In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC₅₀) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC₅₀ (concentration required to inhibit 50% of viral cytopathic effect). CC₅₀s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC₅₀ values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC₅₀ 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.     

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(ΔIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97 kDa adhesion were successfully fused to the downstream of PE(ΔIII) to create a subunit vaccine, i.e. PE(ΔIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97 kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(ΔIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(ΔIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(ΔIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(ΔIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.     

Occurrence of methicillin-resistant Staphylococcus aureus in rats living on pig farms

In The Netherlands, MRSA ST398 has emerged in hospitals and human carriers have been associated with exposure to pigs and cattle. High prevalences of MRSA ST398 in pigs and pig farmers have been determined and the transmission routes of MRSA on pig farms need to be elucidated. In the south of the Netherlands, in recent years, the black rat (Rattus rattus) has emerged as a prominent rodent on livestock farms. From March till May 2008, a survey on MRSA in rats living on livestock farms in the south of The Netherlands and the north of Belgium was conducted. In total, 40 black rats (R. rattus) and 3 brown rats (Rattus norvegicus) were collected on 12 farms including five pig farms, five poultry farms, one mixed pig and veal farm and one goat farm. MRSA ST398 was detected in black rats captured at two of the five pig farms as well as in a black rat living on the mixed pig and veal farm. From one black rat captured at another pig farm MRSA ST 97 was isolated. Considering the behaviour of rats on livestock farms, it is concluded that rats might play a role in the spread and persistence of MRSA on pig farms.     

救必应中药血清与抗菌药联合对产ESBLs细菌抑菌效果的研究

本试验主要探讨救必应中药血清与抗菌药联合诱导细菌传代的体外抑菌活性。制备救必应中药血清,采用二倍微量稀释法体外检测抗菌药的最小抑菌浓度(MIC),然后救必应中药血清与抗菌药联合诱导产超广谱β-内酰胺酶(extended spectrumβ-lactamases,ESBLs)细菌传代。救必应中药血清与抗菌药联合诱导细菌传代对耐药大肠杆菌有不同程度的抑菌活性。救必应中药血清可明显地增强β-内酰胺类抗菌药、氨基糖苷类抗菌药、喹诺酮类抗菌药和卡巴氧类抗菌药对耐药菌的抑制作用。     

Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants

Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46 kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.     

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5 ± 1.5%) and CEC-32 (RIF 7.0 ± 0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R_low. Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2 ± 0.3%) similar to that of R_low (1.1 ± 0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.     

Transmission of methicillin resistant Staphylococcus aureus among pigs during transportation from farm to abattoir

The prevalence of methicillin resistant Staphylococcus aureus (MRSA) in pigs at abattoirs is higher than in pigs sampled on farms. This study investigated whether MRSA negative pigs can become MRSA positive during transportation from the farm to the abattoir after exposure to other pigs and environmental sources of MRSA. Nasal swabs were collected from four batches of pigs during loading at the farm, on arrival at the abattoir and after stunning. Environmental wipes were taken from lorries after transporting pigs and from lairages after holding pigs. All pigs (n = 117) tested MRSA negative before transportation. On arrival at the abattoir, 12/117 (10.3%) pigs in two batches tested MRSA positive. In lorries that tested positive after transportation, the prevalence of MRSA positive pigs was 21.1%, whereas no MRSA was detected in pigs that had been transported in lorries that tested negative after transportation. At stunning, all batches and 70/117 (59.8%) pigs tested MRSA positive. Pigs can become MRSA positive in the short period of time during transportation from the farm to stunning at the abattoir.     

四川省部分地区山羊沙门氏菌健康带菌率及耐药性调查

为了解四川省山羊沙门氏菌带菌情况,本试验采集了四川省部分地区5个规模化山羊养殖场表观健康山羊粪便196份,经BPW预增菌、TTB选择性增菌后,采用靶向invA基因的PCR方法检测沙门氏菌的带菌率;对阳性样本进行细菌分离鉴定,随机选择25株沙门氏菌分离株测定其对15种抗菌药物的敏感性。结果显示,山羊沙门氏菌的平均带菌率为54.59%。药敏试验结果显示,分离株均对丁胺卡那敏感,而对其余14种抗菌药物呈不同程度的耐药,多重耐药菌株占88%,其中耐2~7种药物的占52%,耐9~13种药物的占36%。结果表明,四川省部分地区山羊的沙门氏菌健康带菌率较高,且多重耐药性普遍,其公共卫生意义值得进一步研究。     

Staphylococcus pseudintermedius expresses surface proteins that closely resemble those from Staphylococcus aureus

