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Isolation and characterization of a new biovar of Moraxella bovis from healthy caprine nasal swabs
Small Ruminant Research ELSEVIER
Small Ruminant Research 15 (1994) 87-95
Isolation and characterization of a new biovar of Moraxella bovis from healthy caprine nasal swabs A. Kodjo a'*, A. Dorier b, C. Lerondelle c, Y.
Richard a
aEcole Nationale Vdtdrinaire de Lyon, 1, Av. Bourgelat, BP 83, 69280Marcy l'Etoile, France blUT, Facultd des Sciences, la Doua, Bd. du 11 Novembre 1918, 69100 Villeurbannes, France ClNRA-Labo Associd, 1, Av. Bourgelat BP 83 69280 Marcy I'Etoile, France Accepted 21 January 1994
Abstract
Twenty-four strains of presumptive Moraxella and Branhamella spp. isolated from healthy small ruminant nasal swabs and nine collection strains of the two above genera were compared by means of physiological, enzyme hydrolysing procedures and experimental infectivity tests. Four physiological groups were established. One new biovar of Moraxella, intermediate between Moraxella bovis and Moraxella lacunata was identified from goat nasal isolates. Corneal swabbing and instillation in mice of such Moraxella strain gave a mild transitory photophobia as did the collection strain CIP 7039 Moraxella bovis used in this study. Taxonomic studies are warranted to determine whether it is a new Moraxella species or a Moraxella bovis subspecies. Keywords: Goat; Moraxella bovis new biovar; Keratoconjunctivitis; Bovine pinkeye
I. Introduction Moraxella boris is considered to be the main primary aetiological agent of infectious bovine keratoconjunctivitis (IBK; bovine pinkeye), a contagious disease of cattle occurring perennially in all cattle-raising areas. Disease occurs most often during summer when enhancing factors such as ultraviolet radiation, fly population and microbial agents are increased, and investigations have demonstrated that the number of Moraxella boris isolations is increased particurlarly during summer months (Barber, 1985). These findings, together with the parasitic life of bacteria from the genus Moraxella in mammalians, can be associated with the existence of carriers other than cattle. Some *Corresponding author. 0921-4488/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO921-4488(94)OOOII-U
investigators have reported the existence of such cartiers, especially horses (Hughes and Pugh, 1970; Huntington et al., 1987), sheep (Baker et al., 1965; Travicek et al., 1982; Menoueri et al., 1988; Richard et al., 1989), and goats (Pande and Sekariah, cited by Vedros, in Bergey's Manual, 1984; Richard et al., 1989; Kodjo et al., 1993). These data strongly suggest that Moraxella boris and related bacteria may be widespread among small ruminants. Previous research indicated that the pathogenicity of Moraxella boris could be related to the ability of fimbriated strains to produce haemolysin and other hydrolytic enzymes (Pugh and Hughes, 1968; Frank and Gerber, 1981; Lepper and Power, 1988). The purpose of this study was to investigate the biochemical and hydrolytic enzyme profiles, using two standardised micromethods, the agglutination features
88
A. Kodjo et al. /Small Ruminant Research 15 (1994) 87-95
of 24 bacteria from the genera Moraxella and Branhamella, isolated from healthy small ruminant nasal cavities. Various collection strains of Moraxella and Branhamella were included in the study. The infectivity of one hemolytic strain of Moraxella boris-like isolate from goat and two strains each of Moraxella boris and Branhamella ovis were also tested.
2. Materials and methods
2.1. Bacterial strains The following reference bacterial strains were obtained from 'Collection Institut Pasteur' (CIP), Paris, France: Moraxella boris neotype strain CIP 7040T, Moraxella boris CIP 7039, Moraxella lacunata CIP A 182T, Moraxella nonliquefaciens 6836T, Branhamella ovis CIP 7320T, Branhamella cuniculi 7317T, and Branhamella caviae 7319T. An additional reference strain ofBranhamella ovis ATCC 33078 was purchased from American Type Culture Collection, Rockville, USA. Field strains, listed in Table 1, representing a total of 24 Gram-negative, oxidase-positive, aerobic coccobacilli resembling Moraxella or Branhamella were isolated from healthy goats and sheep nasal swabs in the Rh6ne-Alpes region of France during two surveys,
previously described by Richard et al. (1988) and Menoueri et al. (1988). 2.2. Bacteriology 2.2.1. Biochemical profile Strains were plated onto trypticase soy agar (TSA) containing 5 % defibrinated sheep blood (Bio M6rieux, Lyon) and incubated at 37°C under aerobic atmosphere. After 24 h, the strains were subcultured on brain heart agar infusion agar (BHIA) or brain heart infusion broth (BHIB) (Merck, Darmstadt) for identification in conventional biochemical tests as previously described (Kodjo et al., 1993). A standardised micromethod combining eight conventional and 12 assimilation tests for non-enteric Gram-negative bacteria identification was used for this study (AP120 NE, Bio M6rieux, Lyon). Because this system has not been used extensively in Moraxella species identification, all the reference strains were also tested. 2.2.2. Enzymes profile Constitutive enzymes of all reference strains and field isolates were assayed colorimetrically with a semiquantitative micromethod for enzymatic activity research (API ZYM system, Bio M6rieux, Lyon). Strains were tested according to the manufacturer's instructions. Briefly, microcupules containing various enzyme substrates were filled with a heavy suspension
Table 1 List of microorganisms used in this study Species
No. of strains tested
Source of strains
Designation
Collection strains M. lacunata M. bovis M. nonliquefaciens B. ovis B. cuniculi B. caviae
1 3 1 2 l l
human eyes cattle with IBK human respiratory tract ovine conjunctivitis rabbit oral cavity guinea pig pharynx
CIP A 182T CIP 7039, CIP 7040T, ATCC 17948 CIP 6836 CIP 7320T, ATCC 33078 CIP 7317T CIP 7319T
Field strains Moraxella bovis related Moraxella nonliquefaciens Branhamella spp.
7 8 9
goat nasal swab goat nasal swab goat nasal swab sheep nasal swab
88363A, 88363B. 88364A, 88364B, 88365, 88366, 88369 8897,8898,88100,88101,88103,284, J132, J134 88105,88102 88153,88155, J83,281,282,283,285
CIP = Collection Institut Pasteur, Paris, France; ATCC, American Type Culture Collection, Rockville, USA.
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95
(superior to McFarland No. 5 standard) of a 24-h bacterial culture. Strips were then incubated for 4 h at 37°C in a humidified chamber. The two reagents provided, Zym A and Zym B, were added and the color was allowed to develop for 5 min by exposure to sunlight to remove background color. The intensity of the colorimetric reaction was estimated with the reading scale provided.
2.3. Agglutination tests Autoagglutination ability was determined by suspending growth in a 150 mM NaCI buffer in order to achieve a McFarland No. 3 standard in plastic tests tubes. Haemagglutination (HA) was tested with freshly prepared red blood cells from rabbit, sheep and chicken using a modified method described by Varga et al. (1987). Each blood sample ( 1 ml) was collected in 3 ml of an Alsever buffer (sodium citrate 8%, sodium chloride 4.2% and glucose 20%, pH ajusted to 7 with citric acid), washed three times and suspended in 150 mM NaC1 buffer at a final concentration of 2%. Tests were carried out at room temperature in a 96-well plexi plate by mixing 100/zl of red blood cell suspension with an equal amount of bacterial suspension in, respectively, 150 mM NaC1, 1% d-mannose and 10% MgC1 solution containing approx. 109 cells per ml. Tests were read after 1 h. All strains were used in autoagglutination and haemagglutination tests.
