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Seroprevalence survey for Anaplasma card-test reactors in Louisiana, U.S.A., cattle
Preventive Veterinary Medicine, 6 (1988) 143-153
143
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Seroprevalence Survey for Anaplasma Card-test Reactors in Louisiana, U.S.A., Cattle* M.E. HUGH-JONES, D. BUSCH, C. RABY 1 and F. JONES 2
Department of Epidemiology and Community Health, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) 1Louisiana State Department o/ Agriculture, P.O. Box 1951, Baton Rouge, LA 70821 (U.S.A.) 2CADGIS Laboratory, Department o/Geography and Anthropology, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) (Accepted for publication 27 January 1988 )
ABSTRACT Hugh-Jones, M.E., Busch, D., Raby, C. and Jones, F., 1988. Seroprevalence survey for Anaplasma card-test reactors in Louisiana, U.S.A., cattle. Prey. Vet. Med., 6: 143-153. During 1982-1984 a total of 274 herds, or 11 175 cattle, throughout the State of Louisiana in south central U.S.A. were bled and their serum stored in a serum bank at - 4 0 ° C ; nine of these herds were bled twice. Using the rapid-card agglutination test these sera were tested for Anaplasma antibodies and the overall seroprevalence was 5.64% of the beef cattle seropositive and 4.27% of the dairy cattle.
INTRODUCTION
The economic loss from anaplasmosis results from decreased milk production, severe weight loss, reduced reproductive ability, abortion and death of infected animals (Jones and Brock, 1966). In 1979, the American National Cattlemen's Association considered anaplasmosis to be the second most important cattle disease in the country, which was estimated to have an annual cost of $100 million (McCallon, 1979). The last published estimate of economic loss in Louisiana owing to Anaplasma marginalewas $4 million in 1958 (Mott, 1960). The Mississippi delta region reportedly suffered a $3 million loss the same year (USDA Agricultural Research Service, 1963), but Goodger et al. (1979) estimated that Louisiana and the Mississippi delta area combined, lost more than $28 million in 1958. Alderink and Dietrich (1982) estimated the 1980 cost to Texas at $8.96 million per year. Recently, Morley has * This project was supported by a grant from the Louisiana Department of Agriculture.
0167-5877/88/$03.50
© 1988 Elsevier Science Publishers B.V.
144
estimated the annual cost of anaplasmosis in Louisiana at $1.2 million (Morley, 1985 ). Anaplasma marginale infections are believed to be enzootic in cattle in 40 of the 50 states in the U.S.A. (Goodger et al,, 1979) and a severe problem in three distinct geographic areas, the southeastern U.S.A., the intermountain west and the west coast (Saulman, 1962). No statewide surveys for this disease have been carried out in Louisiana. On the basis of sampling from selected herds, there were approximately 40% card-test seroreactors in 1969 (Henderson, 1969), 8% in 1975 (Hollon and Branton, 1975) and 9% in 1980 (Luther et al., 1980). In Louisiana, mosquitoes and tabanid flies are held to be the only significant (mechanical) vectors of anaplasmosis (Steelman et al., 1968; Wilson, 1960). The card-agglutination test was used as it is inexpensive, fast, requires little skill to perform, requires no special equipment and has been the test of choice for large-scale surveys (Amerault and Roby, 1968; Amerault et al., 1972; Gonzalez et al., 1978; Luther et al., 1980). It has a sensitivity of 84% and a specificity of 98 % for recently-infected cattle (Gonzalez et al., 1978 ); the sensitivity is 96% in the identification of carrier animals (Gonzalez et al., 1978). Because of the risks of neonatal isoerythrolysis, immunization in Louisiana is widely restricted to bulls and the occasional herd with repeated clinical cases. As the disease prevalence was uncertain, the purpose of this paper was to report the results of the card test for anaplasmosis from 274 herds to help determine the statewide distribution of Anaplasma seroreactors in order that there would be an increased knowledge base before a control strategy policy was considered. MATERIALS AND METHODS
Sera As only small volumes of sera (1 ml) are normally collected for brucellosis testing, the 14 state Veterinary Medical Officers (VMO) responsible for brucellosis testing were individually approached and agreed to collect a full tube of blood (10 ml) during their normal duties. They also agreed to attempt to record the actual age of each animal when the handling facilities allowed this to be done with safety. The serum was tested for brucellar reactors in the normal manner by the VMO in the field and then collected, with documentation, from the WE Anderson State Diagnostic Laboratory in Baton Rouge after any card-positive brucellar reactors had been retested; any positive samples were retained by that laboratory and were, therefore, not in this collection. During the period 1982-1984, 561 085 cattle were farm bled and 4.15% brucellar reactors identified (J. Lomme, U.S.D.A., personal communication, 1986). As the epidemiologies of bovine brucellosis and anaplasmosis are totally different, the loss of this small proportion of brucellar-positive sera will not introduce any
145
bias into the anaplasma serological results. Because of the potential for interference with serological tests in future years and specifically with the complement-fixation test, any serum more than slightly hemolysed (light pink) was rejected; if half or more of the samples from a herd were hemolysed, the whole herd sample was rejected. Approximately 20% of the sera were rejected for hemolysis. According to G. Wagner (personal communication, 1986) the blood from Anaplasma seroreactors is not more or less likely to hemolyse and therefore bias is not expected. No more than 100 samples were kept from any single herd bleeding. The samples were split into 1/2 ml aliquots, placed in individually-numbered semen straws (I.M.V. International Corp., 6870 Shingle Creek Pkwy, Minneapolis, 55430) and stored at - 4 0 ° C in a Revco ULT 1685-A-LM. Before testing, an aliquot from each stored sample was drawn and thawed. The sera were tested using the rapid-card agglutination test (Brewer Diagnostic Kit, Hynson, Westcott and Dunning, Inc.) as prescribed by the kit manufacturers.
