HAL Id: hal-00902510https://hal.archives-ouvertes.fr/hal-00902510

Submitted on 1 Jan 1998

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

In vivo studies on lysosubtilin. 2. Efficacy for treatmentof post-partum endometritis in cows

Gediminas Arvydas Biziulevichius, Kazimieras Lukauskas

To cite this version:Gediminas Arvydas Biziulevichius, Kazimieras Lukauskas. In vivo studies on lysosubtilin. 2. Efficacyfor treatment of post-partum endometritis in cows. Veterinary Research, BioMed Central, 1998, 29(1), pp.47-58. �hal-00902510�

Original article

In vivo studies on lysosubtilin. 2. Efficacy fortreatment of post-partum endometritis in cows

Gediminas Arvydas Biziulevichiusa Kazimieras Lukauskasb

&dquo;Sector of Immunobiotechnology, Institute of Immunology, 12 Mokslininku St,2600 Vilnius, Lithuania

bstate Veterinary Service, 19 Gedimino Ave, 2025 Vilnius, Lithuania

(Received 24 March 1997; accepted 03 October 1997)

Abstract - Lysosubtilin is a broad-spectrum preparation of lytic enzymes from Bacillus suhtilisdesigned for veterinary medicine. This study demonstrates its efficacy for the treatment of post-partum endometritis in cows. Prior to the determination of optimal therapeutic doses, samples takenfrom the uterus of sick animals were examined microbiologically. The examinations revealed ahigh incidence of polymicrobial infections (65.0 %) caused by various mixtures of bacteria (bothGram-positive and Gram-negative) and fungi/yeasts. Dose-determination studies involved 160 cowswith clinical signs of post-partum endometritis. For treatment both aqueous and oil-based for-mulations were tested and the optimal dose was found to be 2 x 106 U lysosubtilin dissolved in100 mL of distilled water. When administered intrauterinarily twice a week until recovery this doseresulted in a 100 % clinical recovery rate (versus 90 %) and a statistically significant decrease (73± 5 d), when compared with cows treated with neofur (92 ± 9 d, P < 0.05) in the calving-to-conception interval. These findings were confirmed by results of field trials performed in four For-mer Soviet Union republics (Byelarus, Estonia, Lithuania, Russia) on 932 cows. Increases inclinical recovery (93.7 versus 80.2 %, P < 0.05) and conception rates (64.4 versus 45.8 %, P < 0.01)were found to be statistically significant when compared with cows treated with neofur oruterosan. We therefore conclude that lytic enzyme preparations are prospective antimicrobialdrugs and when used to combat animal diseases they may serve as a possible alternative to com-mon antibiotics. &copy; Inra/Elsevier, Paris

lysosubtilin / cow / post-partum endometritis / enzymotherapy / calving-to-conceptioninterval

* Correspondence and reprintsTel: (370) 2 589 230; fax: (370) 2 729 827

Résumé - Utilisation in vivo de la lysosubtiline. 2. Efficacité pour le traitement des endo-métrites post-puerpérales des bovins. La lysosubtiline est une préparation produite à partird’enzymes lytiques de Bacillus subtilis, qui possède un large spectre d’activités et qui est desti-née à la médecine vétérinaire. Cette étude montre l’efficacité de la lysosubtiline dans le traitementdes endométrites post-puerpérales des bovins. Avant de déterminer les doses optimales théra-

peutiques, les échantillons pris dans l’utérus ont été examinés pour leur qualité microbiologique.Les analyses ont montré un pourcentage élevé d’infections polymicrobiennes (65,0 %) provoquéespar divers mélanges de bactéries (tant Gram-positives, que Gram-negatives) et de fungi/levure.160 bovins présentant des signes cliniques d’endométrites post-puerpérales ont été examinés envue de determiner les doses thérapeutiques. Pour le traitement des endométrites post-puerpé-rales les formulations aqueuses et huileuses ont été examinées et une dose optimale égale à 2 x106 U de lysosubtline diluée dans 100 mL d’eau distillée a été déterminée. Cette dose introduitedans l’utérus 2 fois par semaine jusqu’à la guérison a donné 100 °7o de guérison (contre 90 % chezles contrôles) et le raccourcissement statistiquement significatif de l’intervalle vêlage-inséminationartificielle fécondante (73 ± 5 j contre 92 ± 9 j, p < 0,05). Ces données ont été confirmées par lesrésultats d’expériences sur le terrain faites sur 932 bovins dans les républiques de l’ancienneUnion soviétique (Biélorussie, Estonie, Lituanie, Russie). L’augmentation de taux de guérison cli-nique (93,7 contre 80,2 %, p < 0,05) et de la fécondité (64,4 contre 45,8 %, p < 0,01 ) était sta-tistiquement significative. Ceci permet de conclure que les préparations d’enzymes lytiques sontdes médicaments antimicrobiens possibles et pourraient être une solution de remplacementconvenable des antibiotiques traditionnels. &copy; Inra/Elsevier, Paris

lysosubtiline / bovin / endométrite post-puerpérale / enzymothérapie / intervallevêlage-insemination artificielle fecondante

1. INTRODUCTION

Post-partum endometritis is one of themajor economically important diseases indairy cattle. Although cases of mortalityassociated with post-partum endometritisin cows are rare, subsequent economicloss due to decreased reproductive capac-ity and treatment costs is remarkable [5, 6,30, 54, 76, 92]. ] .

The disease is generally of microbialetiology and a large number of Gram-pos-itive and Gram-negative bacteria are impli-cated as causative agents [9, 19, 38, 40,56, 60, 62, 74, 88]. Fungi, most commonlyyeasts, are also known to be involved ininfections of the uterus in cows [58, 68,77, 96j. Mixed (polymicrobial) infectionsare very common in a number of caseswhere even representatives of various tax-onomic groups are involved. These latterare especially difficult to treat.