89
The three groups of guinea pigs and three groups of mice were inoculated by intra peritoneal route with respectively 1 ml of each prepared bacterial suspension, whereas control groups were given PBS. The cornea (left and right) of the three other mice groups was swabbed with each bacterial suspension, then a further 0.1 ml of each inoculum was deposited on the surface of the cornea. Only mice were used in cornea inoculation because these animals have been used successfully in experimental induction of IBK. Control mice group were treated with PBS. Animals were maintained in a closed specific pathogen-free area with no fly admission, temperature regulated at approx. 20-22°C, and fed ad libitum water and food (Aliments UAR type 114 for guinea pig and type A03 for mice). Clinical signs of conjunctivitis and other clinical pathological signs were evaluated daily during 1 month. At the end of the experiment, blood samples were collected from guinea pigs by intra cardiac puncture in order to evaluate any hematologic or biochemical disorder using automatic systems (Easycell of Hycel, Beecroft-NSW, Australia for hematologic parameters; Reflotron type IV, Boehringer Mannheim, Germany for plasma urea and creatinine). Animals were then sacrified with ether prior to postmortem examinations. Spleen, liver, bone marrow, lung, and cornea were plated on blood agar in order to recover the inoculated bacteria. Testicles were examined histologically.
2.4. Experimental infectivity
3. Results
Three groups of three holoxenic male Dunkin Hartley guinea pigs of approx. 350 g (Elevage de Saint Antoine, Pleudaniel, France) and six groups of five male Ico:OF1-SWISS (lOPS/J) mice (IFFA CREDO, Les Oncins, l'Arbresles, France) of approx. 20-22 g were used for inoculation. Control groups were made with two guinea pigs and two groups of five mice. Moraxella boris-type strain CIP 7039, Branhamella ovis CIP 7320T and one haemolytic and fimbriated caprine field strain of Moraxella boris 80363 A were used for inoculation. Strains were subcultured 24 h at 37°C on TSA containing 5% defibrinated sheep blood then suspended in phosphate buffer saline (PBS) at a concentration of 109 colony forming units (CFU) per ml as indicated by Lepper and Power (1988).
3.1. Bacteriology After 24 h incubation on blood agar, or BHIA, all strains grew aerobically and gave non-pigmented small colonies which became larger within 48 h. Haemolysis on blood agar occurred frequently with large and thick colonies of diplobacilli. Haemolysis was weak or non existent with small, friable and non adherent colonies of cocci. All field strains were catalase- and oxidasepositive and none produced acids from glucose, maltose and lactose, nor urease and indole. All strains failed to grow on McConkey agar or on mineral medium with acetate. Other characteristics determined four groups within field strains (Table 2). Group 1 was identified as Moraxella boris, group 2 as Moraxella nonliquefa-
90
A. Kodjo et al./ Small Ruminant Research 15 (1994) 87-95
Table 2 AP120 NE identification and agglutination tests of reference and field Moraxella strains No. tested/isolates
Gram Oxidase Catalase Haemolysis* AP120 NE substrates Nitrate Tryptophan
Glucose ADH Urease Aesculin Gelatin
Reference strains A
B
+ + -
. + + +
+
C
D
.
.
+ +
. . +
+
. + + w
+ + +
+
+
.
group 1 7/7
group 2 8/8
group 3 5/5
group 4 4/4
rods
rods
cocci
cocci
+ + -
+
+
+
+
+
+
+
+
+
+
-
+
-
+
+
-
+
.
.
.
. .
.
I
.
. .
+
. + w
.
. .
H
.
. .
. .
G
.
. .
F
+
.
. .
.
+
.
. .
+ + -
-
.
.
.
+ + +
-
.
E
.
-
. . . . . -
Field strains
. .
.
All other AP120 NE substrates negative (glucose, arabiuose, mannose, mannitol, NAG, maltose, gluconate, caprate, adipate, malate, citrate phenyl acetate) Agglutination features Autoagglutination HA of chicken RBC sheep RBC rabbit RBC
. . -
+ . .
. . .
+
. . .
-
. . . +
. . .
.
.
. .
. . +
. . . +
+
-
7/7** 0/7 0/7 0/7
0/8 0/8 0/8 0/8
2/5 0/5 0/5 4/5
0/4 0/4 0/4 0/4
A = Moraxella lacunata A182T; B = Moraxella bovis CIP 7039; C = Moraxella bovis CIP 7040T; D= Moraxella bovis ATCC 17948; E = Moraxella nonliquefaciens CIP 6836T; F = Branhamella ovis ATCC 33078; G = Branhamella ovis CIP 7320T; H = Branhamella cuniculi CIP 7317T; 1= Branhamella caviae 7319T; group 1 = Moraxella bovis (88363A, 88363B, 88364A, 88364B, 88365, 88366, 88369); group 2=Moraxella nonliquefaciens (8897, 8898, 88100, 88101, 88103, 284, J132, J134); group 3 (88102, 88105, 88153, 88155, J83) and4 (281, 282, 283,285 ) = Branhamella spp. ADH=arginine dihydrolase; PNPG=para-nitro-phenyl-/3-galactopyranoside; NAG=N-acetyl-glucosamine; *haemolysis on sheep blood; **No. positive/No, tested; w = weak reaction.
ciens, a n d g r o u p s 3 a n d 4 as B r a n h a m e l l a spp. b e c a u s e they d i d n o t fit t h e p r e c i s e f e a t u r e s o f the c o c c a l Branhamella r e f e r e n c e strains i n c l u d e d in the study. Strains of group 1 and Moraxella boris CIP 7039 investigated by n e g a t i v e c o n t r a s t u n d e r the e l e c t r o n m i c r o s c o p y were f i m b r i a t e d . T h e A P I 2 0 N E results c o n f i r m e d t h o s e o b t a i n e d p r e v i o u s l y b y c o n v e n t i o n a l p r o c e d u r e s in w h i c h m o s t o f the b i o c h e m i c a l tests w e r e n e g a t i v e : n o s u g a r was assimilated, a n d t r y p t o p h a n test w a s also n e g a t i v e . T h i s pattern w a s c o m m o n to all strains. G e l a t i n l i q u e f a c t i o n was o b s e r v e d o n l y w i t h r e f e r e n c e a n d field strains o f MoraxeIla b o r i s ( g r o u p 1 s t r a i n s ) . In o t h e r respects,
M o r a x e l l a nonliquefaciens ( c o l l e c t i o n s t r a i n ) , Branhamella ovis ( c o l l e c t i o n s t r a i n ) , B r a n h a m e l l a caviae
( c o l l e c t i o n strain) a n d field isolates o f M o r a x e l l a or B r a n h a m e l l a o f g r o u p s 2, 3 a n d 4 s h a r e d e x a c t l y the s a m e b i o c h e m i c a l pattern. BranhameUa cuniculi was
s o m e w h a t d i f f e r e n t b e c a u s e this strain c o u l d n o t r e d u c e nitrates. G e l a t i n l i q u e f a c t i o n w a s u n e x p e c t e d l y n o t o b s e r v e d w i t h r e f e r e n c e strain o f M o r a x e l l a lacunata A182T (Table 2). Results o f A P I Z Y M tests are r e c o r d e d in T a b l e 3. All the strains tested (field a n d r e f e r e n c e s t r a i n s ) disp l a y e d i n t e n s e c o n s t i t u t i v e C 4 esterase, C 8 e s t e r a s e lipase a n d l e u c i n e a r y l a m i d a s e activities. R e f e r e n c e
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95
91
Table 3 Hydrolytic enzymes profiles of reference and field Moraxella strains No. of isolates tested
Reference strains
Field strains
A
B
C
D
E
F
Control
.
.
.
.
.
.
.
Alkaline phosphatase Esterase C4 Esterase Lipase C8 Lipase C14 Leucine arylamidase Valine arylamidase Trypsin a-Chymotrypsin Acid phosphatase Naphtol-phosphohydrolase a-Galactosidase /3-Galactosidase /3-Glucuronidase a-Glucosidase /3-Glucosidase N-acetyl-glucosamidase
+ + + . + . . . w . . . . . .
