KINETICS OF EXPERIMENTAL SALMONELLA ABORTUS OVIS INFECTION IN EWES

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KINETICS OF EXPERIMENTAL SALMONELLA ABORTUS OVIS INFECTION IN EWES

F Lantier

To cite this version:

F Lantier. KINETICS OF EXPERIMENTAL SALMONELLA ABORTUS OVIS INFECTION IN

EWES. Annales de Recherches Vétérinaires, INRA Editions, 1987, 18 (4), pp.393-396. �hal-00901772�

KINETICS OF EXPERIMENTAL SALMONELLA ABORTUS OVIS INFECTION IN EWES

F LANTIER

Station de Pathologie ^{de la} Reproduction, INRA, Centre de Tours-Nouzilly, 37380, Monnaie, France received

20/12/86 / accepted

15/03/87

Résumé

CINÉTIQUE

D’UNE INFECTION

EXPÉRIMENTALE

PAR SALMONELLA ABORTUS OVlS CHEZ LA BRE- BIS. ^- La

multiplication

^et la dissémination de Salmonella abortus ovis, un

sérotype

de salmonella

spé- cifique

^des

petits

ruminants, ^ont été étudiées sur 17 brebis de réforme, non gestantes. La souche viru- lente

15/5

^a été inoculée par voie sous-cutanée à la dose de 101bactéries par animal, ^une dose

qui

n’a induit chez la

plupart

des animaux que de

légères

^réactions

cliniques (réactions

locales,

hyperthermie).

Les brebis ont été sacrifiées par groupes de 4 ^ou 5 aux

jours 3,

6,

10,

^{et 14}

après

l’inoculation. Des numé- rations

bactériennes,

^{réalisées sur} différents organes ^et

ganglions,

^ont

permis

^de situer le niveau maximal de la colonisation

par S

abortus ovis au 6e

jour après

l’inoculation. A cette date, le

ganglion

drainant le lieu d’inoculation

(le ganglion préscapulaire ^droit),

la rate, le foie, et les poumons étaient

régulièrement

infectés. Seuls le

ganglion préscapulaire

^{droit et} les tissus au lieu d’inoculation l’étaient encore quatorze

jours après

l’inoculation.

Early

Salmonella dissemination and

multiplica-

tion in the host reticuloendothelial system influence the level of infection in

target

organs and

subsequent

evolution of the disease. In

laboratory rodents,

kinetics of infection

by

facultative intracel- lular

parasites

represent ^{the main}

quantitative

^data

for a great number of studies

(Collins

^and

Camp-

bell

1982,

Rosenstreich et

a/ 1982).

Such data are scarce for domestic animals for which clinical

expression

of the disease and excretion of para- sites are the criteria

generally

retained for most models of infection. The aim of the

experiment

related in the present paper ^{was to}

investigate

^for

the

multiplication

and dissemination of Salmonella abortus ovis in

sheep.

^This

particular

serotype ^of Salmonella is

specific

for small ruminants. It pro- vokes abortions

and/or mortality

among newborns

(Jack 19711. A

virulent strain of S abortus

ovis,

that has

previously

been used either for the deve-

lopment

^{of mouse models}

^(Pardon

^and

Marly 1979,

^{Lantier et al}

19831,

^{or as an} abortion indu-

cing

agent ⁱⁿ

sheep (Pardon

^and

Marly 1980),

^was

injected subcutaneously

^{in non} pregnant ^ewes.

These were killed at intervals for bacterial counts of their

lymph

nodes and organs.

Materials and Methods Animals and experimental design

Seventeen culled adult ^ewes from Prealpes ^{or Berrichon-} du-Cher breeds were housed in ^an air-filtered

building.

They ^were inoculated by the subcutaneous route on the

ribs, just behind the right elbow and examined each day for clinical reactions. Rectal temperature, thickness of local reaction, and agglutination titers of serum were measured ^at intervals

(fig

^{1 and} 2). Four to five ewes were killed on day 3, 6, 10, and 14 after inoculation.

Local reaction, lymph nodes, spleen, part of liver,

lungs

and kidneys ^were aseptically removed, weighed, ^and kept frozen at -20 °C until they could be treated.

Serology

Anti-antigene

^{a O} » agglutination titers were measured by ^{means of a} microtitration technique using whole cells coloured with tetrazolium red (Pardon ^et a/ 1983).

