tion in several biochemical test media. (1,2). These organisms frequently grow poorly on enriched media and several tests, particularly the indole reaction are ...
An Evaluation of the API ZYM System as a Means of Identifying Haemophilus somnus and Related Taxa S.C. Groom, M.J. Hazlett and P.B. Little*
ABSTRACT
entre H. somnus et d'autres genres, mais il suggera une relation etroite
The commercially available API ZYM microbiological identification system was evaluated for the
avec celui de trois microorganismes semblables. L'activite enzymatique des souches de H. somnus s'avera rapid identification of Haemophilus similaire a celle des bacteries identisomnus. Eighty-seven isolates of the fiees comme Histophilus ovis et organism had API ZYM profiles Haemophilus agni, ainsi qu'a celle de which were characteristic. The souches UQV d'Actinobacillus actiAPI ZYM profiles demonstrate clear noides et d'A ctinobacillus seminis; elle differences between H. somnus and se revela toutefois clairement difother genera but suggest a close ferente de celle de certaines souches de association to three related organisms. Pasteurella haemolytica, Pasteurella Enzyme activity of H. somnus isolates multocida, Bordetella bronchiseptica were similar to organisms identified as et du groupe EF4. Le systeme Histophilus ovis, Haemophilus agni API ZYM permit d'indentifier plus and strains UQV of Actinobacillus rapidement H. somnus que les actinoides and A ctinobacillus seminis epreuves biochimiques conventionbut was clearly different from isolates nelles; il pourrait meme constituer une of Pasteurella haemolytica, Pasteu- aide aux methodes conventionnelles rella multocida, Bordetella bronchi- utilisees pour l'identification rapide septica and group EF4. The API ZYM des souches de H. somnus. Le test ne system allowed more rapid identifica- presenta pas de differences manifestes tion of H. somnus than conventional entre les souches, independamment biochemical tests and may be a useful des organes d'oiu on les avait isolees. adjunct to conventional methods used for identification of H. somnus Mots cles: Haemophilus somnus, isolates. The test did not reveal activite enzymatique, systeme obvious differences between isolates API ZYM. from various anatomic locations.
Key words: Haemophilus somnus, enzymatic activity, API ZYM sys-
INTRODUCTION
tems.
RESUME Cette experience consistait 'a evaluer le systeme didentification microbiologique API ZYM commercial, comme moyen d'identifier rapidement Haemophilus somnus. Le profil API ZYM de 87 souches de cette bacterie se revela caracteristique et demontra des differences evidentes
Haemophilus somnus is a Gramnegative poorly fermentative coccobacillus which requires high CO2 tension and enriched media for primary isolation (1). Haemin (X factor) and NAD (V-factor) are not required for growth (2), suggesting that it is improperly classified as Haemophilus (3). Antigenic, biochemical and cultural properties have been examined in detail and the exact taxonomic position of H. somnus is confused by
similarities to other bovine and ovine pathogens, particularly Haemophilus agni and Histophilus ovis (2). Haemophilus somnus is frequently associated with a variety of bovine disease syndromes such as thromboembolic meningoencephalitis (4,5), pneumonia (6), abortion, vaginitis, endometritis (7,8) and has also been isolated from the prepuce and semen of clinically normal bulls (9,10). Strain differences in virulence have been demonstrated (9,1 1) but virulence factors and markers have not yet been identified. Traditionally, identification of H. somnus isolates requires 48 h incubation in several biochemical test media (1,2). These organisms frequently grow poorly on enriched media and several tests, particularly the indole reaction are difficult to interpret (2). Additional procedures generally not available in diagnostic laboratories are frequently required for definitive identification (1,2,12,13,14,15). It would be desirable, therefore, to have a rapid, dependable and readily available method of identifying H. somnus. The ability to distinguish between isolates from different anatomic sources would aid in understanding the various diseases caused by H. somnus. The API ZYM system is a rapid semiquantitative procedure which allows the detection of 19 enzymatic reactions. The API ZYM system has been evaluated using animal patho-
including Corynebacterium equi (16) and Treponema spp. (17). It has also been employed to classify human pathogens such as oral streptococci
gens
(18), oral actinomycetes (19) and a variety of Gram-negative aerobic and anaerobic rods (18,20,21,22,23,24).
*Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N I G 2W I. Present address of Dr. Hazlett: Veterinary
Laboratory Services, Ontario Ministry of Agriculture and Food, Box 3612, Guelph, Ontario NIH 6R8. Submitted April 29, 1985.