+ + + + +
+ + + + + . . .
+ + + +
+ + + +
w . . . . . .
-
w -
. . + + . . + . . . . . . w w . . . . . . . . . . . .
. . + + + + . . + + . . . . . . w . w . . . . . . . .
+ + + . . .
. . .
.
. .
. . . . .
.
. . . . w
. . . .
G
.
.
I
.
.
. . . . .
H
Group 1 7 .
. 0/7* 7/7 7/7 0/7 7/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7
group 2 8 .
group 3 5
group 4 4
0/5 5/5 5/5 5/5 1/5(w) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
0/4 2/4 2/4 0/4 2/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
. 0/8 8/8 8/8 0/8 8/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8
A=Moraxella lacunata AI82T; B=Moraxella boris CIP 7039; C=Moraxella bovis CIP 7040T; D=Moraxella bovis ATCC 17948; E = Moraxella nonliquefaciens CIP 6836T; F = Branhamella ovis ATCC 33078; G = Branhamella ovis CIP 7320T; H = Branhamella cuniculi CIP 7317T; I=Branhamella caviae 7319T; group 1 =Moraxella bovis (88363A, 88363B, 88364A, 88364B, 88365, 88366, 88369); group 2=Moraxella nonliquefaciens (8897, 8898, 88100, 88101, 88103, 284, J132, J134); group 3 (88102, 88105, 88153, 88155, J83) and 4 (281, 282, 283,285) = Branhamella spp. *No. positive/No, tested; w = weak reaction.
strain o f Moraxella boris C I P 7 0 3 9 s h o w e d a m i l d n a p h t o l p h o s p h o h y d r o l a s e activity. R e f e r e n c e s t r a i n s o f Branhamella spp.: Branhamella ovis C I P 7 3 2 0 T , A T C C 3 3 0 7 8 , Branhamella caviae 7 3 1 9 T , Branhamella cuniculi 7 3 1 7 T a n d Moraxella lacunata had, in addition, a m i l d a l k a l i n e p h o s p h a t a s e activity. All o t h e r s u b s t r a t e s w e r e n o t h y d r o l y s e d b y a n y strain.
3.2. Agglutination tests A u t o a g g l u t i n a t i o n w a s o b s e r v e d w i t h Moraxella boris C I P 7 0 3 9 a n d all t h e s t r a i n s o f g r o u p 1 a n d t w o Branhamella ( g r o u p 3) field strains, d - M a n n o s e d i d not i n h i b i t this a u t o a g g l u t i n a t i o n , w h e r e a s 1 0 % M g C I did. Haemagglutination was observed only on rabbit red b l o o d cells, b u t n o t o n c h i c k e n n o r s h e e p r e d b l o o d cells. P o s i t i v e r e a c t i o n w a s o b t a i n e d w i t h Moraxella
boris C I P 7 0 3 9 a n d A T C C 17948, Branhamella ovis C I P 7 3 2 0 T , A T C C 33078, Branhamella cuniculi CIP 7317, Branhamella caviae 7 3 1 9 a n d five Branhamella ( g r o u p 3) field strains. N o h a e m a g g l u t i n a t i o n w a s o b s e r v e d w i t h Moraxella field isolates a n d o t h e r Moraxella boris c o l l e c t i o n strains w i t h the e x c e p t i o n o f the t w o cited a b o v e ( T a b l e 2 ) .
3.3. Experimental pathogenicity
O n l y m i c e i n f e c t e d via c o r n e a l r o u t e w i t h Moraxella bovis C I P 7 0 3 9 a n d c a p r i n e isolate 8 0 3 6 3 A e x h i b i t e d a transitory p h o t o p h o b i a w i t h i n 2 d a y s after i n o c u l a t i o n ( T a b l e 4 ) . N o o t h e r clinical s i g n s w e r e r e c o r d e d a n d i n o c u l a t e d strains w e r e n e v e r r e c o v e r e d f r o m a n y organ. H e m a t o l o g i c a l a n d b i o c h e m i c a l p a r a m e t e r s w e r e n o r m a l in i n o c u l a t e d g u i n e a pigs c o m p a r e d to
2
206 47 <0.5
124 51 < 0.5
100 46.8 < 0.5
667
NT
o/5
5 mice i.c. _
NT
o/5
63 0.6
0
76.3 30 6.8 35.7 0.5
2 48.6 6.6 15.3
135 49 co.5
512.3
o/3
3 guinea pigs i.p.
ovis CIP 7320T
5 mice i.p.
Branhamella
*Bach animal was given 1 ml of a PBS suspension containing approx. lo9 CPU per ml by intra peritoneal route (i.p.) or comeal swabbing and instillation (i.c.) in each eye solely on mice. NT = not tested; ESR = erythrocytes sedimentation rate; RBC = red blood cells; MCV = mean corpuscular volume; MCHC = mean corpuscular hemoglobin concentration.
Urea (mg/loO ml) Creatinin mg/ 100 ml)
Biochemical parameters Glucose (mg/ 100 ml)
Platelets ( X 103/mm3)
412.3
0
monocytes
RBC (x 106/mm3) Hemoglobin (g/ 100 ml) MCV ($) MCW (g/100 ml) Leukocytes ( X 103/mm3) neutrophils (96) eosinophils (%)
0 62 1
48.5 6.4 15.3
0 56 1.5 654.6
NT
0 69.5
NT
basophils lymphocytes
2 51
6.3 36.5 0.5
NT
o/3
3 guinea pigs i.p. _
8.6 42 0.5
NT
5 mice i.p. o/5
6 30 0.5
1.5 50.5
5 mice ic. photophobia o/5
bovis 88363A (caprine strain)
Moraxella
74.5 31.5
NT
photophobia o/5
_ o/2
5 mice 3 guinea pigs i.p. i.p. o/5 o/3
bovis CIP 7039
6.7 15.4 75 30.1
NT
o/5
o/5
5 mice i.c.
Morarella
2 guinea pigs i.p.
strains
6.5 15.2 77.3 30.1
(means)
5 mice i.p. _
5 mice i.c. _
Control (PBS)
and Brunhamella infectivity of MoraxelZu
No. animals* Administration route Clinical signs No. deaths Hematology parameters ESR (mm in 2 h) Hematocrit
Table 4 Experimental
7 ;5:
S ,Q
2
R L Q S z
f
z 2 2
S
P * g 8. z R
A. Kodjoet aL / Small RuminantResearch 15 (1994)87-95
control group. Post-mortem and testicular histological examinations revealed no pathological disorders.
