Rev. sci. tech. Off. int. Epiz., 1998,17 (3), 699-712
A r e v i e w of leptospirosis in farm animals in Portugal T. Rocha Research Assistant, Bacteriology Department, Laboratório Nacional de Investigaçâo Veterinària, Estrada de Benfica, 701,1500 Lisbon, Portugal
Summary This paper presents a review of Leptospira infection in farm animals in Portugal which is based mainly on serological results obtained in the National Veterinary Research Laboratory between January 1987 and December 1993. Serum samples were tested by the microscopic agglutination test, at a minimum dilution of 1:100. Positive titres were obtained in 15.3% of the 9,543 bovine samples examined. Sejroe, Pomona, Hebdomadis, Tarassovi and Icterohaemorrhagiae were the principal serogroups which reacted in the tests. A total of 3,195 pigs were tested, of which 20.2% showed positive reactions. The main serogroups which reacted were Australis, Pomona, Cynopteri, Icterohaemorrhagiae and Autumnalis. Field observations on outbreaks of leptospirosis in pigs, along with data obtained from an experimental infection with serovar mozdok in pregnant gilts suggest that this serovar, rather than serovar pomona, may be causing Pomona group infections in pigs. Serum samples from 5,298 sheep were tested and 3.3% gave positive results. The predominant serogroups involved were Canicola, Pomona, Cynopteri, Sejroe and Icterohaemorrhagiae. From the 1,631 goats examined serologically, 5.0% gave positive results, mainly to serogroups Grippotyphosa, Canicola, Pomona, Icterohaemorrhagiae and Pyrogenes. Only 83 serum samples from horses were obtained, of which 43.4% showed positive titres. Serogroups Australis, Autumnalis, Cynopteri and Pyrogenes were those most commonly found. Serological evidence of leptospirosis in farm animals is widespread in Portugal, particularly in cattle and pigs. Leptospirosis in horses needs to be studied further. In an attempt to provide a general view on the occurrence of leptospirosis in these animal species in Portugal, the present results are compared with results obtained in previous studies and are complemented with both previous and recent bacteriological findings.
Keywords Animal health - Leptospirosis - Portugal - Serological techniques.
Introduction The study of animal leptospirosis was neglected in Portugal for several years after the inquiries made by Azevedo in 1966 (1) and Azevedo and Palmeiro in 1972 (6). In 1985, the Laboratório Nacional de Investigação Veterinária (LNIV) (National Veterinary Research Laboratory) established a new
section for the diagnosis of leptospirosis. Since then, efforts have been made to update the information on the occurrence of this zoonosis in animals, namely by conducting serological surveys on several animal species. The first new surveys, performed in collaboration with the Institute of Hygiene and Tropical Medicine, Lisbon, in 1986 and 1987, involved the analysis of serum samples from
Rev. sci. tech. Off. int. Epiz., 17 (3)
700
apparently healthy brucellosis seronegative cattle (8, 2 8 ) . Subsequent research included further serological studies in cattle (25), pigs, sheep and goats (T. Rocha, unpublished data). The LNIV is the only existing veterinary laboratory in Portugal that performs diagnosis of animal leptospirosis, besides a unit established in the Azores Regional Veterinary laboratory for the analysis of local animals, which has been in operation since 1993. The leptospirosis section is located in Lisbon and serves the entire country. The laboratory receives material for serological and bacteriological diagnosis of leptospirosis from several regions, which provides further information. This review presents both the more recent data and the data obtained through the studies by Azevedo and Palmeiro on leptospirosis in Portugal.
Materials and methods Between January 1987 and December 1993, a total of 19,750 sera from various regions of Portugal (Fig. 1) were tested at the LNIV, Lisbon, for leptospiral antibodies. This number does not include the sera examined in previously published surveys undertaken in 1986 and 1987 (8, 25, 2 8 ) . Of the samples tested, 5 9 . 3 % were diagnostic submissions (DS) while 4 0 . 7 % were obtained in abattoir surveys (AS). The sera incorporated a range of farm animals, as follows: 9,543 cattle (all DS), 3,195 pigs ( 4 6 . 4 % DS, 5 3 . 6 % AS), 5,298 sheep ( 7 . 2 % DS, 9 2 . 8 % AS), 1,631 goats ( 1 3 . 7 % DS, 8 6 . 3 % AS) and 8 3 horses (all DS). Sera were tested by the microscopic agglutination test (MAT) with an initial serum dilution of 1:100, using the following 18 live antigens, belonging to 16 serogroups: icterohaemorrhagiae, pomona, grippotyphosa, canicola, bratislava, hardjo, sejroe, saxkoebing, hebdomadis, mini, poi, celledoni, pyrogenes, autumnalis, ballum, bataviae, tarassovi and cynopteri. During the same period, a total of 4 0 0 bacteriological diagnostic submissions (mainly aborted foetuses) were analysed. The samples were obtained from a range of farm animals, comprising 112 cattle, 249 pigs, 30 sheep, 6 goats and 3 horses. The material submitted for bacteriological diagnosis of leptospirosis, which came from several regions, was processed as described earlier (26). The typing of the obtained isolates to serovar level was performed using monoclonal antibodies (30). The interpretation of the serological results took into account the fact that vaccination of farm animals in Portugal against leptospirosis was not practised until the end of this study. Thus, in accordance with Faine, MAT titres of 1:100 or greater were considered as diagnostic and indicative of the occurrence of a current or previous leptospiral infection (32).
A: Ftibatejo e Oeste B: Beira Litoral C: Entre Douro-e-Minho D: Trás-os-Montes E: Beira Interior F: Alentejo G: Algarve H: Terceira Island
Fig. 1 Regions of Portugal from w h i c h sera w e r e e x a m i n e d
For the purpose of the serological surveys it was not relevant to determine whether the seropositive animals were also carriers, but rather, according to Ellis, to use the MAT to determine those serogroups which may infect the above-mentioned animal species in the analysed geographical areas (9).