Antibiotics are the preparations ofchoice in the treatment of cow post-par-tum endometritis [10, 31, 33, 45, 57, 66,73, 90], despite the fact that there are unde-sirable consequences of their regular appli-cation. Evidence for increasing antibiotic

resistance was made in the first part of thisresearch report [ I I J.

Sulphonamides, furans, iodine con-taining preparations, nonsteroidal anti-inflammatory drugs, oxytocin, and

prostaglandins are also often used to com-bat post-partum endometritis [4, 32, 48,50, 53, 59, 64, 71, 81 ]. Immunomodulatorshave been shown to be of great value [ 1, 8,79, 9 1 ], while the future of homeopathictreatments is questionable [41 Recently,the high efficacy of probiotic culturesderived from the vaginal microflora ofhealthy women and used in the prophy-laxis and treatment of cow post-partumendometritis was shown [44, 63, 91].Moreover, modern treatment techniquesother than the use of drugs, such as theuse of ultra-high frequency electromag-netic field and low level laser therapies,have been developed [25, 42, 43, 89].

Antimicrobial or lytic enzymes arepreparations of great interest for veteri-narians [ 13, 47]. It is interesting that theuse of such enzymes in veterinary prac-tice started when hen egg white lysozymewas employed to treat gynaecological dis-eases (post-partum endometritis, vulvo-vaginal infections) in cows [61]. Later dif-

ferent preparations of hen egg whitelysozyme were proposed for the treatmentof bovine mastitis as well 151 Morcover,lysozyme is widely used in the treatmentof bacterial and viral infections in humans

[67, 69, 72]. Lytic enzyme preparationsfor the treatment of local fungal infectionsin animals are also known (22, 23(. [ .

Immobilized proteolytic enzymes,capable of degrading proteinaceous com-ponents of microbial cell walls, are alsopromising preparations for veterinarians[34, 35, 65]. These enzymes have beenshown to be effective devices for the treat-ment of post-partum endometritis in cows[2, 3, 55, 75!.

In addition to their positive features,the above mentioned enzyme preparationshave one essential shortcoming - they areall narrow-spectrum acting antimicrobialagents. We have designed, and in 1983started to produce on an industrial scale, abroad-spectrum action preparation of lyticenzymes for veterinary purposes calledlysosubtilin. Its properties, including itsspectrum of antibacterial and antifungalaction, have already been described! 15-17!. At this point it may be of someinterest to add that first attempts by vet-erinarians to show antibacterial activityof lysosubtilin in vitro were unsuccessful[37!. In vivo, the high efficacy and safetyof lysosubtilin for prophylaxis and treat-ment of gastrointestinal disorders in new-born calves has been described III, 12!as well as the safety of the preparationwhen administered intrauterinarily in cows[93]. Recently we published a short com-munication on the preliminary results ofthe application of an aqueous solution oflysosubtilin for treatment of bovineendometritis [941.

In this paper we provide the final resultsof our investigations on the efficacy oflysosubtilin when applied for treatment ofpost-partum endometritis in cows, includ-ing the results of the field trials.

2. MATERIALS AND METHODS

2.1. Animals and experimentalprotocols

Lysosubtilin therapeutic dose-determina-tion studies were performed in the clinics ofthe Lithuanian Academy of VeterinaryMedicine and its training farm. In the experi-ment, 160 cows of different breeds, predomi-nantly the Lithuanian Black and White breed,were used. The criteria for animal participa-tion were clinical signs of post-partumendometritis (fetid discharge from the uterus,fever, anorexia, depression) confirmed by bac-teriological and mycological investigations.The cows were divided randomly into eightgroups with 20 animals in each. Clinical obser-vation of the cows was performed at least oncedaily throughout the experiment. Seven groupsof animals were treated with different dosesof lysosubtilin ( I x 106 to 3 x I0!’ U) dissolvedin 10(l mL of distilled water (five doses) orsuspended in the same amount of fish oil (twodoses). The eighth group of cows (control) wastreated traditionally with neofur. Lysosubtilinsolutions/suspensions were prepared ex tem-pore.

Prior to treatment the cow genitals werecleaned with water and soap, their uteri wererinsed with 0. (Ye solution of KMnO! and evac-uated by siphoning. The lysosubtilin solu-tions/suspensions, warmed to body tempera-ture, were then applied intrauterinarily withthe help of a syringe connected to an insemi-nation pipette. Neofur was applied in the formof uterine suppositories, three suppositoriesbeing a single dose. Subcutaneous injectionsof 50 IU oxytocin followed in every cow at24, 48 and 72 h post treatment.

The treatment procedures described abovewere repeated at intervals of 3-4 days untilrecovery, but five sets were considered to be alimit. After this time unsuccessfully treatedcows were withdrawn from the experiment andwere further treated in other ways or culled.The cows that remained in the experiment wereinseminated as soon as possible and the result-ing conception was the main indicator of treat-ment success.

The results were verified by field trials.which were performed in the farms of fourFSU republics (Byelarus, Estonia, Lithuania,Russia) on 932 cows. The cows were main-tained under the usual industrial breeding con-ditions. Lysosubtilin was administered in a

dose of 2 x 106 U dissolved in 100 mL of dis-tilled water, except in the Raseiniai region ofLithuania, where an outbreak of post-partumendometritis, characterized by an exaggeratedincidence of fungal pathogens, was observed.In this region the following drug formulation,was used: 2 x 106 U lysosubtilin, 0.5 g nys-tatin, 20 mL trivitamin AD3E , 80 mL fish oil.Control cows were administered neofur oruterosan. The choice was left up to the localveterinarians. The antimicrobial drugs wereadministered twice a week until recovery, butfor no longer than 3 weeks. The other treat-ment procedures were the same as describedabove.