4. Discussion
This study showed the existence of bacteria from the genera Moraxella and Branhamella in the nasal bacterial flora of sheep and goats. These bacteria exhibited relatively homogeneous cultural and biochemical characteristics in which no carbohydrates were assimilated, whatever the system used (conventional or micromethod tests). This agrees with Catlin (1976; 1991) who proposed the genera Moraxella and Branhamella to constitute the family of Branhamaceae due to their asaccharolytic properties. Within the genus Branhamella, strains of groups 3 and 4 were suspected to be Branhamella ovis regarding their coccal morphology but their assignation to a physiologically known species was uncertain, because they did not fit the precise characteristics of the collection strains of Branhamella studied. Nevertheless we believe that these two groups could be identified as two biovars ofBranhamella ovis, even if negative nitrate reductase strains of Branhamella ovis have never been reported. Experimental infectivity of Branhamella ovis has been clearly demonstrated by Elad et ah (1988) to be very low which agrees with our test on laboratory animals. The common occurrence ofBranhamella ovis in the normal eyes of goats has also been reported by Pitman and Reuter (1988). Cases of conjunctivitis in sheep or cattle due to these bacteria seem to be concurrent with other microbial infections, (Rosenbusch, 1983; Elad et al., 1988). We identified diplobacilli with intermediary features between Moraxella lacunata (nitrate was reduced) and Moraxella boris (hemolysis occurred on sheep blood), solely on goat nasal swabs. Such strains bore fimbriae probably responsible for their autoagglutinating properties. In the same way, they produced the same hydrolytic enzymes as Moraxella boris collection strains. There is little information available in the literature concerning these tests and our findings were in agreement with those of Frank and Gerber ( 1981). However, in our study, the Moraxella boris reference strains only showed a weak reaction with acid phosphatase. Such phosphatases may be allergenic and contribute together with different enzymes or toxins production to the path-
93
ogenicity of Moraxella bovis (Frank and Gerber, 1981). Pathogenicity oflBK is still obscure. Only hemolytic and piliated strains of Moraxella bovis could induce IBK (Pugh and Hughes (1968), Lepper and Power (1988) ), and one of the initial steps in the pathogenesis of the disease is the adsorption on the cornea due to the fimbriae of the bacteria. Such features were also detected on the intermediary goat field strains of Moraxella bovis, but both Moraxella bovis reference strains CIP 7039, a virulent known strain and the caprine isolate 80363A failed to induce the complete clinical signs of IB K, when administred to mice or guinea pigs except a mild photophobia on mice inoculated by corneal swabbings and instillations. We believe that this photophobia was due to the adsorption of the fimbriae on the surface of the cornea, but the other steps following the primary adsorption of bacteria on cornea, usually occurring in IBK pathogenesis, were not observed, probably because of lack of enhancer factors such as concurrent microbial infection, corneal damage due to radiation or fly irritation. Therefore, we did not observe any corneal ulceration or discharge. Many workers have attempted with variable success to induce IBK, either on calves or on laboratory animals, particularly on mice, and the success of this experimentally induced IBK depends on preliminary corneal damage either by ultraviolet radiation (Pugh and Hughes, 1968) or microbial agents such as Mycoplasma (Rosenbusch, 1983). Other workers also reported success only after corticosteroid administration (Lepper and Power, 1988). Working in a specific pathogen-free area could explain our unsuccessful attempt. This result agrees with Lepper and Power (1988), who also failed to reproduce IBK on non-corticosteroid-treated mice with full virulent strains of Moraxella bovis. This study on Moraxella strains, isolated from small ruminant nasal swabs, shows the existence of a new biovar of MoraxeUa boris in the nasal flora of healthy goats. Lack of biochemical conventional tests to differentiate the nonfennenter Moraxella and lack of available laboratory models for IBK reproduction indicate the need of other tests to differentiate this new biovar from Moraxella bovis. Other sophisticated tests as genetic transformations (Hoke and Vedros 1982; Bovre, 1984; Juni and Heym, 1988) or DNA/DNA hybridizations (Tonjum et al., 1989; 1993) have indi-
94
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95
cated that Moraxella boris, Moraxella lacunata and MoraxeUa nonliquefaciens shared strong interspecific genetic homologies. Works on ribosomal RNA vs. DNA hybridization (Rossau et al., 1991) have confirmed these findings and proposed these three species to constitute the lacunata group. The lack of conventional characteristics and genetic homologies among strains of the lacunata group represent, therefore, a strong handicap for identification of Moraxella strains based upon traditional parameters. Our Moraxella boris isolates, intermediate between Moraxella boris and Moraxella lacunata, could be a new Moraxella species or a Moraxella boris subspecies. Such a haemolytic strain was described and named MoraxeIla lacunatus by Witers and Davis (1961) in a conjunctivitis outbreak in a cattery. Since that date there is no available information about such particular hemolytic 'Moraxella lacunatus' strain. Taxonomic studies would therefore be of interest to determine whether or not it is a new Moraxella species.
5. Conclusion Moraxella-like bacteria have been isolated from healthy sheep and goat nasal swabs. A new biovar of Moraxella boris isolated from healthy goats on farms with no IBK history has been characterized. The biochemical, constitutive enzymes and infectivity patterns of this biovar were about the same as for the Moraxella boris collection strains used in the study except the ability to reduce nitrate.
References Baker, J.R., Fanl, W.B. and Ward, W.R., 1965. Conjunctivitis in sheep associated with Moraxella (Haemophilus) organisms. Vet. Rec., 77: 402. Barber, D.M.L., 1985. Bacterial population of slaughter cattle. Vet Rec., 115: 169. Bovre, K., 1984. Neisseriaceae. Bergey's Manual of Systematic Bacteriology. The William & Wilkins Co., Baltimore, pp. 288309. Catlin, B.W., 1976. Cellular elongation under the influence of antibacterial agents: Way to differentiate coccobacilli from cocci. J. Clin. Microbiol., 1: 102-106. Catlin, B.W., 1991. Branhamaceae Fam. Nov., a new proposed family to accommodate the genera Branhamella and Moraxella. Int. J. Syst, Bacteriol., 41: 320-323.
Elad, D., Yeruham, i. and Bernstein, N., 1988. Moraxella bovis in cases of Infectious Bovine Keratoconjunctivitis (IB K ) in Israel. J. Vet. Med., 35: 431--434. Frank, K.S. and Gerber, J.D., 1981. Hydrolytic enzymes of Moraxella bovis. J. Clin, Microbiol, 13:269-271. Fraser, J. and Gilmour, N.J.L., 1979. The identification of Moraxella boris and Neisseria ovis from the eyes of cattle and sheep. Res. Vet. Sci., 27: 127-128. Hoke, C. and Vedros, N.A., 1982. Taxonomy of the Neisseriae: Deoxyribonucleic acid base composition, interspecific transformation, and deoxyribonucleic acid hybridization. Int. J. Syst. Bacteriol., 32: 57-66. Hughes, D.E. and Pugh, G.W., 1970. Isolation and description of a Moraxella from horses with conjunctivitis. Am. J. Vet. Res., 31,457--459. Huntington, P.J., Coloe, P.J., Bryden, J.D. and Macdonald, F., 1987. Isolation of a Moraxella sp. from horse with conjunctivitis. Aust. Vet. J., 64: 118-119. Juni, E. and Heym, G.A., 1988. Identification ofMoraxella bovis by quantitative genetic transformation and nutritional assays. Appl. Environ. Microbiol., 54:1304-1306. Kodjo, A., Moussa, A., Borges, E. and Richard, Y., 1993. Identification of Moraxella-like bacteria isolated from ovine and caprine nasal flora. J. Vet. Med. B, 40: 97-104. Lepper, A.W.D. and Power, B.E., 1988. Infectivity of Australian strains of Moraxella bovis for murine and bovine eye in relation to pilus serogroup sub-unit size and degree of piliation. Aust. Vet. J., 65: 305-309. Mattinson, A.D. and COX, P.J., 1982. Use of Tween 80 agar in the study ofMoraxella bovis infection in cattle. Vet. Rec., 111: 395396. Menoueri, N., Richard, Y., Brunet, J. and Oudar, J., 1988. Flore bacttrienne a~robie et atro-anatrobie des cavitts nasales de l'agneau de bergerie (Bacteria flora from nasal fossae of