Staphylococcus pseudintermedius is a commensal of dogs that is implicated in the pathogenesis of canine pyoderma. This study aimed to determine if S. pseudintermedius expresses surface proteins resembling those from Staphylococcus aureus and to characterise them. S. pseudintermedius strain 326 was shown to adhere strongly to purified fibrinogen, fibronectin and cytokeratin 10. It adhered to the α-chain of fibrinogen which, along with binding to cytokeratin 10, is the hallmark of clumping factor B of S. aureus, a surface protein that is in part responsible for colonisation of the human nares. Ligand-affinity blotting with cell-wall extracts demonstrated that S. pseudintermedius 326 expressed a cell-wall anchored fibronectin binding protein which recognised the N-terminal 29 kDa fragment. The ability to bind fibronectin is an important attribute of pathogenic S. aureus and is associated with the ability of S. aureus to colonise skin of human atopic dermatitis patients. S. pseudintermedius genomic DNA was probed with labelled DNA amplified from the serine-aspartate repeat encoding region of clfA of S. aureus. This probe hybridised to a single SpeI fragment of S. pseudintermedius DNA. In the cell-wall extract of S. pseudintermedius 326, a 180 kDa protein was discovered which bound to fibrinogen by ligand-affinity blotting and reacted in a Western blot with antibodies raised against the serine-aspartate repeat region of ClfA and the B-repeats of SdrD of S. aureus. It is proposed that this is an Sdr protein with B-repeats that has an A domain that binds to fibrinogen. Whether it is the same protein that binds cytokeratin 10 is not clear.     

Protective immunity of recombinant Mycobacterium bovis BCG expressing rhomboid gene against Eimeria tenella challenge

Two recombinant Mycobacterium bovis BCG (rBCG) strains carrying the Eimeria tenella rhomboid gene (Rho) delivered by extrachromosomal vector pMV261 and integrative vector pMV361 were evaluated for their ability to protect chickens against E. tenella challenge. The chickens were immunized intranasal with BCG, rBCG pMV261-Rho, or rBCG pMV361-Rho twice at a 2-week interval. All the recombinant BCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4⁺ and CD8⁺ cells compared to the control (P < 0.05). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge. But vaccination with rBCG pMV261-Rho induced higher specific antibody titers and produced greater protection rate (56.04%) than rBCG pMV361-Rho group (P < 0.05). These results indicated that M. bovis BCG is a novel vaccine vector to express and present antigens of E. tenella, and rBCG has a potential as vaccine in chickens.     

Effect of Phenobarbitone Treatment Against Signal Grass (Brachiaria decumbens) Toxicity in Sheep

The effect of phenobarbitone against signal grass (Brachiaria decumbens) toxicity was studied in 26 male crossbred sheep. Grazing on signal grass significantly decreased the concentration of cytochrome P-450 and the activity of drug metabolizing enzymes, viz. aminopyrine-N-demethylase, aniline-4-hydroxylase, UDP- glucuronyltransferase and glutathione-S-transferase in liver and kidneys of affected sheep.Oral administration of phenobarbitone (30 mg/kg body weight) for five consecutive days before grazing on B. decumbens pasture, and thereafter, for three consecutive days every two weeks, resulted in significant increases in hepatic and renal activities of drug-metabolizing enzymes. The induction of drug metabolizing activity in sheep grazing on signal grass group was found to be lower than in animals given phenobarbitone alone. Induction by phenobarbitone provided a degree of protection against the toxic effects of B. decumbens as indicated by the delay in the appearance of signs of toxicity. Furthermore, these were much milder compared to those in the sheep not treated with phenobarbitone. The present study suggests that phenobarbitone-type cytochrome P-450 isoenzyme-induction may increase resistance against signal grass (B. decumbens) toxicity in sheep.     

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K_s) varied for different substrates. Substrate utilization patterns and K_s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.     

Identification of immunogenic proteins associated with protection against haemorrhagic septicaemia after vaccination of calves with a live-attenuated aroA derivative of Pasteurella multocida B:2

Pasteurella multocida serotype B:2 is the causative agent of haemorrhagic septicaemia (HS), a fatal disease of cattle and buffaloes. As a step towards the identification of individual antigens that may protect against HS, proteins present in a sonicated cell extract (SCE) and outer-membrane protein (OMP) preparation of a wild-type P. multocida serotype B:2 were investigated by immunoblotting with sera from calves which had been protected against challenge with a virulent strain of P. multocida B:2 by vaccination with a live-attenuated aroA derivative of the challenge strain. Five proteins in SCE, of approximately 50, 37, 30, 26 and 16 kDa, were recognised by the sera. In an OMP preparation, two bands, at 37 and 50 kDa, were recognised as strongly immunogenic. Mass spectrometry analysis of proteins corresponding in size to those detected by immunoblotting identified the 37 kDa band as OmpA, but the band at 50 kDa was not identified with certainty. A major 30 kDa OMP, identified as OmpH, was not strongly immunogenic.     