2.2. Sampling and microbiologicalprocedures

Samples from cows suspected of havinguterine infections were taken as follows. Gen-itals were cleaned with water and soap anddried. Then an insemination pipette connectedto a disposable syringe (the system being ster-ile) was guided into the cervical canal andpushed into the uterus lumen. A sample (5-10 ()

mL) was obtained by slight suction and thesyringe was recapped.

The samples were kept on ice and sent tothe regional veterinary laboratories within 2h, where they were analysed by qualifiedmicrobiologists. The samples were inoculatedon 5 % sheep blood agar as well as onSabouraud dextrose agar (Difco Laboratories,Detroit, Michigan) plates and were incubated at37 OC for 24 h and at 30 &dquo;C for 72 h, respec-tively. The isolates were identified using stan-dard procedures [18].

2.3. Lysosubtilin and other drugs

Lysosubtilin (more precisely lysosubtilinGIOX) is a commercial preparation of lyticenzymes from Bacilllls .;ilbiili.>. SK-52 and was

acquired from the State Joint-Stock Enterprise’Biosinteze’ formerly know as the VilniusExperimental-Industrial Plant of EnzymePreparations (Vilnius, Lithuania). Lysosubtilinwas obtained by cultivating the producer innutrient medium containing carbon and nitro-gen sources as well as mineral salts. This wasfollowed by steps involving processing cul-ture liquid with calcium phosphate gel, filtra-tion, concentration, enzyme precipitation with

acetone and drying the final product. This finalproduct was a powder of light (greyish or yel-lowish) colour with a lytic activity 1 000 000U’g’’ according to Kislukhina [46]. A unit (U)is defined as follows: one unit generates adecrease in turbidity of 0.001 OD min’’ at520-540 nm in a suspension of E.scherichiacoli K12 dried cells (initial OD 0.6) in I .0 mLof 0.004 M phosphate buffer, pH 7.2 at 30 ° C.

Uterosan (which contains chlorampheni-col, nitrofurazone, polymyxin and sulphanil-amide as its active components) and oxytocinwere obtained from Sanitas (Kaunas, Lithua-nia). Neofur, containing neomycin and fura-zolidone, was a product of Sigfarm (Sigulda,Latvia). Nystatin, vitamin AD 3E, fish oil,KMn04 were acquired from other various phar-maceutical enterprises that supply the FSUMinistry of Health Care.

2.4. Statistical evaluation

Results are reported as mean ± SEM. Meanvalues in the text and the tables were comparedusing the Student t test. The Chi-square testand methodological references to biologicalstatistics [701 were used to evaluate the sig-nificant differences between microbiologicalsample examination results and in the numberof cured as well as impregnated animals ineach treatment group. P < 0.05 was consid-ered statistically significant.

3. RESULTS

Microbiological sample testing resultsare given in table I. Only approximatelyone third of the uterine infections werecaused by a single microorganism, whilethe rest were caused by representatives oftwo or three species. The microbial asso-ciations present in the samples (in orderof decreasing frequency) were: E. coli plusstaphylococci, Actinomyces pyogene.s plusstaphylococci and/or streptococci, E. coliplus Proteus spp., A(-tiiiotn,v(-es pyogenesplus fungi/yeasts, and other combinations.In general, E. coli and Actinomyces pyo-genes were the most frequent isolates andwere found taken together in more thanhalf (53.7 %) of the samples. All fungalinfections caused by a single organism

were due to Candida krusei. All Bacillus

spp. sole infections were caused by Bacil-lus cereus. As the cows were divided into

groups randomly, the distribution ofmicrobial isolates from the uterus betweenthe groups according to the species as wellas quantitatively was also random. Thebacteriological and mycological investi-gations did not indicate any significantdifferences between the various experi-mental groups.

Data of lysosubtilin therapeutic dose-determination studies are provided in tableII. The most effective dose was 2 x 1 O6 U

lysosubtilin dissolved in 100 mL of dis-tilled water. When administered at inter-vals of 3-4 days this dose resulted in a100 % clinical recovery rate and a statis-

tically significant decrease in calving-to-conception interval when compared withthe control. The same dose formulated infish oil was not as efficient.

The high efficacy of the optimal dosewas confirmed by the results of the fieldtrials (table Ill). Increases in clinical recov-ery and conception rates were found to bestatistically significant. Results obtainedfrom the Raseiniai region showed that theoil-based lysosubtilin formulation used in

the non-specific fungal post-partumendometritis outbreak was more efficientthan the control drug. But in general, itsefficacy was lower than that of the aque-ous solution.

4. DISCUSSION

In this paper we provide results con-cerning the efficacy of lysosubtilin, apreparation of lytic enzymes created as analternative to common antibiotics, fortreatment of post-partum endometritis incows. The results supplement our in vitroand in vivo observations Ill, 12, 16, 94! [and confirm previous speculations of thegreat potential of this preparation for vet-erinary applications.

As the disease of interest is of microbial

aetiology, identification of causativeagents was our primary task. This wasthen followed by therapeutic dose-deter-mination studies and evaluation of the pro-posed drug formulations in comparisonwith traditionally used medications.

Among the microorganisms isolated inour studies from uterine samples (table nE. coli was most frequent (28.7 % sam-ples) and this correlated with the results

obtained by other authors [87, 96].Another pathogen frequently found in theexamined uterine samples was Actino-myces pyogenes (25.0 %) also known asCorynebacterium pyogenes. Recently avery high incidence (up to 70 %) of Acti-nomvces pyogene.s in the uterine samplesof cows with post-partum endometritis hasbeen described [20, 21, 78].