lambs). Ann. Rech. Vet., 19: 175-180. Pitman, D.R. and Reuter, R., 1988. Isolation of Branhamella ovis from conjunctivae of Angora goats. Aust. Vet. J., 65: 91. Pugh, G.W. and Hughes, D.E., 1968. Experimental Bovine Infectious Keratoconjunctivifis caused by sunlamp irradiation and Moraxella boris infection: Correlation of hemolytic ability and pathogenicity. Am. J. Vet. Res., 29: 835-839. Pugh, G.W. and Mcdonald, T.J., 1986. Identification of bovine carriers of Moraxella boris by comparative cultural examinations of ocular and nasal secretions. Am. J. Vet. Res., 47: 2343-2345. Richard, Y., Borges, E., Favier, C. and Oudar, J., 1989. Flores nasale et pulmonaire de la ch~vre (Nasal fossae and pulmonary bacterial flora in goats). Ann. Rech. Vet., 2: 269-276. Riou, J.Y., Buissiere, J., Guibourdenche, M., Brault, G. and Carlier, J.P., 1981. Hydrolyse de la tributyrine par les Neisseria et les Branhamella (Hydrolysis of tributyrin by species of Neisseria and Branhamella). Ann. Microbiol. Inst. Pasteur, 132: 159-169. Rosenbusch, R.F., 1983. Influence of Mycoplasma preinfection on the expression of Moraxella pathogenicity. Am. J Vet. Res., 44:1621-1624. Rossau, R., Vanlandsehoot, A., Gillis, M. and De Ley, J., 1991. Taxonomy of Moraxellaxeae Fam Nov, a new Bacteria family to accommodate the genera Moraxella, Acinetobacter, and Psy-
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95 chrobacter and related organism. Int. J. Syst. Bacteriol., 41: 310319. Tonjum, T., Bukolm, G. and Bovre, K., 1889. Differentiation of some species of Neisseriaceae and other bacterial groups by DNADNA hybridization. APMIS, 97: 395-405. Tonjum, T., Caugant, D.A., and Bovre K., 1993. Differentiation of Moraxella nonliquefaciens, Moraxella lacunata, and Moraxella bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes. J. Clin. Microbiol., 30: 3099-3107. Travicek, M., Dravecky, T. and Balascak, J., 1982. Isolation of Chlamydia psittaci and Moraxella boris from infectious keratoconjunctivitis in lambs. Vet. Med., Praha, 27: 491-496. Vedros, N.A., 1984. Neisseria. Bergey's Manual of Systematic Bac-
95
teriology. The William & Wilkins Co., Baltimore, pp. 290-296. Varga, J., Fodor, L., Hajtos, I. and Ratz, F., 1987. Haemagglutination by Neisseria ovis isolated from the eyes of sheep. J. Vet. Med. B, 34:211-215. Wilcox, G.E., 1970a. Bacterial flora of the bovine eye with special reference to the Moraxella and the Neisseria. Aust. Vet. Rec., 46: 153-257. Wilcox, G.E., 1970b. An examination of Moraxella and related genera commonly isolated from the bovine eye. J. Comp. Pathol., 80: 65-74. Witers, A.R. and Davies, E.M, 1961. An outbreak of conjunctivitis in a cattery (caused by Moraxella lacunatus). Vet. Rec., 75: 856--857.
Small Ruminant Research 15 (1994) 87-95
Isolation and characterization of a new biovar of Moraxella bovis from healthy caprine nasal swabs A. Kodjo a'*, A. Dorier b, C. Lerondelle c, Y.
Richard a
aEcole Nationale Vdtdrinaire de Lyon, 1, Av. Bourgelat, BP 83, 69280Marcy l'Etoile, France blUT, Facultd des Sciences, la Doua, Bd. du 11 Novembre 1918, 69100 Villeurbannes, France ClNRA-Labo Associd, 1, Av. Bourgelat BP 83 69280 Marcy I'Etoile, France Accepted 21 January 1994
Abstract
Twenty-four strains of presumptive Moraxella and Branhamella spp. isolated from healthy small ruminant nasal swabs and nine collection strains of the two above genera were compared by means of physiological, enzyme hydrolysing procedures and experimental infectivity tests. Four physiological groups were established. One new biovar of Moraxella, intermediate between Moraxella bovis and Moraxella lacunata was identified from goat nasal isolates. Corneal swabbing and instillation in mice of such Moraxella strain gave a mild transitory photophobia as did the collection strain CIP 7039 Moraxella bovis used in this study. Taxonomic studies are warranted to determine whether it is a new Moraxella species or a Moraxella bovis subspecies. Keywords: Goat; Moraxella bovis new biovar; Keratoconjunctivitis; Bovine pinkeye
I. Introduction Moraxella boris is considered to be the main primary aetiological agent of infectious bovine keratoconjunctivitis (IBK; bovine pinkeye), a contagious disease of cattle occurring perennially in all cattle-raising areas. Disease occurs most often during summer when enhancing factors such as ultraviolet radiation, fly population and microbial agents are increased, and investigations have demonstrated that the number of Moraxella boris isolations is increased particurlarly during summer months (Barber, 1985). These findings, together with the parasitic life of bacteria from the genus Moraxella in mammalians, can be associated with the existence of carriers other than cattle. Some *Corresponding author. 0921-4488/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIO921-4488(94)OOOII-U
investigators have reported the existence of such cartiers, especially horses (Hughes and Pugh, 1970; Huntington et al., 1987), sheep (Baker et al., 1965; Travicek et al., 1982; Menoueri et al., 1988; Richard et al., 1989), and goats (Pande and Sekariah, cited by Vedros, in Bergey's Manual, 1984; Richard et al., 1989; Kodjo et al., 1993). These data strongly suggest that Moraxella boris and related bacteria may be widespread among small ruminants. Previous research indicated that the pathogenicity of Moraxella boris could be related to the ability of fimbriated strains to produce haemolysin and other hydrolytic enzymes (Pugh and Hughes, 1968; Frank and Gerber, 1981; Lepper and Power, 1988). The purpose of this study was to investigate the biochemical and hydrolytic enzyme profiles, using two standardised micromethods, the agglutination features
88
A. Kodjo et al. /Small Ruminant Research 15 (1994) 87-95
of 24 bacteria from the genera Moraxella and Branhamella, isolated from healthy small ruminant nasal cavities. Various collection strains of Moraxella and Branhamella were included in the study. The infectivity of one hemolytic strain of Moraxella boris-like isolate from goat and two strains each of Moraxella boris and Branhamella ovis were also tested.
2. Materials and methods
2.1. Bacterial strains The following reference bacterial strains were obtained from 'Collection Institut Pasteur' (CIP), Paris, France: Moraxella boris neotype strain CIP 7040T, Moraxella boris CIP 7039, Moraxella lacunata CIP A 182T, Moraxella nonliquefaciens 6836T, Branhamella ovis CIP 7320T, Branhamella cuniculi 7317T, and Branhamella caviae 7319T. An additional reference strain ofBranhamella ovis ATCC 33078 was purchased from American Type Culture Collection, Rockville, USA. Field strains, listed in Table 1, representing a total of 24 Gram-negative, oxidase-positive, aerobic coccobacilli resembling Moraxella or Branhamella were isolated from healthy goats and sheep nasal swabs in the Rh6ne-Alpes region of France during two surveys,
previously described by Richard et al. (1988) and Menoueri et al. (1988). 2.2. Bacteriology 2.2.1. Biochemical profile Strains were plated onto trypticase soy agar (TSA) containing 5 % defibrinated sheep blood (Bio M6rieux, Lyon) and incubated at 37°C under aerobic atmosphere. After 24 h, the strains were subcultured on brain heart agar infusion agar (BHIA) or brain heart infusion broth (BHIB) (Merck, Darmstadt) for identification in conventional biochemical tests as previously described (Kodjo et al., 1993). A standardised micromethod combining eight conventional and 12 assimilation tests for non-enteric Gram-negative bacteria identification was used for this study (AP120 NE, Bio M6rieux, Lyon). Because this system has not been used extensively in Moraxella species identification, all the reference strains were also tested. 2.2.2. Enzymes profile Constitutive enzymes of all reference strains and field isolates were assayed colorimetrically with a semiquantitative micromethod for enzymatic activity research (API ZYM system, Bio M6rieux, Lyon). Strains were tested according to the manufacturer's instructions. Briefly, microcupules containing various enzyme substrates were filled with a heavy suspension
Table 1 List of microorganisms used in this study Species
No. of strains tested
Source of strains
Designation
Collection strains M. lacunata M. bovis M. nonliquefaciens B. ovis B. cuniculi B. caviae
1 3 1 2 l l
human eyes cattle with IBK human respiratory tract ovine conjunctivitis rabbit oral cavity guinea pig pharynx
CIP A 182T CIP 7039, CIP 7040T, ATCC 17948 CIP 6836 CIP 7320T, ATCC 33078 CIP 7317T CIP 7319T
Field strains Moraxella bovis related Moraxella nonliquefaciens Branhamella spp.