Seroprevalence and risk factors associated with seropositivity in sows from 67 herds in north-west Germany infected with Mycoplasma hyopneumoniae

Risk factors for the spread of Mycoplasma hyopneumoniae in sows have not been studied although vertical transmission from sows to their offspring is considered a significant risk factor in the development of enzootic pneumonia in growers and finishers. Seropositivity for M. hyopneumoniae in sows, as assessed by commercial ELISA, is a possible indicator of infection pressure among sows. The objective of this study was to estimate seroprevalence and associated risk factors of a sow being seropositive for M. hyopneumoniae. A cross-sectional study was carried out in 2578 sows from 67 herds in north-west Germany. Data concerning general herd characteristics, acclimatisation practices, indoor and outside contacts, as well as data describing the immediate local environment were collected during a herd visit via questionnaire. Blood samples were seropositive in 65% of the 2578 sows, and all herds had ≥14% seropositive sows. Data analysis was performed in two steps. First, univariate analysis of predictor variables for the risk of a sow being seropositive for M. hyopneumoniae was performed using chi-square test. Secondly, all variables associated with the risk of a sow being seropositive (P ≤ 0.25) were included in a multivariate model using a generalised linear model. The risk of a sow being seropositive for M. hyopneumoniae was increased in herds with two- or three-site production (OR 1.50), when piglets were not vaccinated against M. hyopneumoniae (OR 1.81), in herds with a 2-week farrowing intervals (OR 1.84) and in herds without all-in/all-out management of the farrowing units (OR 1.37). The lack of an acclimatisation period for replacement boars was also associated with the risk of a sow being seropositive (OR 2.10). The results indicate that M. hyopneumoniae seropositivity is common in sows in north-west Germany and is influenced by various management factors. It is recommended that evaluation of sow herd management should be included in any strategic health plan to control M. hyopneumoniae infection.     

Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant

The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund's modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund's complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (±S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 ± 97.5) were 77.25% (724 ± 98) in group 1, 88.02% (381 ± 97.5) in group 2, 38.92% (1943 ± 130.3) in group 3, 44.31% (1771.4 ± 102) in group 5, 59.28% (1295.2 ± 99.1) in group 7 and 55.69% (1409.5 ± 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests.In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.     

地顶孢霉培养物对肉仔鸡增重的影响

为了探讨地顶孢霉培养物（culture of Acrermonium Terricola,ATC）对白羽肉仔鸡增重的影响,试验选用200只新生肉仔鸡,随机分成2组。对照组饲喂基础日粮,地顶孢霉组在基础日粮中添加0.20%的ATC,试验期为42 d,期间每周记录体重,分析平均日增重,并统计试验期各组耗料量,计算增重带来的养殖效益。结果表明：试验期内,与对照组相比地顶孢霉组平均体重显著提高13.23%（P<0.01）;平均日增重显著提高13.53%（P<0.01）。地顶孢霉组盈利比对照组提高14.06%。说明饲料中添加ATC可以显著提高肉仔鸡的平均日增重,增加养殖效益,有利于禽类动物的无抗养殖。     

Dietary supplementation with Acanthopanax senticosus extract enhances gut health in weanling piglets

The present study was conducted to investigate the effects of Acanthopanax senticosus extract (ASE) as a dietary additive on gut health in weanling piglets by examining diarrhea frequency, intestinal microbiota and morphology. A total of 96 Duroc× (Landrace × Yorkshire) piglets weaned at 21 days of age with an average initial body weight (BW) of 5.6 ± 0.4 kg were randomly assigned to 3 treatment groups with 4 duplicates of 8 piglets each. The piglets were fed basal diet to which had been added 0 or 1 g/kg of ASE, or 0.7 g/kg antibiotics, respectively. Fecal consistence was monitored twice daily and the frequency of diarrhea was calculated. On day 21 after the initiation of supplementation, 8 piglets were randomly selected from each treatment group (2 piglets per pen) and slaughtered. The jejunum, ileum, colon and cecum were then excised and fixed in 10% neutral formalin solution to determine villus height and crypt depth, after their contents were collected to determine microbiota. The results showed that dietary supplementation with ASE increased (P < 0.05) the density of bacterial populations that co-migrated with Lactobacillus amylovorus, Lactobacillus salivarius, Bacillus subtilis, and Clostridium lituseburens, but decreased (P < 0.05) those co-migrating with Staphylococcus aureus, Salmonella typhimurium, Ruminococcus forques, and E. coli O157:H7 in the PCR-DGGE profiling analysis when compared with the control group. The villus height of the duodenum, jejunum and ileum increased (P < 0.05) by 14.8, 13.7 and 10.0%, while the crypt depth decreased (P < 0.05) by 17.9, 9.1 and 12.1%, respectively, in response to dietary ASE supplementation. Additionally dietary supplementation with ASE or an antibiotic decreased (P < 0.05) the frequency of diarrhea by 55.6 and 52.2%, respectively, compared with the control group. In conclusion, these findings suggest that dietary supplementation with ASE could regulate the microbiota composition and maintain a normal morphology of gut mucosa in weanling piglets, thereby decreasing diarrhea that resulted from weaning stress.