238
Can J Vet Res 1986; 50: 238-244.
TABLE I. Sources and Site of Isolation of the Strains Used. The Total Number of Each Type is Enclosed in Brackets Organism, Strain and Site of Isolation H. somnus Bovine pneumonia (43) 51111; 51130; 51136; 51150; 51747; 51788; 51811; 51855; 51858; 52574; 52688; 53108; 70986; 71898; 72215; 73052; 73118 - 22-83; 416-82; 611-82 6110-83; 6489-83; 6519-83; 6651-83; 6711-83; 6743-83; 6791-83; 6948-83; 7082-83; 7112-83; 7226-83; 7323-83; 7460-83; 7484-83; 7684-83; 7704-83; 7768-83; 7816-83; 7939-83; 7953-83; 7965-83; 7999-83; 9394-83 Bovine encephalitic (20) 43826 23-22; 71; 51372 2426 5819-81; 6728-83; 6988-83; 7286-83; 7291-83; 7509-83; 7606-83; 7653-83; 7735-83; 7754-83; 7757-83; 7807-83; 7809-83; 7929-83; 7964-83 Bovine seminal and preputial (18) 10-2; 10-4; 10-15; 10-16; 11-15; 1 1-16; 14-3; 15-16; 17-17; 25-1; 26-16; 70-1; S-1; S-5; S-70; S-71 139-55-79; 49KB2-78 Bovine abortion and infertility (6) - 78-95, 79-341, 79-566, 79-1360, 80-1096, 83-145 Strain UQV 178+179 (Actinobacillus actinoides) (2) - UQV 178 Bovine lung - UQV 179 Bovine lung Histophilus ovis (5) 70959 ovine septicemia 83-4572 ovine epididymitis 43803 ovine brain
Source
H-989 ovine blood -9L ovine vagina Haemophilus agni (1) - 1314 ovine septicemia Actinobacillus seminis (2) - ATCC 15768 ovine semen - AST ovine epididymis
Riffkinh
-
Untypable (1) - 51947 bovine pneumonia Pasteurella haemolytica (4) -Jl, J2 bovine pneumonia - 53069, 73118 bovine pneumonia Pasteurella multocida (3) -J3 bovine pneumonia - 53008, 53118 bovine pneumonia Bordetella bronchiseptica (2) - 562-82, 1586-82 swine rhinitis EF4 (1) bovine pneumonia
own isolates'
Sidolib
Rawluk'
investigated. MATERIALS AND METHODS BACTERIA
Stephens own isolates
Biberstein' Rawluk
Humphrey' Rawluk Rawluk
University of
Queensland' own isolate Rawluk Stephens & Humphrey
Higgins' Biberstein
ATCC' Headlamk own isolates
PrescottI own isolates
Prescott own isolate
Prescott own isolate
aIsolated by the authors
bL. Sidoli, Istituto Zooprofilattico, Sperimentale della Lombardia, Emilia Sezione di Parma, Via dei Mercati, Parma, Italy
CS. Rawluk, Veterinary Services Laboratory, Edmonton, Alberta, Canada dL. Stephens, Pathology Department, University of Guelph, Canada
The present study was undertaken to evaluate the ability of the API ZYM system to identify isolates of H. somnus and differentiate them from closely related organisms and a variety of unrelated genera. The capability of the system to distinguish between H. somnus isolates from different anatomical sources was also
'E.L. Biberstein, Department of Veterinary Microbiology, University of California, Davis, USA fJ. Humphrey, Pathology Department, University of Guelph, Canada 'Animal Pathogen Culture Collection, Department of Veterinary Pathology and Public Health, University of Queensland, Australia, Courtesy of M.D. Mutimer hG. Riffkin, Department of Agriculture, Regional Veterinary Laboratory, Hamilton, Australia
Table I lists the study strains, the site of isolation, and the source from which they were received. Our own isolates were tentatively identified biochemically as previously described (1) and by immunofluorescence (10). Immunodiffusion was used as the definitive test on isolates tentatively identified as H. somnus (12). Isolate 51947 was included because it was the only organism tentatively identified as H. somnus which was excluded from the group based on the immunodiffusion test. The organism was untypable. All cultures were inoculated into the yolk sacs of seven day old embryonated eggs. After 24 h of incubation yolks were harvested and stored at -70°C. GROWTH CONDITIONS
Bacteria from thawed egg yolk were initially grown on brain heart infusion agar (Difco Laboratories, Detroit, Michigan) supplemented with 5% bovine blood and 0.5% yeast extract (BHIBYE). All cultures were routinely incubated at 37°C in 10% CO2 in air for 48 h. Cultures were transferred to supplemented proteose peptone agar (PPA) as previously described (2) for use in the API ZYM test. API ZYM TESTS
The API ZYM system (API ZYM Analytab Products, Plainsview, New York) comprises enzymatic tests performed on dried substrates in cupules. The enzyme activities tested for are shown in Table II. Growth from one PPA plate was suspended in 2 to 3 mL of sterile distilled water to provide heavy bacterial suspensions with an absorbance of 1.0-2.0 measured at 550 nm in a spectrophotometer (Coleman
239
language. The results were presented as a similarity matrix following previously outlined principles (25).
