VETERINARSKI ARHIV 68 (2), 59-62, 1998

ISSN 0372-5480
Printed in Croatia

Listeria ivanovii from a repeat breeding buffalo

Parag Nigam1, Ramesh C. Katoch2*, Munish K. Batta2,
and Subhash Verma2

1Remount Veterinary Corps, Indian Army, Meerut Cantt., India

2Department of Veterinary Microbiology and Immunology,
College of Veterinary and Animal Sciences, Palampur, India

* Contact address:
Prof. Dr. Ramesh C. Katoch,
Department of Veterinary Microbiology & Immunology, College of Veterinary & Animal Sciences, HPKV, Palampur-176062, H.P. India,
Phone: 91 1894 30304; Fax: 91 1894 30511

NIGAM, P., R. C. KATOCH, M. K. BATTA, S. VERMA: Listeria ivanovii from a repeat breeding buffalo. Vet. arhiv 68, 59-62, 1998.


Thirty-one deep vaginal swabs from 19 normal cyclic repeaters and 12 endometritis cases of buffaloes were screened for isolation of Listeria species. The solitary isolate from a repeat breeding buffalo was identified on the basis of cultural, morphological and physiological behaviour as Listeria ivanovii. Its pathogenicity was tested in mice and 10-day-old embryonated chicken eggs. The main macrolesions in mice were swollen liver with necrotic foci, splenomegaly and slight congestion of lungs. Histopathomorphologically, similar changes were confirmed and the organisms (L. ivanovii) were demonstrated by Brown and Bren staining. The inoculated chicken embryos discerned conspicuous thickening of chorio-allantoic membrane (CAM). The isolate was also serologically identified as L. ivanovii on the basis of agar diffusion test (AGPT), counter immunoelectrophoresis test (CIET) and dot-enzyme linked immunosorbent assay (dot-ELISA). This is the first report of its association with repeat breeding syndrome in a buffalo.

Key words: Listeria ivanovii, buffaloes, repeat breeding, Bubalus bubalis, India


Reproductive disorders with infectious aetiology in female genitalia of buffaloes (Bubalus bubalis) have been considered as one of the most feared financial setback having vicious cycles especially in herds. The role of specific bacterial infections leading to reproductive problems viz. Brucella spp., Campylobacter spp., Leptospira serovars, Chlamydia psittaci, Coxiella burnetii, Mycoplasma bovigenitalium, Listeria monocytogenes has been extensively reported in the literature by RADOSTITS et al. (1994).

L. monocytogenes is considered as a potentially pathogenic species that has diverse distribution in nature. It's role in causation of meningo-encephalitis, septicaemia, endocarditis, abortion, endometritis, cervicitis, diarrhoea, mastitis, keratoconjunctivitis, local purulent lesions etc. has been established beyond doubt (GITTER, 1985). In 1957, Ivanov isolated a phenotypically different strain from aborted ovine foetuses in Bulgaria. The isolate was eventually classified as L. monocytogenes serotype 5 as a separate species, L. ivanovii (BUCHANAN and GIBBONS, 1974).

Listeriosis is also a disease of zoonotic importance. In older children and adults involvement of central nervous system, pneumonia, endocarditis, localized abscesses, skin lesions or conjunctivitis have been reported as the commonest complications (ANONYMOUS, 1991). The present study was contemplated to find the prevalence of Listeria spp. leading to reproductive failure in buffaloes.

Materials and methods

In all 31 deep vaginal swabs accruing from 19 normally cyclic repeaters and 12 endometritis cases in buffaloes (Bubalus bubalis) were aseptically collected. The swabs were transferred into about 5 ml of Listeria enrichment broth at 37 C for 24 h each (DONNELLY and BAIGENT, 1986) also known as University Vermant Medium (UVM) as well as both on potassium tellurite. Suspected Listeria colonies based on differential characteristic were restreaked as well as were subjected to Gram's staining. The isolate exhibited cultural, morphological and physiological behavior of L. ivanovii as described by CARTER (1995). The pathogenicity of isolate was tested in mice and ten day old embryonated chicken eggs. Three mice infected with 0.4 ml of 18 h broth culture intraperitoneally having 106 colony forming units c.f.u./ml and three control mice also received similar volume of normal saline intraperitoneally. All sluggish mice were sacrificed on 13 day post-infection (DPI) for pathological studies as well as reisolation of L. ivanovii from the pooled visceral organs of sacrificed mice. The visceral organs showing gross lesions were preserved in 10% neutral buffered formalin for histopathomorphological alterations. Ten day old embryonated chicken eggs were infected with 0.1 ml of 10-5 dilutions of 18 h broth culture of L. ivanovii via the chorioallantoic membrane (CAM) route.

