential source leading to economic losses in livestock production.

Applicability of Wireless Activity Sensor Network to Avian Influenza Monitoring System in Poultry Farms

Since there are cases that chickens infected with highly pathogenic avian influenza (HPAI) viruses die with almost no fever, body-temperature sensing cannot be effective for the early detection of avian influenza (AI) infection in these cases. In addition, sensors directly attached to the body surface are easily affected by their surroundings, therefore it is not easy to measure the body-temperature of chickens. This paper proposes a new method to detect abnormal states of chickens with only their activity data obtained by wearable wireless sensor nodes. The method is that if its state that the present average activity of a chicken in one hour is smaller than 0.6 times minimum average daytime activity in its life continues for 3 hours, a judgment that the chicken is abnormal state is made. This method can detect the abnormal states twice as early as that with body-temperature sensing does. Since the activity sensor nodes are easily attached to chickens and hardly affected by their surroundings, this system can be reliable.

A gene expression estimator of intramuscular fat percentage for use in both cattle and sheep

Background：The expression of genes encoding proteins involved in triacyglyceride and fatty acid synthesis and storage in cattle muscle are correlated with intramuscular fat（IMF）%.Are the same genes also correlated with IMF%in sheep muscle,and can the same set of genes be used to estimate IMF%in both species？Results：The correlation between gene expression（microarray） and IMF%in the longissimus muscle（LM） of twenty sheep was calculated.An integrated analysis of this dataset with an equivalent cattle correlation dataset and a cattle differential expression dataset was undertaken.A total of 30 genes were identified to be strongly correlated with IMF%in both cattle and sheep.The overlap of genes was highly significant,8 of the 13 genes in the TAG gene set and 8 of the 13 genes in the FA gene set were in the top 100 and 500 genes respertively most correlated with IMF%in sheep,P-value = 0.Of the 30 genes,CIDEA,THRSP,ACSM1,DGAT2 and FABP4 had the highest average rank in both species.Using the data from two small groups of Brahman cattle（control and Hormone growth promotant-treated[known to decrease IMF%in muscle]） and 22 animals in total,the utility of a direct measure and different estimators of IMF%（ultrasound and gene expression） to differentiate between the two groups were examined.Directly measured IMF%and IMF%estimated from ultrasound scanning could not discriminate between the two groups.However,using gene expression to estimate IMF%discriminated between the two groups.Increasing the number of genes used to estimate IMF%from one to five significantly increased the discrimination power;but increasing the number of genes to 15 resulted in little further improvement.Conclusion：We have demonstrated the utility of a comparative approach to identify robust estimators of IMF%in the LM in cattle and sheep.We have also demonstrated a number of approaches（potentially applicable to much smaller groups of animals than conventional methods） to using gene expression to rank animals for IMF%within a single farm/treatment,or to estimate differences in IMF%between two farms/treatments.

Survey of Angiostrongylus cantonensis in rats and giant African land snails in Phitsanulok province,Thailand

Objective:To survey the Angiostrongylus cantonensis(A.cantonensis) or the rat lungworm in a rat,definitive host,and in a giant African land snail(Achatina fulica),the intermediate host,in Phitsanulok,Thailand.Methods:Rats and giant African land snails were captured from Tha Pho sub-district,Phitsanulok,Thailand.Rats were killed and examined for adult A.cantonensis. The artificial digestion method following Baermann technique were used for isolation third stage larvae of A.cantonensis.Results:Sixty-two rats were captured and they were identified as Rattus argentiventer,Rattus rattus(R.rattus),Bandicota savilei,and Bandicota indica but only one animal(R.rattus) of 62 rats(1.61%) was positive with adult worm of A.cantonensis.The third stage larvae of A.cantonensis were examined on 307 Angiostrongylus fulica snails.It was found that the overall infection rate was 12.38%(38 infected out of 307 Achatina snails).Conclusions: This study demonstrates that A.cantonensis is available in the natural hosts of Phitsanulok.This suggests that the transmissions of this parasite to human may occur in this region.

