J.
Med. Ento_ol.
Vol. 14,
DO.
25 Nove_ber
2: 146-151
1977
PRECIPITATING ANTIBODIES IN CATTLE INFESTED BY PSOROPTES OVIS (ACARINA: PSOROPTIDAE)1,2 By WilliaDl F. Fisher3,4 and Grant I. Wilson3
Precipitating antibodies have been detected by agar-gel diffusion tests in the sera of animals infested with mites of the family Psoroptidae. Serum precipitins were found by Fox et al. (1967) in rabbits infested with Psoroptes cuniculi (Delafond), by Fisher (1972) in sheep infested with Psoroptes ovis (Hering), by Butler (1968) in cattle infested with Chorioptes bovis (Hering), and by Weisbroth et al. (1974) in cats infested with Otodectes 0Jnotis (Hering). Information on the antibody response to P. ovis in cattle and the immunochemical identification of those antibodies is needed. Such information may be useful in treatment and control of diseases caused by these mites. Because many cattle may be concomitantly parasitized by a number of arthropods (lice, grubs, and ticks), the antibody response to these parasites must be taken into account when they occur in animals being tested for antibodies to P. ovis. This paper presents the results of agar-gel diffusion tests on the sera of cattle infested with P. ovis, Hypoderma bovis (Linnaeus), Hypoderma lineatum (de Villiers), Otobius megnini (Duges), Haematopinus eurysternus (Denny), Linognathus vituli (Linnaeus), Bovicola bovis (Linnaeus), Demodex bovis (Stiles), and combinations of these parasites, against extracts of several of these arthropods. lMention of a trade name, a proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 'Address reprint requests to Fisher in Kerrville. ·Parasite Research Laboratory, Western Region, Agricultural Research Service, U.S. Department of Agriculture, P. O. Box 705, Albuquerque, New Mexico 87103, U.S.A. 'Present address: U.S. Livestock Insects Laboratory, USDA, ARS, P.O. Box 232. Kerrville, Texas 78028, U.S.A.
MATERIALS
AND METHODS
Experimental animals In the 1st experiment, sera were collected from 12 cattle (10 calves, 2 cows) with P. ovis infestations and 12 cattle (6 calves, 6 cows) without P. ovis infestations. The P. ovis infestations were from 1 to 8 months duration and were moderate to heavy at the time of sampling. Most of the infestations were the result of manually transferring mites to clean animals and allowing the infestations to develop until they became suitable subjects for the testing of experimental acaricides. In the 2nd experiment, sera were collected from 26 cattle (24 calves, 2 cows) with P. ovis infestations and 42 cattle (34 calves, 8 cows) without P. ovis infestations. The P. ovis infestations were from 2 to 9 months duration and were light to heavy at the time of sampling. The sera from 21 of the uninfested calves were the preinfestation samples of 21 of the P. ovis-infested calves. The sera from the 8 mature cows came from cattle in Beltsville, MD, U.S.A. Most of the cattle of both experiments were Hereford or Hereford-Angus crosses. The 8 mature cows of the 2nd experiment were dairy cows. Some of the cattle from both experiments (P. ovis-infested and uninfested) were parasitized with H. eurysternus (10 cows), L. vituli (12 calves), Hypoderma spp. (7 calves, 1 cow), O. megnini (17 calves, 1 cow), B. bovis (4 calves) and D. bovis (35 calves, 10 cows) at the time of bleeding. Thirty-three were found to be free of H. eurysternus, 21 free of L. vituli, 8 free of Hypoderma spp., 20 free of O. megnini, and 27 free of B. bovis at the time of sampling. All sera were stored at -18°C until needed.
Preparation of extracts P. ovis mites were collected from cattle and P. cuniculi mites from rabbits, as described by Fisher (1972). P. ovis extract (PO) was prepared from mites that had been freshly killed by freezing and from others that had been stored frozen from 4 to 10 months. The mites were thawed, dried (a portion at 37°C for 5 hr and a portion at 39°C for 16 hr) and ground in a Ten Broeck tissue grinder for 5 min. in 1: 5000 merthiolated saline [20 ml of 1: 1000 thimerosal solution (MerthiolateiB> solution, Eli Lilly & Company, Indianapolis, Indiana 46206,
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Abstract: Precipitating antibodies, as demonstrated by agar-gel diffusion tests, were found in the sera of 23 of 38 Psoroptes ovis-infested cattle when tested against a P. ovis extract and in the sera of 9 of 17 cattle when tested against a P. cUlliculi extract. Sera from 5 of 54 P. ovis-uninfested cattle reacted to the P. ovis extract and 3 of 22 reacted to the P. cUlliculi extract. Analysis of the P. ovis and the P. CUlliculiextracts indicates that they share common antigens. The reaction between the sera of mite-infested cattle and the above antigens is a specific antigenantibody reaction. Extracts of Hypoderma bovis, Otobius megllilli, Haematopinus eurystemus, Demodex hovis, cattle tissue, and cattle blood were made and tested with some of the above sera. Only the H. bovis extract gave a specific reaction with the serum of a grub-infested calf. Reactions with the O. megllilli and blood extracts were considered nonspecific.