The number of uterine samples fromwhich fungi/yeasts were isolated was rel-atively small (6.9 %). The causative agentswere mainly yeasts (Candida spp.), buttwo individuals belonging to filamentousfungi (A.spergillu.s spp.) were isolated aswell. Serious consequences of Candida

spp. infection within the bovine reproduc-tive system, including infertility, have beendescribed by several authors [48, 82-85]. ] .

Veterinarians have developed differentapproaches for controlling and prevent-ing post-partum endometritis (some ofthese are discussed in the Introduction).The use of antibiotics remains the methodof choice. The latter are applied by dif-ferent routes (systemic, local or combined)and debates concerning the optimal path-way have continued over the years [7, 31,

36, 80]. When antimicrobials are usedintrauterinarily, the choice of the vehiclemay be of some importance and in thisrespect at least two opinions exist as well.One is for the application of oil-based for-mulations [77, 95 the other for the appli-cation of aqueous solutions [7]. A finalessential feature of antibiotics or similar

substances, i.e. the spectrum of theirantimicrobial activity, needs to be kept inmind. Most antibiotics are effective againstdefinite microbial communities. However,the number of complex polymicrobialinfections (for example, the experimentalresults presented in this paper), includingantibiotic resistant organisms, are tendingto increase.

With all this in mind we started lyso-subtilin efficacy evaluation studies. Withrespect to the application route, theinjectable form is not possible, becauseheterologous proteins, such as lysosub-tilin, are immunogenic. lntrauterinaryapplication can involve both oil-based andaqueous formulations and the results werein favour of the latter (tables II and III).

Our previous in vitro studies on lyso-subtilin showed that the preparation pos-

sesses a very broad antimicrobial spec-trum [ 16, 17]. It lyses both Gram-positiveand Gram-negative bacteria (Bacillus,Corynebacterium, Escherichia, Staphylo-coccus spp.) as well as fungi (Alternaria,Aspergillus, Fusarium, Penicillium, Scle-rotinia, Stemphylium spp.) and yeasts(Candida, Cryptococcus, Han.renula, Sac-charomyces spp.). This feature of lyso-subtilin allowed us to hope that the treat-ment of encountered polymicrobialinfections would be successful.

We began the evaluation of lysosub-tilin as a post-partum endometritis thera-peutic treatment in 1977, shortly after wefirst published [14] our theoretical ideasabout the possibilities of using lysosub-tilin as a veterinary preparation. In the pre-liminary studies the 106 U lysosubtilin oilsuspension was only used for the deter-mination of a clinical cure rate and theresults were similar to those shown in table

II, line 2. This formulation has recentlybeen modified several times. Lysosubtilintherapeutic dose-determination studies,using both oil-based and aqueous formu-lations, are shown in table II. It is quiteclear that a 2 x 106 U lysosubtilin dosedissolved in 100 mL of distilled water was

optimal and the results achieved (100 %clinical recovery and 85 % conception rateof the animals as well as a statistically sig-nificant reduction in calving-to-concep-tion interval when compared with the con-trol) were satisfactory. Undoubtedly, theoxytocin included in the treatment regi-men, gave it additional impact. The bene-ficial effect of this drug in the treatment ofpost-partum endometritis is widelydescribed [24, 29, 80]. Recently the effi-ciency of intrauterine antimicrobial treat-ment in reducing calving-to-conceptioninterval, the latter being one of the mainindicators of reproductive performance[26-28, 39, 52], has been shown by Thur-mond et al. [86].

The results were verified by the positiveoutcome in field trials performed in four

FSU republics (table III). As a conse-quence, the Chief Veterinary MedicineBoard of FSU Ministry of Agriculture in1991 approved the use of lysosubtilin fortreatment of bovine post-partumendometritis throughout the Union.

As for the property rights regarding thepost-partum endometritis treatment meth-ods in cows with the help of lysosubtilin,we have been granted three inventions(patents) by the FSU Invention Commit-tee. The first (priority dated 1977) wasgranted for the already mentioned 106 Uoil-based formulation. The second (prior-ity dated 1986) was granted for the 2! 106U oil-based suspension supplemented withnystatin and vitamins (for details see tableIII Raseiniai region). The third (priorityalso dated 1986) was granted for the 2 %aqueous solution (optimal variant in thepresent study). Because of the policy ofthe FSU Invention Committee, the detailsof these inventions were considered to be

unpublishable. It is only now that we areable to present them to the scientific com-munity. The same situation was true forthe use of lysosubtilin in the prophylaxisand treatment of gastrointestinal disordersin newborn calves only recently publishedby us [I I], although the invention wasgranted with priority in 1981.

In the first part of this research report[ I I we mentioned that about I 500 kg oflysosubtilin had already been used for thetreatment of reproductive system diseasesin cows. Approximately 1 200 kg of thisamount (the exact number cannot bechecked) were used throughout the FSUfor the treatment of post-partumendometritis and the rest (300 kg) wasused for the treatment of superticial lesionsof the udder and teats.

The results of this study (table II) showthat up to three optimal doses, i.e. 2 x LO6U x 3 = 6 x 106 U or 6 g of lysosubtilin areneeded to ensure recovery from post-par-tum endometritis. By dividing the above-mentioned amount (1 200 kg) by 6 g we

obtain the number of cows with post-par-tum endometritis having been treated withlysosubtilin, namely 200 000. If we willadd to this the 675 000 calves that havebeen protected or treated for gastroin-testinal disorders [I I] as well as the 600000 cows having been treated for superfi-cial lesions of the udder and teats (Biz-iulevichius, unpublished), we find thatabout 1.5 million animals have received

lysosubtilin in order to reduce their mor-bidity and mortality.

