Indian Journal of Experimental Biology Vol. 40, March 2002, pp. 296-303

Evaluation of guinea pig model for experimental Salmonella serovar Abortusequi infection in reference to infertility

B R Singh, Javed Alarn, D Hansda, J C Verma, V P Singh & M P Yadav

National Salmonella Centre (Vet), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar 243 122, India Received 1 August 2001; revised 12 December 2001

The present study conclusively revealed the role for Salmonella enterica subspecies enterica serovar Abortusequi in conception failure. None of the 1 2 guinea pigs conceived when orally exposed to sublethal dose of the pathogen during breeding, while 66.67% of animals in control group were found pregnant during same period of observation under similar conditions. Salmonella carrier animals also had drastic reduction in conception rate ( 16.67%). During mid pregnancy, S. Abortusequi exposure to guinea pigs through intravaginal, intramuscular and subcutaneous routes induced fetal death fol­lowed by resorption. While 2 out of 6 orally inoculated and 3 out of 6 intraperitonially inoculated guinea pigs aborted, in rest of the animals fetal death was followed by meceration and resorption. It was interesting to note that S. Abortusequi could not persist longer than a week in males while in pregnant females it could be detected for >10 weeks after inoculation. In late pregnancy, most of the exposed animals aborted and non aborting animals though had normal parturition, survival rate of their babies was nearly zero in comparison to the control group. The study revealed role for S. Abortusequi in impair­ing conception, abortion, early fetal deaths, fetal meceration and resorption. Further studies are required to identify factors responsible for increased susceptibility of females particularly during pregnancy.

Infertility is a complex syndrome either caused by defects in anatomy, disturbed physiology or pathology of the genital tract. Among the pathological causes, infectious diseases are important. Of these, bacterial infections viz. listeriosis, brucellosis and campylobac­teriosis are quite frequent and well studied I . Salmo­nellosis, specially caused by host adapted serovars has often been reported to be associated with infertility. However, firm evidences are lacking to prove the role for Salmonella in infertility in absence of evidences to reveal affinity of this pathogen to tissues of the genital tract. Although S. enterica subspecies enterica se­rovar Abortusequi (written as S. Abortusequi) 1 has been shown to affect the gravid uterus of equines, it has rarely b�en shown to be associated with non­gravid uteri2•3 . Some studies have indirectly revealed that salmonellosis due to wide host range serovars viz. S. typhimurium may also be associated with infertil­ity4.5. Therefore, to determine the role for salmonello­sis in infertility, this study was conducted with S. Abortusequi, an equine host adapted Salmonella, in guinea pigs, used earlier as an experimental model6 for S. Abortusequi induced abortions.

Materials and Methods Bacterial strain-Reference S. Abortusequi strain

(E 1 56), pathogenic to mouse (MLD50 _ 107 CFU) and

resistant to nalidixic acid (MIC-50 Ilg mrl), main­tained at the National Salmonella Centre (Vet) was used throughout the study.

Animal model-Adult (5 to 6 months old, -500g in weight) male and female guinea pigs were procured from the Laboratory Animal Resource (LAR) Section of the Institute, having no detectable (with ELISA and micro-agglutination test) S. Abortusequi antibodies, were used in the experiment. All the animals were maintained in pans (75 x 1 25 x 35 cm) on sterilised rice husk padding, in groups of 6-7, on sufficient green fodder and concentrate ration recommended for breeding animals by LAR Section. All the animals were screened for faecal excretion of Salmonella for 7 dIlys continuously by processing faecal swabs for selective enrichment and plating (described later) . Salmonella isolates were characterized through mor­phological, cultural, biochemical and serological characteristics7• Only Salmonella free animals were included in the study.

Breeding -Six adult females were kept with an adult male for 1 7 days and thereafter male was sepa­rated. Females were palpated for pregnancy at 40th

and 55th day of breeding. Non-pregnant animals were sacrificed to collect visceral organs (liver, spleen, gall bladder), contents from intestine (ileum-jejunum) and uterus for isolation of Salmonella7•

'- \ ..

