KPMG IFRG Limited Tel +44 (0) 20 7694 8089 Fax +44 (0) 20 7694 8429 8 Salisbury Square DX 38050 Blackfriars London EC4Y 8BB [email protected] Technical Director International Auditing and Assurance International Federation of Accountants 545 Fifth Avenue, 14th Floor New York, New York 10017 Contact Sylvia Smith IAASB Exposure Draft, Proposed International Standard on Review Engagements: ISRE 2400 (Revised), Engagements to Review Historical Financial Statements
Ijaah-latin 2.inddIranian Journal of Aquatic Animal Health
2 (1) 54-65
Antibacterial activity, antibiotic susceptibility and probiotic use of lac-
tic acid bacteria (LAB) in Persian sturgeon (Acipenser persicus)
M Soltani 1, A Shenavar Masouleh 1,2, M Ahmadi 2, M Pourkazemi 1 and A Taherimirghaed 2
1Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran; Center of excel-lence of aquatic animal health, University of Tehran, Iran2International Sturgeon Research Institute, Rasht, Iran Recived: June 2015 Accepted: November 2015 Growth behavior of five lactic acid bacteria pre-viously isolated from Persian sturgeon ( Nowadays, a high request for consumption of aqua- Acipenser culture production has been significantly increased persicus), were evaluated at different pH, tempera-ture and salt concentrations. Also, antibacterial ac- together with a remarkable decrease in the wild tivity of extracellular products (ECPs) of this LAB aquatic animal stock resources. Appllication of new were assessed to commercial aquatic animal species and aquaculture Aeromonas hydrophila. Further, their antibiotic susceptibility was determined with methods are generally raised some new diseases and some commonly used antibiotics in aquaculture. economical problems (Oidtmann, Thrush, Denham In an in vivo work the effect of & Peeler 2011). Therefore, there is a highly demand Lactococcus lactis as the supplementary diet was evaluated on growth for new methods for prevention and treatments of performance of Persian sturgeon for a period of 56 fish diseases in aquaculture industry in order to in- days. Strong growth of all LAB were seen at 20 and crease the cost-benefit for the farmers.
30˚C, as well as 40 and 80 g L-1 NaCl. The LAB Application of antibiotics for treatment and con- ECPs exhibited varied results of antagonism to the trol of fish diseases can cause a harmful effect for both the environment and consumer. The residual A. hydrophila with maximum activity observed at temperature between 25 and 30˚C. Also, the higher of chemical substances such as antibiotics could be antagonistic activity was observed for ECPs of appeared as some drug resistances in both animal and human bacterial flora. Hence to improve the op- cibaria, P. pentosaceus, and L. lactis at pH 9. Both tional tools for prevention of fish diseases it should W. cibaria and E. faecalis were resistance to oxy-tetracycline, erythromycin, trimethoprim sulfame- be deflected to find some new health managements thoxazol, enrofloxacin, florfenicol, and flumequine, such as enhancing the husbandry, the water quality, decreasing the fish densities, improveding food ef- L. lactis was sensitive to oxytetracycline, erythromycin, enrofloxacin, florfenicol. These re- ficacy, vaccines, probiotics and immunostimulants sults showed that use of (Sharifuzzaman & Austin 2009). Although vac- L. lactis can act as a posi- tive probiotic in Persian sturgeon feed via improve- cines and chemotherapeutic treatments have been ment of fish growth performance, feed efficiency used for protection of fish from bacterial diseases and fish health.
but sometimes need more therapeutics to prevent or treat fish infections because vaccination process Keywords: sturgeon, health, Lactococcus lactis,
is frequently incapable in immature fish (Balcazar, Vendrell, Blas, Ruiz-Zarzuela, Múzquiz & Gironés 2008).
Correspondence M Soltani, Department of Aquatic
Intensive sturgeon culture are exposed to stressful Animal Health, Faculty of Veterinary Medicine, conditions and culture may result in incidences of University of Tehran (e-mail: msol[email protected]) some diseases, which have caused acute economic M Soltani et al., LAB in Persian sturgeon (Acipenser persicus)
losses (Soltani & Kalbassi 2001; Khoshbavar-Rost- Merrifield & Masouleh 2013). A. hydrophila (A4) ami, Soltani & Hassan 2006,2007; Yang & Li 2009; strain was originally isolated from diseased Persian Meng, Xiao & Zeng 2011). Persian sturgeon, Acip- Sturgeon (Department of Aquatic Animal Health, enser persicus, is one of the most important com- Faculty of Veterinary Medicine, University of Teh- mercial fish species in the South Caspian Sea. ran, Iran). LAB strains were grown on MRS broth The beneficial effects of some probiotics, prebiotics, (Merck, Germany) incubated for overnight at 30 immunostimulants, and vitamin have been demon- °C before analysis. A. hydrophila in TSB (Merck, strated in a number of previous studies in sturgeon Germany) incubated for overnight at 25 °C ahead (Falahatkar, Soltani, Abtahi, Kalbassi, & Pourka- zemi 2006; Jalali, Ahmadifar, Sudagar & Takami 2009; Akrami, Abdolmajid, Abbas & Abdolmo- Growth Ability at Different Salt Concentration,
hammad 2009; Jafaryan et al. 2010; Hoseinifar et al. Temperature, and pH
2011a,b,c; Askarian, Kousha, Salma & Ringø 2011) Growth ability of LAB at different environmental However, there is no information for the dietary ef- conditions was performed following method de- fect of lactococcus lactis as a probiotic in sturgeon. scribed by Reddy (2007), with slight modifications. Also, despite isolation and in some contents charac- The growth behavior of LAB was assessed at five terization of some lactic acid bacteria (LAB) from different temperatures of 4, 10, 20, 30, and 40°C by alimentary tract of sturgeons have reported, there is inoculating into 5 mL MRS broth (Merck, Germa- no information regarding the physiological respons- ny) incubated for 24 h.