Animal data and analysis The following data were recorded in a computerized database: VMO; herd identification; date of bleeding; map reference of the bleeding site; herd purpose (dairy, beef or mixed); and for each animal, its ear tag number, serum sample number, age, breed and sex. The sampling was begun in April 1982, and when terminated in September 1984, a total of 274 herds had been sampled, nine more than once. Although each VMO was given a specific number of herds of various sizes for sampling, for various reasons this was seldom achieved and TABLE 1 C T seroprevalences of anaplasmosis reactors in Louisiana by herd size Herd size
Cows Number tested
1-25 26-50 51-75 76-100 101-125 126-150 151-175 176-200 > 200 Totals
Herds Number positive
Percent positive
Number tested
Number positive
Percent positive
1213 2285 2145 1595 1284 818 829 300 706
76 122 190 59 66 35 56 8 19
6.27 5.34 8.86 3.40 5.14 4.28 6.76 2.67 2.69
97 75 41 22 14 8 8 3 6
30 35 33 14 8 6 8 1 2
31 47 80 64 57 75 100 33 33
11 175
631
5.64
274
137
50
146
•
..:
+o
( .+..
o o ~.....
else
•
•
2"
~_.~•.
-
t-"l[~
OO O O 0 _
w /' •
+
Fig. 1. Distribution of beef and dairy herds sampled in Louisiana; dairy herds (open squares); mixed herds (open triangles); beef herds (solid circles); dots=single herds; intermediate circles = 2-5 herds; large circles-- > 5 herds. therefore this collection must be regarded as a convenience sample of the state herd. The sample cannot be claimed to be statistically unbiased. The data was statistically analyzed on the University IBM 370/3081 computer using the Statistical Analysis System (SAS Institute Inc., Cary, NC). The computer-drawn contour map of seroprevalence was constructed on the LSU D e p a r t m e n t of Geography VAX 730 using the " I N T E R G R A P H " D T M N software on a special subset of the original data. RESULTS The overall seroprevalence was 5.64% of cattle tested and 50% of herds had one or more reacting animals; for dairy cows the seroprevalence was 4.27% (31% of dairy herds) and 5.92 % for beef cows (53 % of beef herds had at least one reactor). This was independent of herd size (Table 1 ). This difference in
147
k ¢.7
2
\
) J 41"
Fig. 2. Computer-drawn contour map of bovine seroprevalence for Anaplasma reactors with contours starting at 10% and increasing at 10% intervals. seroprevalence is statistically significant ( P < 0.05) but may be more related to the lower seroprevalences in the southeastern or Florida parishes (counties) and the northwestern parishes, where the majority of dairy herds are located, than to the herd function (Fig. 1). The prevalence for each herd sampled is shown in the computer-drawn contour map (Fig. 2). While it must be interpreted with caution because of: ( 1 ) the irregular distribution of sampled herds; (2) in some of the apparently infection-free areas there are some known unsampled herds with regular clinical disease (i.e. about Oakdale in south-central Louisiana); (3) certain parts of the state have few or no herds, such as along the Mississippi River border with the state of Mississippi, it is, however, clear that the seroprevalence is clumped with intervening areas of low or absent prevalence. A careful examination of the maps showed that these apparent high-prevalence areas were centered on single herds and the contours included few other surrounding herds. Less than 40% of the cattle bled had their ages recorded. Therefore, there
148 TABLE 2 Seroprevalence of bovine Anaplasma card-test reactors in Louisiana, by age
Age (years)
Beefherds
Dairy herds
Number tested 1 2 3 4 5 6 7 8 >8
187 494 434 423 567 489 348 319 314
Totals
3575
Percent positive
Number tested
Percent positive
3.7 4.3 4.1 7.1 8.6 7.6 7.2 9.4 11.8
12 63 89 100 128 150 85 54 46
0 1.6 0 3.0 7.0 6.0 1.2 1.9 8.7
727
3.85
7.10
16 14
o o--o :- : ~__A
1982 1983 1984
#f
\
f
\
o
o
12 •
~
10
8
Q.
8
6
~
4 2
o
\
1
A
/
2
~,
3
\
,,&/ 4
5
6
7
I
\
"~ 8
18
Age ( Y e a r s )
Fig. 3. Seroprevalence of
Anaplasma ~reactor beef cattle,
by age, 1982-1984.
must be a measure of bias in these data as they largely applied to medium herds and under-represent large herds, where the seroprevalence may have been less. Taking the data as they are, however, there was a sudden upward shift in seroprevalence for 5-year-old dairy cows and for 4-year-old beef cows (Table 2). However, when these data for beef animals are broken down for the individual years (Fig. 3), it can be seen that in 1982 the rise in seroprevalence began with the 2-year-old animals. In 1983, the rise in seroprevalence was with the 4-5year-olds and there was a dip in the prevalence for 7-8-year-olds, reflecting the 1982 trough for 6-year-olds. In 1984, there was a decreased prevalence with no obvious pattern, except for the high prevalence in the aged animals. This
149 TABLE 3 CT seroprevalence of all cattle by month of sampling, April 1982 to September 1984 Sampling month
Beef cattle Number tested
Dairy cattle Percent positive
Number tested
Percent positive
Jan. Feb. Mar.
635 831 1180
5.5 5.9 5.8
~
378
2.9
Apr. May Jun.
1563 1710 1214
4.9 4.3 8.0
I
916
6.0
Jul. Aug. Sep.
730 481 302
6.3 4.8 14.2
I
47
Oct. Nov. Dec.