1977
Fisher
& Wilson:
Precipitating
TABLE
infested
SERUM NO. I
2 3 4 5 6 7 8 9 10 11 12 13-18 19-20 21 22 23 24
in P. ovis-infested J.
147
cattle
Agar-gel reactions in the sera of Psoroptes ovisand P. ovis-uninfested cattle with P. ovis (PO) and P. cuniculi (PC) extracts (Experiment I). DEGREE OF DURATION PARASITE INFESOF INFESTATION TATION· INFESTATION
P. ovis
5 5 5 5 4 4 4
3-4 3 3 t t
H. eurysternus Uninfested ttt ttt
I I
5 5
3 mo. 2 mo. 2 mo. 2 mo. 5 mo. 1.75 mo. 1.75 mo. 8 mo. 1.75 mo. 1.75 mo. 2-3 mo. 2-3 mo. -tt
EXTRACTS PO
PC
No. of bands
0 0 2 0
I··· I I·· 2 I
I···
I"
I··
0 0 2 0 0
I I
2
I
I
2 0
0 0 0 0 0
I
0 0 I
I
0
0
·5, heavy; 4, moderate to heavy; 3, moderate; 2, light to moderate; I, light infestation. • ·Positive on test; negative on retest or vice versa. ···Faint reaction. tConcurrent H. eurysternus infestation (heavy). ttField infestation; duration unknown. ttt Bovicola bovis present.
setting-20 kc, Bronson Sonic Power, Div. Bronson Instruments, Inc., Mirybrook Rd, Danbury, Conn. 06810, U.S.A.) for 5 min. The mixture was placed at 4°C for 2 days and centrifuged at 900 g for 15 min. The supernatant was removed and frozen. The tissue extract was prepared from pieces of cattle hide that were dehaired with lime and sodium sulfide. Portions of the tissue were placed in 0.9% saline and some in 10% formalin. The specimens were removed from their respective solutions and dried at 37-38 DC for 24 hr, weighed (saline treated-635.3 mg; formalin treated-776.8 mg), and homogenized in phosphate-buffered saline (PBSpH 7.2) using a Virtis blender. An additional 8 ml of PBS was added to the mixture to obtain a better homogenization. The tissue suspension was further ground in a tissue grinder and centrifuged in a refrigerated centrifuge at 17,000 rpm for 30 min. The supernatant was removed, concentrated to 10 ml with an ultrafilter [Ami con Diaflow Ultrafilter (UM-2), 1000 mol.wt. cut off, Amicon Corporation, Lexington, Mass. 02173, U.S.A.] and frozen at -18°C until needed. A blood extract was prepared from whole heparinized cattle blood. The sample was taken from 2 cows that were heavily parasitized with H. eurysternus. The blood was then frozen at -18 DC; when
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U.S.A.) and 80 ml of 0.9% saline] at a ratio of 100 mg of dried mites per 1.5 ml of saline. The mixture was left standing, with periodic shaking, at 6 DC for 2 to 3 days. It was then centrifuged at 900 g for 20 min. The supernatant was removed and used as the test antigen. The P. cuniculi extract (PC) was prepared using the same method as for the PO extract. The Hypoderma bovis extract (HB) was prepared from 4 mature, 3rd-stage H. bovis larvae. The larvae were collected from the backs of several cattle, rinsed in saline and frozen at -18 DCfor 2 months. When needed, they were thawed and dried for 5 hr at 37°C, and prepared in the same manner as the PO extract except that the ratio of tissue to saline was 1 g larval tissue to 4 ml of saline. The O. megnini extract (OM-I) was prepared from 28 nymphal ear ticks that were collected from 1 cow. The ticks were kept at 22-24 DCfor 6 days and then frozen at -18 DCfor 9 months. When needed, they were thawed, dried at 37 DCfor 5 hr, and prepared in the same manner as the HB extract except that the ratio of the tick material to saline was 100 mg dried tick per 1 ml of saline. A 2nd extract (OM-2) was prepared in the same manner as the OM-l extract from 9 nymphal ear ticks obtained from goats' ears. The H. eurysternus extract (HE) was prepared from lice obtained from heavily parasitized cattle. The lice were separated from the hair and frozen