Studies on the Anti-inflammatory Activity of Three Extraction Processes of Bidens alba(L.) DC

[Objectives] To compare the anti-inflammatory activities of three extraction processes of the exotic plant Bidens alba(L.) DC in Lingnan(south of the Five Ridges in China).[Methods]The alcohol extracts of B.alba(L.) DC were extracted and separated with petroleum ether,chloroform and ethyl acetate respectively.The anti-inflammatory activity experiment of these three extracts was carried out with the RAW264.7 inflammatory model of mouse macrophages in vitro.[Results]When the concentration was 0.05-1.6 mg/L,the petroleum ether extract of B.alba(L.) DC produced strong inhibitory effects on nitric oxide(NO) release of RAW264.7 macrophages induced by LPS;the inhibition of ethyl acetate extract was weak,and the chloroform extract showed no significant inhibition.[Conclusions]Petroleum ether is the best extractant for active anti-inflammatory ingredients of B.alba(L.) DC.

High Prevalence of Eimeria Infection in Domestic Pigeons(Columba Livia Domestica) in Guangdong Province, Southern China

[Objective] This study aimed to make a survey of domestic pigeons for infection with Eimeria species of coccidia was conducted in Guangdong Province, southern China. [Method] A total of 244 fecal samples(102 from Huizhou, 83 from Jiangmen, 35 from Chaozhou and 24 from Guangzhou, respectively) in four pigeon breeding farms between June 2012 and March2013 were collected and examined microscopically. [Results] Eimeria oocysts were seen in 223(91.4%) fecal samples, with three species, namely E. labbeana, E.columbae and E. kapotei. E. labbeana was the most common species in Guangdong provinces with an overall prevalence of 91.4%, while a slightly lower incidence of E. columbae and E. kapotei species was detected with11.1% and 6.6%, representatively. The prevalence in different months varied ranging from 83.3% to 91.7%, with the highest prevalence in summer. Nestling groups showed obviously high infection than adult pigeons. [Conclusion] The present survey indicated the wide and severe prevalence of Eimeria infection in Guangdong domestic pigeons, which suggested that integrated measures should be taken to control and prevent coccidiosis of pigeons in this province.

LipL21 mRNA expression in lungs of hamsters infected with pathogenic Leptospira

Objective:Pulmonary haemorrhage is an increasing cause of death in leptospirosis patients.However,molecular mechanism underlying pathologies in this organ is not clearly understood.It has been shown that sodium transport was disturbed following Leptospira infection.LipL21 is the second abundant outer membrane protein found only in pathogenic Leptospira.Its expression in vivo has been shown which suggests that this protein may be involved in survival in hosts or pathogenesis.However,the expression of this protein in host organs and its role in lung pathology has not been demonstrated.In this study we demonstrated the expression of LipL21 in lungs of hamsters infected with pathogenic Leptospira.Methods:Lung tissues were collected from Golden Syrian hamsters injected with Leptospira interrogans serovar Pyrogenes at days 3,5 and 7 post-infection.Four hamsters were used for each time point.Lungs from non-infected hamsters were collected as a control group.LipL21 mRNA expression in lung tissues was investigated by reverse transcription and nested PCR.Results:LipL21 mRNA expression was detected in all lung tissues from hamsters infected with pathogenic Leptospira.No PCR product was detected when tissues from non-infected hamsters were investigated.Conclusion:Our data demonstrated that LipL21 is expressed in lungs of hamsters infected with pathogenic Leptospira.Additional experiments such as quantitation and localization of LipL21 expression in lungs will provide further information whether this protein is involved in pathogenesis.

Studies on the Analgesic Activity and Acute Toxicities of Bidens alba(L.) DC

[Objectives]This study was conducted to investigate the analgesic effects and acute toxicities of Bidens alba (L.) DC.[Methods]The alcohol extract of B.alba (L.) DC was extracted and separated with petroleum ether and chloroform successively.The acute toxicities of the two extracts on mice were measured,and then the analgesic effects were measured with writhing pain model induced by acetic acid.[Results]No mice died when the crude dosages of B.alba (L.) DC from petroleum ether extract and chloroform extract were 5 016 and 5 100 mg/kg,respectively.When the petroleum ether extract was 60.0 mg/kg,the percentage of twisted mice induced by acetic acid was 40%,the analgesic rate was 77.5%,and the time of the first writhing was (294.0±165.8) s;when the chloroform extract was 20.0 mg/kg,the percentage of twisted animals was 55.6%,the analgesic rate was 51.5%,and the time of the first writhing was (273.8 ±153.4) s;and when the chloroform extract was 4.0 mg/kg,the percentage of twisted animals was 40%,and the analgesic rate was 62.1%,and the time of the first writhing was (370.6±231.3) s.[Conclusions]The petroleum ether extracts and chloroform extracts of B.alba (L.) DC have good analgesic effects and no acute toxicities.