Results and discussion Previous surveys on the occurrence of leptospirosis in farm animals in Portugal, conducted by Azevedo in 1966 (1) and Azevedo and Palmeiro in 1972 (6), reported a low prevalence of infection and a reduced pathogenicity of the infecting serovars. Table I shows their results on the percentages of positive sera, belonging to farm animals in several regions, which were randomly collected in abattoir surveys.
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Table I Distribution of positive titres to
Leptospira
antibodies in sera c o l l e c t e d in abattoir surveys from farm a n i m a l s in 1966 and 1972
Sera from the 1966 abattoir survey
(1,6)
Sera from the 1972 abattoir survey
Species Cattle
No. tested 1.309
No. positive 6
Percentage 0.5
No. tested 1,133
No. positive 7
Percentage 0.6
Sheep
649
18
2.7
134
0
0.0
Goats
182
10
5.4
154
0
0.0
Pigs
610
36
5.9
NT
Horses
939
14
1.4
NT
NT: Not tested
Although Azevedo and Palmeiro claimed always to have used serovars icterohaemorrhagiae, pomona, grippotyphosa, canicola, ballum, tarassovi and sejroe in the MAT (5), the serological tests in the 1966 survey (1) were performed with only the six serovars then recognised in Portugal, that is:
due to contamination by other bacteria and to post-mortem changes. The material rarely reaches the laboratory in ideal aseptic and refrigeration conditions and within a short time after the death of the animal. Autolysis is known to greatly impair the viability of leptospires ( 3 1 ) , even if it occurs in the
icterohaemorrhagiae, pomona, grippotyphosa, canicola, ballum and tarassovi. However, the 1972 survey (6) was performed with an enlarged battery of antigens very similar to that used in the present paper and in more recent surveys of cattle sera in 1986 and 1987, except that serovars hardjo, mini, pyrogenes, celledoni (25, 2 8 ) , and later cynopteri (8), were additionally used in the later surveys.
uterus after the death of the foetus and prior to its expulsion. This has been observed in an experimental infection of three pregnant gilts with serovar mozdok (29), performed at the LNIV, which resulted in the abortion of a total of 22 piglets. Isolates were obtained from only 12 of the piglets, which corresponded to those that were b o m alive and those expelled dead but with no signs of autolysis. Temperature also affects the viability of leptospires; they survive well at 40°C but are rapidly killed in tissues held at 20°C, and at 30°C-40°C, a range of temperature at which dead foetuses are held before being expefled by their dams (11).
With regard to the bacteriological research on Leptospira, the majority of the existing studies in farm animals were performed by Azevedo and colleagues on kidneys randomly collected in abattoir surveys (1). The only more recent similar study was performed by Collares-Pereira in 1987 using cattle kidneys (7). At the LNIV, the cultivation of samples sent for bacteriological analysis of leptospirosis seldom yields positive results. Of the 4 0 0 analyses performed between 1987 and 1993 only 2 . 3 % gave positive results, while 1 5 . 3 % were discarded due to contamination by other bacteria and 8 2 . 5 % gave negative results. The low percentage of positive isolations does not necessarily correspond to a low prevalence of Leptospira infection in the analysed animals but rather, to a degree, is related to difficulties in isolating leptospires, mainly
The results of the present survey will be discussed for each animal species and then compared with those of previous surveys in an attempt to review the available information on the occurrence of leptospirosis in the given species in Portugal.
Leptospirosis in cattle Of the 9,543 DS bovine sera tested, 1 5 . 3 % gave positive results for leptospirosis and Leptospira infection was serologically detected in all the examined regions (Table II).
Table II Distribution of positive titres in cattle sera from diagnostic submissions (1987-1993) and comparison w i t h sera collected in abattoir surveys (1986-1987), by region ( 8 , 2 5 , 2 8 ) Sera from 1987-1993 diagnostic submissions No. tested Ribatejo e Oeste (A)
No. positive
Percentage
Sera from 1986-1987 abattoir surveys No. tested
No. positive
Percentage 19.8
5,808
766
13.2
561
111
Beira Litoral (B)
753
48
6.4
301
5
1.7
Entre-Douro-e-Minho (C)
281
43
15.3
190
24
12.6
Trás-os-Montes (D)
488
21
4.3
1,077
40
3.7
NT
-
-
1,145
33
2.9
Alentejo (F)
1,176
302
25.7
370
6
1.6
Algarve (G)
25
1
4.0
363
3
0.8
Beira Interior (E)
Terceira Island (H)
1,012
282
27.9
64
12
18.8
Total
9,543
1,463
15.3
4,071
234
5.8
NT: Not tested
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Rev. sci. tech. Off. int. Epiz., 17 (3)
Table III Distribution of positive titres in cattle sera from diagnostic submissions (1987-1993), by leptospiral serovar and region Positive titres by region Serogroup
Serovar
No. positive
Percentage A
B
C
D
E*
F
G
H
-
36 39 12
0 0 0
216 175 132
172
0
0
-
18
0
83
10
1
0
1
19 19
Sejroe
hardjo sejroe saxkoebing
737 417 224
30.5 17.3 9.3
452 188 61
8 0 0
14 15 6
11 0 13
Pomona
pomona
389
16.1
196
4
17
0
Hebdomadis
hebdomadis
176
7.3
67
1
1
6
Tarassovi
tarassovi
123
5.1
107
4
0
1
-
-
Icterohaemorrhagiae
icterohaemorrhagiae
85
3.5
27
29
7
1
-
1
Australis
bratislava
63
2.6
13
3
3
1
-
24
0
Cynopteri
cynopteri
58
2.4
13
0
6
0
39
0 .