The selling price of the preparation inJanuary 1997 was 20 $ US per kg (sub-ject to change), thus the cost of the post-partum endometritis treatment by lyso-subtilin is 0.12 $ US per animal. Thereader can judge for himself whether thecost is high or low.

In conclusion, lytic enzyme prepara-tions are prospective antimicrobial drugsand when used to combat animal diseases

they may serve as a good alternative tocommon antibiotics. One can only specu-late, that in conjunction with probiotics,immunomodulators or modern treatment

techniques, such as electromagnetic fieldsand laser therapies, their efficacy may beeven higher, but this remains to be demon-strated experimentally.

ACKNOWLEDGEMENTS

We wish to thank all those, whose enthu-siasm and inextinguishable belief in the futuresuccess of antimicrobial enzymotherapyallowed us to introduce lysosubtilin into vet-erinary practice. In this respect, the efforts ofI.G. Arestov from Byelarus, A. Nummert fromEstonia, L.A. Kalinichenko from Russia, S.Laurusevichius, R. Marushka, H. Medvetskas,A. Tseplis, B. Znaidauskas (all from Lithua-nia) are especially appreciated.

REFERENCES

[1] ] Aknazarov B.K., Humoral factors of localprotection of uterus in cows, Veterinariya(Moscow) 8 (1988) 41-43 (in Russian,English abstract).

[2J ] Anyulis E., Proteolytic enzymes in treatmentof endometritis in cows, LVA mokslo dar-

bai. Veterinariya/Proceedings of the Lithua-nian, Academy of Veterinary Medicine, Vet.Med. 19 (1989) 117-122 (in Lithuanian, Rus-sian abstract).

[31 Anyulis E., Antimicrobial preparations andproteolytic enzymes in treatment ofendometritis in cows, LVA mokslo darbai.

Veterinariya/Proceedings of the LithuanianAcademy of Veterinary Medicine, Vet. Med.20 (1991) 3-7 (in Lihuanian, English andRussian abstrats).

[4] Aurich J.E., Grunert E., Pathogenese undTherapie postpuerperaler Endometritidenbeim Rind, Collegium. Vet. 26 (1996) 28-30.

151 Bach S., Haase H., Zur Bedeutung genitalerInfektionen beim Rind, Monatshefte fur Vet-erin5rmedizin 44 (1989) 86-89.

[6] Barlett P.C., Kirk J.H., Wilke M.A., KaneeneJ.B., Mather E.C., Metritis complex in Michi-gan Holstein-Friesian cattle: incidence,descriptive epidemiology and estimated eco-nomic impact, Prev. Vet. Med. 4 (1986)235-248.

[7! BmTagry T., Therapy of bovine metritis, Irish.Vet. J 49 (1996) 303-305.

[8] Bazhenova N.B., Davydov V.U., StepanovG.S., Treatment of acute post-partumendometritis in cows, Veterinariya (Moscow)2 (1989) 42-43 (in Russian, Englishabstract).

191 Bekana M., Jonsson P., Kindahl H., Intrauter-ine bacterial findings and hormonal profiles inpost-partum cows with normal puerperium,Acta. Vet. Scand. 37 (1996) 251-263.

[10! Bezborodin V.V., Niyatbekov A., Yan-galichev Ya Kh., Treatment of post-partumendometritis, Veterinariya (Moscow) 6 (1984)48-49 (in Russian).

[I I ] Biziulevichius G.A., Arestov I.G., In vivostudies on lysosubtilin. 1. Efficacy for pro-phylaxis and treatment of gastrointestinal dis-orders in newborn calves, Vet. Res. 28 (1997)19-35.

[ 12J Biziulevichius G.A., Arestov LG., Safety oflysosubtilin per os in mice, rabbits, and calves,Vet. Rcs. 28 (1997) 385-395.

[13] Biziulevichius G.-A.S., Kislukhina O.V.,Lytic enzymes in veterinary medicine. Areview, Veterinariya (Moscow) 3 (1988)56-59 (in Russian).

[141 Biziulevichius G.S., Kislukhina O.V.,Avizhienis V.J., On the technology of receiptand specificity of action of the preparationlysosubtilin G10X, Trudy VNII prikladnoienzimologii/Proceedings of the All-UnionResearch Institute of Applied Enzymology’Production and application of microbialenzymatic preparations’ 3. Mokslas, Vilnius(in Russian, English abstract) (1976)105-116.

[15J Biziulevichius G.-A.S., Shablinskas A.J.,Zhukaite V.P., Kislukhina O.V., Lysosub-tilin G 10X, a new antimicrobial agent fortreatment and prophylaxis. Physicochemicalproperties, Antibiotiki i meditsinskayabiotekhnologiya/Antibiotics and MedicalBiotechnology 32 (1987) 331-333 (in Rus-sian, English abstract).

! 16! Biziulevichius G.-A.S., Shablinskas A.J.,Zhukaite V.P., Kislukhina O.V., Antibacterialspectrum of lysosubtilin G10X, Antibiotikii khimioterapiya/Antibiotics and Chemother-apy 34 (1989) 579-581 (in Russian, Englishabstract).

! 17J J Biziulevichius G.A., Zhukaite W., Kislukhina0., Lysosubtilin - a broad-spectrum actionpreparation of lytic enzymes for veterinaryuse: in vitro studies on activators and

inhibitors, complex structure and antifungalaction, Biomed. Lett. 52 (1995) 43-57.

[ 18J ] Carter G.R., Cole J.R.Jr., Diagnostic Proce-dures in Veterinary Bacteriology and Mycol-ogy ( 1990), 5th ed, Academic Press, NewYork.