7 8 9
goat nasal swab goat nasal swab goat nasal swab sheep nasal swab
88363A, 88363B. 88364A, 88364B, 88365, 88366, 88369 8897,8898,88100,88101,88103,284, J132, J134 88105,88102 88153,88155, J83,281,282,283,285
CIP = Collection Institut Pasteur, Paris, France; ATCC, American Type Culture Collection, Rockville, USA.
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95
(superior to McFarland No. 5 standard) of a 24-h bacterial culture. Strips were then incubated for 4 h at 37°C in a humidified chamber. The two reagents provided, Zym A and Zym B, were added and the color was allowed to develop for 5 min by exposure to sunlight to remove background color. The intensity of the colorimetric reaction was estimated with the reading scale provided.
2.3. Agglutination tests Autoagglutination ability was determined by suspending growth in a 150 mM NaCI buffer in order to achieve a McFarland No. 3 standard in plastic tests tubes. Haemagglutination (HA) was tested with freshly prepared red blood cells from rabbit, sheep and chicken using a modified method described by Varga et al. (1987). Each blood sample ( 1 ml) was collected in 3 ml of an Alsever buffer (sodium citrate 8%, sodium chloride 4.2% and glucose 20%, pH ajusted to 7 with citric acid), washed three times and suspended in 150 mM NaC1 buffer at a final concentration of 2%. Tests were carried out at room temperature in a 96-well plexi plate by mixing 100/zl of red blood cell suspension with an equal amount of bacterial suspension in, respectively, 150 mM NaC1, 1% d-mannose and 10% MgC1 solution containing approx. 109 cells per ml. Tests were read after 1 h. All strains were used in autoagglutination and haemagglutination tests.
89
The three groups of guinea pigs and three groups of mice were inoculated by intra peritoneal route with respectively 1 ml of each prepared bacterial suspension, whereas control groups were given PBS. The cornea (left and right) of the three other mice groups was swabbed with each bacterial suspension, then a further 0.1 ml of each inoculum was deposited on the surface of the cornea. Only mice were used in cornea inoculation because these animals have been used successfully in experimental induction of IBK. Control mice group were treated with PBS. Animals were maintained in a closed specific pathogen-free area with no fly admission, temperature regulated at approx. 20-22°C, and fed ad libitum water and food (Aliments UAR type 114 for guinea pig and type A03 for mice). Clinical signs of conjunctivitis and other clinical pathological signs were evaluated daily during 1 month. At the end of the experiment, blood samples were collected from guinea pigs by intra cardiac puncture in order to evaluate any hematologic or biochemical disorder using automatic systems (Easycell of Hycel, Beecroft-NSW, Australia for hematologic parameters; Reflotron type IV, Boehringer Mannheim, Germany for plasma urea and creatinine). Animals were then sacrified with ether prior to postmortem examinations. Spleen, liver, bone marrow, lung, and cornea were plated on blood agar in order to recover the inoculated bacteria. Testicles were examined histologically.
2.4. Experimental infectivity
3. Results
Three groups of three holoxenic male Dunkin Hartley guinea pigs of approx. 350 g (Elevage de Saint Antoine, Pleudaniel, France) and six groups of five male Ico:OF1-SWISS (lOPS/J) mice (IFFA CREDO, Les Oncins, l'Arbresles, France) of approx. 20-22 g were used for inoculation. Control groups were made with two guinea pigs and two groups of five mice. Moraxella boris-type strain CIP 7039, Branhamella ovis CIP 7320T and one haemolytic and fimbriated caprine field strain of Moraxella boris 80363 A were used for inoculation. Strains were subcultured 24 h at 37°C on TSA containing 5% defibrinated sheep blood then suspended in phosphate buffer saline (PBS) at a concentration of 109 colony forming units (CFU) per ml as indicated by Lepper and Power (1988).
3.1. Bacteriology After 24 h incubation on blood agar, or BHIA, all strains grew aerobically and gave non-pigmented small colonies which became larger within 48 h. Haemolysis on blood agar occurred frequently with large and thick colonies of diplobacilli. Haemolysis was weak or non existent with small, friable and non adherent colonies of cocci. All field strains were catalase- and oxidasepositive and none produced acids from glucose, maltose and lactose, nor urease and indole. All strains failed to grow on McConkey agar or on mineral medium with acetate. Other characteristics determined four groups within field strains (Table 2). Group 1 was identified as Moraxella boris, group 2 as Moraxella nonliquefa-
90
A. Kodjo et al./ Small Ruminant Research 15 (1994) 87-95
Table 2 AP120 NE identification and agglutination tests of reference and field Moraxella strains No. tested/isolates
Gram Oxidase Catalase Haemolysis* AP120 NE substrates Nitrate Tryptophan
Glucose ADH Urease Aesculin Gelatin
Reference strains A
B
+ + -
. + + +
+
C
D
.
.
+ +
. . +
+
. + + w
+ + +
+
+
.
group 1 7/7
group 2 8/8
group 3 5/5
group 4 4/4
rods
rods
cocci
cocci
+ + -
+
+
+
+
+
+
+
+
+
+
-
+
-
+
+
-
+
.
.
.
. .
.
I
.
. .
+
. + w
.
. .
H
.
. .
. .
G
.
. .
F
+
.
. .
.
+
.
. .
+ + -
-
.
.
.
+ + +
-
.
E
.
-
. . . . . -
Field strains
. .
.
All other AP120 NE substrates negative (glucose, arabiuose, mannose, mannitol, NAG, maltose, gluconate, caprate, adipate, malate, citrate phenyl acetate) Agglutination features Autoagglutination HA of chicken RBC sheep RBC rabbit RBC
. . -
+ . .
. . .
+
. . .
-
. . . +
. . .
.
.
. .
. . +
. . . +
+
-
7/7** 0/7 0/7 0/7
0/8 0/8 0/8 0/8
2/5 0/5 0/5 4/5
0/4 0/4 0/4 0/4
A = Moraxella lacunata A182T; B = Moraxella bovis CIP 7039; C = Moraxella bovis CIP 7040T; D= Moraxella bovis ATCC 17948; E = Moraxella nonliquefaciens CIP 6836T; F = Branhamella ovis ATCC 33078; G = Branhamella ovis CIP 7320T; H = Branhamella cuniculi CIP 7317T; 1= Branhamella caviae 7319T; group 1 = Moraxella bovis (88363A, 88363B, 88364A, 88364B, 88365, 88366, 88369); group 2=Moraxella nonliquefaciens (8897, 8898, 88100, 88101, 88103, 284, J132, J134); group 3 (88102, 88105, 88153, 88155, J83) and4 (281, 282, 283,285 ) = Branhamella spp. ADH=arginine dihydrolase; PNPG=para-nitro-phenyl-/3-galactopyranoside; NAG=N-acetyl-glucosamine; *haemolysis on sheep blood; **No. positive/No, tested; w = weak reaction.