mini
45
1.9
42
0
0
0
-
Mini
3
0
0
Canicola
canicola
36
1.5
21
2
4
2
-
3
1
3
2
0
3
-
0
0
15
-
0
0
0
-
0
1
1
0
0
0
-
0
0
0
0
0
1
Bailum
bailum
18
0.8
6
3
4
0
Autumnalis
autumnalis
16
0.7
0
0
1
0
Grippotyphosa
grippotyphosa
14
0.6
10
1
1
2
Pyrogenes
pyrogenes
10
0.4
3
1
0
4
Javanica
poi
2
0.1
1
1
0
0
Celiedoni
celiedoni
1
0.04
1
0
0
0
Bataviae
bataviae
1
0.04
0
0
0
0
-
-
-
0
* Not tested
Regions A, C, F and H (Fig. 1) presented the highest percentages of positive reacting sera, while region G showed a particularly low percentage. The predominant serogroups found (Table III) were Sejroe, Pomona, Tarassovi and Icterohaemorrhagiae. Multiple litres were often observed, especially between hardjo and related serovars (sejroe, saxkoebing, mini and hebdomadis), which are probably due to hardjo infections (27). Terceira Island (H) in the Azores exhibited the highest prevalence of positive sera ( 2 7 . 9 % ) , which agrees with the recognised importance of leptospirosis in that region (25; T. Rocha, unpublished data). However, Pomona and Tarassovi antibodies were not detected on the island, which contrasts with the significant prevalences found for these serogroups on the continent. The results agree with those of the 1 9 8 6 - 1 9 8 7 survey of apparently healthy cattle (8, 2 5 , 2 8 ) , in which the percentage of positive titres in 4,071 examined sera was 5 . 8 % (Table II) and was particularly high in regions A ( 1 9 . 8 % ) , C (12.6%) and H ( 1 8 . 8 % ) , as observed in DS sera. In both studies, the Sejroe group is the predominant serogroup (Tables III and IV). The main differences are the higher overall seroprevalence in region F (Table II) and the higher representation of Pomona (16.1%) and Tarassovi (5.1%) serogroups in DS sera, analysed between 1987 and 1993, while in the AS sera examined in the 1 9 8 6 - 1 9 8 7 survey, the highest prevalences other than the Sejroe group (Table IV) were observed in the Australis ( 6 . 3 % ) , Canicola (5.8%) and Pomona (5.3%) serogroups.
Serovar hardjo titres have proved to be the most frequently found in cattle. This serovar was isolated for the first time in Portugal from bovine kidneys in an abattoir survey conducted in 1987 (7). These findings are particularly significant considering the recognised importance of hardjo infection in cattle, which may cause serious outbreaks of mastitis and is a major cause of abortion and premature calving (21). Hardjo infections in
Table IV Distribution of positive titres in cattle sera collected in abattoir surveys (1986-1987), by leptospiral serovar ( 8 , 2 5 , 2 8 ) Serogroup
Serovar
Sejroe
No. positive
Percentage
saxkoebing hardjo sejroe
89 88 51
22.5 22.2 12.9
Australis
bratislava
25
6.3
Canicola
canicola
23
5.8
Pomona
pomona
21
5.3
Hebdomadis
hebdomadis
19
4.8
Mini
mini
19
4.8
Icterohaemorrhagiae
icterohaemorrhagiae
13
3.3
Pyrogenes
pyrogenes
11
2.7
Javanica
poi
9
2.3
Tarassovi
tarassovi
8
2.0
Grippotyphosa
grippotyphosa
8
2.0
Autumnalis
autumnalis
6
1.5
Ballum
ballum
6
1.5
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Rev. sci. tech. Off. int Epiz., 17 (3)
cattle have become established in many parts of the world
In the present study, a high prevalence of positive titres in DS
after first being recognised in the United States of America and
sera was also shown for serogroup Pomona (Table III). This
Canada in the 1960s ( 2 1 ) . Whether the 'apparent' spread to
serogroup has also demonstrated some importance in cattle
Australia, New Zealand and Europe was due to the movement
leptospirosis in Portugal. The high seroprevalence ( 2 5 . 7 % )
of cattle or to the advent of better diagnostic tests is uncertain,
observed in DS sera from region F (Table II) is mainly due to
although cattle from North America are definitely thought to
the occurrence of a serious outbreak of Pomona infection in
be responsible for the introduction of this serovar into
1990, which affected several cattle herds and resulted in a
Hungary ( 2 0 ) . Some of the first European reports of hardjo
high mortality rate in calves and considerable economic losses
infections occurred in the United Kingdom (UK), where Ellis
for farmers. Another Pomona group outbreak of a smaller
et al. reported bacteriological evidence of the role of hardjo in
dimension had already been observed in 1 9 8 5 in cattle herds
bovine abortion in 1976 ( 2 1 ) . Other European countries,
from this region, where animals presented symptoms such as
such as The Netherlands and Germany, have also reported
haemoglobinuria and sudden death of calves, and where other possible aetiological agents were excluded (8). Several
high prevalences of serovar hardjo in cattle (20).