Bacteria

Salmonella abortus ovis strain 15/5 ^{was described} previously (Pardon and Marly 19791. II was passaged in mice and stored at - 70 °C in infected spleens. An over-

night culture from a homogenate of ^a thawed spleen

was subcultured (24 h, 37 °C) ^onto trypticase soy agar slopes (TSA, BioMerieux, Lyon France). ^{II was used to} prepare ^a suspension in phosphate-buffered saline

(pH

6.9) that was

turbidimetrically

standardized and tested for purity and number of viable organisms by

plating

serial dilutions. Each ewe was inoculated subcutaneou- sly ^{with 2 x 109} bacteria in 2 ml of saline.

Research of bacteria in lymph nodes and organs.

Thawed spleens and lymph nodes were homogenized in saline (1/3 dilution) in a blender (Colworth Stomacher, Cofralab, Bordeaux).

Homogenates

and ten-fold dilutions of homogenates ^were plated (2 ^x 0.2 ml _per plate, ^one plate per dilution) ^onto

Salmonella-Shigella

Agar (SSA, Institut Pasteur Production, Paris, France) for enumera-

tion of bacteria.. Local reaction, liver,

lung

^and kidneys

were

homogenized

^{with a Kenwood}

mixer (Woking,

^UK).

Dilutions of liver

homogenates

^were plated ^{as above but} local reaction and samples of lungs ^and kidneys ^homo- genates ^were plated ^{on two} plates of SSA for qualitative

assessment of bacterial colonization. Cultures ^were incu- bated for 48 hours ^at 37 °C before colonies were

counted.

Expression ^{of results}

Viable counts of S abortus ovis were expressed ^as

tog 10

values, but for

lungs

^and kidneys for which only qualitative results could be obtained. Means and stan- dard errors of the means were calculated from the

loglo

⁰

values.

Organs

wich gave ^no positive ^{cultures were} regarded ^{as infected} by ^one bacterium, ^{and means were} calculated from all ewes of the experimental groups.

Results Clinical results

Ewes were not

severely

^affected

by

^Salmonella

abortus ovis infection except ^{one that had a rectal} temperature

equal

^or

superior

^to 42 °C and diar- rhea. Mean rectal temperatures ^{were at} their maxi-

mum

(40.8 °C)

^on

day

^one after inoculation but

some animals

presented

^a second

peak

^on

days

3-5

(fig 11.

Some ewes had

pronounced

^{local reac-}

tions at the site of inoculation

(fig 11. ).

Serological

^results

For most animals, ^a

significant

increase in

agglutination

titers could be observed from

day

⁵

after inoculation

(fig ^2).

Bacteriological

^results

From

day

3 after

inoculation,

^a

large

^{number or}

S abortus ovis could be recovered from the local reactions and from the

right prescapular lymph

node that drains the site of inoculation. Maximal level of infection was on

day

6 after inoculation

(fig ^3).

^{At that time, the}

right prescapular lymph

node, the

spleen,

^the

lungs

and the liver were regu-

larly

infected. On

day

10, ^the

global

level of infec- tion was reduced ;

however,

salmonella coloniza- tion of the

right precrural lymph

node reached its maximum. On

day 14,

infection had

considerably

decreased. But three

exceptions (1

liver, 1 left

prescapular lymph

node, and 1

right precrural lymph node),

^{it was limited to} the local reaction and the

right prescapular lymph

^node.

Whatever the

day

^of autopsy, ^{the more}

frequen- tly

^infected

samples

^were the local reaction, the

right prescapular

^and

precrural lymph

^{nodes, the} liver, the

spleen

^{and the}

lung,

ⁱⁿ

decreasing

^order.

Salmonella were

rarely

reisolated from left presca-

pular

^and

precrural lymph

^{nodes, and}

only

^from

highly

infected animals.

Kidneys

^were found infec- ted in two cases, with few Salmonella.

Discussion

The subcutaneous route of inoculation allowed

us to obtain a

reproducible

infection of the organs of the central reticuloendothelial system

(this paper),

^{as well as} abortion of pregnant ^ewes

(Par-

don and

Marly 1980,

Sanchis and Pardon

1984).

As

previously

described in our

laboratory (Pardon

et a/,

1 983),

the dose used in this

experiment (ie

²

x 109salmonella per

ewe)

induced moderate clini- cal reactions. Kinetics of infection

appeared

^remar-

kably

similar in mice

(Pardon

and

Marly

1979, Lantier et al

1983)

^and

sheep.

^{In both cases at} least three different

phases

^of

pathogenesis

^{can be}

distinguished.

In the first

phase,

dissemination of

bacteria occurred from subcutaneous tissues to the organs of the central RES

by passing through draining lymph

nodes, ie

through popliteal lymph

node in mice inoculated in their

footpad

and pre-

scapular lymph

node in

sheep

inoculated on their chest. This dissemination

phase

^can be followed

by

enumeration of S abortus ovis in local inflamma- tory reaction and

draining lymph

nodes. In the second

phase, multiplication

of bacteria occurred in

spleen,

liver and

lungs.