334 681 217
6.6 6.5 4.6
f
393
0
3.0
age analysis of the data by year is not presented for the dairy animals as the numbers are so small. The seroprevalence of beef cattle bled in 1982 was 7.63%, 7.92% in 1983, and 3.73% in 1984; for dairy cattle the seroprevalences were 2.75, 2.67 and 9.05%. The sudden change in dairy prevalence in 1984 is a sampling effect owing to including, by chance, one severely affected herd near Monroe in the northeast among the seven herds and 486 dairy cows bled that year. In 1984, the most heavily sampled areas for beef herds were the southwest and northeast quadrants of the state, and both these areas had many infected herds (see Fig. 2). Although some individual months, such as September 1982, showed very high reactor rates (17%), there was no overall obvious seasonality (Table 3). However, in 1984, the months of June, July and August were in the range 6.16.7% and significantly higher than the previous 5 months, January (4.4%), February (0.9%), March (0.9%), April (1.4%) and May (4.1%), against an overall downward trend. A total of nine herds happened to be bled twice, 7 herds in 1984, and 2 at 12month intervals in 1983 and 1984. The data on these herds where identified animals were sampled and retested are given in Table 4. The visiting interval in 1984 was not constant and, therefore, the apparent seroconversion, either from CT positive to CT negative or CT negative to CT positive, is given both as an animal rate but also on the basis of cow-months of exposure. If the anal-
150 TABLE 4 Apparent CT seroconversions of identified cattle in 9 herds sampled twice Herd
Samplings
Exposure'
CT positive to CT negative CT negative to CT positive
(cow-months) (a) Within 1984 1 2 3 4 5 6 7
Totals
/cm 1
No reactorsat first test No reactors at first test No reactorsat first test 2/3 1/6.9 4/5 1/3.7 4/4 1/2.3 No reactors at first test
11/10 0/28 5/44 8/55 0/9 6/73 0/13
1/43.7 0/135.3 1/14.1 1/31.6 0/26.4 1/24.7 0/26.9
740.7 464.5 276.3
10/12 1/3.7 10/12 1/3.7 No reactors at first test
8.6% 10.2% 6.3%
1/35.2 1/30.5 1/46.1
229.8 307.5
No reactorsat first test No reactorsat first test
0/18 1/25
0/229.8 1/307.5
537.3
No reactors at first test
2.3%
1/537.3
2
/Cows + v e
Jan. Feb. Apr. Apr. May Jun. Jun.
May 43.7 Jul. 135.3 Jun. 70.4 A u g . 266.8 Aug. 41.1 A u g . 156.6 Aug. 26.9
Totals All herds Initial reactors (4,5,6) Remainder (1,2,3,7)
(b) 1983-1984 8 9
/Cows - v e
1
May
May
Apr.
May
/cm ~
~Exposure was calculated in cow-months (cm); differences are owing to rounding to 1/10th of month.
ysis is limited to those herds revisited within 1984 which had evidence of an initial herd infection (herds 4,5,6), 10/12 animals reverted from CT positive to CT negative or I cow per 3.7 cow-months of exposure; 10.2% converted from CT negative to CT positive or 1 cow per 31 cow-months of exposure. For the herds without any apparent reactors in the first test, the conversion for CT negative to CT positive was 6.3% of seronegative cows or one cow per 46 cowmonths. For the two herds with a 12-month interval between bleedings, there were no original reactors to negative convert, and 2.3% became seroreactors at a rate of 1 cow per 537 cow-months. In summary, cattle were reverting to CT negative 7-10 times more rapidly than they were converting to CT positive, with the latter dependent on the prior presence or absence of CT reactors in the herd.
DISCUSSION
As these herds were initially selected as part of the brucellosis eradication policy, they are certainly biased in relation to that disease; these sera were collected from herds undergoing brucellosis eradication and adult Brucella abortus strain 19 vaccination. The secondary use of control-program serum for epidemiological surveys is traditional and held to be essentially unbiased if the
151
new serological tests are epidemiologically unrelated to the primary purpose of the initial sampling. This is the case in this instance. However, the results must be accepted with caution because of possible but maybe unrealized statistical biases. If the herds tested are taken as representative of the state herd, the results can be recalculated to estimate the state prevalence based on the latest available census (USDA, 1981 ) relating to 1978 and prevalence with herd size. The statewide prevalence was, therefore, estimated at 4.9%, 5.5% for beef cattle, and 2.2% for dairy cattle. However, as the number of cattle in the state has been falling each year, and as the VMOs reported that the census figures bore scant relation to their knowledge of herds in their respective areas, especially in relation to the relative frequency of small herds, this estimate should be regarded with caution. The ability to blood-test aged animals fortunately coincided with an apparent radical change in the pattern of infection. In 1982, the age distribution of prevalences increased with age and presumably exposure. In 1983, the increase did not come until 4 or 5 years of age. The change would seem to be real because of the further changes in 1984 which cannot be explained by sampling in low seroprevalence areas. The reason, or reasons, for this change must remain speculative, if only because age analyses on this scale are few and there is nothing to compare with this Louisiana experience. Among 8175 beef cattle bled in northern California in 1969-1970, the seroprevalence rates were 4% for 1-year-olds, 5% for 2-year-old cattle, 11% for 3-year-old cattle, 18% for 4-yearold cattle and 31% for 5-year-old cattle (Utterbeck et al., 1972). The report by Haynes and his colleagues (1983) in Alabama on 8 years, 1975-1982, of card testing cattle would lead one normally to expect a relatively constant seroprevalence, but maybe fluctuations do occur even in randomly-bled herds; the apparent sudden drop in seroprevalence in Alabama in 1977 was merely caused by a change in sampling. However, year to year changes have been noted in Illinois (R. Smith, personal communication, 1986). A new survey is needed in Louisiana to determine whether a lower endemic state now exists. The data are unclear in demonstrating a major month for the spread of infection. This is to be expected with a persistent carrier infection of this kind, the method of testing, which does not distinguish between recent and persistent infections, and analysis of essentially single samples. The raised seroprevalence in September would indicate a possible increased incidence in the immediately preceding months, when there is increased tabanid activity (Wilson, 1960, 1963; Tidwell, 1973 ). The 1984 monthly data indicated an increasing chance of infection as the summer progressed. Within infected herds, the frequency of a seronegative cow converting to seropositive, using the card test, was one cow per 31 cow-months of exposure during the first half of the 1984 fly season but only one cow/46 cow-months in herds initially without reactors. The clumped nature of the reactor distribution in Louisiana matches one's
152 e x p e c t a t i o n s o f a m e c h a n i c a l v e c t o r - b o r n e i n f e c t i o n as t h e t a b a n i d v e c t o r h a s a l i m i t e d flight r a n g e ( W i l s o n , 1983). H i g h p r e v a l e n c e s are r e l a t e d to high i n c i d e n c e s o f o v e r t disease ( A l d e r i n k a n d D i e t r i c h , 1982 ) and, t h e r e f o r e , t h e s e p r e v a l e n c e p e a k s p o s s i b l y reflect l i m i t e d s p r e a d f r o m clinical cases, as well as local t a b a n i d a c t i v i t y a n d h a b i t a t . ACKNOWLEDGEMENTS I t is a p l e a s u r e to a c k n o w l e d g e t h e e x t e n s i v e s u p p o r t b y M a r g a r e t Erwin, Programmer, Jennifer Broussard, Laboratory Technician, and Michael Kearney, S t a t i s t i c i a n , a n d t h e following V M O s : Drs. B o s t w i c k , Bourgeois, Carloss, Foley, Good, H a r g i s , H i g g i n b o t h a m , H o f f p a u i r , J o s e p h , K n i g h t , K o l b , Lee, Lewy, Low, M o r e a u , Miller, R a t i n e r , S m i t h a n d T h o m a s .