similar isolated cases of Pomona infection were reported Considering the known origin and spread of serovar hardjo, it
around 1990 in some other regions, namely regions A, B and
is reasonable to assume that hardjo infection probably hardly
C. These outbreaks of Pomona infections tend to be observed
existed in Portugal prior to 1972, the year in which Azevedo
at varying time intervals, which is probably linked to
and Palmeiro conducted their last survey (6). In that study,
ecological relations in certain areas leading to the formation of
only two positive titres to the Sejroe group were found in
endemic foci of leptospirosis, as has been referred by Faine
1,133 AS cattle sera (6), and these are the first reference to the
(32), and to fluctuations in herd immunity. Leptospires may
detection of this serogroup in Portugal. The observed low
circulate in these areas for many years ( 3 2 ) , particularly those
prevalence contrasts with the high Sejroe group prevalences
belonging to the Pomona group, which is one of the most
reported in the present paper and already detected in previous
resistant groups of pathogenic leptospires (17). Cattle from
surveys of apparently healthy cattle undertaken in 1986 and
region F are at particular risk since in that region a large
1987 (8, 2 5 , 2 8 ) . There is no certainty as to when
hardjo
proportion of animals are husbanded outdoors, thus may
infections started but the results suggest that these infections
easily come into contact with other animals which may be
have been spreading in Portugal, as observed in other
carriers of leptospires, such as the populations of wild small
European countries, especially if the foflowing points are
mammals and pigs. The absence of regular control and
taken into account:
prophylactic measures against leptospirosis facilitates the
- the knowledge that cattle act as maintenance hosts for serovar hardjo (20)
reappearance of outbreaks. The results of the present study suggest that leptospirosis has
- the importation of cattle, without frontier control for this
increased in cattle in Portugal since the 1972 survey, in which
disease, from countries which are known to have high
the seroprevalence upon the examination of 1,133 cattle sera
prevalences of hardjo
was as low as 0 . 6 % (Table I) (6). Positive titres in the 1972
in cattle, namely The Netherlands,
Germany and the UK (8)
survey were distributed as follows: two sejroe,
- the absence of control measures for leptospirosis in farm animals in Portugal - the detection in Portugal of recently imported cows (from the UK) with indications of a recent hardjo infection (8) - the detection of some focuses of endemic hardjo infection in two regions (8) - the isolation of serovar hardjo in an abattoir survey from kidneys of cows with positive titres to antigens of the Sejroe group (7, 8) - the transit of cattle between the continent and the islands, especially the Azores (8). The clinical importance of serovar hardjo
infections in
Portugal has not been estimated, although there have been several complaints from farmers about the occurrence of abortions in their herds which, upon serological testing for leptospirosis, presented high prevalences of Sejroe (mainly serovar hardjo) titres.
and four
one
pomona
tarassovi.
There is also evidence that Leptospira infection, particularly that of serogroup Pomona, has some clinical importance, while the earlier studies could report no information about any clinical manifestations in cattle in Portugal that might be due to leptospirosis ( 1 , 6 ) . After the Sejroe serogroup, the Australis group presented the highest seroprevalence (6.3%) in the 1 9 8 6 - 1 9 8 7 abattoir survey cattle sera (Table IV). This observation is important, since it was the first detection of Australis in this animal species in Portugal, and since the serogroup has been considered important in farm animals, although it is not yet clearly associated with disease in cattle (23). Its widespread appearance in domestic animals may be a reflection of a reported dynamic change in the distribution of leptospires of this group (20). The data available give no indication that either the Australis or the Tarassovi serogroups may have any clinical significance in cattle in Portugal.
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Rev. sci. tech. Off. int. Epiz., 17 (3)
The main reacting serogroups in DS sera were Australis, Pomona, Cynopteri and Autumnalis. Both in DS and AS sera, but especially in the latter, there was a marked predominance of bratislava titres ( 3 8 . 7 % and 7 9 . 6 % , respectively). The main difference between DS and AS samples was that in DS sera, pomona titres also showed a high percentage ( 2 3 . 9 % ) , whereas in AS sera pomona titres were as low as 0.3%. Titres to cynopteri and autumnalis often appeared in bratislava positive sera and may have been due to cross-reactions ( 1 5 ) .
The percentage of Canicola serogroup titres in the AS sera of the 1 9 8 6 - 1 9 8 7 survey (Table IV) was also noticeable (5.8%) and may reflect the continued low level of vaccination against canicola infection in dogs, the maintenance host for this serovar (8). From the 112 bacteriological analyses performed on diagnostic submissions from 1987 to 1993, 17 (15.1%) were discarded due to contamination by other bacteria, 9 4 ( 8 4 % ) gave negative results and only one isolate was obtained, from a calf that died during the above-mentioned Pomona group outbreak in region F in 1990. This strain proved not to be pomona when typed by monoclonal antibodies (MAbs), but rather had an agglutination profile that resembled mozdok and proechymis to some extent (H. Korver, personal communication).
Serogroup Australis is evidently the most widespread in pigs in Portugal, as in other European countries (10). This is a relevant finding considering the fact that pigs can be maintenance hosts for leptospires of the Australis group, and that these leptospires have been implicated in reproductive wastage (10) as a cause of infertility, late-term abortion and low viability litters. The appearance of a widespread Australis serogroup infection in domestic animals in Europe may be a reflection of a reported dynamic change in the distribution of .leptospires of this group (20). The hedgehog was for many years considered the maintenance host for bratislava, but in the 1970s and 1980s bratislava was isolated from other animal species, namely pigs, in several European countries (12, 2 0 ) . The possibility that bratislava or related serovars could be a problem in pigs emerged in the 1980s (10). Considering the above, it is likely that bratislava hardly existed in Portugal when Azevedo conducted his last survey of pig sera in 1966 (1), although this cannot be verified since the Australis group was not included in the antigen battery. However, the group was included in the 1972 survey of cattle sera and no titres to this antigen were detected (6), in contrast to the present findings in cattle which suggest the widespread occurrence of this serogroup in Portugal (Tables III and IV). Other authors have verified the emergence of Australis serogroup infection in the 1970s and 1980s: in 1981 Hathaway and Little found a prevalence of over 2 4 % of antibodies to the Australis serogroup strains in pigs in England, upon the introduction of serovars lora and bratislava in the antigen battery in 1 9 7 8 (15), while earlier serological surveys performed in Scotland by Michna and Campbell in 1969 and in Ireland by McErlean in 1 9 7 3 , as described by
Until the end of 1993, besides this isolate, only serovar hardjo was isolated from cattle in Portugal, in 1987 (7). The early studies by Azevedo and Palmeiro did not attempt the isolation of leptospires in cattle ( 1 , 6).