- �

SINGH et al. : EVALUATION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELlA SEROVAR 29'7

Salmonella carriers-Forty-two adult female guinea pigs were orally inoculated with 4.3 x 107 CFU of S. Abortusequi (E- 1 56) overnight broth culture and were monitored for Salmonella excretion as above for 75 days of inoculation by taking faecal swabs, twice weekly . Animals excreting Salmonella, even after a month of inoculation but having no sign of salmonel­losis were designated as carriers.

Experimental plan for breeding efficiency­Three groups of adult female guinea pigs consisting of 12 Salmonella free animals in group 1 and 2 each and 12 Salmonella- carrier animals in group 3 were set to breed as above. Group 1 of Salmonella free animals was exposed to oral infection of S. Abortuse­qui (-4.3 x 107 CFU per animal) on the first day of breeding while the other group 2 was kept as control.

Effect of Salmonella on pregnant guinea pigs­To assess the effect of S. Abortusequi infection on pregnant guinea pigs, groups of 6 pregnant guinea pigs (-40 days and -55 days), procured from LAR Section, were exposed to S. Abortusequi (E- 156) through oral (4.3 x 109CFU per animal), intra vaginal (4.3 x 109 CFU per animal), intra muscular (4.2 x 107

CFU per animal), intra peritoneal (4.2 x 107 CFU per animal) and subcutaneous (4.2 x 107 CFU per animal) routes. Control group was kept separately without any exposure to Salmonella. Each group was observed for 75 days for parturition, resorption of fetus, live births (litter size), faecal Salmonella excretion and survival of baby guinea pigs up to one month of age.

Three aborted animals were monitored for faecal and vaginal excretion of Salmonella by processing rectal and vaginal swabs, respectively, taken on

. 0, 1 ,3,7 , 15,21 ,30 and 45th day of abortion. Salmonella carriage in male guinea pigs-Male

animals inoculated with S. Abortusequi through oral (4.3 x 109 CFU per animal), subcutaneous (4.2 x 1 07

CFU per animal) or intra muscular (4.2 x 107 CFU per animal) routes were also monitored for faecal excre­tion of the pathogen up to 2 1 days of infection and then sacrificed to isolate Salmonella from visceral organs.

Salmonella carriage in female guinea pigs-To assess the carriage and excretion of S. Abortusequi by adult female guinea pigs, groups of 1 2 adult female guinea pigs were exposed to S. Abortusequi (E- 1 56) through oral (4.3 x. 109CFU per animal), intra vaginal (4.3 x 1 09 CFU per animal), intra muscular (4.2 x 107

CFU per animal), intra peritoneal (4.2 x 1 07 CFU per animal) and subcutaneous (4.2 x 1 07 CFU per animal) routes while control group was kept separately with­out any exposure to Salmonella. Each group was ob­served for 75 days for faecal excretion and survival of Salmonella in liver, spleen, gall b1addt!r, kidneys and uteri.

Isolation of Salmonella-To isolate Salmonella from faeces and different visceral organs of experimen­tal animals, faecal swabs/tissue homogenates were enriched in Rappaport Vassiliadis (RV) broth contain­ing nalidixic acid (30 ).l.g ml-I ) and incubated at 42°C for 1 8 hr and then plated on to Enteric hektoen agar (Hi-Media, Mumbai), containing nalidixic acid (30 ).l.g mn) plates and suspected isolated colonies were characterised through biochemical and serological methods7 •

ELISA for Salmonella antibodies-The test was conducted as described by Hudson and Hal using sonicated antigen (200 ).l.g rnI-l) prepared from over­night culture of S. Abortusequi (E156) according to Singh and Sharma9.