REFERENCES Alderink, F.J. and Dietrich, R., 1982. Anaplasmosis in Texas: epidemiology and economics data from a questionnaire survey. Proc. 7th Nat. Anaplasmosis Conf., 86: 66-75. Amerault, T.E. and Roby, T.O., 1968. A rapid card agglutination test for anaplasmosis. J. Am. Vet. Med. Assoc., 153: 1828-1834. Amerault, T.E., Rose, J.E. and Roby, T.O., 1972. Modified card-agglutination test for bovine anaplasmosis. Proc. Annu. Meet. U.S. Anim. Health Assoc., 76: 736-744. Gonzales, E.F., Long, R.F. and Todorovic, R.A., 1978. Comparisons of the complement fixation, indirect fluorescent antibody and card agglutination tests for the diagnosis of bovine anaplasmosis. Am. J. Vet. Res., 39: 1538-1541. Goodger, W.J., Carpenter, T. and Riemann, H., 1979. Estimation of economic loss associated with anaplasmosis in California beef cattle. J. Am. Vet. Med. Assoc., 174: 1333-1336. Haynes, T.R., Lauerman, L.H. and Shugart, J.L., 1983. Anaplasmosis in Alabama: A serologic profile (1975-1982) and cattle market survey (1980). Proc. 87th Annu. Meet. U.S. Anim. Health Assoc., Las Vegas, NE, 16-21 Oct 1983, pp. 66-75. Henderson, F.E., 1969. Anaplasmosis. Proc. U.S. Anim. Health Assoc., 73: 113-114. Hollon, H.C. and Branton, C., 1975. Performance of Holstein and cross-bred dairy cattle in Louisiana: III. Health and viability. J. Dairy Sci., 58: 93-101. Jones, E.W. and Brock, W.E., 1966. Bovine anaplasmosis: its diagnosis, treatment, and control. J. Am. Vet. Med. Assoc., 149: 1624-1633. Luther, D.G., Cox, H.U. and Nelson, W.O., 1980. Comparison of sero-tests with inoculation for detection of carriers in anaplasmosis-vaccinated cattle. Am. J. Vet. Res., 41: 2085-2086. McCallon, B.R., 1979. Prevalence and economic aspects of anaplasmosis. Proc. 6th Natl. Anaplasmosis Conf., pp. 1-3. Morley, R.S., 1985. Epidemiology and economic implications of anaplasmosis in Louisiana. Ph.D. Thesis, Louisiana State University, Baton Rouge, Louisiana, August 1985. Mott, L.O., 1960. Anaplasmosis experimental field trial activities. Proc. 6th Annu. Meet. U.S. Livestock Sanitary Assoc., pp. 95-101. Saulman, E.E., 1962. Changes in the anaplasmosis map. Proc. 4th Natl. Anaplasmosis Conf., pp. 2-3. Steelman, C.D., Foote, L.E., Roby, T.O. and Hollon, B.F., 1968. Seasonal occurrence of mosqui-
153 toes feeding on dairy cattle and the incidence of anaplasmosis in southern Louisiana. Proc. 5th Natl. Anaplasmosis Conf., pp. 178-182. Tidwell, M.A., 1973. The Tabanidae (Diptera) of Louisiana. Tulane Stud. Zool. Bot., 18 (1 & 2 ): 1-95. USDA Agricultural Research Service, 1963. Anaplasmosis: a costly disease. USDA Agricultural Research Service, Washington, DC, U.S. Government Printing Office, Document 22-86. USDA, 1981. Census of Agriculture, Vol 1, State and County Data Part 18. Louisiana U.S. Department of Commerce, Washington. Utterbeck, W.W., Stewart, L.M., Beals, T.L. and Franti, C.E., 1972. Anaplasmosis survey in northern California 1969-1970; prevalence of complement fixing antibodies in cattle in herd location. Am. J. Vet. Res., 33: 357-367. Wilson, B.H., 1960. A study of the seasonal occurrence and relative abundance of horse flies (Tabanidae ) in Louisiana. M.S. Thesis, Louisiana State University Library, Baton Rouge, 57 pp. Wilson, B.H., 1963. Seasonal occurrence and relative abundance of horse flies (Tabanidae) in Louisiana. Ph.D. Thesis, Louisiana State University.