Leptospirosis in pigs Prior to the present survey, the last serological survey on the occurrence of leptospirosis in pigs in Portugal was conducted by Azevedo in 1966 (1). Azevedo reported a prevalence of 5 . 9 % of positive sera, mainly involving serovar pomona (4.7%), in 610 animals tested (Table I). As mentioned above, this survey tested for six serovars only, which did not include the Australis group. The present study examined a total of 3,195 pigs DS and AS sera, of which 2 0 . 2 % showed positive results. Both DS and AS sera exhibited a significant percentage of positive titres ( 2 0 . 6 % and 19.8%, respectively). The highest percentages were found in regions A, C, F and G. The percentages of positive sera and the distribution of serological reactors to various leptospiral serovars in DS and AS samples are presented in Tables V, VI and VII.
Table V Distribution of positive titres in pig sera from diagnostic submissions and sera collected in abattoir surveys (1987-1993), by region
Region Ribatejo e Oeste (A) Beira Litoral (B) Entre-Dotiro-e-Minho (C) Beira Interior (E) Alentejo (F) Algarve (G) Total NT: Not tested
Sera from diagnostic submissions
Sera from abattoir surveys
Total
No. tested
No. positive
Percentage
1,084
208
19.2
57
8
14.0
135
27
20.0
NT
35
1
2.9
NT
138
52
37.7
508
32
9
28.1
287
48
16.7
319
57
17.9
1,481
305
20.6
1,714
340
19.8
3.195
645
20.2
No. tested
No. positive
Percentage
No. tested
No. positive
Percentage
919
206
22.4
2,003
414
20.7
NT
-
-
57
8
14.0
135
27
20.0
35
1
2.9
86
16.9
646
138
21.4
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Rev. sci. tech. Off. int. Epiz., 17 (3)
Table V I Distribution of positive titres in pig sera from diagnostic submissions (1987-1993), by leptospiral serovar and region Positive titres by region Serogroup
Serovar
Percentage
No. positive
A
B
C
D
E
F
Australis
bratislava
212
38.7
141
4
19
1
38
9
Pomona
pomona
131
23.9
91
4
6
0
30
0
Cynopteri
cynopteri
94
17.2
58
1
12
0
23
0
Icterohaemorrhagiae
icterohaemorrhagiae
46
8.4
20
1
7
0
18
0
Autumnalis
autumnalis
21
3.8
20
0
1
0
0
0
Grippotyphosa
grippotyphosa
19
3.5
9
0
1
0
9
0
Canicola
canicola
9
1.6
6
0
1
0
2
0
Ballum
ballum
9
1.6
6
0
1
0
2
0
Tarassovi
tarassovi
3
0.6
1
1
0
0
1
0
Javanica
poi
3
0.6
0
0
2
0
1
0
Pyrogenes
pyrogenes
1
0.2
1
0
0
0
0
0
Hathaway and Little, in which bratislava was used as an antigen, had found no evidence of infection ( 1 5 ) . Similarly, Weber and Fenske documented a dramatic increase in the prevalence of bratislava titres in boars in Germany between 1968 and 1 9 7 3 , also described by Hathaway and Little ( 1 5 ) . The clinical importance of Australis infection in pigs in Portugal has not been estimated. The previous bacteriological survey of leptospirosis in pigs in Portugal was performed by Azevedo and colleagues in 1955 (1), using 154 pigs, from several regions, that were collected in an abattoir in Lisbon. Of these animals, 17 ( 1 1 % ) were carriers of leptospires, and of the 15 strains that were typed, 9 (60%) were identified as serovar pomona, while six ( 4 0 % ) were tarassovi. 2 9 . 8 % of the carriers had positive titres to pomona and 2 6 . 6 % to tarassovi. In spite of the relevant
Pomona serogroup findings, clinical disease was not reported and the pomona strain which was isolated, although pathogenic for hamsters, was not pathogenic for 6 inoculated pigs (2). The authors concluded that the Portuguese strain was probably not host-adapted pomona serovar, which is known to cause endemic infections in pigs that can result in serious outbreaks of reproductive disease ( 1 7 ) , but rather another biotype (3). In contrast to the early report provided by Azevedo, there is evidence that the Pomona group is clinically relevant in pigs in Portugal. Outbreaks of Pomona infection are regularly observed as a cause of late-term abortions with considerable economic losses for farmers. Of the 2 4 9 analyses performed for the bacteriological search for Leptospira in pigs between 1987 and 1 9 9 3 , which used
Table V I I Distribution of positive titres in pig sera from diagnostic submissions and abattoir surveys (1987-1993), by leptospiral serovar and region Positive titres by region Serogroup
Australis Cynopteri
Serovar
bratisiava cynopteri
No. positive
Percentage A
F
G 44
305
79.8
186
75
29
7.6
18
6
5
9
5
3 0
Autumnalis
autumnalis
17
4.5
Icterohaemorrhagiae
icterohaemorrhagiae
11
2.9
6
5
2.1
7
0
1
3
0 0 0
Panama
panama*
8
Javanica
poi
4
1.0
1
Sejroe
sejroe saxkoebing
2 1
0.5 0.3
2 1
0 0
Shermani
shermani*
1
0.3
0
0
1
Pomona
pomona
1
0.3
0
1
0
Pyrogenes
pyrogenes
1
0.3
1
0
0
Canicola
canicola
1
0.3
1
0
0
Ballum
ballum
0.3
1
0
0
1
* Forthe testing of sera from the abattoir surveys, serovars panama and shermani were included
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Rev. sci. tech. Off. int. Epiz., 17 (3)
mainly aborted foetuses, 4 3 (17.3%) were discarded due to contamination by other bacteria, 198 (79.5%) yielded negative results and 8 (3.2%) gave positive results. The strains obtained were typed by MAbs ( 3 0 ) : 1 was serovar canicola and 7 were serovar mozdok. After the isolation of serovar mozdok from aborted pig foetuses for the first time in Portugal in 1 9 8 9 ( 2 6 ) , an experimental challenge study was conducted in pregnant gilts with this strain (29). The serological response was monitored at 3 - to 4-day intervals. The infection produced abortion in all gilts and an interesting serological response with cross-reactions to bratislava, valbuzzi, icterohaemorrhagiae and cynopteri antigens (Fig. 2 ) .