es of this LAB to environmental and chemical vari- Striled MRS broth (Merck, Germany) with different ables. The aim of the present study was to assess pHs values of 3.7, 4.5, 5, and 6.7 (adjusted with 1N the growth behavior of five species of LAB con- HCL) (Merck, Germany) and pH 8 and 9 (adjust- sisting of Lactococcus garvieae, Weissella cibaria ed with 1N NaOH) (Merck,Germany) were used , Pediococcus pentosaceus, Lactococcus lactis and to evaluate the LAB growth. A volume of 100μl Enterococcus facealis isolated from Persian stur- of overnight culture was inoculated to broth tubes geon fingerling intestine at different salt concentra- maintained at different pH incubated at 30°C for 24 tions, temperatures and pHs. Also, the antibacterial h. Also, the LAB growth was assessed at four differ- activity of the extracellular products of this LAB ent salinity consisting of 40, 80, 120 and 300 g L-1 was evaluated to a virulent strain of Aeromonas hy- NaCl prepared in MRB broth (Merck, Germany) drophila. The antibiotic resistance profiles of these inoculated with 100μl of 24 h culture of each LAB LABs were also tested using some commonly used incubated at 30°C for 24h. The growth behavior of antibiotics in the aquaculture. Moreover, potential LABs was measured by turbidimetric assay. use of Lactococcus lactis (JF831150) was assessed on some growth performance, haematological and Antibacterial Activity of LAB Extracellular
nonspecific immune response parameters of Persian The agar well diffusion assay was used to detect the possible antimicrobial activity of the LAB su- Material and Methods
pernatant cultures according to procedure described by Casla, Requena & Gómez (1996) with slight Microorganisms and Culture Conditions
modifications. Briefly, wells of 5 mm diameter of Lactococcus garvieae (JF831155), Weissella cibaria TSA (Merck,Germany) were loaded with 50 μl of (JF831160), Pediococcus pentosaceus (JF831149), the LAB culture supernatants obtained by cen- Lactococcus lactis (JF831150), and Enterococcus trifugation (Hettich,Germany) at 10,000 ×g for 10 faecalis (JF831161) were originally isolated from min at 4°C, filtered through a 0.2 μm millipore fil- intestine of Persian sturgeon (Acipenser persicus) ter (Whatman®, Schleicher & Schuell) buffered at (Soltani, Pourkazemi, Ahmadi, Taherimirghead, Iranian Journal of Aquatic Animal Health
pH 6.7. The supernatant of each LAB isolate was John (2008 b) with slight modification. Briefly, the prepared with four levels of pH of 6.7, 7.0, 8.0, bacterium was inoculated in MRS broth (Merck,- and 9.0. Lawns of the A. hydrophila were prepared Germany) incubated for 24 h at 30 ˚C and centri- by inoculating (ca 108cells/well) in 15 mL of the fuged at 5000 g for 10 min at 4 ˚C,. The bacterial soft-overlay (7.5 g L-1 agar) medium. The antibac- cells were then washed three times with sterile sa- terial activity of LAB ECPs against A. hydrophila line to obtain a suspension of ca 1010 cells mL-1.
(108 cells/well) was also examined at four different The bacterial suspension was the sprayed into the temperatures of 4, 25, 30, and 60°C provided at pH commercial food [Biomar, 470 g kg-1 protein, 140 g 6.7 for 30 min using well diffusion agar and zones kg-1 lipid, 81 g kg-1 ash , 31 g kg-1 cellulose/fibre, of growth inhibition were measured after an over- 8.8 g kg-1 phosphorous, 23.4 g kg-1 calcium, 2.7 g night incubation.
kg-1 sodium, vitamin A 7500(Ui kg-1), vitamin D3 1500(Ui kg-1), copper 1.6 (mg kg-1), Magnesium Antibiotic Resistance Profiles
12.6 (mg kg-1), Zinc 78.6(mg kg-1), Iodine 1.9 (mg The antibiotic resistance profiles were measured kg-1), and ethoxyquin 1.9 (mg kg-1)] slowly, mix- following methods described by Bello, Cocol- ing part by part in a drum mixer to give experimen- in, Zeppa, Field, Cotter & Hill (2012), with slight tal diets containing L. lactis at 106, 107, and 108 cfu modifications. The LAB were grown in MRS broth g-1 diets. The pellets were air-dried at 25 ˚C under (Merck,Germany) for an overnight at 30 °C. A vol- sterile conditions for 12 h, packed and stored in a ume of 20 mL of TSA agar (Merck, Germany) was refrigerator (4˚C) until used within 3 days. The via- inoculated with revitalized LAB strains (1% v/v) bility of the incorporated bacterial cells in the feed and permitted to solidify. Antibiotic resistance pat- was assessed by CFU counting via spreading onto tern was determined by the disk diffusion method MRS agar (Merck, Germany). Three groups of fish using oxytetracycline (30 μg/disc), erythromycin (each group containing 15 fish) were fed with diets (15 μg /disc), enrofloxacin (5 μg/disc), florfenicol containing 106 (Treatnebt one=T1), 107 (Treatnebt (30 μg/disc), flumequine (30 μg/disc), and trimetho- one=T2), and 108 (Treatnebt one=T3) cfu g-1 of prim -sulfamethoxazol (200 μg/disc)(Padtan-Teb, Lactococcus lactis for 56 days. The forth group was Iran).The plates were incubated at 30 °C for 24 h considered as control . Fish were fed 3% biomass and the zone of inhibition was measured.
per day provided in equal rations at 09.00 and 17.00 h.
Fish and rearing condition
Apparently healthy Persian sturgeon (Acipenser
Growth performance and carcass composition
persicus) weighing 114±5.47g were acclimated to Growth performance was assessed in terms of con- the rearing conditions for 2 weeks at the Interna- dition factor (CF), specific growth rate (SGR), feed tional Sturgeon Research Institute, Guilan Province, conversion ration (FCR), protein efficiency ration Iran prior to the experiment. The fish were randomly (PER), hepatosomatic index (HSI), weight gain divided into 4 groups each group containing 15 fish. (WG), initial body weight (IBW), and feed efficien- Water quality parameters of freshwater consisting cy (FE). The calculations were performed using the of temperature, dissolved oxygen content, and pH following formulae (Merrifield, Dimitroglou, Brad- were 20.48± 1.02 ˚C, 6.62±0.43 mg L-l, and 6.8- ley, Baker & Davies 2009 a): 7.25, respectively. CF = W×100 / L3; SGR= (LnFW-LnIW) × 100/T; FCR= feed intake/ (Wf-Wi); PER=(Wf-Wi)/PI; Preparation of feed with the Lactococcus lactis
HSI= LW × 100/W; WG= (Wf-Wi); IBW = (Wf - and feeding fish
Wi) × 100 / BWi ; × 100 ( ; FE= [(Wf-Wi)/FI]×100, Preparation of feed with the Lactococcus lactis where W is the weight in g, FW is the final weight, (JF831150) was according to Aly, Mohamed & IW is the initial weight, L is the length in mm, T M Soltani et al., LAB in Persian sturgeon (Acipenser persicus)
is the duration of feeding (in days), WG, is the wet One-Way ANOVA (SPSS 17.0; SPSS Inc., Chicago, weight gain, LW is the liver weight, FI is the feed IL, USA) and Duncan's test were conducted to find intake and PI is the protein ingested. Fish were ana- significant differences between treated and control lysed according to AOAC (1995) protocols to deter- trials at P<0.05 level.
mine carcass composition.