Phylogenetic Analysis Revealed Concurrent Circulation of Two Clusters of Duck Tembusu Virus in South China

Eleven strains of duck Tembusu virus(TMUV)were isolated from diseased ducks at different duck farms in South China during 2011–2015,and their whole genomes were sequenced.The 11 isolated strains shared high sequence identity from 95.9%to 99.5%.Phylogenetic analysis revealed that all TMUV strains isolated after 2010 are clustered into two distinct branches,one branch comprising 2 Malaysian strains,and the other branch comprising all Chinese and Thai strains forming Chinese cluster 1 and Chinese cluster 2.Five of the 11 isolated strains were closely related to classical Chinese strains(BYD)belonging to Chinese cluster 1,and the remaining 6 isolated strains were closely related to strains newly isolated in South China and Thailand,belonging to Chinese cluster 2(a mixed China-Thailand cluster).Phylogenetic analysis of the partial E and NS 5 gene of TMUVs also showed the similar results.The results collectively showed a new trend of TMUV disease that two Chinese clusters of TMUV have been concurrently circulating in South China.This study provides practical guidance for preparing vaccines against TMUV in South China.

Suicide Vector Construction of Haemophilus parasuis hhd B Gene Marker-free Deleted

To construct the suicide vector of hhd B gene marker-free mutant in Haemophilus parasuis( HPS),two pairs of specific primers were designed and synthesized according to the hhd B gene upstream and downstream sequences of HPS published in Gen Bank. The hhd B gene upstream and downstream sequences were amplified by PCR,which were further ligated( hhd B-up + down) through overlapping PCR method. NotⅠand SalⅠrestriction enzyme sites were introduced on both ends of the ligated sequence. After the corresponding digestion,the hhd B-up + down sequence was directionally cloned to the suicide plasmid vector p EMOC2. Results showed that the suicide vector of hhd B gene marker-free deleted( p EMOC2Δhhd B) with stable inheritance in E. coli β2155 strain was successfully obtained,thereby laying the foundation for construction of HPS-hhd B gene marker-free mutant strain.

Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives on Ehrlich Ascites Tumor [EAT] Cells in-Vivo

In search of synthetic chemotherapeutic substances capable of inhibiting, retarding, or reversing the process of multi-stage carcinogenesis, we synthesized a series of novel 5-amino pyrazole derivatives 11(a-h) by a nucleophilic substitution reaction and characterized by 1H nuclear magnetic resonance (NMR), liquid chromatography mass spectrometry (LC/MS), Fourier-transform infrared (FTIR), and elemental analysis. These novel compounds were evaluated for their efficacy in inhibiting Ehrlich ascites tumor [EAT] cells in-vivo. In the present study we designed, synthesized, characterized and investigate the anti-angiogenic effects of these compounds, on Ehrlich ascites tumor [EAT] cells in-vivo. The compounds were subsequently tested for their ability to inhibit neovascularisation in chorio allantoin membrane (CAM) model. From the Structure Activity Relationship (SAR) studies, it reveals that, the substitution at N-terminal in pyrazole ring plays key role in the antitumor and anti-angiogenic effects.

Sero-Prevalence of Paratuberculosis (Johne's Disease) in Cattle Population of South-Western Bangalore Using ELISA Kit

Johne's disease or paratuberculosis is a chronic mycobacterial infection that affects cattle, sheep, goats and other ruminants, adversely, leading to huge economic losses throughout the world. The estimation of sero-prevalence of this disease in the cattle population of south-western Bangalore, Karnataka, using an immunological assay and statistical analyses, was the objective of this study. One of the diagnostic tools used to detect an antigen or an antibody in animal serum or milk is the Enzyme Linked Immuno-Sorbent Assay, which has been widely used in the research and diagnosis of animal and human diseases as its accuracy is of nanogram-picogram/milliltre level. In the present study, indirect-ELISA was used to diagnose and estimate the sero-prevalence of paratuberculosis in cattle showing diarrhoea and/or anaemia, at 5 local dairy farms in south-west Bangalore, India. Out of 350 bovine serum samples, 53 (15.14%) were positive, 55 milk samples out of 300 were found positive (18.33%) for antibody against Johne's disease by indirect ELISA. The positive samples were then confirmed by direct smear examination of dung by Ziehl-Neelsen staining. Statistical analyses were carried out to indicate the seroprevalence of Johne's disease in the cattle population of this region to be 15 ± 10%, taking a confidence interval of 95%. The results emphasize the need to prevent the further spread of infection to other susceptible animals and humans as the causative organism, Mycobacterium avium subsp. paratuberculosis is implicated in Crohn's disease, an irritable bowel syndrome in humans.