The drug sensitivity pattern of the isolated strain was tested against 12 chemotherapeutics.

The standard strain of L. ivanovii (ATCC 10119) procured from J. M. Rocourt, Central Nationalle de Reference des Listeria, Institut Pasteur, 28, Rue Du Dr Roux 75724, Paris, Cedex for raising antiserum. The antiserum was raised with cold extracted antigen in two 3-4 months old New Zealand white rabbits following the protocol of JAIN and CHANDIRAMANI (1978).

Agar gel precipitation test (AGPT) was performed following the technique of JAIN and CHANDIRAMANI (1978) whereas counter-immunoelectrophoresis test (CIET) and dot Enzyme linked immunosorbent (dot ELISA) were performed as per methods of KORDE (1992) and ANONYMOUS (1994), respectively.


The isolation of L. ivanovii was successful from one repeat breeding buffalo that was not conceiving for the last one and half years. The identification of the isolate was established as L. ivanovii on the basis of cultural, morphological, physiological and serological behavior. The isolate was catalase positive, motile at 25 C and b haemolytic on sheep blood agar. The isolate metabolized glucose, maltose, xylose, salicin, lactose and trehalose but not rhamnose and mannitol but hydrolyzed aesculin. The isolate manifested positive reaction for methyl red, Voges Proskauer test and negative reaction for indole production, nitrate reduction, urea hydrolysis and gelatin liquefaction. The isolate was sensitive to gentamicin, chloramphenicol, penicillin G, ampicillin, chlortetracycline and moderately sensitive to ciprofloxacin, lomofloxacin, cotrimazine, tetracycline, spiramicyn and resistant to nalidixic acid and furaxone.

Serologically the isolate was identified as L. ivanovii on the basis of AGPT, CIET and dot ELISA.

The macrolesions in mice comprised of swollen liver with necrotic foci, splenomegaly and slight congestion of lungs. Histopathomorphologically, liver displayed congestion, degeneration of hepatic cords, neutrophil and mononuclear cell infiltration and the organisms (L. ivanovii) were demonstrable by Brown and Brenn staining, while spleen manifested slight congestion, depletion of lymphoid cells and red pulp degeneration, with slight mononuclear cell infiltration. The heart, lungs and kidneys revealed mild to moderate interstitial congestion. The focal areas of tubular degeneration were evident in renal tissues. The inoculated chicken embryos displayed conspicuous thickening of CAM.


A careful scrutiny of the literature reveals association of L. ivanovii with repeat breeding syndrome in a buffalo for the first time. Earlier, SING et al. (1995) reported the isolation of L. denitrificans from nondescript repeat breeding buffaloes. ALEXANDER et al. (1992) has recommended inclusion of L. ivanovii as a potential cause of abortion in bovines and encountered its prevalence to the extent of 1.64%. IVANOV and MASALASKI (1979) cited that L. ivanovii infection accounted for 10% of animal listeriosis in Bulgaria. This study has, of course,  been conducted on small available number of samples but it acts as a pointer for future detailed investigation into its role as a causative agent of genital tract infections in buffaloes.

Listeriosis can occur in humans in otherwise healthy adults and children. However, the most commonly affected population has been reported to be pregnant women, neonated, elderly people, cancer patients, those with cirrhosis, kidney dialysis patients, people with AIDS and persons who are immuno-suppressed through medication or illness (ANONYMOUS, 1988).

Thanks are due to the Dean, College of Veterinary and Animal Sciences, HPKV, for providing the necessary facilities, as well as to the Indian Council of Agricultural Research, New Delhi for the grant of a Junior fellowship to the first named author.