Sample collection and analysis
Fish samples and analysis were collected to proce-
Bacterial growth behaviar
dures described by Aly, Ahmed, Ghareeb & Mo- All LAB isolates were able to grow at 20 and 30˚C hamed 2008a) and Harikrishnan, Kim, Kim, Bal- while no growth occurred at 4, 10 and 40˚C. Also, asundaram & Heo (2011) with slight modification. all LAB isolates were able to grow at salinites 4, After 8 weeks feeding, nine fish were randomly and 8 g L-1 (w/v) NaCl with no growth at 12 and 30 collected from each treatment and blood samples g L-1 (w/v) NaCl. Furthermore, both W. cibaria and were obtained from caudal vein after fish being an- E. faecalis were able to grow at pH 3.7-9, while L. aesthetized with solution of tricaine methane-sul- lactis and L. garvieae were able to grow at pH 5-9 fonate (MS-222, Sigma Chemical Co., USA). The and pH 6-9, respectively. Interstingly the P. pentosa- sera samples were pooled and preserved at -20 ˚C cus could not grow above pH 7. or used immediately for analysis. Haematocrit lev-els (expressed as % packed cell volume: % PCV), white and red blood cells (WBC: ×103 mm-3 and Both W. cibaria and E. faecalis were resistance to RBC: ×106 mm-3), haemoglobin (Hb: g dL-1), the examined antibiotics while L. lactis exhibited mean corpuscular volume (MCV: fl), mean corpus- sensitivity to oxytetracycline (23mm), erythromy- cular haemoglobin (MCH:Pg), mean corpuscular cin (22mm), enrofloxacin (21mm) and florfenicol haemoglobin concentration (MCHC: %) and leuko- (18mm). L. garvieae was also sensitive to all of cytes were determined as lymphocytes (LYM: %), used antibiotic (13-26mm) except erythromycin. P. monocytes (MON:%), eosinophils (EOS;%), and pentosaceus exhibited sensitive only to oxytetracy- neutrophils (NEU:%) following standard methods cline (11mm).
described by Merrifield et al. (2009a) and Rawling, Merrifield & Davies (2009). The serum lactate de- Anti- Aeromonas hydrophila activity of ECPs
hydrogenate (LDH: IU mL-1), alanine aminotrans- The maximum antagonistic activity of 9-10 mm of ferase (ALT: IU mL-1), aspartate aminotransferase ECPs against A. hydrophila were observed at tem- (AST: IU mL-1) activities, glucose (mM), and con- perature between 25 and 30˚C for the ECPs of P. centration of total protein (g L-1) were determined pentosaceus, L. lactis, E. faecalis while W. cibaria using an autoanalyzer (Biotecnica Instruments, Ita- showed moderate activity (7-8mm) at the examined ly). Also, serum lysozyme activity was determined temperatures and L. garvieae showed highest activ- according to Sahoo, Mahapatra, Saha, Barat, Sahoo, ity(10mm) at 30˚C. The highest antagonistic activ- Mohanty, Gjerde, Ødegard, & Salte (2008). Alter- ities (9-10mm) were observed for the ECPs of W. native complement activity (ACH50) was assayed cibaria, P. pentosaceus, and L. lactis at pH 9 while using the method of Matsuyama, Tanaka, Nakao & L. garvieae and E. faecalis showed moderate activ- Yano (1988). The level of IgM was measured using ity (7-8mm) at the examined pH. analysis kits (Binding Site Company, www.binding-site.co.uk) in MININEPH auto analyzer (Binding Haematological and immunological parameteres
Haematocrit levels (%PCV), MCHC, MON re-mained unaffected in the experimental groups. WBC and RBC counts were significantly higher in T3 than control. On the other hand, the highest HB, Iranian Journal of Aquatic Animal Health
Table 1 Changes of hematological parameters of Acipenser persicus fed Lactococcus lactis (JF831150) for 56 days
Haemoglobin (g dl-1) Haematocrit (% PCV) Mean Corpuscular Volume (ft) Mean Corpuscular Haemoglobin Concentration (%) Mean Corpuscular Haemoglobin (Pg) Basal diet without bacteria (Control), basal diet containing 106 CFUg-1 Lactococcus lactis (JF831150) (T1), basal diet containing 107 CFUg-1 Lactococcus lactis (JF831150) (T2) and basal diet containing 108 CFUg-1 Lactococcus lactis (JF831150) (T3).
Table 2 Changes in some biochemical and immunological parameters of Acipenser persicus fed Lactococcus lactis (JF831150) for 56
Alternative complement activity (U mL-1) Lactate dehydrogenase (IU mL-1 Alanine aminotransferase (IU mL-1) Aspartate aminotransferase (IU mL-1) Basal diet without bacteria(Control), basal diet containing 106 CFUg-1 Lactococcus lactis (JF831150) (T1), basal diet containing 107 CFUg-1 Lactococcus lactis (JF831150) (T2) and basal diet containing 108 CFUg-1 Lactococcus lactis (JF831150) (T3).
MCH, NEU and EOS were obtained in T3, while tained in T1 (1.11). The CF and HSI of the experi- the lowest levels were obtained in control (P<0.05). mental groups were not significantly different from Moreover, the MCV and LYM levels were signifi- the control. Also, the highest SGR was obtained in cantly decreased in T3, while the highest levels T3 (1.46), while the lowest one was obtained in T1 were obtained in control (Table 1).
(1.28) (P < 0.05). The highest PER was obtained in The total protein, LDH, IgM, ALT, AST, and glu- T3 (0.48), while the lowest one was obtained in T1 cose of the experimental groups were not signifi- (0.42) (P < 0.05). In addition, the highest FE was cantly different from the control. Also, lysozyme, obtained in T3 (103), while the lowest one was ob- and ACH50 increased significantly in groups com- tained in T1 (89.92) (P<0.05) (Table 3).
pared to control one (Table 2).
The highest moisture was obtained in T2, while the lowest one was obtained in the control (P<0.05). Growth performance of fish
The crude protein content was significantly higher The growth performance of fish fed with 3 levels in control than experimental groups. The highest to- of L. lactis is shown in Table 4. The highest growth tal lipid was obtained in T2, while the lowest one rate was obtained in T3, while the lowest one was was obtained in control. Meanwhile, the lowest and obtained in T1 (P<0.05). The best FCR was ob- the highest ash-content were significantly obtained tained in T3 (0.96), while the highest one was ob- in the control and T1, respectively (Table 4).