be rare (17), and mozdok was not considered to be a pathogen of domestic animals ( 2 2 ) . However, more recent findings indicate that serovar mozdok can have clinical importance in pigs and cattle. The serovar has been recovered from cattle affected by a clinical syndrome suggestive of leptospirosis (17, 18), was isolated from the kidneys of a sow which had aborted (24) and has been recovered from aborted pig foetuses (19, 2 6 ) . When infected with strains of serovar mozdok isolated from a rodent from the UK ( 1 6 , 18) and from an aborted pig foetus from Portugal (26, 2 9 ) , pregnant gilts aborted infected foetuses. In northern Europe, endemic pomona infection in pigs does not occur (20). The situation appears to be one of occasional accidental infection of pigs and cattle with a serovar maintained by another species. T h e same happens in Great Britain, where mozdok is probably the infecting serovar ( 2 0 ) . Pomona group infections in pigs in Portugal would thus present similarities European countries.
cynopteri
infections
observed
in
other
The occurrence of Leptospira infection in pigs with serogroups other than Australis and Pomona in Portugal seems to be sporadic and, besides one detected case of abortion demonstrated to be due to infection by serovar canicola, which was recovered from the aborted foetuses, there is no evidence that such infections have relevant clinical importance.
Days post-inoculation - • - mozdok
with
pomona
--«-.
bratislava
—
valbuzzi icterohaemorrhagiae
Fig. 2 Serological response of three gilts to e x p e r i m e n t a l infection w i t h
Leptospira interrogans serovar mozdok (29) This pattern of cross-reactivity is frequently observed in DS sera from aborting sows. Serovar mozdok has been isolated from foetuses aborted by sows in two Pomona outbreaks that occurred in different regions (26). It is possible that mozdok, rather than host-adapted pomona, is causing Pomona infection in pigs in Portugal. This agrees with the above-mentioned suggestion by Azevedo and Faro (3) and would explain the usual appearance of outbreaks in geographically separated herds that have some contact with small wild mammals. Infection with serovar mozdok should be studied further. Although this serovar is adapted to wildlife (20), its recovery from the kidneys of the three inoculated gilts suggests that there is the potential for pig-to-pig transmission (29).
Leptospirosis in sheep The serological survey of leptospirosis in sheep performed in 1966 by Azevedo (1) involved the six Leptospira serovars then considered to exist in Portugal. Results revealed a low prevalence (2.7%) of leptospiral infection, with positive titres observed for serovars tarassovi ( 1 . 5 % ) , ballum (0.8%) and icterohaemorrhagiae ( 0 . 5 % ) . The survey undertaken in 1 9 7 2 , which tested only 134 sheep using an enlarged battery of antigens (including sejroe), revealed no positive titres (Table I) (6). In 1989 a new serological study of leptospirosis in sheep was begun. The results obtained for 5 , 2 9 8 tested sera ( 9 2 . 8 % AS) are presented in Tables VIII and IX. The percentage of positive sera was low (3.3%), although higher than the one observed Table VIII Distribution of positive titres in sheep sera (1989-1993), by region Region
The serovars of the Pomona group are antigenically closely related and can be classified only with difficulty by conventional (cross-agglutination absorption test) typing methods (30). The situation in Europe with regard to Pomona group infections is confused because of the difficulty in identifying strains, many of which have been identified as pomona (20). For a long time the infection of domestic stock by other serovars of the Pomona serogroup was considered to
Ribatejo e Oests (A) Beira Litoral (B) Entre-Douro-e-Minho (C) Trás-os-Montes (D)
No. tested
No. positive
Percentage
3,462
122
3.5
21
0
0.0
155
15
9.7
1,417
35
2.5 3.0
Alentejo (F)
33
1
Algarve(G)
210
0
0.0
5.298
173
3.3
Total
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Table IX Distribution of positive titres in s h e e p sera (1989-1993), by leptospiral serovar and region Positive titres by region Serogroup
Serovar
No. positive
Percentage A
B
c
D
F
G
Canicola
canicola
38
15.6
17
0
12
9
0
0
Pomona
pomona
36
14.8
34
0
0
2
0
0
Cynopteri
cynopteri
26
10.7
25
0
0
1
0
0
Sejroe
hardjo sejroe saxkoebing
25 20 1
10.3 8.2 0.4
25 18 0
0 0 0
0 0 0
0 2 1
0 0 0
0 0 0
Icterohaemorrhagiae
icterohaemorrhagiae
22
9.1
18
0
2
2
0
0
Grippotyphosa
grippotyphosa
20
8.2
4
0
0
16
0
0
Tarassovi
tarassovi
17
7.0
16
0
1
0
0
0
6.6
13
0
1
2
0
0
2
0
0
Australis
bratisiava
16
Pyrogenes
pyrogenes
8
3.3
1
0
5
Ballum
ballum
6
2.5
2
0
3
0
1
0
Mini
mini
5
2.1
2
0
3
0
0
0
Hebdomadis
hebdomadis
2
0.8
2
0
0
0
0
0
Autumnalis
autumnalis
1
0.4
0
0
0
1
0
0
in the 1966 survey (1). Region C showed the highest percentage of positives, followed by regions A and F. Most sheep in Portugal are still kept outdoors, and the positive reacting serovars reflect their contact with free-living species and the dogs used by shepherds, which may explain the higher percentages of positive titres to canicola ( 1 5 . 6 % ) . The vaccination of dogs against leptospirosis in Portugal is still not widely performed. In addition to the pomona (14.8%) and cynopteri ( 1 0 . 7 % ) titres, the relatively high percentage of hardjo titres ( 1 0 . 3 % ) was noteworthy, since sheep may be acting as maintenance hosts for this serovar ( 2 0 ) , and this result may be a reflection of the above-mentioned spread of serovar hardjo in the country. This is the first time that hardjo has been serologically detected in sheep in Portugal, as well as a number of other antigens. Multiple titres were often found, some of which may be due to cross-reactions, as often happens with cynopteri titres ( 1 5 ) . Of the 649 sheep examined by Azevedo in 1966 (1), 5 0 0 kidneys were collected for bacteriological culturing of leptospires. Only one isolate was obtained, which was typed as pomona and was not pathogenic for 3 experimentally infected sheep. This is the only isolation in sheep in Portugal up to now, since the 3 0 analyses of DS material performed at the LNIV between 1987 and 1993 all gave negative results. Up until the end of 1 9 9 3 , no report was made of any clinical cases of leptospirosis in sheep in Portugal. Leptospira infection in this animal species seems to be less significant than in cattle and pigs.