Micro agglutination test for Salmonella aggluti­nins-The test was conducted in V well microtitre plates8 using Salmonella (E156) '0' 4, 12 and 'H' enx antigens7 •

Results ]'lone of the guinea pigs, received from LAR Section

had detectible '0'4, 1 2 and 'H'enx agglutinin titre in their serum samples. Background opacity was detect­able with ELISA in undiluted serum (0.22±0.0 13) but at 1 : 100 dilution all samples induced opacity similar to blank control (0.03 ± 0.0 1 1 ).

Table I -Faecal excretion of S.Abortusequi in adult male guinea pigs experimentally infected through various routes

Mode of inoculation No. of animals No. of animals excreting S.Abortusegui o DPI I DPI 3 DPI 7 DPI 15 DPI 21 DPI

Oral 6 0 •

5 2 0 0 0 sc 6 0 2 0 I 0 0 ip 6 0 3 0 0 0

DPI-days post inoculation; sc-subcutaneous; ip-intraperitoneal.

298 INDIAN J EXP BIOL, MARCH 2002

Carriage of Salmonella Abortusequi in experimen­tally inoculated male guinea pigs- Observations on S. Abortusequi inoculated males revealed (Table 1 ) fae­cal excretion of Salmonella only up to 7 days in sub­cutaneously inoculated animals and only up to 3 days in orally and intra muscularly inoculated animals. None of the animal harbored S. Abortusequi in vis­ceral organs when sacrificed on day 21 of infection.

Carriage of Salmonella Abortusequi in experimen­tally inoculated female guinea pigs-Results of fae­cal excretion of S. Abortusequi in non pregnant fe­male animals (Fig. I ) revealed faecal excretion of Salmonella up to 75 days of inoculation through ei­ther of the route except oral route. Salmonella could not be isolated from any of the orally inoculated ani­mals from rectal swabs on or after 55 days of inocula­tion. On sacrificing the animals on day 75 of inocula­tion, S. Abortusequi could be isolated from spleen of

� .8 � (,) x Q) '0 CD .0 E ::l Z

1 4

1 2

1 0

8

6

4

2

o o 3 7 1 5 21

Days post inoculation

1 , 3, 3, 2 and 2 animals inoculated through oral, intra vaginal, intra muscular, intra peritoneal and subcuta­neous routes, respectively. Salmonella could not be isolated from any of the liver, gall bladder or kidneys. However, one uterus each from intra vaginally and subcutaneously inoculated animals contained S. Abor­tusequi.

Salmonella Abortusequi in experimentally inocu­lated breeding female guinea pigs-Of the 1 2 control animals, 8 were palpated positive for pregnancy at day 40 of breeding while only 2 of the 1 2 animals Salmonella carrier became pregnant. None of the 1 2 animals exposed to S. Abortusequi during breeding was found pregnant even after 55 days of observation.

Salmonella Abortusequi in experimentally inocu­lated guinea pigs with -40 days of gestation­Results of Salmonella isolation from rectal swabs of pregnant guinea pigs (Fig. 2) showed faecal excretion

D Oral • Intra vaginal

II Intra muscular • Intra peritoneal

• Subcutaneous

28 37 45 55 65 75

Fig. I -Faecal excretion of S. Abortusequi by adult female guinea pigs experimentally inoculated through different routes.

Table 2 - lmpact of Salmonella Abortusequi infection on guinea pigs at about 40 days of gestation

Observations Control Route of inoculation (Animals ex�sed) Oral IVG 1M IP SC

Animals aborted 0 2 0 0 3 0 Neither aborted nor parturited 0 3 6 6 3 6 Faecal excretors at 75 days post infection 0 0 0 1 0 0 Harboring Salmonella in spleen 0 1 0 2 2 2 Li ve babies born 1 8 2 0 0 0 0 Babies died in 21 days of life 1 0 0 0 0 0 Dead babies having Salmonella in spleen 0 2 0 0 0 0

IVG, intra-vaginal; 1M, intra-muscular; IP, intra-peritoneal; SC, subcutaneous. Note, None of the animal yielded Salmonella from uteruslliverl gall bladder or kidneys when sacrificed at 75th day of inoculation. For each treatment 6 animals were taken.