[ 19] Chaffaux S., Lakhdissi H., Thibier M., Etude6pid6miologique et clinique des endometritespost-pucrp6rales chez les vaches laitières,Rccl. M6d. V6t. Ec Alfort 167 (1991)349-358.

[20] Cohen R.O., Bernstein M., Ziv G., Isolationand antimicrobial susceptibility of A. Pyo-genes recovered from the uterus of dairy cowswith retained fetal membranes and post par-turient endometritis, Theriogenology 43(1995) 1389-1397.

[21 1 ] Cohen R.O., Colodner R., Ziv G., Keness J.,Isolation and antimicrobial susceptibility ofobligate anaerobic bacteria recovered fromthe uteri of dairy cows with retained fetalmembranes and post parturient endometritis,J. Vet. Med. B 43 (1996) 193-199.

[22] Davies D.A.L., Method for treating fungalinfections using cell lytic enzymes, US patent4062941, 1979.

[23] ] Davies D.A.L., Pope A.M.S., Antimicrobialcomposition comprising lytic enzymes fromphysarum and an antimycotic agent, USpatent 4144327, 1979.

1241 ] De Kruif, Postpariale Endometritis bcim Rind,Prakt. Tierarzt. 12 (1994), 1071-1078.

125] ] Demidova L.D., Yurkov V.M., Balkovoi LL,Inozemtsev V.P., Cherkasov A.S., SvirinV.N., Application of laser veterinary appa-ratus &dquo;Vega-MV&dquo; for mastitis in cows, Vet-erinariya (Moscow) 5 (1996) 9-12 (in Rus-sian).

[26] Dhaliwal D.S., Murray R.D., Dobson H.,Effects of milk yield, and calving to first ser-vice interval, in determining herd fertility in

dairy cows, Anim. Reprod. Sci. 41 (1996)109-I17.

[27! ] Diskin M.G., Factors affecting conceptionrate in cows, Irish. Vet. J. 49 (1996) 245-251. 1.

[28] Diskin M.G., Regulation of post-partum inter-val in cattle, Irish Vet. J. 50 (1997) 238-247.

[29] Eiler H., Hopkins F.M., Armstrong-BackusC.S., Lyke W.A., Uterotonic effect ofprostaglandin FZa and oxytocin on the postpartum cow, Am. J. Vet. Res. 45 (1984)1011-1014.

[30] Esslemont R.J., Kossaibati M.A., Culling in50 dairy herds in England, Vet. Rec. 140(1997)36-39.

131 ! 1 ] Fraser C.M. (ed), The Merck Veterinary Man-ual. A Handbook of Diagnosis, Therapy andDisease Prevention and Control for the Vet-

crinarian, 7th ed., Merck and Co Inc, Rah-way, New Jersey 1991, pp. 692-693.

[321 Gavrish V.G., Rodin V.P., Semenov S.V.,Hyslerofur for therapy of cow endometritis,Veterinariya (Moscow) 5 (1996) 40-43 (inRussian).

[331 Gilbert R.O., Bovine endometritis: the bur-den of proof, Cornell Vet. 82 (1992) 1 1-13.

[34J ] Gonchar A.M., Semenov B.S., Perspectives ofapplication of preparations of immobilizedbacterial proteases, Veterinariya (Moscow)7 ( 1988) 57-59 (in Russian).

[35) ] Gonchar A.M., Shelyugina Z.G., BogdanovaL.A., Salganik R.L, Avdeenko V.S., Appli-cation of immobilized proteases for treatmentof purulent processes, Veterinariya (Moscow)4 ( 1989) 52 55 (in Russian, English abstract).

1361 ] Grigorieva T.E., Treatment and Prophylaxisof Endometritis in Cows, Rosseljkhozizdat,Moscow (in Russian) 1988.

[37] Grubliauskas L., Znaidauskas B., Antibacte-rial effect of lytic enzyme preparation lyso-subtilin G10X and its influence on activityof antibiotics, Trudy LVA i LNIIV/Proceed-ings of the Lithuanian Academy of Veteri-nary Medicine and the Lithuanian ResearchInstitute of Veterinary Medicine 18 ( 1987)62-65 (in Russian).

[38 ! ] Gudimova T.E., Genital diseases and masti-tis, Veterinariya (Moscow) 8 (1986) 62 (inRussian).

[39! ] Harman J.L., Grohn Y.T., Erb H.N., CasellaG., Event-time analysis of the effect of 60-day milk production on the parturition-to-conception interval in dairy cows, Am. J. Vet.Res. 57 ( 1996) 634-639.

[40] Hussain A.M., Daniel R.C.W., Boyle 0.,Post-partum uterine flora following normaland abnormal puerperium in cows, Theri-ogenology 34 (1990) 291-302.

[41 1 ] lliynskii Ye V., Troshin A.N., Nazarov M.V.,New medicines of plant origin for treatment

of uteri, Veterinariya (Moscow) 8 (1996)56-58 (in Russian, English abstract).

[421 Inozemtsev V.P., Balkovoi 1.1., Sochnev V.V.,Samodelkin A.G., UHF electromagnetic fieldfor endometritis therapy in cows, Veterinariya(Moscow) 11 (1996) 29-31 (in Russian,English abstract).

[43] Inozemtsev V.P., Balkovoi LL, Krylova T.V.,Siniaev V.A., Kuklin A.D., Kosorlukova Z.Ya, Miakin N.A., Batrakova Ye A., NikulinD.M., Pakhomov A.P., Gusev V.A.,Doszhanov Yu D., Experience of using laserapparatus STP-3 in veterinary practice, Vet-erinariya (Moscow) 5 ( 1997) 32-36 (in Rus-sian).

[44] Ivanovskii A.A., New probiotic bactocel-lolactin for treatment of various pathologiesin animals, Veterinariya (Moscow) 11 (1996)34-35 (in Russian, English abstract).