Leptospirosis in goats There is little information available about leptospiral infection in goats. The serological survey conducted in 1966 (1), which involved 182 animals, revealed a prevalence of 5 . 4 % sera which gave positive reactions, with titres to ballum ( 2 . 7 % ) , tarassovi (2.2%) and canicola (0.5%) serovars, while in 1972 no positive titres were detected (Table I) (6). Tables X and XI show recent results for 1,631 goat sera, of which the majority (86.3%) were obtained from abattoir surveys.
Table X Distribution of positive titres in goat sera (1987-1993), by region Region
No. tested
No. positive
Percentage
90
21
23.3
Entre-Douro-e-Minho (C)
372
18
4.8
Trás-os-Montes (D)
986
41
4.2
13
0
0.0
170
1
0.6
1,631
81
5.0
Ribatejo e Oeste (A)
Beira Interior (E) Algarve (G) Total
The percentage of positive serological results (5.0%) is close to the percentage observed in the 1966 survey (1). Region A showed the highest percentage of positive titres ( 2 3 . 3 % ) . The predominant serogroups were Grippotyphosa, Canicola, Pomona, Icterohaemorrhagiae and Pyrogenes.
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Rev. sci. tech. Off. int. Epiz., 17 (3)
Table X I Distribution of positive titres in goat sera (1987-1993), by leptospiral serovar and region Positive titres by region Serogroup
Serovar
No. positive
Percentage A
C
D
E
G 0
Grippotyphosa
grippotyphosa
26
21.3
0
1
25
0
Canicola
canicola
23
18.9
0
11
11
0
1
Pomona
pomona
21
17.2
20
0
1
0
0
Icterohaemorrhagiae
icterohaemorrhagiae
16
13.1
0
5
10
0
1
Pyrogenes
pyrogenes
13
10.7
0
3
10
0
0
Mini
mini
8
6.6
0
6
2
0
0
Ballum
ballum
5
4.1
0
2
3
0
0
Sejroe
sejroe
4
3.3
2
0
2
0
0
hardjo
2
1.6
2
0
0
0
0
Australis
bratislava
3
2.5
0
2
1
0
0
Cynopteri
cynopteri
1
0.8
0
0
1
0
0
In the recent survey, a number of antigens, namely hardjo and pomona, were serologically detected for the first time in goats in Portugal. As with sheep, there is no information about the existence of clinical cases of leptospirosis in goats. Following the culturing of kidneys from 123 goats collected in an abattoir survey by Azevedo and Palmeiro in 1963, only one isolate was obtained, which was not typed due to the loss of the culture by contamination, but which the authors deemed to be ballum, since the goat from which it was obtained had a positive titre to that antigen (4). This was the only isolate ever obtained from goats in Portugal to date. There have been only six bacteriological analyses of goats between 1987 and 1993, from which one was discarded due to contamination by other bacteria and five yielded negative results.
Leptospirosis in horses In the 1966 serological survey of 9 3 9 horse sera (1), a low percentage (1.4%) of positive titres was obtained, involving serovars tarassovi ( 0 . 7 % ) , ballum (0.5%) and pomona (0.2%). Previously, Azevedo and Palmeiro had performed serological and bacteriological examinations of 163 horse samples collected in an abattoir (4). All cultures showed negative results, though positive titres to tarassovi and ballum serovars were observed in four animals (2.5%). Between 1987 and 1993, only 83 sera from horses, all DS and mainly (80.7%) from region A, were available for leptospirosis testing at the LNIV. Of these, 4 3 . 4 % showed positive results. The distribution of positive serological reactions to various leptospiral serovars is presented in Table XII. It is interesting to observe the higher percentage of the Australis group, which may be maintained by horses (20). Despite the fact that Pomona infection is widespread in Portugal and is considered important in horses (14), no titres to this antigen were detected. Multiple titres were frequently found, which often happens in horse sera, thus making it difficult to determine the infecting serogroup(s) (13).
Between 1987 and 1993 only three bacteriological analyses for Leptospira were performed, all of which gave negative results. There is no information on the possible occurrence of clinical signs attributable to leptospirosis in horses, such as periodic ophthalmia and abortion. Leptospirosis in horses in Portugal needs to be studied on a larger scale.
Table XII Distribution of positive titres in horse sera (1987-1993), by leptospiral serovar Serogroup
Serovar
No. positive
Percentage
Australis
bratislava
17
24.3
Autumnalis
autumnalis
12
17.1
Cynopteri
cynopteri
8
11.4
Pyrogenes
pyrogenes
8
11.4
Grippotyphosa
grippotyphosa
5
7.1
Icterohaemorrhagiae
icterohaemorrhagiae
5
7.1
Tarassovi
tarassovi
5
7.1
Canicola
canicola
3
4.3
Ballum
ballum
2
2.9
Sejroe
saxkoebing sejroe
2 1
2.9 1.4
Celledoni
celledoni
1
1.4
Javanica
poi
1
1.4
Conclusion Previous surveys by Azevedo and Palmeiro on the occurrence of leptospirosis in farm animals in Portugal reported a low prevalence of infection and a reduced pathogenicity of the infecting serovars ( 1 , 4 , 6). In contrast, the more recent information presented in this paper shows that Leptospira infection is widespread in farm animals in Portugal, and is particularly significant in cattle and pigs. The results suggest that the prevalence of Leptospira
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infection for these two animal species has increased considerably since the earlier studies by Azevedo and Palmeiro. This may be the result of a lack of control measures against leptospirosis, which favours the spreading of infection, in particular of host-adapted serovars hardjo and bratisiava. These serovars of the Sejroe and Australis serogroups were the predominant serovars found in cattle and pigs, respectively, and it is probable that they started spreading in Portugal after the 1970s.