ciens, a n d g r o u p s 3 a n d 4 as B r a n h a m e l l a spp. b e c a u s e they d i d n o t fit t h e p r e c i s e f e a t u r e s o f the c o c c a l Branhamella r e f e r e n c e strains i n c l u d e d in the study. Strains of group 1 and Moraxella boris CIP 7039 investigated by n e g a t i v e c o n t r a s t u n d e r the e l e c t r o n m i c r o s c o p y were f i m b r i a t e d . T h e A P I 2 0 N E results c o n f i r m e d t h o s e o b t a i n e d p r e v i o u s l y b y c o n v e n t i o n a l p r o c e d u r e s in w h i c h m o s t o f the b i o c h e m i c a l tests w e r e n e g a t i v e : n o s u g a r was assimilated, a n d t r y p t o p h a n test w a s also n e g a t i v e . T h i s pattern w a s c o m m o n to all strains. G e l a t i n l i q u e f a c t i o n was o b s e r v e d o n l y w i t h r e f e r e n c e a n d field strains o f MoraxeIla b o r i s ( g r o u p 1 s t r a i n s ) . In o t h e r respects,
M o r a x e l l a nonliquefaciens ( c o l l e c t i o n s t r a i n ) , Branhamella ovis ( c o l l e c t i o n s t r a i n ) , B r a n h a m e l l a caviae
( c o l l e c t i o n strain) a n d field isolates o f M o r a x e l l a or B r a n h a m e l l a o f g r o u p s 2, 3 a n d 4 s h a r e d e x a c t l y the s a m e b i o c h e m i c a l pattern. BranhameUa cuniculi was
s o m e w h a t d i f f e r e n t b e c a u s e this strain c o u l d n o t r e d u c e nitrates. G e l a t i n l i q u e f a c t i o n w a s u n e x p e c t e d l y n o t o b s e r v e d w i t h r e f e r e n c e strain o f M o r a x e l l a lacunata A182T (Table 2). Results o f A P I Z Y M tests are r e c o r d e d in T a b l e 3. All the strains tested (field a n d r e f e r e n c e s t r a i n s ) disp l a y e d i n t e n s e c o n s t i t u t i v e C 4 esterase, C 8 e s t e r a s e lipase a n d l e u c i n e a r y l a m i d a s e activities. R e f e r e n c e
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95
91
Table 3 Hydrolytic enzymes profiles of reference and field Moraxella strains No. of isolates tested
Reference strains
Field strains
A
B
C
D
E
F
Control
.
.
.
.
.
.
.
Alkaline phosphatase Esterase C4 Esterase Lipase C8 Lipase C14 Leucine arylamidase Valine arylamidase Trypsin a-Chymotrypsin Acid phosphatase Naphtol-phosphohydrolase a-Galactosidase /3-Galactosidase /3-Glucuronidase a-Glucosidase /3-Glucosidase N-acetyl-glucosamidase
+ + + . + . . . w . . . . . .
+ + + + +
+ + + + + . . .
+ + + +
+ + + +
w . . . . . .
-
w -
. . + + . . + . . . . . . w w . . . . . . . . . . . .
. . + + + + . . + + . . . . . . w . w . . . . . . . .
+ + + . . .
. . .
.
. .
. . . . .
.
. . . . w
. . . .
G
.
.
I
.
.
. . . . .
H
Group 1 7 .
. 0/7* 7/7 7/7 0/7 7/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7
group 2 8 .
group 3 5
group 4 4
0/5 5/5 5/5 5/5 1/5(w) 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5
0/4 2/4 2/4 0/4 2/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
. 0/8 8/8 8/8 0/8 8/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8
A=Moraxella lacunata AI82T; B=Moraxella boris CIP 7039; C=Moraxella bovis CIP 7040T; D=Moraxella bovis ATCC 17948; E = Moraxella nonliquefaciens CIP 6836T; F = Branhamella ovis ATCC 33078; G = Branhamella ovis CIP 7320T; H = Branhamella cuniculi CIP 7317T; I=Branhamella caviae 7319T; group 1 =Moraxella bovis (88363A, 88363B, 88364A, 88364B, 88365, 88366, 88369); group 2=Moraxella nonliquefaciens (8897, 8898, 88100, 88101, 88103, 284, J132, J134); group 3 (88102, 88105, 88153, 88155, J83) and 4 (281, 282, 283,285) = Branhamella spp. *No. positive/No, tested; w = weak reaction.
strain o f Moraxella boris C I P 7 0 3 9 s h o w e d a m i l d n a p h t o l p h o s p h o h y d r o l a s e activity. R e f e r e n c e s t r a i n s o f Branhamella spp.: Branhamella ovis C I P 7 3 2 0 T , A T C C 3 3 0 7 8 , Branhamella caviae 7 3 1 9 T , Branhamella cuniculi 7 3 1 7 T a n d Moraxella lacunata had, in addition, a m i l d a l k a l i n e p h o s p h a t a s e activity. All o t h e r s u b s t r a t e s w e r e n o t h y d r o l y s e d b y a n y strain.
3.2. Agglutination tests A u t o a g g l u t i n a t i o n w a s o b s e r v e d w i t h Moraxella boris C I P 7 0 3 9 a n d all t h e s t r a i n s o f g r o u p 1 a n d t w o Branhamella ( g r o u p 3) field strains, d - M a n n o s e d i d not i n h i b i t this a u t o a g g l u t i n a t i o n , w h e r e a s 1 0 % M g C I did. Haemagglutination was observed only on rabbit red b l o o d cells, b u t n o t o n c h i c k e n n o r s h e e p r e d b l o o d cells. P o s i t i v e r e a c t i o n w a s o b t a i n e d w i t h Moraxella
boris C I P 7 0 3 9 a n d A T C C 17948, Branhamella ovis C I P 7 3 2 0 T , A T C C 33078, Branhamella cuniculi CIP 7317, Branhamella caviae 7 3 1 9 a n d five Branhamella ( g r o u p 3) field strains. N o h a e m a g g l u t i n a t i o n w a s o b s e r v e d w i t h Moraxella field isolates a n d o t h e r Moraxella boris c o l l e c t i o n strains w i t h the e x c e p t i o n o f the t w o cited a b o v e ( T a b l e 2 ) .
3.3. Experimental pathogenicity
O n l y m i c e i n f e c t e d via c o r n e a l r o u t e w i t h Moraxella bovis C I P 7 0 3 9 a n d c a p r i n e isolate 8 0 3 6 3 A e x h i b i t e d a transitory p h o t o p h o b i a w i t h i n 2 d a y s after i n o c u l a t i o n ( T a b l e 4 ) . N o o t h e r clinical s i g n s w e r e r e c o r d e d a n d i n o c u l a t e d strains w e r e n e v e r r e c o v e r e d f r o m a n y organ. H e m a t o l o g i c a l a n d b i o c h e m i c a l p a r a m e t e r s w e r e n o r m a l in i n o c u l a t e d g u i n e a pigs c o m p a r e d to
2
206 47 <0.5
124 51 < 0.5
100 46.8 < 0.5
667
NT
o/5
5 mice i.c. _
NT
o/5
63 0.6
0
76.3 30 6.8 35.7 0.5
2 48.6 6.6 15.3
135 49 co.5
512.3
o/3
3 guinea pigs i.p.
ovis CIP 7320T
5 mice i.p.
Branhamella
*Bach animal was given 1 ml of a PBS suspension containing approx. lo9 CPU per ml by intra peritoneal route (i.p.) or comeal swabbing and instillation (i.c.) in each eye solely on mice. NT = not tested; ESR = erythrocytes sedimentation rate; RBC = red blood cells; MCV = mean corpuscular volume; MCHC = mean corpuscular hemoglobin concentration.
Urea (mg/loO ml) Creatinin mg/ 100 ml)
Biochemical parameters Glucose (mg/ 100 ml)
Platelets ( X 103/mm3)
412.3
0
monocytes
RBC (x 106/mm3) Hemoglobin (g/ 100 ml) MCV ($) MCW (g/100 ml) Leukocytes ( X 103/mm3) neutrophils (96) eosinophils (%)
0 62 1
48.5 6.4 15.3
0 56 1.5 654.6
NT
0 69.5
NT
basophils lymphocytes
2 51
6.3 36.5 0.5
NT
o/3
3 guinea pigs i.p. _
8.6 42 0.5
NT
5 mice i.p. o/5
6 30 0.5
1.5 50.5
5 mice ic. photophobia o/5
bovis 88363A (caprine strain)
Moraxella
74.5 31.5
NT
photophobia o/5
_ o/2
5 mice 3 guinea pigs i.p. i.p. o/5 o/3
bovis CIP 7039
6.7 15.4 75 30.1
NT
o/5
o/5
5 mice i.c.