ALEXANDER, A. V., R. L. WALKER, B. J. JOHNSON, B. R. CHARLTON, L. W. WOODS (1992): Bovine abortions attributable to Listeria ivanovii: four cases (1988-1990). J. Amer. Vet. Med. Assoc. 200, 711-714.

ANONYMOUS (1988): Food borne listeriosis (update). Bull. WHO. 66, 421-428.

ANONYMOUS (1991): Listeria monocytogenes. Recommendation by the National Advisory Committe on microbiological criteria for foods. Inst. J. Food Microbiol. 14, 185.

ANONYMOUS (1994): Epidemiology and immunodiagnosis of Chlamydia psittaci infections in sheep and goats in Himachal Pradesh. Department of Veterinary Microbiology and Immunology, College of Veterinary and Animal Sciences, H.P.K.V. Palampur, submitted to ICAR, New Delhi, India.

BUCHANAN, R. E., N. GIBBONS (1974): Bergey's manual of determinative bacteriology. 8th ed. Williams and Wilkins Co. Baltimore.

CARTER, G. R. (1995): Diagnostic procedures in veterinary bacteriology and mycology. 6th ed. (Carter, G. R., J. R. Cole, Jr., Eds.), pp. 457-467. Academic Press Inc. California.

DONNELLY, C. W., G. J. BAIGENT (1986): Methods for flow cytometric detection of Listeria monocytogenes in milk. Appl. Envirol. Microbiol. 52, 689-695.

GITTER, M. (1985): Listeriosis in farm animals in Great Britain. In: Isolation and identification of micro-organisms of medical and veterinary importance (C. H. Collins, J. M. Grangel, Eds.). pp. 191-200. Society for Applied Bacteriology Technical Series. No. 21. Academic Press. London, U. K.

IVANOV, L., N. MASALASKI (1979): Listeriosis in Bulgaria. In: Problems of listeriosis (Ivanov, I., Ed.). pp. 254-263. National Agroindustrial Union. Sofia.

JAIN, N. C., N. K. CHANDIRAMANI (1978): Studies on listeria antigens by agar gel double diffusion tests. Indian J. Anim. Sci. 48, 167-172.

KORDE, N. M. (1992): Studies on seroprevalence of Listeria monocytogenes infection in man and animals. M. V. Sc. Thesis submitted to Deemed University, IVRI. Izatnagar, U. P.

RADOSTITS, O. M., D. C. BLOOD, C. C. GAY (1994): Veterinary medicine, 8th ed. ELBS Bailliere Tindall. London.

SINGH, N. P., V. K. CHATURVEDI, D. P. SINGH (1995): Possible etiological role of Listeria denitrificans in repeat breeding in bovines. Indian J. Comp. Microbiol. Immunol. Infect. Dis. 16, 156-157.

Received: 12 December 1997
Accepted: 12 January 1998

NIGAM, P., R. C. KATOCH, M. K. BATTA, S. VERMA: Listeria ivanovii izdvojena iz bivolice koja se preganjala. Vet. arhiv 68, 59-62, 1998.


Duboko iz rodnice uzet je 31 obrisak od 19 bivolica s normalnim spolnim ciklusom i od 12 s endometritisom. Obrisci su bili pretrazeni na prisutnost vrsta iz roda Listeria. Jedan izolat izdvojen iz bivolice koja se preganjala identificiran je na osnovi kulturalnih, morfoloskih i fizioloskih osobina kao Listeria ivanovii. Njezina patogenost je provjerena na misevima i desetdnevnim kokosjim zametcima. Glavne makroskopske promjene u miseva bile su otecena jetra s nekroticnim zaristima, povecana slezena i blaga punokrvnost u plucima. Navedene promjene potvrdene su patohistoloski, a bakterija (L. ivanovii) je dokazana Brownovim i Brenovim bojenjem. U inokuliranim kokosjim zametcima uocena su napadna zadebljanja korioalantoisne opne. Izolat je bio i seroloski identificiran kao L. ivanovii reakcijom imunodifuzije u gelu, imunoelekroforezom i dot-imunoenzimnim testom. Ovo je prvo izvjesce o povezanosti te bakterije sa sindromom preganjanja u bivola.

Kljucne rijeci: Listeria ivanovii, bivol, Bubalus bubalis, preganjanje, Indija