143
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Seroprevalence Survey for Anaplasma Card-test Reactors in Louisiana, U.S.A., Cattle* M.E. HUGH-JONES, D. BUSCH, C. RABY 1 and F. JONES 2
Department of Epidemiology and Community Health, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) 1Louisiana State Department o/ Agriculture, P.O. Box 1951, Baton Rouge, LA 70821 (U.S.A.) 2CADGIS Laboratory, Department o/Geography and Anthropology, Louisiana State University, Baton Rouge, LA 70803 (U.S.A.) (Accepted for publication 27 January 1988 )
ABSTRACT Hugh-Jones, M.E., Busch, D., Raby, C. and Jones, F., 1988. Seroprevalence survey for Anaplasma card-test reactors in Louisiana, U.S.A., cattle. Prey. Vet. Med., 6: 143-153. During 1982-1984 a total of 274 herds, or 11 175 cattle, throughout the State of Louisiana in south central U.S.A. were bled and their serum stored in a serum bank at - 4 0 ° C ; nine of these herds were bled twice. Using the rapid-card agglutination test these sera were tested for Anaplasma antibodies and the overall seroprevalence was 5.64% of the beef cattle seropositive and 4.27% of the dairy cattle.
INTRODUCTION
The economic loss from anaplasmosis results from decreased milk production, severe weight loss, reduced reproductive ability, abortion and death of infected animals (Jones and Brock, 1966). In 1979, the American National Cattlemen's Association considered anaplasmosis to be the second most important cattle disease in the country, which was estimated to have an annual cost of $100 million (McCallon, 1979). The last published estimate of economic loss in Louisiana owing to Anaplasma marginalewas $4 million in 1958 (Mott, 1960). The Mississippi delta region reportedly suffered a $3 million loss the same year (USDA Agricultural Research Service, 1963), but Goodger et al. (1979) estimated that Louisiana and the Mississippi delta area combined, lost more than $28 million in 1958. Alderink and Dietrich (1982) estimated the 1980 cost to Texas at $8.96 million per year. Recently, Morley has * This project was supported by a grant from the Louisiana Department of Agriculture.
0167-5877/88/$03.50
© 1988 Elsevier Science Publishers B.V.
144
estimated the annual cost of anaplasmosis in Louisiana at $1.2 million (Morley, 1985 ). Anaplasma marginale infections are believed to be enzootic in cattle in 40 of the 50 states in the U.S.A. (Goodger et al,, 1979) and a severe problem in three distinct geographic areas, the southeastern U.S.A., the intermountain west and the west coast (Saulman, 1962). No statewide surveys for this disease have been carried out in Louisiana. On the basis of sampling from selected herds, there were approximately 40% card-test seroreactors in 1969 (Henderson, 1969), 8% in 1975 (Hollon and Branton, 1975) and 9% in 1980 (Luther et al., 1980). In Louisiana, mosquitoes and tabanid flies are held to be the only significant (mechanical) vectors of anaplasmosis (Steelman et al., 1968; Wilson, 1960). The card-agglutination test was used as it is inexpensive, fast, requires little skill to perform, requires no special equipment and has been the test of choice for large-scale surveys (Amerault and Roby, 1968; Amerault et al., 1972; Gonzalez et al., 1978; Luther et al., 1980). It has a sensitivity of 84% and a specificity of 98 % for recently-infected cattle (Gonzalez et al., 1978 ); the sensitivity is 96% in the identification of carrier animals (Gonzalez et al., 1978). Because of the risks of neonatal isoerythrolysis, immunization in Louisiana is widely restricted to bulls and the occasional herd with repeated clinical cases. As the disease prevalence was uncertain, the purpose of this paper was to report the results of the card test for anaplasmosis from 274 herds to help determine the statewide distribution of Anaplasma seroreactors in order that there would be an increased knowledge base before a control strategy policy was considered. MATERIALS AND METHODS
Sera As only small volumes of sera (1 ml) are normally collected for brucellosis testing, the 14 state Veterinary Medical Officers (VMO) responsible for brucellosis testing were individually approached and agreed to collect a full tube of blood (10 ml) during their normal duties. They also agreed to attempt to record the actual age of each animal when the handling facilities allowed this to be done with safety. The serum was tested for brucellar reactors in the normal manner by the VMO in the field and then collected, with documentation, from the WE Anderson State Diagnostic Laboratory in Baton Rouge after any card-positive brucellar reactors had been retested; any positive samples were retained by that laboratory and were, therefore, not in this collection. During the period 1982-1984, 561 085 cattle were farm bled and 4.15% brucellar reactors identified (J. Lomme, U.S.D.A., personal communication, 1986). As the epidemiologies of bovine brucellosis and anaplasmosis are totally different, the loss of this small proportion of brucellar-positive sera will not introduce any
145
bias into the anaplasma serological results. Because of the potential for interference with serological tests in future years and specifically with the complement-fixation test, any serum more than slightly hemolysed (light pink) was rejected; if half or more of the samples from a herd were hemolysed, the whole herd sample was rejected. Approximately 20% of the sera were rejected for hemolysis. According to G. Wagner (personal communication, 1986) the blood from Anaplasma seroreactors is not more or less likely to hemolyse and therefore bias is not expected. No more than 100 samples were kept from any single herd bleeding. The samples were split into 1/2 ml aliquots, placed in individually-numbered semen straws (I.M.V. International Corp., 6870 Shingle Creek Pkwy, Minneapolis, 55430) and stored at - 4 0 ° C in a Revco ULT 1685-A-LM. Before testing, an aliquot from each stored sample was drawn and thawed. The sera were tested using the rapid-card agglutination test (Brewer Diagnostic Kit, Hynson, Westcott and Dunning, Inc.) as prescribed by the kit manufacturers.