Maximum level of infec- tion was reached 6

days

after subcutaneous inocu- lation, both in mice and ewes. At that time, ^anti- bodies directed

against

the cell wall of S abortus ovis could be

easily

detected in serum

by

^{means of}

a

microagglutination technique.

In the third

phase

the infection _may evolve toward bacteria elimina- tion from the infected host or toward a chronic infectious state. As indicated

by

bacterial counts, S abortus ovis is

spontaneously

cleared from most

experimental

animals, mice and ewes as well.

However,

chronic infection of some

susceptible sheep

^is

likely

to account for the survival of the disease in flocks (Jack

197 1 It

can be

reproduced

with murine models

by

^{use of some Salmonella}

susceptible

inbred lines

(Lantier, unpublished data),

^{but has not} yet been studied in the natural host.

To each of these three

phases

^of

pathogenesis probably correspond

different mechanisms of the host response. Their

analysis,

eg

by

studies of their

genetic

^{control as}

actually

realised in mice (Rosenstreich et al

1982,

^{Skamene 1983).}

requires

^an

experimental

model of infection allo-

wing

^{the measure of an accurate} parameter ^direct-

ly

correlated with the infectious process. Bacterial

counts per organ represents such ^a basic parame- ter with which other correlates of the infectious process

(level

of antibodies and

complement,

^cel- lular

reactions)

should be

compared

^{in order to}

investigate host-parasite

interactions.

Acknowledgments

This work was

supported by

the INRA-ATP

«

G6n6tique

^de I’h6te et resistance aux maladies infectieuses et

parasitaires

^».

I am

grateful

^to TT Kramer for his critical review of the

manuscript .

^{I thank R} Fensterbank, M Plom- met, P

Pardon,

^M

P!pin

^{and J}

Marly

for

helpful

dis- cussions. We are indebted to P

Léchopier

^and

G

Bourgy

^for

solving

financial and technical pro- blems and to J

Chardron,

^R

Delaunay,

^D Musset, and W Piémont for care and maintenance of animals.

Abstract

The

multiplication

and dissemination of Salmonella abortus ovis, a Salmonella serotype which is

specific

for small ruminants, were studied in 17 culled ^non

pregnant

^ewes. The virulent strain

15/5,

that has pre-

viously

been used either for the

development

^{of mouse models or as an abortion}

inducing agent

ⁱⁿ

sheep,

was

injected by

the subcutaneous ^{route at a} sublethal dose of 10.salmonella per animal. In ^most ani- mals, it induced

only

^transient

hyperthermia

^and

pronounced

local reactions.

Groups

^{of four to five ewes}

were killed on

days 3, 6, 10,

and 14 for bacterial counts of their

lymph

nodes and organs. Maximal level of infection was on

day 6,

^at which time the

lymph

^node

draining

the site of inoculation

(right prescapular lymph node), spleen,

^{liver and}

lungs

^were

regularly

^{infected. On}

day

fourteen, S abortus ovis were regu-

larly

reisolated

only

^from

right prescapular lymph

nodes and local reactions.

Reference

Collins FM, Campbell SG, 1982. Immunity ^to intracellular bacteria. Vet Immunol Immunopathot 3:5-66 Jack EJ. 1971. Salmonella abortion in sheep. Vet Annu 12:57-63.

Lantier F, Pardon P, Marly J, 1983.

Immunogenicity

^{of a} low vaccinal strain against Salmonella aborrus ovis infection in mice. Infect Immun 40:601-607

Pardon P, Marly J, ^1979. Experimental Salmonella abortus ovis infection of normal or primo-infected CD-1 mice. Ann Microbiol (Inst Pasteur) 130B:21-28

Pardon P, Marly J, 1980. Experimental Salmonella abortus ovis infection in ewes. Vet Rec 106:389-390 Pardon P, Marly J, Sanchis R, Fensterbank R, 1983. Influence des voies et doses d’inoculation ^avec Salmonella abor- tus ovis ^sur 1’effet abortif et la réponse s6rologique des brebis. Ann Rech Vet 14:129-139

Rosenstreich DL, ^Weinblatt AC, O’Brien A, 1982. Genetic control of resistance to infection in mice. CRC Crit Rev Immunol 3:263-330

Sanchis R, ^Pardon P, 1984. Infection expérimentale de la brebis avec Salmonella abortus ovis : influence du stade de gestation. Ann Rech Vet 15:97-103

Skamene E, 1983. Genetic

regulation

of host resistance to bacterial infection. Rev Infect Dis 5:823-832