Quantitative Microbiological Evaluation of <i>Salmonella</i>Typhimurium Shed in Diarrhea, Loose, and Normal Stools of Infected Pigs

Control of within-herd transmission of Salmonella is important for reducing the prevalence of this organism on pig farms and for preventing Salmonella-contamination of pork. At the farm level, understanding the within-herd transmission of Salmonella can lead to more effective control. Salmonella infection is dependent on the inoculation dose;hence, quantitative evaluation of Salmonella shed in feces would provide useful information for developing effective measures. In this study, to reproduce and evaluate the number of Salmonella shed in diarrhea, loose stools, and normal feces, weaned pigs were inoculated with 3.2 × 109, 3.2 × 107, and 3.2 × 105 cfu of Salmonella Typhimurium, respectively. The number of S. Typhimurium shed in the feces peaked within 1 week post-inoculation in every group and the most amount of diarrhea and loose stools were observed within 2 weeks post-inoculation. Diarrhea occurred 10 times (six pigs), and loose stools were observed 25 times (11 pigs). The average concentration of S. Typhimurium shed in diarrhea, loose stools, and normal feces was 1.0 × 108, 1.6 × 104, and 7.1 × 101 cfu/g feces, respectively. These data suggest that diarrhea and loose stools are significant sources of within-herd transmission of Salmonella. Moreover, as some of the normal feces contained >1.0 × 106 cfu/g of S. Typhimurium, even normal feces could be a source of within-herd transmission of Salmonella. At Salmonella-positive farms, reduction of the amount of Salmonella shed even in normal feces would lead to better control of within-herd transmission of Salmonella. These data can contribute to the control of within-herd transmission of Salmonella, particularly during the weaning period.

Avian influenza virus ecology in wild birds of Western Siberia

Background:The aim of the study was to explore the ecological diversity of wild birds in Siberia, which are the natural reservoir of avian influenza virus(AIV).Methods:Cloacal swabs and intestinal fragments were collected from wild migratory birds from 2007-2014. Isolated viruses were grown in the allantoic cavity of embryonated chicken eggs. The presence of virus was determined using hemagglutination assays. Primary identification and subtyping of influenza viruses was confirmed by RT-PCR.Results:A total of 2300 samples obtained from wild migratory birds of 8 orders were collected and tested. Influenza was detected in 185 birds of 3 orders. Species of family Anatidae(order Anseriformes) such as European Teal(Anas crecca), Garganey Teal(A. querquedula), and Shoveler(A. clypeata) play the main role in AIV circulation in the south of Western Siberia. The proportion of viral carriers among waterfowl ranged from 5.6 to 20% in 2007-2014. The order Charadriiformes had lower virus isolation rates of not more than 1.4%.Conclusions:Wild migratory waterfowl of orders Anseriformes and Charadriiformes are the main reservoir of AIV in the south of Western Siberia. This area plays a key role in persistence, evolution, and geographical distribution of avian influenza.

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

The evolution,pathogenicity and transmissibility of quadruple reassortant H1N2 swine influenza virus in China:A potential threat to public health

Swine are regarded as“intermediate hosts”or“mixing vessels”of influenza viruses,capable of generating strains with pandemic potential.From 2020 to 2021,we conducted surveillance on swine H1N2 influenza(swH1N2)viruses in swine farms located in Guangdong,Yunnan,and Guizhou provinces in southern China,as well as Henan and Shandong provinces in northern China.We systematically analyzed the evolution and pathogenicity of swH1N2 isolates,and characterized their replication and transmission abilities.The isolated viruses are quadruple reassortant H1N2 viruses containing genes from pdm/09 H1N1(PB2,PB1,PA and NP genes),triple-reassortant swine(NS gene),Eurasian Avian-like(HA and M genes),and recent human H3N2(NA gene)lineages.The NA,PB2,and NP of SW/188/20 and SW/198/20 show high gene similarities to A/Guangdong/Yue Fang277/2017(H3N2).The HA gene of swH1N2 exhibits a high evolutionary rate.The five swH1N2 isolates replicate efficiently in human,canine,and swine cells,as well as in the turbinate,trachea,and lungs of mice.A/swine/Shandong/198/2020 strain efficiently replicates in the respiratory tract of pigs and effectively transmitted among them.Collectively,these current swH1N2 viruses possess zoonotic potential,highlighting the need for strengthened surveillance of swH1N2 viruses.