Animal data and analysis The following data were recorded in a computerized database: VMO; herd identification; date of bleeding; map reference of the bleeding site; herd purpose (dairy, beef or mixed); and for each animal, its ear tag number, serum sample number, age, breed and sex. The sampling was begun in April 1982, and when terminated in September 1984, a total of 274 herds had been sampled, nine more than once. Although each VMO was given a specific number of herds of various sizes for sampling, for various reasons this was seldom achieved and TABLE 1 C T seroprevalences of anaplasmosis reactors in Louisiana by herd size Herd size
Cows Number tested
1-25 26-50 51-75 76-100 101-125 126-150 151-175 176-200 > 200 Totals
Herds Number positive
Percent positive
Number tested
Number positive
Percent positive
1213 2285 2145 1595 1284 818 829 300 706
76 122 190 59 66 35 56 8 19
6.27 5.34 8.86 3.40 5.14 4.28 6.76 2.67 2.69
97 75 41 22 14 8 8 3 6
30 35 33 14 8 6 8 1 2
31 47 80 64 57 75 100 33 33
11 175
631
5.64
274
137
50
146
•
..:
+o
( .+..
o o ~.....
else
•
•
2"
~_.~•.
-
t-"l[~
OO O O 0 _
w /' •
+
Fig. 1. Distribution of beef and dairy herds sampled in Louisiana; dairy herds (open squares); mixed herds (open triangles); beef herds (solid circles); dots=single herds; intermediate circles = 2-5 herds; large circles-- > 5 herds. therefore this collection must be regarded as a convenience sample of the state herd. The sample cannot be claimed to be statistically unbiased. The data was statistically analyzed on the University IBM 370/3081 computer using the Statistical Analysis System (SAS Institute Inc., Cary, NC). The computer-drawn contour map of seroprevalence was constructed on the LSU D e p a r t m e n t of Geography VAX 730 using the " I N T E R G R A P H " D T M N software on a special subset of the original data. RESULTS The overall seroprevalence was 5.64% of cattle tested and 50% of herds had one or more reacting animals; for dairy cows the seroprevalence was 4.27% (31% of dairy herds) and 5.92 % for beef cows (53 % of beef herds had at least one reactor). This was independent of herd size (Table 1 ). This difference in
147
k ¢.7
2
\
) J 41"
Fig. 2. Computer-drawn contour map of bovine seroprevalence for Anaplasma reactors with contours starting at 10% and increasing at 10% intervals. seroprevalence is statistically significant ( P < 0.05) but may be more related to the lower seroprevalences in the southeastern or Florida parishes (counties) and the northwestern parishes, where the majority of dairy herds are located, than to the herd function (Fig. 1). The prevalence for each herd sampled is shown in the computer-drawn contour map (Fig. 2). While it must be interpreted with caution because of: ( 1 ) the irregular distribution of sampled herds; (2) in some of the apparently infection-free areas there are some known unsampled herds with regular clinical disease (i.e. about Oakdale in south-central Louisiana); (3) certain parts of the state have few or no herds, such as along the Mississippi River border with the state of Mississippi, it is, however, clear that the seroprevalence is clumped with intervening areas of low or absent prevalence. A careful examination of the maps showed that these apparent high-prevalence areas were centered on single herds and the contours included few other surrounding herds. Less than 40% of the cattle bled had their ages recorded. Therefore, there
148 TABLE 2 Seroprevalence of bovine Anaplasma card-test reactors in Louisiana, by age
Age (years)
Beefherds
Dairy herds
Number tested 1 2 3 4 5 6 7 8 >8
187 494 434 423 567 489 348 319 314
Totals
3575
Percent positive
Number tested
Percent positive
3.7 4.3 4.1 7.1 8.6 7.6 7.2 9.4 11.8
12 63 89 100 128 150 85 54 46
0 1.6 0 3.0 7.0 6.0 1.2 1.9 8.7
727
3.85
7.10
16 14
o o--o :- : ~__A
1982 1983 1984
#f
\
f
\
o
o
12 •
~
10
8
Q.
8
6
~
4 2
o
\
1
A
/
2
~,
3
\
,,&/ 4
5
6
7
I
\
"~ 8
18
Age ( Y e a r s )
Fig. 3. Seroprevalence of
Anaplasma ~reactor beef cattle,
by age, 1982-1984.
must be a measure of bias in these data as they largely applied to medium herds and under-represent large herds, where the seroprevalence may have been less. Taking the data as they are, however, there was a sudden upward shift in seroprevalence for 5-year-old dairy cows and for 4-year-old beef cows (Table 2). However, when these data for beef animals are broken down for the individual years (Fig. 3), it can be seen that in 1982 the rise in seroprevalence began with the 2-year-old animals. In 1983, the rise in seroprevalence was with the 4-5year-olds and there was a dip in the prevalence for 7-8-year-olds, reflecting the 1982 trough for 6-year-olds. In 1984, there was a decreased prevalence with no obvious pattern, except for the high prevalence in the aged animals. This
149 TABLE 3 CT seroprevalence of all cattle by month of sampling, April 1982 to September 1984 Sampling month
Beef cattle Number tested
Dairy cattle Percent positive
Number tested
Percent positive
Jan. Feb. Mar.
635 831 1180
5.5 5.9 5.8
~
378
2.9
Apr. May Jun.
1563 1710 1214
4.9 4.3 8.0
I
916
6.0
Jul. Aug. Sep.
730 481 302
6.3 4.8 14.2
I
47
Oct. Nov. Dec.