at -18°C; when needed they were thawed and dried (a portion at 37°C for 5 hr and a portion at 39°C for 16 hr) and prepared in the same manner as OM-I. A D. bovis extract (DB) was prepared from mites obtained from cattle hides that had been chemically dehaired in a lime-sodium sulfide solution (Fisher 1973). The mites were removed from the nodules, rinsed in saline, transferred to a blender and homogenized at high speed for 60 sec. The suspension was passed through a strainer (4.65 mesh per cm2) to remove tissue debris and entrapped mites. The mites were sedimented by centrifugation and then frozen at -18 DC; when needed they were thawed, placed at 4°C overnight, and suspended in 0.9% saline the next day. The suspension was passed through a strainer and the entrapped mites and remaining tissue were collected in saline and placed at 4°C for several days. The suspension was then centrifuged and the mites resuspended in 32 ml of saline so that 1620 mites were contained in each ml of solution. Sixteen ml of the suspension was removed and the mites were disrupted with a sonicator (Bronson Sonicator, Model LS-75, #6
antibodies
J. Med.
148
needed it was thawed and used without any further treatment.
Serological methods
TABLE 2. Agar-gel reactions in the sera of Psoroptes ovis-infested and P. ovis-uninfested cattle with P. ovis (PO) and P. cuniculi
(PC) extracts (Experiment 2).
SERUM NO. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52-59 60 61 62-92
DEGREE EXTRACTS OF DURATION PO PC PARASITE INFESOF INFESTATIONTATION*INFESTATION No. of bands P.ovis
Uninfested·"
5 5 5 5 4 4 4 4 3-4 3-4 3 3 3 3 3 3 3 2-3 2 2 1 I 1 I 1 1 0 0 0 0 0
6-7 mo. 4mo. 3 mo. 3 mo. 9 mo. 6-7 mo. 4 mo. 2 mo. 4 mo. 2 mo. 6-7 mo. 4 mo. 2 mo. 2 mo. 2 mo. 2 mo. 2 mo. 4 mo. 4 mo. 2 mo. 4 mo. 4 mo. 4 mo. 4 mo. 2 mo. 9 mo.
2 2 0 0 2 1 I 0 1 0 0 I I I 1 0
1** 0 0 1 0 0 0 2 I 0 I 0 0 I 0
2
3 0
Vo!. 14, no. 2
1% agar dissolved in glycine-buffered saline (Kagan & Norman 1970). The pH was adjusted to 7-8. Sixteen ml (large plates) or 8 ml (small plates) of melted agar was poured into the plates, allowed to harden for 0.5 hr at room temperature and placed in the refrigerator at 4°0 overnight. Wells, 5 mm in diam., were cut with a No.2 cork borer so that 6 peripheral wells were grouped around a central well. The center-to-center distance between the central well and each peripheral well was 10 mm. The wells were sealed with a drop of melted agar. The center well was either filled with test serum and the peripheral wells with the test antigens, or the central well was filled with the test antigen and the peripheral wells with the test sera. The plates were placed in a humid chamber at 22-24°0. The plates were usually set up in the afternoon and following incubation at 22-24 °0 overnight were refilled with the appropriate antigens or sera the next morning. In the case of 2 plates, the wells were filled in the morning and refilled in the late afternoon. In a few cases the plates were not refilled due to a shortage of a particular reagent. These 2 modifications in the procedure will be referred to in the results as the modified procedure. Observations of the results obtained were made over a period of 4 to 6 days with the aid of a 7 X hand lens over an illuminated dark background. Mter the final observation the plates were soaked in glycine buffer for 36 hr and then flooded with 0.002% amido black as described by Kagan & Norman (1970). Sera were usually tested against each extract once, but in several cases some of the sera were tested twice with certain of the extracts. The absorbed sera were treated in the same way as the unabsorbed sera.