M Soltani et al., LAB in Persian sturgeon (Acipenser persicus)
Table 3 Growth performance, protein efficiency ratio, hepatosomatic index, condition factor, food conversion ratio, and feed efficiency
of Acipenser persicus fed Lactococcus lactis (JF831150) for 56 days
Initial weight (g) Initial body weight (%) Feed Conversion Ratio Hepatosomatic Index Specific growth rate (% day -1) Protein efficiency ration (%) Feed efficiency 101.0±0.38b Basal diet without bacteria (Control), basal diet containing 106 CFUg-1 Lactococcus lactis (JF831150) (T1), basal diet containing 107
CFUg-1 Lactococcus lactis (JF831150) (T2) and basal diet containing 108 CFUg-1 Lactococcus lactis (JF831150) (T3).
Table 4 Body proximate composition after 8 weeks feeding of Acipenser persicus fed containing Lactococcus lactis (JF831150)
Composition (g kg-1) Basal diet without bacteria(Control), basal diet containing 106 CFUg-1 Lactococcus lactis (JF831150) (T1), basal diet containing 107 CFUg-1 Lactococcus lactis (JF831150) (T2) and basal diet containing 108 CFUg-1 Lactococcus lactis (JF831150) (T3). a maximum inhibitory activity to A. hydrophila at 20-30˚C indicating an optimum temperature for the production of the highest ECPs. However, L. lactis The in vitro growth of LAB at 20-30˚C together showed the highest antimicrobial activity against A. with their resistance to acidity and alkaline condi- hydrophila similar to the results obtained by Balca- tions is correlated to the natural environmental con- zar et al. (2008) who demonstrated a strong inhibi- dition of the fish intestine suggesting the isolated tory activity by L. lactis CLFP 101 against both A. LAB are the representatives of the bacterial flora hydrophila and A. salmonicida. As the best inhib- that are resident in the intestine of Persian sturgeon. itory activity due to L. lactis occurred at optimum For instance, the in vitro ability of some LAB in- physiological temperature of A. hydrophila then it is cluding E. faecalis, P. pentosaceus, and W. cibaria possible to recommend L. lactis as a suitable poten- to grow at low pH of 3.7-4.5 is similar to the fish tial probiotic against motile Aeromonas septicemia intestine condition that is an acidic environment. caused by A. hydrophila in Persian sturgeon (Soltani However, these results showed that all of isolat- & Kalbassi 2001). Such anti- A. hydrophila may be ed LAB had the best growth in almost neutral pH in part due to production of organic acids, hydrogen condition which is preferable for almost all LAB. peroxide, carbon dioxide, acetic acid, bacteriocins, Similar results have been reported by Holt, Krieg, diacetyl, acetaldehyde, ethanol, and low molecular Staley & Willams (1994) and Balcazar et. al (2008) weight antimicrobial compounds e.g reuterin from who assessed the environmental responses of some some LAB (Balcazar, Vendrell, Blas, Ruiz-Zarzu- LAB such as E. faecalis, L. lactis, L. garviae and P. ela, Gironés & Múzquiz 2007; Lee & Salminen pentosaceus in the in vitro condition.
2009). In addition, all examined LAB were able The results of present study also showed that the ex- to keep their anti-A. hydrophila activities at pH 7 tracelluar products of most of isolated LAB showed to 9, a property that may be a benefit tool to pass Iranian Journal of Aquatic Animal Health
the gastrointestinal tract of fish. Such a character is licheniformis increase protein and decrease lipid an important factor for a bacterial strain to become contents (El-Haroun et al. 2006). Moreover, Merri- a suitable probiotic because the high rate of its vi- field et al. (2009 a,b) also reported that commercial ability and its capacity of colonization in the fish probiotics such as B. subtilis, B. licheniformis, and intestine is necessary (Goktepe, Juneja & Ahmedna Enterococcus faecium had no effect on carcass fac- 2006; Lee & Salminen 2009). tors. Variation in lipid and protein contents in fish A number of probiotics have been incorporated into could be due to their synthesis and stock in muscles aquatic animal feeds to increase the growth perfor- (Abdel-Tawwab, Khattab, Ahmad & Shalaby 2006). mances (Ghosh, Sen, & Ray 2003; Son, Chang, Wu, Improvement of immunity using dietary enriched Guu, Chi & Cheng 2009; Merrifield et al. 2009a,b; probiotic could be attributed to the fish species, ge- Abd El-Rhman, Khattab, & Shalaby 2009; Askarian netic, difference in consumption time and quantity et al. 2011).
of probiotics supplementation diet, and probiotic Manipulation of intestinal microflora via probiot- origins (Nikoskelainen, Ouwehand, Bylund, Salm- ics plays an important mechanism for increase of inen & Lilius 2003; Panigrahi, Kiron, Puangkaew, growth performances and survival (Vendrell, Bal- Kobayashi, Satoh & Sugita 2005; Salinas, Cuesta, cazar, Blas, Ruiz-Zarzuela, Girones & Muzquiz Esteban. & Meseguer 2005; Kim & Austin 2006; 2008). However, probiotics are also effectively Pieters, Brunt, Austin & Lyndon 2008; Son et al. important of feed with detoxification and produc- 2009). The present study revealed that administra- tion of some hydrolytic enzymes such as amylase, tion of probiotic in diets significantly enhanced the protease and some vitamins like biotin as well as WBC, RBC, haemoglobin, MCH, neutrophils and vitamin B12 (El-Haroun, Goda & Chowdhury eosinophils. Immunocompetent cells are important 2006). In the present study, after 8 weeks adminis- for assessment of fish health and their activation and tration of the supplemented diets had a significant proliferation is stimulated by probiotics enriched di- increase in final weight, FCR, SGR, PER, and FE ets (Irianto & Austin 2002; Brunt & Austin 2005). of Persian sturgeon. This result is in agreement with Leukocytes are the sources of lysozymes produc- Askarian et al. (2011) after feeding Persian sturgeon tion (Akrami Ghelichi & Ahmadifar 2011), and in (Acipenser persicus) and Beluga (Huso huso) with this study a significant increase in WBC population Chironomidae incorporated with Lactobacillus cur- was correlated with the enhancement of lysozyme vatus and Leuconostoc mesenteroides for 50 days. level in fish sera. The present study also indicates Mechanisms of the improvement of growth perfor- that probiotics diet could decrease the Haematocrit mances have not completely clear but production of levels (%PCV) similar results seen Abd El-Rhman some vitamins like vitamin k, and B12 (Martens, et al. (2009) who feed Nile tilapia with Micrococcus Barg, Warren & Jahn 2002) as well as extracellu- luteus and Pseudomonas species as probiotics for lar enzymes such as esterase, protease, and amylase 90 days. Also, in this study, alternative complement (Azokpota, Hounhouigan, Nago & Jakobsen 2006) activity (ACH50) was enhanced significantly in fish could have important role. Administration of probi- fed with the probiotic supplementation diet after 8 otics enriched diets helps to improve feed utility and weeks. Similar results were seen in works reported digestion of proteins, as well as increase the digest- by Panigrahi, Kiron, Kobayshi, Puangkaew, Satoh& ibility of feed resulting in increase of growth and FE Sugita (2004); Son et al. (2009) ; Harikrishnan et al. (Lara-Flores, Olvera-Novoa, Guzma´n-Me´ndez & (2011) and Geng et al. (2011) used some probiotics Lo´pez-Madrid 2003). The results presented in this in the diets of rainbow trout (Oncorhynchus mykiss), study revealed a significant increase in the lipid con- grouper (Epinephelus coioides), rock bream (Ople- tent compare to control. On the contrary, in Nile tila- gnathus fasciatus), cobia (Rachycentron canadum) pia, Oreochromis niloticus (L.), fed with commercial and tilapia (Oreochromis niloticus). probiotics including Bacillus subtilis and Bacillus An increase in complement components in treated M Soltani et al., LAB in Persian sturgeon (Acipenser persicus)
fish sera is because of an increase in WBC popu- aby A.M.E.(2006) Compensatory growth, feed utilization, lation as observed in this study. Also, an increase whole body composition and hematological changes in in level of total IgM has been seen in the treated starved juvenile Nile tilapia, Oreochromis niloticus (L). fish compared to control one (P>0.05) indicating Journal of Applied Aquaculture 18, 17–36.