./ I

SINGH et al.: EVALUATION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELLA SEROVAR 299

of the pathogen by animals inoculated through par­enteral (subcutaneous, intra muscular and intra perito­neal) routes but rectal swabs from those inoculated either through oral or intra vaginal routes had no Sal­monella on day 75 of inoculation. Salmonella could not be isolated from, uteri liver, gall bladder or kid­neys. S. Abortusequi could be isolated from spleen of 1 , 3, 3, 3 and 3 animals inoculated through oral, intra­vaginal, intra-muscular, intra-peritoneal and subcuta­neous routes, respectively. Results of abortion and post abortion observations (Table 2) revealed either resorption (Fig. 3) or abortion in most of the animals with no living baby guinea pigs from S. Abortusequi inoculated animals. However, control group of guinea pigs had 17 healthy babies in the end of experiment.

7

6

5

4 l!? � 3 0 x Q) '0 Qj 2 .c E ::l Z

o +------,-L-o 3 7 1 5 21

Days post inoculation

Both the babies born live from orally inoculated guinea pigs died on day 4 of parturition having wa­tery diarrhoea. Salmonella could be isolated from fe­ces as well as from spleen of the dead baby guinea pigs. In contrast, the only baby died in control group had no Salmonella in its feces or in spleen.

Salmonella Abortusequi in experimentally inocu­lated guinea pigs with -55 days of gestation­Salmonella could be isolated from rectal swabs of pregnant guinea pigs inoculated through intra vaginal, intra-muscular and intra peritoneal routes but rectal swabs from those inoculated either through oral or subcutaneous routes had no Salmonella on day 75 of inoculation. Salmonella could be isolated from one uterus each from animals inoculated through intra

D Oral

fi htra rruscular

• Subcutaneous

28 37 45

• ntra vaginal

• ntra peritoneal

55 65 75

Fig. 2-Faecal excretion of S. Abortusequi by adult female guinea pigs experimentally inoculated through different routes during pregnancy.

Table 3-lmpact of Salmonella Abortusequi infection on guinea pigs with -55 days of gestation Observations Control Route of inoculation (Animals exposed)

Oral IVG 1M IP SC

Animals died before parturition 0 0 0 I I 2 Animals aborted 0 3 5 4 5 4 Neither aborted nor parturited 0 0 0 0 0 0 Faecal excretors at 75 days post infection 0 0 I I 2 0 Harboring Salmonella in spleen on 75lh day 0 2 3 4 4 4 Harboring Salmonella in uterus on 75th day 0 0 I I I 0 Live babies born 2 1 1 2 3 4 0 0 Babies died in 2 1 days of life 3 8 I 2 0 0 Dead babies having Salmonella in spleen 0 7 1 2 0 0

IVG, intra-vaginal; 1M, intra-muscular; IP, intra-peritoneal; SC, subcutaneous Note, None of the animal yielded Salmonella from liver! gall bladder or kidneys when sacrificed at 75th day of inoculation. For each treatment 6 animls were taken.

300 INDIAN J EXP BIOL, MARCH 2002

Table 4-Faecal/vaginal excretion of S. Abortusequi in aborted guinea pigs following experimental infection through various routes at -55 days of pregnancy

Days post Salmonella positive rectal swabs from animals inoculated Salmonella positive vaginal swabs from animals inoculated abortion through through

Oral Iva 1M IP SC Oral Iva 1M IP SC (3) (5) (4) (5) (4) (3) (5) (4) (5) (4)