0 0
RESULTS
The results of the 1st experiment are presented in Sera from 9 of the 12 mite-infested catde formed precipitin bands against the P. ovis extract and 7 against the P. cuniculi extract. Sera from 2 of the 8 cattle infested with H. eurystemus but not P. ovis and 1 of the 4 uninfested catde also reacted against the P. ovis extract; serum from the latter uninfested calf also reacted against the P. cuniculi extract. The sera from the 10 cattle infested with H. eurysternUJ did not react with the H. eurystemus extract nor did those of the 2 infested with B. bovis nor the 2 uninfested. The Hypoderma extract did not react with the sera of 5 grub-infested nor with those of 5 grub-free cattle. The serum from 1 of TABLE 1.
I 0 1
·5, heavyj 4, moderate to heavyj 3, moderate; 2, light to moderatej I,light infestation. • ·Positive on 1st test, negative on retest. ••• See text.
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Several sera were absorbed with some of the extracts in the following manner. Equal volumes of the serum were mixed with the test extract in a small test tube and placed at 4°0 for approximately 24 hr (some of the sera were absorbed for 2 days). Duplicate samples of the sera were mixed with saline instead of the extract and placed at 4 °0. Mter the prescribed time the mixture was centrifuged at 900 g for 10 min. and the supernatant removed. The saline-serum mixture provided a control reaction at the same dilution as the serum which had been absorbed with the extract. In experiments I and 2, agar-gel diffusion tests were performed in either 100 mm X 15 mm or 60 mm X 15 mm glass or plastic petri dishes with
Entomo!.
1977
Fisher & Wilson:
Precipitating antibodies in P. avis-infested cattle
treated serum. This same serum (31) reacted against the HB extract. The reaction was removed when the serum was absorbed with the HB extract but not when the serum was absorbed with the PO extract. Serum 8 (P. ovis-infested) showed similar results as serum 31, except that the absorbing of the
TABLE
3.
SERUM NO.
Agar-gel reactions in selected sera after absorption with several of the test extracts. No. OF BANDS TEST ABSORBING Before After EXTRACT EXTRACT absorption absorption PO PO 1 0 PO PC 1 0* PO Saline 1 1 PO HB I 1 PO HE 1 1 PO OM-I 1 1 PO OM-2 1 1 PO Blood 1 1* PO Tissue 1 1* PO DB 1 1* HB HB I 0 HB PO 1 1** HB Saline 1 1*** PO PO 1* 1** PO PC 1* 0* PO HE 1* 1* PO OM-l 1* 1* PO OM-2 1* 1* PO HB 1* 1* PO Blood 1* 1* PO Tissue 1* 1* PO DB 1* 1* PO Saline 1* 1* PO PO 1* 0* PO PC 1* 0* PO Saline 1* 1* PO PO 1 0 PC PO 1 0 PO Saline 1 0 OM-I OM-I 1 1 OM-2 OM-2 1 I OM-l OM-2 1 1 OM-l OM-2 1 1
31 31 31 31 31 31 31 31 31 31 31 31 31 8 8 8 8 8 8 8 8 8 8 23 23 23 51 51 51 84 84 84 84 *Modified procedure-see text. **Very faint reaction. ***Extremely faint reaction.