of stimulating of lymphocyte population for IgM production as already reported by other researchers Akrami R., Abdolmajid H., Abbas M. & Abdolmoham- using some teleost fish (Salinas Abelli, Bertoni, Pic- mad A.K.(2009) Effect of dietary prebiotic inulin on chietti, Roque, Furones, Cuesta, Meseguer & Este- growth performance, intestinal microflora, body compo- ban 2008; Sun, Yang, Ma & Lin 2010). sition and hematological parameters of juvenile beluga, Huso huso (Linnaeus, 1758). Journal of the World Aqua- culture Society 40, 771–779.
In conclusion, the in vitro environmental responses Akrami R., Ghelichi A. & Ahmadifar E. (2011) Effect of of LAB recovered from the intestine of Persian stur- dietary prebiotic inulin on hematological and biochem- geon are in correlation with the physiological condi- ical parameters of cultured juvenile Beluga(Huso huso). tions of fish gastrointestinal tract making it possible Journal of Veterinary Research 66(2), 131-136.
to use some of these LABs such as L. lactis as a po-tential probiotic against A. hydrophila septicemia. Aly S.M., Ahmed YA-G., Ghareeb AA-A. & Mohamed However, examination of the pathogenicity of these M.F. (2008a) Studies on Bacillus subtilis and Lactobacil- LAB bacterial species as probiotic candidates is re- lus acidophilus, as potential probiotics, on the immune quired prior to judging on their probiotic activity in response and resistance of Tilapia nilotica (Oreochromis fish. This is particularly true in case of L. garvieae niloticus) to challenge infections. Fish and Shellfish Immu- that has become a universal pathogen for many fish nology 25, 128-136.
species under different environmental conditions. Also, the results of in vitro and in vivo works here Aly S.M., Mohamed M.F. & John G. (2008b) Effect of pro- in this study clearly show that supplementation of biotics on the survival, growth and challenge infection in Persian sturgeon feed with L. lactis as a native pro- Tilapia nilotica (Oreochromis niloticus). Aquaculture Re- biotic is able to enhance both fish growth perfor- search 39, 647-656.
mances and some immunophysiological responses.
AOAC (1995) Official methods of analysis of AOAC In- ternational, 16th edn. AOAC International, Arlington, This work was funded by the grant of research coun-cil of the University of Tehran, Center of Excellence Askarian F., Kousha A., Salma W. & Ringø E. (2011)The of Aquatic Animal Health, University of Tehran, In- effect of lactic acid bacteria administration on growth, ternational Sturgeon Research Institute and Iranian digestive enzyme activity and gut microbiota in Persian Fisheries Science Research Institute.
sturgeon (Acipenser persicus) and beluga(Huso huso) fry. Aquaculture Nutrition 17, 488-497.
Abd El-Rhman A.M., Khattab Y.A.E. & Shalaby A.M.E. Azokpota P., Hounhouigan D.J., Nago M.C. & Jakobsen (2009) Micrococcus luteus and Pseudomonas species as M. (2006) Esterase and protease activities of Bacillus probiotics for promoting the growth performance and spp. from afitin, iru and sonru; three African locust bean health of Nile tilapia, Oreochromis niloticus. Fish and (Parkia biglobosa) condiments from Benin. African Jour- Shellfish Immunology 27, 175–180.
nal of Biotechnology 5, 265–272.
Abdel-Tawwab M., Khattab Y.A.E., Ahmad M.H. & Shal- Iranian Journal of Aquatic Animal Health
Balcázar J.L., Vendrell D., Blas I., Ruiz-Zarzuela I., and Shellfish Immunology 31, 400-406.
Múzquiz J.L. & Gironés O.(2008) Characterization of probiotic properties of lactic acid bacteria isolated from Ghosh K., Sen S.K. & Ray A.K. (2003) Supplementation intestinal microbiota of fish. Aquaculture 278, 188–191.
of an isolated fish gut bacterium, Bacillus circulans, in formulated diets for rohu, Labeo rohita, fingerlings. Israeli Balcázar J.L., Vendrell D., Blas I., Ruiz-Zarzuela I., Gi- Journal of Aquaculture-Bamidgeh 55, 13–21.
ronés O. & Múzquiz J.L.(2007) In vitro competitive adhe- sion and production of antagonistic compounds by lactic Goktepe I., Juneja V.K. & Ahmedna M. (2006) Probiotics acid bacteria against fish pathogens. Veterinary Microbiol- in food safety and human health. CRC press, Taylor and ogy 122, 373–380.
Francis Group, USA.