[45] Khiljkevich N.M., Timoshenko O.V., Dzhu-maniazov K. Kh., Khiljkevich S.N., Use ofanitimicrobial preparations in combinationwith oxygen for mastitis and some uterus dis-

eases in cows, Veterinariya (Moscow) 3(1994) 37-39 (in Russian, English abstract).

1461 Kislukhina O.V., Determination of lytic activ-ity of lysosubtilin by action towardsE.rcherichia coli cells, Mikrobiologicheskayapromyshlennostj/Mikrobiological industry141 (1976) 43-45 (in Russian).

[47] Kondrakhin I.P., Perspectives of applicationof enzymes in veterinary practice, Veteri-nariya (Moscow) 8 (1990) 61-64 (in Rus-sian, English abstract).

[48] Kremlev E.P., Prophylaxis of infertility incows, Veterinariya (Moscow) 3 (1982) 46-48(in Russian).

[49] Kremlev E.P., Banakova L.A., TkachenkoYu G., Iodine containing preparations fortherapy of endometritis, Veterinariya(Moscow) 1 ( 1985) 49-51 (in Russian).

[50] Kuklin A.D., Kosorlukova Z. Ya, Treatmentof cows with endometritis, Veterinariya(Moscow) I (1987) 47-48 (in Russian).

[51 ! Lacombe J.-F.P.L., Nouveau medicamentv6t6rinaire destine au traitment des mammitesinfectieuses des bovins et des caprins a basede polymixine, d’eritromycine et de

lysozyme, French patent 2528703, 1983.[52] Mac Gregor R.G., Evaluation of calving date

and calving interval as measures of repro-ductive efficiency in beef cows, J. S. Afr.Vet. Assoc. 66 (1995) 235-238.

[53] Malashenko N.P., Furazolidone sticks ’Ultra’for treatment of inflammatory processes inuterus, Veterinariya (Moscow) 3 (1997)44-45 (in Russian, English abstract).

[54] Markusfeld O., Factors responsible forpost-parturient metritis in dairy cattle, Vet.Rec. 1 14 (1984) 539-542.

[55! ] Marushka R., Treatment of cow uterus withenzyme and antimicrobial preparations, LVAmokslo darbai. Veterinariya/Proceedings ofthe Lithuanian Academy of VeterinaryMedicine, Vet. Med. 19 (1989) 153-156 (inLithuanian, Russian abstract).

[561 Miroliubov M.G., Borskov A.A., Vagina anduterus diseases in cows, Veterinariya(Moscow) 7 ( 1984) 51-52 (in Russian).

157] Money I.A.L., Efficacy of pefloxacin treat-ment for endometritis in cow, Indian Vet. J.73 ( 1996) 1072-1073.

!58J ] Murtazin B.F., Pulatov G.S., On etiology ofendometritis in cows, Veterinariya (Moscow)4 ( 1995) 41!44 (in Russian, English abstract).

[59! Nakao T., Moriyoshi M., Kawata K., Effect ofpost-partum intrauterine treatment with 2 per-cent polyvinylpyrrolidone-iodine solution onreproductive efficiency in cows, Theri-ogenology 30 (1988) 1033-1043.

[60] ] Nezhdanov A.G., Koniaev M.T., Treatmentof cows with endometritis and subinvolutionof the uterus, Veterinariya (Moscow) I ( 1982)45-46 (in Russian). ).

[61) 1 ] Nikolaevskii LL, Lysozyme in treatment ofgynaecological diseases of agricultural ani-mals, Doct Diss Vet Sci., Moscow Veteri-nary Academy, Moscow (in Russian). ).

[62] Noakes D.E., Till D., Smith R.G., Bovineuterine flora post partum: a comparison ofswabbing and biopsy, Vet. Rec. 124 (1989)563-564.

[63] Opekunov K.A., Ivanovskii A.A., VeprevaN.S., Dry biosan for treatment of endometri-tis in cows, Veterinariya (Moscow) 2 (1994)39-40 (in Russian, English abstract).

[641 Ott R.S., Gustafsson R.K., Use of

prostaglandins for the treatment of bovinepyometra and post-partum infections, Areview. Compendium on Continuing Educa-tion for the Practicing Veterinarian 3 (1981)184-188.

[ 65! ] Plakhotin M.V., Gafurov D.S., Filippov Yu I.,Enzymotherapy of purulent-necrotic pro-cesses, Veterinariya (Moscow) 11 (1985), ),58-59 (in Russian).

[661 Prescott J.F., Baggot J.D., AntimicrobialTherapy in Veterinary Medicine, 2nd ed.,Iowa State University Press, Ames, Iowa,1993.

[67! ] Proctor V.A., Cunningham F.E., The chem-istry of lysozyme and its use as a food preser-vative and a pharmaceutical, Crit. Rev. Food.Sci. Nutr. 26 (1988) 359-395.

[68] Rabochev V.K., Rodina A.I., Serov V.M.,Volkov V.L, Alimbekov M., Akbaev A.,Getas for therapy of genital organ diseases,Veterinariya (Moscow) 3 (1983) 50-51 (inRussian).

[69] Reynolds J.E.F. (ed), Martindale the ExtraPharmacopoeia, 28th ed., PharmaceuticalPress, London, 1982, pp. 651-652.

[70J J Rokitskii P.F., Biological Statistics Vysheishashkola , Minsk (in Russian) 1973.

[71 ] Rubanets L.N., Desoxyfur for therapy of post-partum endometritis, Veterinariya (Moscow)6 (1986) 51-53 (in Russian).