serum with PO extract only removed part of the reaction. A faint band still was found when the absorbed serum was tested against the PO extract. Absorbing the serum with PC extract completely removed the reaction between the serum and the PO extract. Serum 23 (P. ovis-uninfested) reacted against the PO extract in the test proper. Absorbing this serum with the PO and PC extract removed the reaction between the serum and the PO extract. The saline-diluted serum still was reactive. Serum 51 (P. ovis-uninfested) reacted against the PO extract in the test proper. When the serum was absorbed with PO and PC extracts, no reaction occurred between the extract and the absorbed serum. The saline-treated serum also did not react. Serum 84 reacted against OM-l and OM-2 in the test proper. This animal was not infested with ear ticks. When this serum was absorbed with OM-l and OM-2 and retested with these extracts, the precipitin was
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the 8 cattle with tick-infested ears reacted with the O. megnini extract (OM-I). None of these 8 sera reacted with the OM-2 extract. The sera from the 4 cattle free of ear ticks did not react with either the OM-lor OM-2 extract. None of the sera from the 24 Demodex-infestedcattle reacted with the Demodex extract. None of the 24 sera (12 P. ovis-infested and 12 P. ovis-free cattle) reacted with the tissue extract but all 24 reacted against the blood extract. A thin precipitin line formed between the sera and blood extract. This line was not seen until after the plates had been treated with the stain. The results of the 2nd experiment are presented in TABLE 2. Sera from 14 of the 26 P. ovis-infested cattle formed precipitin bands against the P. ovis extract, and those from 2 of the 5 P. ovis-infested cattle reacted against the P. cuniculi extract. Sera from 2 of the P. ovis-uninfested cattle reacted with the P. ovis extract, and those from 2 of the 10 uninfested animals (51-60) reacted against the P. cuniculi extract. No precipitin bands Were formed between the H. eurysternus extract and the sera of 12 L. vituli-infested and 17 L. vituli-free cattle, nor between this extract and the sera of 2 B. bovisinfested and 25 B. bovis-uninfested cattle. The serum from 1 of the 3 grub-infested cattle formed a band against the Hypoderma extract; sera from the 3 grub-uninfested animals did not react. A precipitin band was formed between the serum of 1 of the 16 cattle without ear ticks with both the OM-l and OM-2 extracts. The sera from 10 animals infested with ear ticks did not react with the 1st extract but 1 reacted with the 2nd extract. None of the sera from 21 Demodex-infested cattle reacted against the Demodex extract, and no reaction occurred between 41 of the sera and the tissue extract. Only 2 of the 41 sera tested reacted against the blood extract. The results of the absorption tests are found in TABLE 3. The reaction that occurred with serum 31 (P. ovis-infested) and the PO extract was removed when the serum was absorbed with PO and PC extracts. Absorbing the serum with the other extracts did not remove the precipitin band that was formed between the serum and the PO extract. The serum was still reactive when diluted with the extracts as shown by the reaction of the saline-
149
J.
150
Med. Entomol.
still present in the absorbed serum. This test was repeated at a later date. The serum (unabsorbed) did not react to the O. megnini extracts the 2nd time. The results of the tests on cross-reactions (FIG. I) show (a) common antigens between P. ovis and P. cuniculi (FIG. Ia), and (b) no cross-reaction between H. bovis and P. ovis extracts (FIG. Ib), or P. cuniculi extracts (FIG. Ic). DISCUSSION
centrifuging the serum to remove the precipitated antigen-antibody complex. Only the P. ovis and P. cuniculi extracts could remove the reaction that occurs between the serum and the PO extract. The other extracts tested failed to remove the reaction. In 1 case (Serum 8, TABLE 3), the PO extract only partially removed the reaction but the PC extract removed the reaction completely. The difference between this serum and Serum 31 is that the 2 sera were tested with 2 different batches of PO antigen. In preparation of the extracts, the 2nd batch did not seem to be as dryas the 1st batch and may have resulted in a slightly different dilution of the antigen. The absorption tests were carried out by using equal amounts of serum and antigen. In the 1st example (Serum 31) enough antigen may have been present and all the antibody precipitated out, whereas in the 2nd example (Serum 8) there may not have been enough antigen to completely remove the reaction. The PC extracts were in sufficient strengths to remove the reaction completely. The reactions between the sera of the mite-free cattle and the P. ovis and P. cuniculi extracts are difficult to interpret. Two of the animals (51 & 61) were from herds with no known history of P. ovis infestation. The other cattle were bought from cattle traders and were free of the infestation when purchased but they may have been exposed previously. The reaction between serum 51 and the P. ovis extract could be removed by absorption, indicating that it was an antigen-antibody reaction. Perhaps the animal had been exposed to a parasite that was antigenically related to P. ovis. Only 1 of the 8 cattle with grub cysts in their backs reacted to the 3rd-stage larval H. bovis extract. These results agree with those of Beesley (1970)
abc FIG. I. Agar-gel diffusion reaction between selected cattle sera and P. avis, P. cuniculi, H. eurysternus, O. megnini, and H. bovis extracts. (a) Reaction between extracts and serum showing relationship between P. avis and P. cuniculi. (b) Reaction between serum and extracts showing no relationship between H. bovis and P. Dvis extracts. (c) Reaction between extracts and serum showing no relationship between H. bovis and P. cuniculi
extracts.