Bello B.D., Cocolin L., Zeppa G., Field D., Cotter P.D. Harikrishnan R., Kim M., Kim J., Balasundaram C. & & Hill C.(2012)Technological characterization of bacte- Heo M. (2011a) Protective effect of herbal and probiotics riocin producing Lactococcus lactis strains employed to enriched diet on haematological and immunity status of control Listeria monocytogenes in Cottage cheese. Inter- Oplegnathus fasciatus (Temminck & Schlegel) against Ed- national Journal of Food Microbiology 153, 58–65.
wardsiella tarda. Fish and Shellfish Immunology 30, 886-
Brunt J. & Austin B. (2005) Use of a probiotic to control Holt J. G., Krieg N.R., Staley J.T. & Willams S.T. (1994) lactococcosis and streptococcosis in rainbow trout, On- Bergeys manual of determinative bacteriology, 9th edn. corhynchus mykiss (Walbaum). Journal of Fish Disease 28,
Willams and Wilkins Puble.
Hoseinifar S.H., Mirvaghefi A. & Merrifield D.L. (2011c) Casla D., Requena T. & Gómez R. (1996) Antimicrobial The effects of dietary inactive brewer's yeast Saccharomy- activity of lactic acid bacteria isolated from goat's milk ces cerevisiae var. ellipsoideus on the growth, physiologi- and artisanal cheeses: characteristics of a bacteriocin pro- cal responses and gut microbiota of juvenile beluga (Huso duced by Lactobacillus curvatus IFPL105. Journal of Ap- huso). Aquaculture 318, 90–94.
plied Bacteriology 81, 35–41.
Hoseinifar S.H., Mirvaghefi A., Merrifield D.L., Mojazi EL-Haroun E.R., Goda A. M. & Chowdhury M.A.K. Amiri B., Yelghi S. & Darvish Bastami K.( 2011a) The (2006) Effect of dietary probiotic Biogens supplementa- study of some haematological and serum biochemical pa- tion as a growth promoter on growth performance and rameters of juvenile beluga (Huso huso) fed oligofructose. feed utilization of Nile tilapia Oreochromis niloticus (L.). Fish Physiology and Biochemistry 37, 91-96.
Aquacultre Research 37, 1473-1480.
Hoseinifar S.H., Mirvaghefi A., Mojazi Amiri B., Khosh- Falahatkar B., Soltani M., Abtahi B., Kalbassi M.R. & bavar Rostami H. & Merrifield D.L.( 2011b)The effects Pourkazemi M. (2006) Effects of dietary vitamin C sup- of oligofructose on growth performance, survival and au- plementation on performance, tissue chemical composi- tochthonous intestinal microbiota of beluga (Huso huso) tion and alkaline phosphatase activity in great sturgeon juveniles. Aquacultre Nutrition 17, 498–504.
(Huso huso). Journal of Applied Ichthyology 22, 283–286.
Irianto A. & Austin B. (2002) Probiotics in aquaculture. Geng X., Dong X., Tan B., Yang Q., Chi S., Liu H. & Liu Journal of Fish Disease 25, 633–642.
X. (2011) Effects of dietary chitosan and Bacillus subtilis on the growth performance, non-specific immunity and Jafaryan H., Taati M.M. & Makhtoumi N.M. (2010) The disease resistance of cobia, Rachycentron canadum. Fish effects of probiotic bacillus for promotion of growth and M Soltani et al., LAB in Persian sturgeon (Acipenser persicus)
feeding parameters in beluga (Huso huso) larvae via feed- ing by bioencapsulated Artemia. Aquaculture, Aquarium, Meng Y., Xiao H.B. & Zeng L.B. (2011) Isolation and Conservation & Legislation 3(4), 273–280.
identification of the hemorrhagic septicemia pathogen from Amur sturgeon, Acipenser schrenckii. Journal of Jalali M.A., Ahmadifar E., Sudagar M. & Takami G.A. Applied Ichthyology 27, 799–803.
(2009) Growth efficiency, body composition, survival and haematological changes in great sturgeon (Huso huso Lin- Merrifield D.L., Bradley G., Baker R.T.M. & Davies S.J. naeus, 1758) juveniles fed diets supplemented with differ- (2009b) Probiotic applications for rainbow trout (Onco- ent levels of Ergosan. Aquaculture Research 40, 804–809.
rhynchus mykiss Walbaum) II. Effects on growth perfor- mance, feed utilization, intestinal microbiota and related Khoshbavar-Rostami H.A., Soltani M. & Hassan H.M.D. health criteria postantibiotic treatment. Aquaculture Nu- (2006) Immune response of great sturgeon (Huso huso) trition 16, 496–503.
subjected to long-term exposure to sublethal concentra- tion of the organophosphate, diazinon. Aquaculture 256,
Merrifield D.L., Dimitroglou A., Bradley G., Baker R.T.M. & Davies S.J. (2009a) Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) I. Effects on Khoshbavar-Rostami H.A., Soltani M. & Hassan M.D. growth performance, feed utilization, intestinal microbi- (2007) Immune responses of great Sturgeon (Huso huso) ota and related health criteria. Aquaculture Nutrition 16,
to Aeromonas hydrophila. Journal of Fish Biology 70(6),
Nikoskelainen S., Ouwehand A.C., Bylund G., Salminen Kim D. & Austin B. (2006) Innate immune responses in S. & Lilius E.M. (2003) Immune enhancement in rainbow rainbow trout (Oncorhynchus mykiss, Walbaum) induced trout (Oncorhynchus mykiss) by potential probiotic bacte- by probiotics. Fish and Shellfish Immunology 21, 513-524.
ria (Lactobacillus rhamnosus). Fish and Shellfish Immunol- ogy 15, 443-452.
Lara-Flores M., Olvera-Novoa M.A., Guzma´n-Me´ndez Oidtmann B.C., Thrush M.A., Denham K.L. & Peeler B.E. and Lo´pez-Madrid W. (2003) Use of the bacteria E.J. (2011) Review. International and national biosecuri- Streptococcus faecium and Lactobacillus acidophilus, and ty strategies in aquatic animal health. Aquaculture 320,
the yeast Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus). Aquaculture 216,
Panigrahi A., Kiron V., Puangkaew J., Kobayashi T., Satoh S. & Sugita H. (2005) The viability of probiotic bacteria Lee Y.K., & Salminen S.( 2009) Handbook of probiotics as a factor influencing the immune response in rainbow and prebiotics (second edition). John Wiley and Sons, trout Oncorhynchus mykiss. Aquaculture 243, 241-254.
Inc., Hoboken, New Jersey.
Panigrahi A., Kiron V., Kobayshi T., Puangkaew J., Satoh Martens J.H., Barg H., Warren M.J. & Jahn D. (2002) Mi- S. & Sugita H. (2004) Immune responses in rainbow trout crobial production of vitamin B-12. Applied Microbiology Oncorhynchus mykiss induced by a potential probiotics and Biotechnology 58, 275–285.
bacteria Lactobacillus rhamnosus JCM 1136. Veterinary Immunology and Immunopathology 102, 379–388.