0 I 3 0 0 I 3 5 4 5 4 I 3 5 4 2 2 2 4 4 4 4 3 2 5 4 5 3 3 5 4 4 3 7 2 5 4 5 4 3 4 4 5 4

15 3 2 4 5 4 3 5 4 5 4 2 1 2 2 4 5 4 3 5 4 5 4 30 0 I 2 5 4 3 5 3 3 4 38 2 3 4 2 2 4 2 3 3 45 2 0 0 0 1 3 2 2 1

Iva, intra-vaginal; 1M, intra-muscular; IP, intra-peritoneal; SC, subcutaneous Figures in parenthesis indicate the total number of animals swabbed

Fig. 3-Normal (left most), gravid (55 days gestation, right most) and uteri showing resorption of foetuses (3 in the centre) on oral Salmonella Abortusequi inoculation in pregnant (around 40 days of gestation) guinea pigs. All infected and control pregnant ani­mals were sacrificed at 55'h day of gestation.

vaginal, intra-muscular and intra peritoneal routes but not from liver, gall bladder or kidneys. S. Abortusequi could be isolated from spleen of 2, 3, 4, 4 and 4 ani­mals inoculated through oral, intra-vaginal, intra­muscular, intra-peritoneal and subcutaneous routes, respectively. Results of inoculation of Salmonella in -55 days pregnant guinea pigs (Table 3) revealed either death or abortion in most animals inoculated parenterally. However, orally and intra-vaginally inoculated with Salmonella and control groups had 4, 2 and 18 healthy babies, respectively in the end of the experiment. All the babies born live but died early in infancy, had diarrhoea. Salmonella could be isolated from spleen of all dead baby guinea pigs except one born from orally inoculated and 3 from control group. From feces of all the babies born of Salmonella inocu-

- lated mothers, the pathogen could be isolated either in pure culture (in one baby each from intra-vaginally and intra-muscularly inoculated groups) or as mixed infection in association with E. coli or Klebsiella

aerogenes (in babies of orally and intra-muscularly inoculated groups). None of the 3 babies died in con­trol group had Salmonella either in feces or in spleen. However, Enterobacter agglumerans could be iso­lated from feces but not from spleen.

S. Abortusequi could be isolated from rectal as well as vaginal swabs of all the aborted animals up to a month of abortion and thereafter variably from rectal swabs of animals inoculated through oral and intra vaginal routes but rarely from parenterally inoculated animals. Vaginal swabs were positive for S. Abor­tusequi for longer period than rectal/faecal swabs (Table 4)

Discussion Salmonella enterica subspecies enterica compris­

ing around 1 500 serovars includes most of the Salmo­nella which cause disease in humans and animals. Pathology of Salmonella is largely an outcome of host-bacteria interaction and varies with specific se­rovar in different host species 10. Salmonella Abor­tusequi, though an equine host adapted serovar, may infect other animals and humansl l . In equines, it often causes abortion in late pregnancy and perinatal foal mortality due to enteritis, omphalophlebitis, pol­yarthritis and septicemia12• 1 3 • It rarely affects non breeding adult females and males to cause only mild infection or orchitis, respectively. In earlier studies in different laboratory animals, it rarely produces classi­cal abortion, the main outcome in natural infection in equines 14• It is of utmost importance to standardize a laboratory model because i'n most places experimenta­tion on horses is not permissible or feasible. More­over, cost of the horse also interferes the studies,

1

. )1

SINGH et al. : EV ALVA TION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELLA SEROVAR 301

which are essential, particularly for determining safety and potency of vaccines. Besides, mere isola­tion of S. Abortusequi from equines or other sources may not be enough to conclude it as a primary cause of disease. Therefore, pathogenicity testing in a suit­able model is important I 5 . Most of the rodents and lagomorphs are born comparatively immature and abortions are rare even in late pregnancy. Therefore guinea pigs which give birth to quite mature babies and have gestational physiology comparable to equine l4, 16 appears to be a suitable model for S. Abor­tusequi. Therefore, this study was undertaken to stan­dardise the guinea pig model for future studies.