334 681 217
6.6 6.5 4.6
f
393
0
3.0
age analysis of the data by year is not presented for the dairy animals as the numbers are so small. The seroprevalence of beef cattle bled in 1982 was 7.63%, 7.92% in 1983, and 3.73% in 1984; for dairy cattle the seroprevalences were 2.75, 2.67 and 9.05%. The sudden change in dairy prevalence in 1984 is a sampling effect owing to including, by chance, one severely affected herd near Monroe in the northeast among the seven herds and 486 dairy cows bled that year. In 1984, the most heavily sampled areas for beef herds were the southwest and northeast quadrants of the state, and both these areas had many infected herds (see Fig. 2). Although some individual months, such as September 1982, showed very high reactor rates (17%), there was no overall obvious seasonality (Table 3). However, in 1984, the months of June, July and August were in the range 6.16.7% and significantly higher than the previous 5 months, January (4.4%), February (0.9%), March (0.9%), April (1.4%) and May (4.1%), against an overall downward trend. A total of nine herds happened to be bled twice, 7 herds in 1984, and 2 at 12month intervals in 1983 and 1984. The data on these herds where identified animals were sampled and retested are given in Table 4. The visiting interval in 1984 was not constant and, therefore, the apparent seroconversion, either from CT positive to CT negative or CT negative to CT positive, is given both as an animal rate but also on the basis of cow-months of exposure. If the anal-
150 TABLE 4 Apparent CT seroconversions of identified cattle in 9 herds sampled twice Herd
Samplings
Exposure'
CT positive to CT negative CT negative to CT positive
(cow-months) (a) Within 1984 1 2 3 4 5 6 7
Totals
/cm 1
No reactorsat first test No reactors at first test No reactorsat first test 2/3 1/6.9 4/5 1/3.7 4/4 1/2.3 No reactors at first test
11/10 0/28 5/44 8/55 0/9 6/73 0/13
1/43.7 0/135.3 1/14.1 1/31.6 0/26.4 1/24.7 0/26.9
740.7 464.5 276.3
10/12 1/3.7 10/12 1/3.7 No reactors at first test
8.6% 10.2% 6.3%
1/35.2 1/30.5 1/46.1
229.8 307.5
No reactorsat first test No reactorsat first test
0/18 1/25
0/229.8 1/307.5
537.3
No reactors at first test
2.3%
1/537.3
2
/Cows + v e
Jan. Feb. Apr. Apr. May Jun. Jun.
May 43.7 Jul. 135.3 Jun. 70.4 A u g . 266.8 Aug. 41.1 A u g . 156.6 Aug. 26.9
Totals All herds Initial reactors (4,5,6) Remainder (1,2,3,7)
(b) 1983-1984 8 9
/Cows - v e
1
May
May
Apr.
May
/cm ~
~Exposure was calculated in cow-months (cm); differences are owing to rounding to 1/10th of month.
ysis is limited to those herds revisited within 1984 which had evidence of an initial herd infection (herds 4,5,6), 10/12 animals reverted from CT positive to CT negative or I cow per 3.7 cow-months of exposure; 10.2% converted from CT negative to CT positive or 1 cow per 31 cow-months of exposure. For the herds without any apparent reactors in the first test, the conversion for CT negative to CT positive was 6.3% of seronegative cows or one cow per 46 cowmonths. For the two herds with a 12-month interval between bleedings, there were no original reactors to negative convert, and 2.3% became seroreactors at a rate of 1 cow per 537 cow-months. In summary, cattle were reverting to CT negative 7-10 times more rapidly than they were converting to CT positive, with the latter dependent on the prior presence or absence of CT reactors in the herd.
DISCUSSION
As these herds were initially selected as part of the brucellosis eradication policy, they are certainly biased in relation to that disease; these sera were collected from herds undergoing brucellosis eradication and adult Brucella abortus strain 19 vaccination. The secondary use of control-program serum for epidemiological surveys is traditional and held to be essentially unbiased if the
151
new serological tests are epidemiologically unrelated to the primary purpose of the initial sampling. This is the case in this instance. However, the results must be accepted with caution because of possible but maybe unrealized statistical biases. If the herds tested are taken as representative of the state herd, the results can be recalculated to estimate the state prevalence based on the latest available census (USDA, 1981 ) relating to 1978 and prevalence with herd size. The statewide prevalence was, therefore, estimated at 4.9%, 5.5% for beef cattle, and 2.2% for dairy cattle. However, as the number of cattle in the state has been falling each year, and as the VMOs reported that the census figures bore scant relation to their knowledge of herds in their respective areas, especially in relation to the relative frequency of small herds, this estimate should be regarded with caution. The ability to blood-test aged animals fortunately coincided with an apparent radical change in the pattern of infection. In 1982, the age distribution of prevalences increased with age and presumably exposure. In 1983, the increase did not come until 4 or 5 years of age. The change would seem to be real because of the further changes in 1984 which cannot be explained by sampling in low seroprevalence areas. The reason, or reasons, for this change must remain speculative, if only because age analyses on this scale are few and there is nothing to compare with this Louisiana experience. Among 8175 beef cattle bled in northern California in 1969-1970, the seroprevalence rates were 4% for 1-year-olds, 5% for 2-year-old cattle, 11% for 3-year-old cattle, 18% for 4-yearold cattle and 31% for 5-year-old cattle (Utterbeck et al., 1972). The report by Haynes and his colleagues (1983) in Alabama on 8 years, 1975-1982, of card testing cattle would lead one normally to expect a relatively constant seroprevalence, but maybe fluctuations do occur even in randomly-bled herds; the apparent sudden drop in seroprevalence in Alabama in 1977 was merely caused by a change in sampling. However, year to year changes have been noted in Illinois (R. Smith, personal communication, 1986). A new survey is needed in Louisiana to determine whether a lower endemic state now exists. The data are unclear in demonstrating a major month for the spread of infection. This is to be expected with a persistent carrier infection of this kind, the method of testing, which does not distinguish between recent and persistent infections, and analysis of essentially single samples. The raised seroprevalence in September would indicate a possible increased incidence in the immediately preceding months, when there is increased tabanid activity (Wilson, 1960, 1963; Tidwell, 1973 ). The 1984 monthly data indicated an increasing chance of infection as the summer progressed. Within infected herds, the frequency of a seronegative cow converting to seropositive, using the card test, was one cow per 31 cow-months of exposure during the first half of the 1984 fly season but only one cow/46 cow-months in herds initially without reactors. The clumped nature of the reactor distribution in Louisiana matches one's
152 e x p e c t a t i o n s o f a m e c h a n i c a l v e c t o r - b o r n e i n f e c t i o n as t h e t a b a n i d v e c t o r h a s a l i m i t e d flight r a n g e ( W i l s o n , 1983). H i g h p r e v a l e n c e s are r e l a t e d to high i n c i d e n c e s o f o v e r t disease ( A l d e r i n k a n d D i e t r i c h , 1982 ) and, t h e r e f o r e , t h e s e p r e v a l e n c e p e a k s p o s s i b l y reflect l i m i t e d s p r e a d f r o m clinical cases, as well as local t a b a n i d a c t i v i t y a n d h a b i t a t . ACKNOWLEDGEMENTS I t is a p l e a s u r e to a c k n o w l e d g e t h e e x t e n s i v e s u p p o r t b y M a r g a r e t Erwin, Programmer, Jennifer Broussard, Laboratory Technician, and Michael Kearney, S t a t i s t i c i a n , a n d t h e following V M O s : Drs. B o s t w i c k , Bourgeois, Carloss, Foley, Good, H a r g i s , H i g g i n b o t h a m , H o f f p a u i r , J o s e p h , K n i g h t , K o l b , Lee, Lewy, Low, M o r e a u , Miller, R a t i n e r , S m i t h a n d T h o m a s .