[72] Sava G., Pharmacological aspects and thera-peutic applications of lysozymes, in: JollesP. (Ed.), Lysozymes: Model Enzymes in Bio-chemistry and Biology, Birkhauser Verlag,Basel, 1996, pp. 433-449.

[73j ] Selivanova A.S., Morozov A.P., KovalevV.F., Avsjukevich V.S., Antibiotics for prac-tice, Veterinariya (Moscow) 4 (1984) 53-56(in Russian). ).

[74] ] Shatalov V.F., Nekhaev E.E., KhrapkovskayaL.P., Susceptibility of bacteria from genitalorgans of animals to preparations, Veteri-nariya (Moscow) 9 (1985) 46-47 (in Rus-sian). ).

[75] Shelyugina Z.G., Avdeenko V.S., GoncharA.M., Salganik R.I., Pankov B.G., GoncharovV.P., Stradina Yu N., Immobilized proteolyticenzymes for post-partum purulent-catarrhalendometritis in cows, Veterinariya (Moscow)6 (1989) 41-42 (in Russian, English abstract).

[76] Sherenas A.J., Struggle Against Infertility inCows, Komi Knizhnoe lzdateljstvo, Syk-tyvkar (in Russian) 1984.

[77] Sherenas A.J., Treatment and Prophylaxis ofGynaecological Diseases in Cows, KomiKnizhnoe Izdateljstvo, Syktyvkar (in Rus-sian) 1989.

[78] Slimane N., Ahmadi C., Ouali F., Kachti M.,Thibier M., Analyses épidémiologique et clin-ique des endométrites post-puerpérales chezles bovins laitiers en Tunisie, Recl. M6d. Vet.Ec Alfort 170 (1994) 823-832.

[79J ] Slipchenko S.N., Timchenko L.D., Lactobrilfor therapy of acute purulent-catarrhalendometritis in cows, Veterinariya (Moscow)11 (1994) 34-36 (in Russian). ).

[80] Studentsov A.P., Shipilov V.S., SubbotinaL.G., Preobrazhenskii O.N., VeterinaryObstetrics and Gynaecology, 6th ed., Agro-promizdat, Moscow (in Russian) 1986.

!81] 1 ] Sundlof S.F., Kaneene J.B., Miller R.A.,National survey on veterinarian-initiated druguse in lactating dairy cows, J. Am. Vet. Med.Assoc. 207 (1995) 347-352.

[82] Sutka P., Candida guillermondii var-guiller-mondii infection in cows slaughtered becauseof infertility and udder inflammation, Acta.Vet. (Belgrade) 5-6 (1983) 287-289.

[83] Tainturier D., Fieni F., Bruyas J.F., Beloeil V.,Lyoen A., Candidose et troubles de la repro-duction chez la vache. 1. Premiers essais d’untraitement de desensibilisation en fin de ges-

tation, Rev. Med. Vet. (Toulouse) 144 (1993)411-414.

[84] Tainturier D., Chatagnon G., Fieni F., BruyasJ.F., Batut L, Candidose et troubles de lareproduction chez la vache: enquêteserologique, Rev. Med. Vet. (Toulouse) 146(1995)35-40.

[85] Tainturier D., Fieni F., Bruyas J.F., Batut I.,Candidose et troubles de la reproduction chezla vache. 3. Essais d’un traitement de déscn-sibilisation chez les vaches inf6condes àchaleurs régulières, Rev. Méd. Vet.

(Toulouse) 146 (1995) 497-500.186] ] Thurmond M.C., Jameson C.M., Picanso J.P.,

Effect of intrauterine antimicrobial treatmentin reducing calving-to-conception interval incows with endometritis, J. Am. Vet. Med.Assoc. 203 (1993) 1576-1578.

[87J ] Trishkina E.T., Galushko L. Kh., Microbialfactor in etiology of post-partum endometri-tis in cows, Veterinariya (Moscow) 1 I (1985)52-53 (in Russian).

[88] Vallet A., Carteau M., Salmon A., ChatelinY., tpid6miologie des endométrites desvaches laitieres, Recl. Med. Vet. Ec Alfort163 (1987) 189-194.

[89] Veremei E.L, Magnetotherapy in clinical vet-erinary medicine, Veterinariya (Moscow) 5(1996) 45-48 (in Russian, English abstract).

[90] Voskoboinik V.F., Treatment of post-partumendometritis in cows, Veterinariya (Moscow)5 (1987) 54-56 (in Russian).

[91 ! [ Yarushin A.D., Prophylaxis of reproductionorgan diseases in cows, Veterinariya(Moscow) 3 (1992) 41-43 (in Russian,English abstract).

[92] Youugquist R.S., Fertility and Infertility inVeterinary Practice, Baliere Tindall, London,1988.

[93] Znaidauskas B., Marushka R., MalakauskasV., Laurusevichius S., Toxicity assessmentof enzyme preparation lysosubtilin G10X,LVA mokslo darbai. Veterinariya/Proceed-ings of the Lithuanian Academy of Veteri-nary Medicine, Vet. Med. 19 (1989) 184-187(in Lithuanian, Russian abstract).

[94 [ Znaidauskas B., Marushka R., BiziulevichiusG., Laurusevichius S., Application of enzymepreparation lysosubtilin G10X for treatmentof endometritis in cows, LVA ir LVI mok-slo darbai. Veterinariya/Proceedings of theLithuanian Academy of Veterinary Medicineand the Lithuanian Institute of VeterinaryMedicine, Vet. Med. 21 (1994) 85-88 (inLithuanian, English and Russian abstracts).

[95] Zyubin LN., Bovine Metritis, Agropromiz-dat, Moscow (in Russian) 1988.

[96J Zyubin LN., Zyubina M.F., Microflora ofgenitals in cows, Veterinariya (Moscow) 9(1982) 50-52 (in Russian).