'R. Higgins, Departement de Pathologie et Microbiologie, Faculte de medicine VWtfrinaire, Universite de Montreal, Saint-Hyacinthe, Quebec, Canada 'American Type Culture Collection, Rockville, Md., USA kp. Headlam, Department of Agriculture, Attwood Veterinary Research Laboratory, Westmeadows, Victoria, Australia 'J. Prescott, Department of Veterinary Microbiology and Immunology, University of Guelph, Canada
RESULTS
Reactions of the API ZYM were not affected by atmospheric conditions or the type of media used for culture of test organisms. Repeated tests of the API ZYM system for known H. somnus isolates exhibited little or no variation between testings. Saline controls frequently produced weak alkaline phosphatase, esterase lipase and phosphoamidase activity. The color intensity of the reactions varied to some extent with the density of the bacterial test suspension. The enzymatic profiles of the 108 isolates tested using the API ZYM are shown in Table III and Table IV. All isolates of Haemophilus somnus and the related taxa of H. ovis, H. agni, and strains UQV of A. actinoides
Ten H. somnus isolates were subculJunior 11, Model 6/20, Illinois). The turbidity was approximately equal to tured onto BHIBYE media and a McFarland No. 5 standard as retested to see if results were similar suggested by the manufacturer. Each when the bacteria were grown on a cupule was inoculated with 0.04 mL of different medium. The same ten bacterial suspension. Because prelimi- isolates were tested twice at different nary studies showed no differences times and at different densities of between API ZYM tests incubated bacterial test suspensions to evaluate under normal atmospheric conditions repeatability of tests. Distilled water or under 10% CO2 in air, tests were flooded on the surface of noninocucarried out under normal atmospheric lated agar plates was collected for use conditions. After incubation at 37°C as controls. The organisms were compared by for four hours in the dark, 0.02 mL each of reagent A and B was added to calculating the percentage similarity each cupule following the manufactur- between isolates using an ATARI ers directions. The reactions were Computer (Atari Co. Ltd., California) allowed to develop for five minutes and a program written in basic and were exposed for 20 seconds under a 750-watt lamp at a distance of 10 cm to remove nonspecific yellowing. The reactions were blindly graded 0-5 according to the color intensity compared to the manufacturers 2 interpretation scheme. Tests given grade 0 and I were regarded as G 3 95III negative and 2, 3, 4 and 5 were considered moderate to strong reacR 4 995 tions and were regarded as positive. 9585 0 n i pm %
.
190
U
TABLE II. Enzyme Activity Evaluated Using API ZYM System No. I
2 3 4
5 6 7 8 9
10 II 12 13 14
15 16 17 18 19
20
2AO
Enzyme Assayed For Control Phosphatase alkaline Esterase (C4) Esterase Lipase (C8) Lipase (C14) Leucine aminopeptidase Valine aminopeptidase Cystine aminopeptidase Trypsin
Chymotrypsin Phosphatase acid Phosphoamidase alpha galactosidase beta galactosidase beta glucuronidase alpha glucosidase beta glucosidase N-acetyl-beta-glucosaminidase alpha mannosidase alpha fucosidase
p
6 90i 95 95 01901
85185 -,r- 90E
m
-
85185 90190 U 8 85L90 90 80 95195185 .
85 90180 95
N
9
U M
10r85!
85 95 90f
-9 901801 9018Sf 9.5 85 90f 80
_
i t ]1~~~~ 1
0
ML-
@
@
--.
r --_ I
80 85 85,75i 990.90] 95i95 85 .