reported for the first time in those species, namely Leptospira
hardjo.
infection in sheep and goats seems to be less
important than in cattle and pigs. Leptospirosis in horses needs to be studied further. The recent findings also show that Leptospira
infection can be
of clinical significance in Portugal, particularly Pomona group infections in cattle and pigs.
t h e prevalences of leptospirosis in sheep and goats in Portugal are similar to prevalences obtained in the earlier surveys, although a number of antigens have now been
Étude sur la leptospirose chez les animaux de ferme au Portugal T. Rocha Résumé L'auteur présente une étude conduite au Portugal sur l'infection à Leptospira chez les animaux de ferme, essentiellement fondée sur les analyses sérologiques conduites au Laboratoire national de recherche vétérinaire de ce pays entre janvier 1987 et décembre 1993. Des prélèvements de sérum ont été soumis à l'épreuve d'agglutination microscopique, à une dilution minimale au 1/100 . Des résultats positifs ont été obtenus pour 15,3 % des prélèvements de bovins (sur un total de 9 543 sérums). Les principaux groupes sérologiques détectés étaient Sejroe, Pomona, Hebdomadis, Tarassovi et Icterohaemorrhagiae. En tout, 3 195 porcins ont été soumis à l'épreuve, dont 20,2 % ont présenté des réactions positives. Les principaux groupes sérologiques retrouvés étaient Australis, Pomona, Cynopteri, Icterohaemorrhagiae et Autumnalis. Des observations sur le terrain de cas de leptospirose chez les porcins, ainsi que les données obtenues à partir d'une infection expérimentale de truies pleines par la variété sérologique mozdok, suggèrent que c'est ce sérovar, et non pomona, qui serait à l'origine des infections du groupe Pomona chez les porcins. Des prélèvements de sérum provenant de 5 298 ovins ont été soumis à l'épreuve et 3,3 % ont donné des résultats positifs. Les sérogroupes prédominants étaient Canicola, Pomona, Cynopteri, Sejroe et Icterohaemorrhagiae. Sur les 1 631 chèvres examinées, 5,0 % ont donné des résultats positifs, essentiellement vis-à-vis des groupes sérologiques Grippotyphosa, Canicola, Pomona, Icterohaemorrhagiae et Pyrogenes. En ce qui concerne les équins, 83 sérums seulement ont pu être examinés, dont 43,4% présentaient des titres positifs. Les groupes sérologiques Australis, Autumnalis, Cynopteri et Pyrogenes étaient les plus répandus. La leptospirose est fréquemment décelée lors d'examens sérologiques des animaux de ferme au Portugal, en particulier chez les bovins et les porcins. Chez les équidés, des études plus approfondies sont nécessaires. e
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Rev. sci. tech. Off. int. Epiz., 17 (3)
Afin de donner un aperçu général sur la fréquence de la leptospirose chez ces espèces animales au Portugal, l'auteur compare ces résultats avec ceux obtenus lors d'études précédentes, qu'il complète par des données bactériologiques antérieures et récentes.
Mots-clés Leptospirose - Portugal - Santé animale - Techniques sérologiques.
Panorámica de la leptospirosis en granjas pecuarias de Portugal T. Rocha Resumen Basándose sobre todo en los resultados serológicos obtenidos por el Laboratorio Nacional de Investigación Veterinaria entre enero de 1987 y diciembre de 1993, el autor repasa los casos de infección por Leptospira de animales de granja en Portugal. Se examinaron muestras de suero por el método de aglutinación microscópica, a una dilución mínima de 1:100. De las 9.543 muestras bovinas analizadas, se obtuvieron resultados positivos en el 15,3% de los casos. Sejroe, Pomona, Hebdomadis, Tarassovi e Icterohaemorrhagiae fueron los principales serogrupos que reaccionaron. Se analizaron un total de 3.195 cerdos, un 20% de los cuales ofreció reacciones positivas. Los principales serogrupos que reaccionaron fueron Australis, Pomona, Cynopteri, Icterohaemorrhagiae y Autumnalis. De las observaciones efectuadas sobre el terreno de brotes de leptospirosis en cerdos, así como de los datos obtenidos a partir de la infección experimental con serovar mozdok de cerdas jóvenes preñadas, se desprende que tal vez sea este serovar, en lugar del pomona, la causa de las infecciones del grupo Pomona en el cerdo. Se analizaron muestras séricas de 5.298 ovejas, de las cuales un 3,3% dio resultado positivo. Los serogrupos implicados fueron principalmente Canicola, Pomona, Cynopteri, Sejroe e Icterohaemorrhagiae. De las 1.631 cabras analizadas, un 5% resultaron positivas, sobre todo a los serogrupos Grippotyphosa, Canicola, Pomona, Icterohaemorrhagiae y Pyrogenes. Sólo se consiguieron 83 muestras de suero de caballo, un 43,4% de las cuales presentaba títulos positivos. Los serogrupos observados con mayor frecuencia fueron Australis, Autumnalis, Cynopteri y Pyrogenes. En Portugal, los animales de granja, especialmente el ganado vacuno y el porcino, presentan frecuentemente indicios serológicos de leptospirosis. Es necesario estudiar con más detenimiento la leptospirosis en los caballos. En un intento de ofrecer una visión general sobre la incidencia de la leptospirosis en las mencionadas especies animales en Portugal, los resultados de este estudio se comparan con los de estudios anteriores, complementándolos además con datos bacteriológicos antiguos y recientes.
Palabras clave Leptospirosis - Portugal - Sanidad animal - Técnicas serológicas. •
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