Matsuyama H., Tanaka K., Nakao M. & Yano T. (1988) Characterization of the alternative complement pathway Pieters N., Brunt J., Austin B. & Lyndon A.R. (2008) Ef- of Carp. Developmental and Comparative Immunology 12,
ficacy of in-feed probiotics against Aeromonas bestiarum and Ichthyophthirius multifiliis skin infections in rain- Iranian Journal of Aquatic Animal Health
bow trout (Oncorhynchus mykiss, Walbaum). Journal of diversity of lactic acid bacteria in the intestine of Persian Applied Microbiology 105, 723-732.
sturgeon fingerlings. Journal of Applied Ichthyology 29(3),
Rawling M.D., Merrifield D.L. & Davies S.J. (2009) Pre- liminary assessment of dietary supplementation of San- Son V.M., Chang C., Wu M., Guu Y., Chiu C. & Cheng grovit on red tilapia (Oreochromis niloticus) growth per- W.(2009) Dietary administration of the probiotic, Lac- formance and health. Aquaculture 294, 118–122.
tobacillus plantarum, enhanced the growth, innate im- mune responses, and disease resistance of the grouper Reddy K.B.P.K. (2007) Effect of drying processes on via- Epinephelus coioides. Fish and Shellfish Immunology 26(5),
bility and selected functional properties of probiotic lactic acid bacteria. PhD thesis, University of Mysore, India.
Sun Y, Yang H, Ma R. & Lin W.(2010) Probiotic applica- Sahoo P.K., Mahapatra K. D., Saha J.N., Barat A., Sahoo tions of two dominant gut Bacillus strains with antago- M., Mohanty B.R., Gjerde B., Ødegard J., Rye M. & Salte nistic activity improved the growth performance and im- R. (2008) Family association between immune parameters mune responses of grouper Epinephelus coioides. Fish and and resistance to Aeromonas hydrophila infection in the Shellfish Immunology 29, 803-809.
Indian major carp, Labeo rohita. Fish and Shellfish Immu- nology 25, 163-169.
Vendrell D., Balcazar J.L., Blas I., Ruiz-Zarzuela I., Gi- rones O. & Muzquiz J.L.(2008) Protection of rainbow Salinas I., Abelli L., Bertoni F., Picchietti S., Roque A., trout (Oncorhynchus mykiss) from lactococcosis by pro- Furones D., Cuesta A., Meseguer J.& Esteban M.A.(2008) biotic bacteria. Comparative Immunology, Microbiology & Monospecies and multispecies probiotic formulations Infectious Diseases 31, 337–345.
produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.). Fish Yang W. & Li A. (2009) Isolation and characterization and Shellfish Immunology 25, 114-123.
of Streptococcus dysgalactiae from diseased Acipenser schrenckii. Aquaculture 294, 14–17.
Salinas I., Cuesta A., Esteban M.A. & Meseguer J. (2005) Dietary administration of Lactobacillus delbrueckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish and Shellfish Im- munology 19, 67-77.