Absence of '0' 4, 1 2 and ' H' enx agglutinating an­tibodies in serum samples of guinea pigs before in­oculation of Salmonella, not only reveals absence of S. Abortusequi infection but also that of related se­rovars in the guinea pig colony. Positive results using cell lysate antigen in ELISA with many of the undi­luted sera or diluted below 1 : 1 00, indicated either over-sensivity of the ELISA or presence of cross re­acting antibodies to some antigens, widely present in nature like enterobacterial common antigen present in many of the intestinal commensals? Therefore, for ELISA, all serum samples were diluted with PBS to 1 :200 to avoid the detection of cross-reacting anti- . bodies.

Under this study, male guinea pigs appeared to be quite resistant to experimental S. Abortusequi infec­tion not only with respect to clinical or overt disease but also for colonization of the pathogen in gastroin­testinal tract. None of the male guinea pig, either in­oculated orally or parenterally with pathogenic S. Abortusequi, excreted the pathogen for more than 7 days, while many of the females continued to excrete S. Abortusequi for more than 2.5 months. S. Abor­tusequi was also absent from visceral organs of the male guinea pigs indicating some sex factors, yet to be identified, playing role in establishment of S. Abortusequi in females. Adult male guinea pigs ap­pear to behave similar to stallions which are often

f I · +' • I I re ractory to natura InlectlOn . Oral as well as parenteral inoculation of S. Abor­

tusequi in female guinea pigs led to development of Salmonella carriage state in the host up to the level of >30% which is common in host adapted Salmonella in their favoured hosts17, therefore guinea pigs appears to mimic as right type of model for S. Abortusequi.

In general, parenteral infection was more long last­ing than oral infection and converted more animals to carrier of S. Abortusequi which may be due to lack of

competition for parenterally inoculated Salmonella encountered in intestine by orally inoculated S. Abor­tusequi.

Not only sex but the age and physiological state of animals also appeared to be important factors to de­cide the course of S. Abortusequi infection in guinea pigs. Pregnant animals were found to be more sensi­tive, as comparatively more animals turned carrier and died particularly in advance pregnancy probably due to in utero death of fetuses not leading to abor­tions. Such deaths were more common with parenteral inoculation of the pathogen. Similar susceptibility in

h b b d . . 1 8·20 late pregnancy as een 0 serve In equmes too

Besides, during pregnancy gastrointestinal tract ap­pears to be more receptive to S. Abortusequi as on

.oral infection >66% females inoculated either in mid or late pregnancy kept on excreting Salmonella even after 2 months of infection while only 16% of those infected during non-pregnant stage turned excretor in the same period of observation under similar mainte­nance and environmental conditions. S. Abortusequi could be isolated from uterus of those animals only inoculated late in gestation. This difference of suscep­tibility to S. Abortusequi in breeding and non­breeding animals was less significant on parenteral infection. Alam2 1 and Hansda22 have reported similar observations on aroA and aroA-htrA mutants of S. Abortusequi in guinea pigs.

S. Abortusequi infection in mid gestation led to death of fetuses and resorption while abortions took place in guinea pigs infected in late pregnancy. It may be either due to absence of hard tissues in fetuses of mid pregnancy which might have made them easy subject for lysis and resorption or due to acuteness of infection as few of those animals fnfected through oral or intra-peritoneal routes aborted even in mid pregnancy. However, more detailed studies, to quanti­tate infection are necessary to reach at final conclu­sion.

Few baby guinea pigs delivered alive by Salmo­nella infected mothers either at early or late preg­nancy, died of salmonellosis and S. Abortusequi could be isolated from their spleens. Though, babies (5.5-14 . 1 %) of control groups died with similar clinical sign of diarrhoea and prostration as in S. Abortusequi infected groups, Salmonella could not be isolated from their faeces or spleens. Similar deaths have been reported in foals born on farms with prevalence of S. Abortusequi infection in mares 1 3 revealing suitability of the experimental host systems.