-
B 12~ 801 85 E 13 801 85 R T4AL751 80 S 701 75 _
T
-.-
161
1 12
Mo.-
85 75j 901 95 1I 85 8Sf 90E 75 75 801__ 80 90 75j 87 18 85U080 70j 85 85 85 90 90 80] 9 85W ml-75 6S 80 8080j 8S 85 75f 90 801 70f 85 7 n7575t651Qnr70 60_5_9 i ,
901801
I
I ,-
I
t~~~~~~~~~~~~~~~~~~~1
6 G R 0 U P
3 14 I5
7
,,
18 9 10 U'12.113 N UMB E R
I
I
14115,,16 It
Fig. 1. Similarity matrix of organisms grouped by the API ZYM system (see Table IV).
(26) characteristically exhibited strong leucine aminopeptidase and Bglucuronidase activity. Four other enzymes often demonstrated in this group were acid and alkaline phosphatase, esterase-lipase, and phosphoamidase. Actinobacillus seminis exhibited a similar pattern to H. somnus isolates and thus was not distinguishable from it. The untypable isolate 51947 and group EF4 were clearly different from H. somnus, producing a greater number of enzymes. Pasteurella spp. and Bordetella bronchiseptica demon-
strated the same enzymes as H. somnus, except for complete absence of B-glucuronidase. Bordetella bronchiseptica also produced chymotrypsin. The percentage similarity between groups in Table IV is illustrated in Figure 1.
DISCUSSION Although traditional criteria used for the identification of H. somnus (1) and taxonomic classification (2,27)
have been reported previously, little is known about the enzyme activity of this and closely related organisms. The API system has successfully been utilized to produce an enzymatic profile useful in the identification of a number of animal pathogens (16,17). This information has also been utilized in the taxonomic classification and virulence evaluation of these organisms (16,17). Our results indicate that the system may be a useful adjunct for identification of H. somnus and for distinguishing it from
TABLE III. Enzymatic Characterization of H. somnus and other Test Organisms by the API ZYM System
2 H. somnus pneumonia
11 11 6 4
3 2 2 2 Ia
3
43 6 6 3 2
6
7
8
9
10
++
+ +
++
+ +
+
11
12
13
14
15
+
+
+
+
+
+
+
16
17
18
19
20
+
+
+ +
+
+
+
+
+
+
+
+
+
+ + +
+
+
+
+
+
+
+
I
5
+ 4-
Ia
encephalitis
4
+
+
+
l
+ +
+
+
+
+
+
+
+
20 seminal
7 4 3 2
+
+
+
+ + + +
+
+
+
+
+
+
+
+
1
abortion/ infertility
H. ovis
UQV
+
+
2 2
6 5 2
H. agni A. seminis
+
+
+
+
+ +
+
++
+
+
+
+
+
+
+
+
+ +
+ +
P. haemolytica P. multocida Bordetella spp.
EF4 51947 aItalian strains, L. Sidoli
4
+
+
+
3 2
+ + +
+
+
+
+
+
+ +
+
+
+
+
+
+
+
241
TABLE IV. Grouping of Organisms by Enzyme Demonstration with the API ZYM System
1
Positive Tests 6,15
2
6,11,15
3
2,6,15
4
6,15,20
5
2,6,11,15
6
6,11,12,15
7
4,6,11,15
8
2,6,11,12,15
9
2,4,6,11,15
10
6,11,12,15,20
11
2,4,6,11,12,15
12 13 14 15 16
2,6,11,12 4,6,10,11 2,4,6,11,12 2,3,4,6,11,12,14,15 2,3,4,6,11,12,14,15,16,18
Group
other common Gram-negative bacterial pathogens. Haemophilus somnus produced seven enzymes useful in identification of the organism (Table III). The reactions were frequently weak but were constantly repeatable. The number of enzymes produced by H. 242
Isolates (Number) H. somnus (23) pneumonic (11): 6110-83, 6711-83, 6743-83, 7112-83, 7226-83, 7965-83, 52574, 53108, 71898, 73052, 73118 encephalitic (6): 71, 7754-83, 7757-83, 7929-83, 7964-83, 51372 seminal and preputial (4): 11-16, 14-3, 49KB2-78, S71 abortion (2): 79-566, 79-1360 H. somnus (16) pneumonic (11): 7460-83, 7484-83, 7953-83, 7999-83, 51111, 51130, 51136, 51855, 51858, 52688, 72215 encephalitic (2): 7286-83, 7807-83 seminal and preputial (3): 11-15, 15-16, 139-55-79 H. ovis (5): 83-4572, 43803, 70959, H-989, 9L H. agni (1): 1314 H. somnus (2) seminal and preputial (1): 17-17 abortion (1): 79-341 H. somnus (1) pneumonic (1): 22-83 H. somnus (20) pneumonic (6): 6489-83, 7684-83, 7704-83, 7816-83, 7939-83, 9394-83 encephalitic (6): 5819-81, 6988-83, 7291-83, 7606-83, 7735-83, 7809-83 seminal and preputial (7): 10-2, 10-4, 10-15, 10-16, S-I, S-5, S-70 abortion (1): 78-95 A. actinoides (2): UQV 178, UQV 179 A. seminis (1): AST H. somnus (4) pneumonic (2): 51747, 51811 encephalitic (1): 2426 seminal and preputial (1): 26-16 H. somnus (2) pneumonic (2): 6651-83, 7768-83 H. somnus (8) pneumonic (3): 6519-83, 6948-83, 70986 encephalitic (1): 43826 seminal and preputial (2): 25-1, 70-1 abortion (2): 80-1096, 83-145 H. somnus (7) pneumonic (4): 6791-83, 7082-83, 7323-83, 51150 encephalitic (3): 6728-83, 7509-83, 7653-83 H. somnus (1) pneumonic (1); 416-82 H. somnus (3) pneumonic (2): 611-82, 51788 encephalitic (1): 23-22 A. seminis (1): ATCC 15768 P. hemolytica (4): 53069, 73118, J 1, J2 B. bronchiseptica (2): 562-82, 1586-82 P. multocida (3): 53008, 53118, J3 Untypable (1): 51947 EF4 (1) EF4