Sharifuzzaman S.M. & Austin B. (2009) Influence of pro- biotic feeding duration on disease resistance and immune parameters in rainbow trout. Fish and Shellfish Immunol- ogy 27, 440–445.
Soltani M. & Kalbassi M.R.(2001)Protection of Persian sturgeon (Acipenser persicus) fingerling against Aero- monas hydrophila septicemia using three different anti- gens. Bulletin of the European Association of Fish Patholo- gists 21(6), 235 – 240.
Soltani M., Pourkazemi M., Ahmadi M.R., Taherimirg- head A., Merrifield D.L. & Masouleh A.S. (2013) Genetic Iranian Journal of Aquatic Animal Health
ﺪﯿﺳا یﺮﺘﻛﺎﺑ ﻚﯿﺗﻮﯿﺑوﺮﭘ هدﺎﻔﺘﺳا و ﯽﻜﯿﺗﻮﯿﺑ ﯽﺘﻧآ ﺖﯿﺳﺎﺴﺣ ،ﯽﯾﺎﯾﺮﺘﻛﺎﺑﺪﺿ ﺖﯿﻟﺎﻌﻓ
(Acipenser persicus) ﯽﻧاﺮﯾا ﯽھﺎﻤﺳﺎﺗ شروﺮﭘ رد ﻚﯿﺘﻛﻻ
1ﺪﺋﺎﻗﺮﯿﻣ ىﺮﻫﺎﻃ ﻰﻠﻋ ،2ﻰﻤﻇﺎﮐرﻮﭘ ﺪﻤﺤﻣ ،1ىﺪﻤﺣا ﺎﺿرﺪﻤﺤﻣ ،2،1ﻪﻟﻮﺳﺎﻣروﺎﻨﺷ ﺎﺿﺮﯿﻠﻋ ،*1ﻰﻧﺎﻄﻠﺳ ىﺪﻬﻣ
ناﺮﻬﺗ هﺎﮕﺸﻧاد نﺎﯾﺰﺑآ ىﺎﻬﯾرﺎﻤﯿﺑ و ﺖﺷاﺪﻬﺑ ﺐﻄﻗ .ناﺮﻬﺗ هﺎﮕﺸﻧاد ﻰﮑﺷﺰﭙﻣاد هﺪﮑﺸﻧاد نﺎﯾﺰﺑآ ىﺎﻬﯾرﺎﻤﯿﺑ و ﺖﺷاﺪﻬﺑ هوﺮﮔ 1 ﺖﺷر ،ىزروﺎﺸﮐ ﺞﯾوﺮﺗ و شزﻮﻣآ ،تﺎﻘﯿﻘﺤﺗ نﺎﻣزﺎﺳ ،رﺰﺧ ىﺎﯾرد نﺎﯿﻫﺎﻤﺳﺎﺗ ﻰﻠﻠﻤﻟا ﻦﯿﺑ تﺎﻘﯿﻘﺤﺗ ﻪﺴﺳﻮﻣ نﺎﯾﺰﺑآ ىﺎﻬﯾرﺎﻤﯿﺑ و ﺖﺷاﺪﻬﺑ ﺶﺨﺑ 2 هﺪﯿﮑﭼ
ﺖﯿﻟﺎﻌﻓ ﻦﯿﻨﭽﻤه .ﺖﻓﺮﮔ مﺎﺠﻧا یرﻮﺷ و ﯽﯾﺎﻣد ، pH توﺎﻔﺘﻣ ﻂﯾاﺮﺷ رد ﯽﻧاﺮﯾا ﯽهﺎﻤﺳﺎﺗ زا ﻩﺪﺷ ﯽﯾﺎﺳﺎﻨﺷ ﮏﯿﺘﮐﻻ ﺪﯿﺳا یﺮﺘﮐﺎﺑ ۵ ﺪﺷر رﺎﺘﻓر ﯽﺑﺎﯾزرا ﺐﻟﺎﻏ یﺎه ﮏﯿﺗﻮﯿﺑ ﯽﺘﻧآ ﻪﺑ ﺖﺒﺴﻧ ﺎﻬﻧآ ﺖﯿﺳﺎﺴﺣ ﺰﯿﻧ و Aeromonas hydrophilaﻪﯿﻠﻋ ﻩﺪــﺷ ﯽﯾﺎﺳﺎﻨﺷ یﺎه یﺮﺘﮐﺎﺑ ﯽﻟﻮﻠﺳ جرﺎﺧ ﯽﺤــﺷﺮﺗ تﺎﺒﯿﮐﺮﺗ تﺎﻌﻟﺎﻄﻣ .ﺪﯾدﺮﮔ ﻦﯿﯿﻌﺗ ﯽـ ﺷروﺮﭘ ﻂﯾاﺮــﺷ رد ﺰﯿﻧ ﻩزور ۵۶ ﻩرود ﮏﯾ رد ﯽﯾاﺬﻏ ﻞﻤﮑﻣ ﮏﯾ ناﻮﻨﻋ ﻪﺑ L. lactis ﺮﺛا .ﺪــﺷ ﯽﺑﺎﯾزرا نﺎﯾﺰﺑآ رد فﺮــﺼﻣ درﻮــﻣ ﯽﺤﺷﺮﺗ تﺎﺒﯿﮐﺮﺗ ﺖﯿﻟﺎﻌﻓ ﯽﺑﺎﯾزرا .ﺪﻧراد ﺪﺷر ﺖﯿﻠﺑﺎﻗ ٪ ۴-٨ یرﻮﺷ و داﺮﮔ ﯽﺘﻧﺎﺳ ﻪﺟرد ٢٠-٣٠ ﯽﯾﺎﻣد ﻩدوﺪﺤﻣ رد ﺎه یﺮﺘﮐﺎﺑ ﻦﯾا ﻪﮐ داد نﺎﺸﻧ ﯽهﺎﮕﺸﯾﺎﻣزآ .ﺪﻧرادرﻮﺧﺮﺑ A. hydrophila ﺎﺑ ﻪﻠﺑﺎﻘﻣ ﯽﯾﺎﻧاﻮﺗ زا داﺮﮔ ﯽﺘﻧﺎــﺳ ﻪﺟرد ٢۵-٣٠ ﯽﯾﺎﻣد ﻪﻨﻣاد رد ﺎه یﺮﺘﮐﺎﺑ ﻦﯾا ﻪﮐ داد نﺎــﺸﻧ ﺎه یﺮﺘﮐﺎﺑ ﻦﯾا ﯽﻟﻮﻠــﺳ جرﺎﺧ ٩ pH رد A. hydrophila ﻪﯿﻠﻋ L. lactis و W. cibaria ،P. pentosaceus یﺎه یﺮﺘﮐﺎﺑ ﯽﻟﻮﻠــﺳ جرﺎﺧ تﺎﺒﯿﮐﺮﺗ ﯽﺘــﺴﯿﻧﻮﮔﺎﺘﻧآ ﺖﯿﻟﺎﻌﻓ ﻦﯾﺮﺘــﺸﯿﺑ ،لوزﺎﺴﮐﻮﺘﻣﺎﻔﻟﻮﺳ ﻢﯾﺮﭘﻮﺘﻣ یﺮﺗ،ﻦﯿﺴﯾﺎﻣوﺮﺘﯾرا ،ﻦﯿﻠﮑﯾﺎﺳاﺮﺘﺗ ﯽﺴﮐا یﺎه ﮏﯿﺗﻮﯿﺑ ﯽﺘﻧآ ﻪﺑ ﺖﺒﺴﻧ E. faecalis و W. cibaria یﺎه یﺮﺘﮐﺎﺑ .ﺖﺳا ﻩدﻮﺑ و ﻦﯿﺳﺎﺴﮐﻮﻠﻓوﺮﻧا ،ﻦﯿﺴﯾﺎﻣوﺮﺘﯾرا ،ﻦﯿﻠﮑﯾﺎﺳاﺮﺘﺗ ﯽﺴﮐا ﻪﺑ ﺖﺒــﺴﻧ L. lactis یﺮﺘﮐﺎﺑ ﻪﮑﯿﻟﺎﺣرد ﺪﻧا ﻩدﻮﺑ موﺎﻘﻣ ﻦﯿﺋﻮﮑﻣﻮﻠﻓ و ﻞﮑﯿﻨﻓرﻮﻠﻓ ،ﻦﯿــﺳﺎﺴﮐﻮﻠﻓوﺮﻧا ﯽهﺎﻤﺳﺎﺗ ﺖﺷاﺪﻬﺑ و اﺬﻏ ﯽﯾآرﺎﮐ ،ﺪﺷر یﺎه ﺺﺧﺎﺷ دﻮﺒﻬﺑ رد ﺪﻧاﻮﺗ ﯽﻣ L. lactis فﺮﺼﻣ ﻪﮐ داد نﺎﺸﻧ ﻪﻌﻟﺎﻄﻣ ﻦﯾا ﺞﯾﺎﺘﻧ .داد نﺎﺸﻧ ﺖﯿﺳﺎﺴﺣ ﻞﮑﯿﻨﻓرﻮﻠﻓ .ﺪﺷﺎﺑ ﺮﺛﺆﻣ ﯽﻟﺎﻤﺘﺣا ﮏﯿﺗﻮﯿﺑوﺮﭘ ﮏﯾ ناﻮﻨﻋ ﻪﺑ ﯽﻧاﺮﯾا .ﻚﯿﺗﻮﯿﺑوﺮﭘ ،ﺲﯿﺘﮐﻻ سﻮﮐﻮﮐﻮﺘﮐﻻ ،ﺖﺷاﺪﻬﺑ ،یرﺎﯾوﺎﺧ نﺎﯿهﺎﻣ :یﺪﯿﻠﮐ یﺎه ﻩژاو
Non-toxic tumortherapy (NTT) for advanced colorectal cancer with liver metastasis, a pilot study E.Valstar M.D., M. Sc. Stadhouderslaan 30, 2517 HZ The Hague, Holland Eighty-three patients with livermetastases from colorectal cancer and in many cases also metastases elsewhere consulted me in 1988-1997 for non-toxic tumortherapy (NTT). No selection was made. Two regressions during