302 INDIAN J EXP BIOL, MARCH 2002

Most of the aborted guinea pigs continued for long to excrete Salmonella not only in faeces but in vaginal discharges too, indicating the danger of spread of Salmonella in their environment and possibly to mat­ing males. This post abortion vaginal and faecal ex­cretion has commonly been reported in S. Abortuse­qui infected mares. Thus female guinea pig seems to behave similar to the natural host, the pregnant mares.,

From the results, i t is evident that S. Abortusequi can cause infertility in carrier animals as well as in those exposed to sublethal infection without any clini­cal sign of salmonellosis. Normal conception rate in control group was 66.67% (8/ 12) while i t remained nil in those infected orally during breeding and 1 6.67% in Salmonella carriers. However, further investiga­tions are necessary to reveal the mechanism of caus­ing infertility as Salmonella could not be isolated from the uterus of those females which failed to breed and sacrificed after 55 days of breeding. Conclusion can be drawn that S. Abortusequi play a significant role in causing infertility and repeat-breeding. The same may be true for salmonellosis induced by other Salmonella strains in domestic animals and should be considered while treating and handling repeat­breeding problem, quite prevalent in all types of l ive­stock similar to prevalence of salmonellosis.

Variations in susceptibility may be associated with immune response. It has frequently been reported that antibody titres in pregnancy goes down with advanc­ing of gestation9, either it is low serum level of anti­bodies or immunomodulatory effect of sex and preg­nancy hormones23,24 or something else leading to pre­cipitation of Salmonella infection, only further inves­tigations can pinpoint the exact factor(s) making ani­mals more susceptible during pregnancy. Earlier stud­ies too have shown difference i n susceptibility to S. Abortusequi at different stages of breeding and in dif­ferent sexes. Alam21 and Hansda22 have shown that male animals mount more pronounced delayed type of hypersensitivity response to S. Abortusequi than fe­males, which may be an important factor in early clearance of S. Abortusequi by males. The study con­cludes that S. Abortusequi is an i mportant pathogen which affects breeding in guinea pigs and probably in its natural host, the equine mares too.

Acknowledgement Authors are thankful to Dr M C Saxena, Incharge,

Laboratory Animal Research, for providing Salmo­nella free guinea pigs and helping in diagnosis of pregnancy.

References 1 Popoff MY & LeMinor L, Antigenic Formulas of Salmonella

Serovars (WHO Collaborating Centre for Reference and Re­search on Salmonella, Pasture Institute Paris) 1997, 1 50.

2 Vaissaire J, Plateau E & Collobar-langier C, Emergence of so called saprophytic bacteria of the genera Aeromonas en­terobacter (Erwinia) and Serratia in cases of bacterial abor­tion in mares, Bul Acad Vet France, 59 ( 1 986) 1 1 3.

3 Manchanda V P, Garg D N, Gupta R C & Chandiramani N K, .The bacterial spectrum of uterus of infertile mares, In­dian J Comp Microbiol Immunol Infect Dis, 4 ( 1983) 82.

4 Kindahl H, Daels P F, Odensvik K, Daunt D, Fredricksson G, Stabenfeldt G & Hughes J P, Experimental models of en­dotoxeamia related to abortion in the mare, J Repro Fertil Supp No 44 ( 199 1 ) 509.

5 Donahue J M, Emergence of antibiotic resistant Salmonella agona in horses in Kentucky, J Am Vet Med Assoc, 188 ( 1986) 592.

6 Singh B R, Verma J C & Singh V P, Standardization of guinea pig model for Salmonella Abortusequi, in: National symposium on trends in vaccinology for animal diseases (In­dian Veterinary Research Institute, Izatnagar) 2000, 37.

7 Edwards P R & Ewing W H, Identification of enterobacteri­aceae (Burgees Publishing Co. Minnepolis, Minnesota) 1972, 146.

8 Hudson L & Hay F C, Practical immunology, 3'd edition (Ox­ford Blackwell Scientific Publications, London) 199 1 , 123.

9 Singh B R & Sharma V D, Isolation and characterisation of four distinct cytotoxins of Salmonella Weltevreden, Zentt Blatt Bacteriol, 289 ( 1999) 457.