somnus isolates varied considerably but was restricted to the same seven enzymes. The usefulness of this system in identifying H. somnus was demonstrated when compared to the profile of isolate 51947 which was culturally, morphologically and biochemically identified as H. somnus but was shown
to be different antigenically using the gel immunodiffusion tests. Conventional biochemical identification procedures are frequently difficult to perform and interpret. Antigenic identification is not readily available to diagnostic laboratories. The API ZYM system is available com-
mercially and may prove useful in identification of H. somnus. A close relationship between the enzymatic profile of H. somnus and the organisms H. ovis, H. agni, strains UQV of A. actinoides and A. seminis was demonstrated using the API ZYM system. This group of organisms produced six enzymes produced by H. somnus and had similar negative reactions. Based on the enzymatic profile there is no clear difference between these organisms and the 87 known isolates of H. somnus tested. Previous investigators have shown that H. somnus and these organisms are similar if not identical (2). The use of the API ZYM test confirms the close relationship of these organisms. The API ZYM system clearly distinguished other genera from H. somnus. All genera including P. haemolytica, P. multocida, B. bronchiseptica and group EF4 had enzymatic reactivity which differed from H. somnus. These organisms lacked enzymes or produced enzymes not characteristic for H. somnus. The data presented in Table IV demonstrates that most H. somnus isolates are arranged in three major groups (groups 1, 2 and 5) with no pattern suggesting a difference between strains isolated from different anatomical sites. The organisms H. ovis, H. agni, strains UQV and A. seminis are closely associated with the H. somnus organisms. Remaining genera, P. haemolytica, P. multocida, B. bronchiseptica and group EF4 formed uniform and clearly separate groups from H. somnus isolates. The API ZYM test was not able to detect differences between H. somnus isolates from different anatomic sites. Seminal isolates recovered from clinically normal bulls and isolates associated with abortion/ infertility had complete absence of esteraselipase activity. Isolates from sources associated with clinical cases of encephalitis and pneumonia had weak and inconsistent activity of this enzyme. This study did not identify an enzyme which may be used as a marker of virulence. Pathogenic organisms did not possess different complements of enzymes than isolates from clinically normal animals. Two of three Italian strains tested were the only isolates to produce alpha fucosi-
dase, however the general usefulness of this enzyme for identifying possible biotypes of H. somnus was not demonstrated in this study. The results of this study illustrate that the API ZYM system produces a variable but identifiable pattern of enzyme activity for H. somnus. This system provides another parameter for identifying H. somnus and closely related genera. Little difference was demonstrated between H. somnus, H. ovis, H. agni, strains UQV of A. actinoides and A. seminis. The test could not reliably distinguish between isolates of H. somnus from different anatomic sites or type of secretions. The system is rapid and readily available, and reproducible using organisms grown on different media. Standardizing bacterial suspension to a high density is important for reproducibility.
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