Morarella
2 guinea pigs i.p.
strains
6.5 15.2 77.3 30.1
(means)
5 mice i.p. _
5 mice i.c. _
Control (PBS)
and Brunhamella infectivity of MoraxelZu
No. animals* Administration route Clinical signs No. deaths Hematology parameters ESR (mm in 2 h) Hematocrit
Table 4 Experimental
7 ;5:
S ,Q
2
R L Q S z
f
z 2 2
S
P * g 8. z R
A. Kodjoet aL / Small RuminantResearch 15 (1994)87-95
control group. Post-mortem and testicular histological examinations revealed no pathological disorders.
4. Discussion
This study showed the existence of bacteria from the genera Moraxella and Branhamella in the nasal bacterial flora of sheep and goats. These bacteria exhibited relatively homogeneous cultural and biochemical characteristics in which no carbohydrates were assimilated, whatever the system used (conventional or micromethod tests). This agrees with Catlin (1976; 1991) who proposed the genera Moraxella and Branhamella to constitute the family of Branhamaceae due to their asaccharolytic properties. Within the genus Branhamella, strains of groups 3 and 4 were suspected to be Branhamella ovis regarding their coccal morphology but their assignation to a physiologically known species was uncertain, because they did not fit the precise characteristics of the collection strains of Branhamella studied. Nevertheless we believe that these two groups could be identified as two biovars ofBranhamella ovis, even if negative nitrate reductase strains of Branhamella ovis have never been reported. Experimental infectivity of Branhamella ovis has been clearly demonstrated by Elad et ah (1988) to be very low which agrees with our test on laboratory animals. The common occurrence ofBranhamella ovis in the normal eyes of goats has also been reported by Pitman and Reuter (1988). Cases of conjunctivitis in sheep or cattle due to these bacteria seem to be concurrent with other microbial infections, (Rosenbusch, 1983; Elad et al., 1988). We identified diplobacilli with intermediary features between Moraxella lacunata (nitrate was reduced) and Moraxella boris (hemolysis occurred on sheep blood), solely on goat nasal swabs. Such strains bore fimbriae probably responsible for their autoagglutinating properties. In the same way, they produced the same hydrolytic enzymes as Moraxella boris collection strains. There is little information available in the literature concerning these tests and our findings were in agreement with those of Frank and Gerber ( 1981). However, in our study, the Moraxella boris reference strains only showed a weak reaction with acid phosphatase. Such phosphatases may be allergenic and contribute together with different enzymes or toxins production to the path-
93
ogenicity of Moraxella bovis (Frank and Gerber, 1981). Pathogenicity oflBK is still obscure. Only hemolytic and piliated strains of Moraxella bovis could induce IBK (Pugh and Hughes (1968), Lepper and Power (1988) ), and one of the initial steps in the pathogenesis of the disease is the adsorption on the cornea due to the fimbriae of the bacteria. Such features were also detected on the intermediary goat field strains of Moraxella bovis, but both Moraxella bovis reference strains CIP 7039, a virulent known strain and the caprine isolate 80363A failed to induce the complete clinical signs of IB K, when administred to mice or guinea pigs except a mild photophobia on mice inoculated by corneal swabbings and instillations. We believe that this photophobia was due to the adsorption of the fimbriae on the surface of the cornea, but the other steps following the primary adsorption of bacteria on cornea, usually occurring in IBK pathogenesis, were not observed, probably because of lack of enhancer factors such as concurrent microbial infection, corneal damage due to radiation or fly irritation. Therefore, we did not observe any corneal ulceration or discharge. Many workers have attempted with variable success to induce IBK, either on calves or on laboratory animals, particularly on mice, and the success of this experimentally induced IBK depends on preliminary corneal damage either by ultraviolet radiation (Pugh and Hughes, 1968) or microbial agents such as Mycoplasma (Rosenbusch, 1983). Other workers also reported success only after corticosteroid administration (Lepper and Power, 1988). Working in a specific pathogen-free area could explain our unsuccessful attempt. This result agrees with Lepper and Power (1988), who also failed to reproduce IBK on non-corticosteroid-treated mice with full virulent strains of Moraxella bovis. This study on Moraxella strains, isolated from small ruminant nasal swabs, shows the existence of a new biovar of MoraxeUa boris in the nasal flora of healthy goats. Lack of biochemical conventional tests to differentiate the nonfennenter Moraxella and lack of available laboratory models for IBK reproduction indicate the need of other tests to differentiate this new biovar from Moraxella bovis. Other sophisticated tests as genetic transformations (Hoke and Vedros 1982; Bovre, 1984; Juni and Heym, 1988) or DNA/DNA hybridizations (Tonjum et al., 1989; 1993) have indi-
94
A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95
cated that Moraxella boris, Moraxella lacunata and MoraxeUa nonliquefaciens shared strong interspecific genetic homologies. Works on ribosomal RNA vs. DNA hybridization (Rossau et al., 1991) have confirmed these findings and proposed these three species to constitute the lacunata group. The lack of conventional characteristics and genetic homologies among strains of the lacunata group represent, therefore, a strong handicap for identification of Moraxella strains based upon traditional parameters. Our Moraxella boris isolates, intermediate between Moraxella boris and Moraxella lacunata, could be a new Moraxella species or a Moraxella boris subspecies. Such a haemolytic strain was described and named MoraxeIla lacunatus by Witers and Davis (1961) in a conjunctivitis outbreak in a cattery. Since that date there is no available information about such particular hemolytic 'Moraxella lacunatus' strain. Taxonomic studies would therefore be of interest to determine whether or not it is a new Moraxella species.
5. Conclusion Moraxella-like bacteria have been isolated from healthy sheep and goat nasal swabs. A new biovar of Moraxella boris isolated from healthy goats on farms with no IBK history has been characterized. The biochemical, constitutive enzymes and infectivity patterns of this biovar were about the same as for the Moraxella boris collection strains used in the study except the ability to reduce nitrate.
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A. Kodjo et al. / Small Ruminant Research 15 (1994) 87-95 chrobacter and related organism. Int. J. Syst. Bacteriol., 41: 310319. Tonjum, T., Bukolm, G. and Bovre, K., 1889. Differentiation of some species of Neisseriaceae and other bacterial groups by DNADNA hybridization. APMIS, 97: 395-405. Tonjum, T., Caugant, D.A., and Bovre K., 1993. Differentiation of Moraxella nonliquefaciens, Moraxella lacunata, and Moraxella bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes. J. Clin. Microbiol., 30: 3099-3107. Travicek, M., Dravecky, T. and Balascak, J., 1982. Isolation of Chlamydia psittaci and Moraxella boris from infectious keratoconjunctivitis in lambs. Vet. Med., Praha, 27: 491-496. Vedros, N.A., 1984. Neisseria. Bergey's Manual of Systematic Bac-
95
teriology. The William & Wilkins Co., Baltimore, pp. 290-296. Varga, J., Fodor, L., Hajtos, I. and Ratz, F., 1987. Haemagglutination by Neisseria ovis isolated from the eyes of sheep. J. Vet. Med. B, 34:211-215. Wilcox, G.E., 1970a. Bacterial flora of the bovine eye with special reference to the Moraxella and the Neisseria. Aust. Vet. Rec., 46: 153-257. Wilcox, G.E., 1970b. An examination of Moraxella and related genera commonly isolated from the bovine eye. J. Comp. Pathol., 80: 65-74. Witers, A.R. and Davies, E.M, 1961. An outbreak of conjunctivitis in a cattery (caused by Moraxella lacunatus). Vet. Rec., 75: 856--857.