REFERENCES Alderink, F.J. and Dietrich, R., 1982. Anaplasmosis in Texas: epidemiology and economics data from a questionnaire survey. Proc. 7th Nat. Anaplasmosis Conf., 86: 66-75. Amerault, T.E. and Roby, T.O., 1968. A rapid card agglutination test for anaplasmosis. J. Am. Vet. Med. Assoc., 153: 1828-1834. Amerault, T.E., Rose, J.E. and Roby, T.O., 1972. Modified card-agglutination test for bovine anaplasmosis. Proc. Annu. Meet. U.S. Anim. Health Assoc., 76: 736-744. Gonzales, E.F., Long, R.F. and Todorovic, R.A., 1978. Comparisons of the complement fixation, indirect fluorescent antibody and card agglutination tests for the diagnosis of bovine anaplasmosis. Am. J. Vet. Res., 39: 1538-1541. Goodger, W.J., Carpenter, T. and Riemann, H., 1979. Estimation of economic loss associated with anaplasmosis in California beef cattle. J. Am. Vet. Med. Assoc., 174: 1333-1336. Haynes, T.R., Lauerman, L.H. and Shugart, J.L., 1983. Anaplasmosis in Alabama: A serologic profile (1975-1982) and cattle market survey (1980). Proc. 87th Annu. Meet. U.S. Anim. Health Assoc., Las Vegas, NE, 16-21 Oct 1983, pp. 66-75. Henderson, F.E., 1969. Anaplasmosis. Proc. U.S. Anim. Health Assoc., 73: 113-114. Hollon, H.C. and Branton, C., 1975. Performance of Holstein and cross-bred dairy cattle in Louisiana: III. Health and viability. J. Dairy Sci., 58: 93-101. Jones, E.W. and Brock, W.E., 1966. Bovine anaplasmosis: its diagnosis, treatment, and control. J. Am. Vet. Med. Assoc., 149: 1624-1633. Luther, D.G., Cox, H.U. and Nelson, W.O., 1980. Comparison of sero-tests with inoculation for detection of carriers in anaplasmosis-vaccinated cattle. Am. J. Vet. Res., 41: 2085-2086. McCallon, B.R., 1979. Prevalence and economic aspects of anaplasmosis. Proc. 6th Natl. Anaplasmosis Conf., pp. 1-3. Morley, R.S., 1985. Epidemiology and economic implications of anaplasmosis in Louisiana. Ph.D. Thesis, Louisiana State University, Baton Rouge, Louisiana, August 1985. Mott, L.O., 1960. Anaplasmosis experimental field trial activities. Proc. 6th Annu. Meet. U.S. Livestock Sanitary Assoc., pp. 95-101. Saulman, E.E., 1962. Changes in the anaplasmosis map. Proc. 4th Natl. Anaplasmosis Conf., pp. 2-3. Steelman, C.D., Foote, L.E., Roby, T.O. and Hollon, B.F., 1968. Seasonal occurrence of mosqui-
153 toes feeding on dairy cattle and the incidence of anaplasmosis in southern Louisiana. Proc. 5th Natl. Anaplasmosis Conf., pp. 178-182. Tidwell, M.A., 1973. The Tabanidae (Diptera) of Louisiana. Tulane Stud. Zool. Bot., 18 (1 & 2 ): 1-95. USDA Agricultural Research Service, 1963. Anaplasmosis: a costly disease. USDA Agricultural Research Service, Washington, DC, U.S. Government Printing Office, Document 22-86. USDA, 1981. Census of Agriculture, Vol 1, State and County Data Part 18. Louisiana U.S. Department of Commerce, Washington. Utterbeck, W.W., Stewart, L.M., Beals, T.L. and Franti, C.E., 1972. Anaplasmosis survey in northern California 1969-1970; prevalence of complement fixing antibodies in cattle in herd location. Am. J. Vet. Res., 33: 357-367. Wilson, B.H., 1960. A study of the seasonal occurrence and relative abundance of horse flies (Tabanidae ) in Louisiana. M.S. Thesis, Louisiana State University Library, Baton Rouge, 57 pp. Wilson, B.H., 1963. Seasonal occurrence and relative abundance of horse flies (Tabanidae) in Louisiana. Ph.D. Thesis, Louisiana State University.