10 Bispham J, Tripathi B N, Watson P R & Wallis T S, Salmo­nella pathogenicity island 2 influences both systemic salmo­nellosis and Salmonella induced enteritis in calves, Infect Immun, 69 (2001 ) 367.

1 1 Stable forth A W & Galloway J A, Salmonellosis, in Infec­tious diseases of animals vol. 2. edited by A W Stable forth and J A Galloway (Butterworth Scientific Publications, Lon­don) 1959, 1 56.

12 Condoleo R, Roperto F, Amaddeo D, Guarino G, Fontanelli G & Troncone A, Abortion and perinatal mortality in horses, microbiological and virological observations and histopa­thological findings, Aft Soc Italiano Sci Vet, 37( 1983) 525.

13 Pace L W, Wilson D A, Fales W H, Messer T, Lattimer J C & Mathews G, Salmonella septiceamia with pulmonary ab­scesses and osteomyilitis in a foal, Equine Vet Edu, 7 ( 1995) 64.

14 Rolle M & Kolvastic T, Salmonellosis, in Infectious diseases of animals vol. 2, edited by A W Stable forth and J A Gallo­way (Butterworth Scientific Publications, London) 1 947, 179.

15 Vasava K A, Studies on comparative efficacy of various vac­cines against Salmonella Abortusequi infection, MVSc The­sis submitted to Deemed University, Indian Veterinary Re­search Institute, Izatnagar, India, ( 1 999) 148.

16 Harkness J E & Wagner J E, The biology and medicine of rabbits and rodents, 2nd edition (Leonard Febiger, Philadel­phia) 1 963, 247.

17 Park B K, Yoon H S, Heo Y. An S H. Yoon Y D. Jeon Y & Kim Y H. Survey of horse diseases of veterinary importance in the Repulic of Korea. Res Rep Rural Develop Admin Vet Korea Republic, 30 (1988) 5 1 .

f

SINGH et al.: EVALUATION OF GUINEA PIG MODEL FOR EXPERIMENTAL SALMONELLA SEROVAR 303

18 Madic J, Hajsig D, Sostaric B, Curic S, Seol B, Naglic T & Cvetnic Z, An outbreak of abortions in mares associated with Salmonella AboT1usequi infection, Equine Vet J, 29 ( 1997) 230.

19 Fumoso E, Soto P, Jaca D, Sparo M, Aracil E & Bellusci J, Outbreak of abortion in mares due to SalmoneLLa Abortuse­qui, Rev Med Vet B Aires, 78 ( 1 997) 86.

20 Fumoso E A, Soto P, Gogorza L M & Auza N, Pathogenesis, diagnosis and control of epizootic abortion due to SalmoneLLa in mares, Rev Med Vet B Aires, 79 ( 1 998) 329.

2 1 Alam J , Characterisation of aro A mutant of SalmoneLLa Abor­tusequi, MVSc Thesis submitted to Deemed University, Indian Veterinary Research Institute, Izatnagar, India (2001) 1 10.

22 Hansda D, Studies on immunogenicity and safety of double mutant (aro A-htr A) of SalmoneLLa AboT1usequi, MVSc The­sis submitted to Deemed University, Indian Veterinary Re­search Institute, Izatnagar, India (200 1 ) 1 2 1 .

23 Daels P F, Stabenfeldt G H , Hughes J P, Odensvik K & Kin­dahl fl, Effect of flunixin neglumine as endotoxin induced prostaglandill f-2-alpha secretion during early pregnancy in mares, Am J Vet Res, 52 ( 1991 ) 276.

24 Daels P F, Stabenfeldt G H, Hughes J P, Odensvik K & Kin­dahl H, Evaluation of progestron deficiency as a cause of fe­tal death in mares with experimentally induced endotoxemia. Am J Vet Res, 52 ( 199 1 ) 282.