Quercetin Alleviates Lipopolysaccharide-Induced Cardiac Inflammation via Inhibiting Autophagy and Programmed Cell Death

Objective The aim of this study is to explore the potential modulatory role of quercetin against Endotoxin or lipopolysaccharide(LPS)induced septic cardiac dysfunction.Methods Specific pathogen-free chicken embryos(n=120)were allocated untreated control,phosphate buffer solution(PBS)vehicle,PBS with ethanol vehicle,LPS(500 ng/egg),LPS with quercetin treatment(10,20,or 40 nmol/egg,respectively),Quercetin groups(10,20,or 40 nmol/egg).Fifteenday-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity.At embryonic day 19,the hearts of the embryos were collected for histopathological examination,RNA extraction,real-time polymerase chain reaction,immunohistochemical investigations,and Western blotting.Results They demonstrated that the heart presented inflammatory responses after LPS induction.The LPS-induced higher mRNA expressions of inflammation-related factors(TLR4,TNFα,MYD88,NF-κB1,IFNγ,IL-1β,IL-8,IL-6,IL-10,p38,MMP3,and MMP9)were blocked by quercetin with three dosages.Quercetin significantly decreased immunopositivity to TLR4 and MMP9 in the treatment group when compared with the LPS group.Quercetin significantly decreased protein expressions of TLR4,IFNγ,MMP3,and MMP9 when compared with the LPS group.Quercetin treatment prevented LPS-induced increase in the mRNA expression of Claudin 1 and ZO-1,and significantly decreased protein expression of claudin 1 when compared with the LPS group.Quercetin significantly downregulated autophagyrelated gene expressions(PPARα,SGLT1,APOA4,AMPKα1,AMPKα2,ATG5,ATG7,Beclin-1,and LC3B)and programmed cell death(Fas,Bcl-2,CASP1,CASP12,CASP3,and RIPK1)after LPS induction.Quercetin significantly decreased immunopositivity to APOA4,AMPKα2,and LC3-II/LC3-I in the treatment group when compared with the LPS group.Quercetin significantly decreased protein expressions of AMPKα1,LC3-I,and LC3-II.Quercetin significantly decreased the protein expression to CASP1 and CASP3 by immunohistochemical investigation or Western blotting in treatment group when compared with LPS group.Conclusion Quercetin alleviates cardiac inflammation induced by LPS through modulating autophagy,programmed cell death,and myocardiocytes permeability.

Hydroelectrolytic and Energetic Replenisher in Horses Undergoing Marcha Training

The study aimed to assess the clinical, laboratory, and blood gas analysis of horses undergoing Marcha training and the effects of voluntary ingestion of hydroelectrolytic and energy replenishers after exercise. Eight horses of both genders aged between 5 and 10 years, were included in the study. The exercise consisted of a 10-min warm-up followed by 45 min uninterrupted Marcha on a flat dirt track in the morning. After exercise, the horses received one of the following treatments: Drinking water (control group);Hydroelectrolytic and energy replenisher containing sodium chloride, potassium chloride, calcium acetate, magnesium chloride, sodium citrate, dextrose, maltodextrin, and sucrose in three different concentrations (Replenishers A, B, and C). The horses were distributed across the four treatments in a 4 × 4 Latin Squares design using a Split-plot system with 48-hr intervals. Clinical and laboratory evaluations were conducted at four time points: T0 - 5 min before exercise;T1 - up to 5 min after exercise;T2 - 2 hr after starting treatment;and T4 - 4 hr after beginning treatment. Concentrations of urea, creatinine, lactate, phosphorus, and ionized calcium significantly changed after exercise. An increase in blood pH and a decrease in chloride concentrations were observed when replenishers B and C were offered after exercise. The replacements were ingested spontaneously by the animals in a volume greater than that of the control group (water). Replacement B was the most ingested by the animals, demonstrating its greatest potential.

Prion Protein Binds to Aldolase A Produced by Bovine Intestinal M Cells

Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.