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The present results indicate that precIpItating antibodies are present in the sera of P. ovis-infested cattle and that these antibodies can be detected by the use of either P. ovis or P. cuniculi extract. Some of the sera only reacted to one or the other of the extracts while others reacted to both extracts. Common antigens are shared by these 2 mites as shown in FIG. I. These results confirm previous studies (Fisher 1972) on common antigens between P. ovis and P. cuniculi. At least 2 antigens are present in the P. ovis extract and 3 in the P. cuniculi extract. A possible reason that only 23 of the 38 P. ovisinfested cattle reacted to the P. ovis extract and 9 of the 17 reacted to the P. cuniculi extract is that most of the animals were less than I year old and some of them may not have been able to respond to the same degree as others. These animals may have produced antibodies but at a low level which was not detectable by agar-gel diffusion tests. In previous tests with sheep (Fisher 1972) infestations were from older animals, over 1 year old. The reaction between the P. ovis extract and the sera of the P. ovis-infested cattle is considered an antigen-antibody reaction, as the reaction correlates well with the infestation and can be removed by absorbing the serum with the test extract, and
Vol. 14, no. 2
1977
Fisher & Wilson:
Precipitating antibodies in P. ouis-infested cattle
extract did not affect the reaction between the serum and the P. ovis extract. Acknowledgments: We wish to thank Dr S. M. Gaafar, Purdue University, Lafayette, Indiana, U.S.A., and Dr 1. Fox, University of Puerto Rico, San Juan, Puerto Rico, for kindly reviewing this manuscript. We also thank Dr S. Tokuda, University of New Mexico, Albuquerque, New Mexico, U.S.A., for technical advice during these experiments; Mr C. James, University of New Mexico, Albuquerque, New Mexico, U.S.A., for disrupting the demodectic mites; Dr R. W. Miller, Agricultural Environmental Quality Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville,MD, U.S.A., for supplying sera from 8 dairy cows; and Dr R. D. Romanowski, Animal Parasitology Institute, Agricultural Research Service, for preparation of the tissue extract. LITERATURE CITED
Beesley, W. N. 1970. Observations on the biology of the ox warble, Hypoderma (Diptera: Oestridae). IV. Some serological reactions against the parasite in natural and artificial hosts. Ann. Trop. Med. Parasitol. 64: 277-81. Boulard, C. 1970. Etude pri:liminare d'une collagenase brute extraite de la larve de premier stade d'Hypoderma lineatum (de Villers). C. R. Acad. Sci., Paris 270: 1349-51. Buder, J. F. 1968. Population dynamics of Chorioptes bouis (Hering): As affected by seasonal conditions in the microclimate and host-parasite interactions. PhD thesis, Cornell University, Ithaca, New York, U.S.A. 218 p. Fisher, W. F. 1972. Precipitating antibodies in sheep infested with Psoroptes ouis (Acarina: Psoroptidae), the sheep scab mite. j. Parasitol. 58: 1218-19. 1973. Natural transmission of Demodex bouis Stiles in cattle. J. Parasitol. 59: 223-24. Fox, I., I. G. Bayona, C. C. Umpierre & J. M. Morris. 1967. Circulating precipitating antibodies in the rabbit from mite infection as shown by agar-gel tests. J. Parasitol. 53: 4Q2-05.
Kagan, I. G. & L. Norman. 1970. Serodiagnosisof parasitic diseases.p. 453-86. In: Blair,J. E., E. H. Lennette &J. P. Truant, eds., Manual of clinical microbiology, 1sted. American Society for Microbiology, Bethesda, MD, U.S.A. Weisbroth, S. H., M. B. Powell, L. Roth & S. Scher. 1974. Immunopathology of naturally occurring otodectic otoacariasis in the domestic cat. j. Am. Vet. Med. Assoc. 165: 1088-93.
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who found that only 2 of 15 naturally infested cattle reacted with a Hypoderma antigen in his agar-gel diffusion tests. Boulard (1970), in similar experiments, did not find any precipitins in cattle sera by the immunoelectrophoresis technique. The reaction between the serum of an animal infested with both grubs and P. ovis and the HB extract was considered a specific reaction, as it could be removed by absorption (TABLE 3) and there was no evidence of cross-reaction between the grub extract and the P. ovis extract (FIG. I). No reactions occurred between the sera of H. eurysternus- or L. vituli-infested cattle and the H. eurysternus extract, although some of these cattle carried heavy burdens oflice. Under the conditions of this test, precipitins were not present in the sera of cattle infested with these species of lice. Zaugg (pel's. commun.), similarly, did not find precipitins in cattle infested with sucking lice. Several sera reacted to the O. megnini extracts, but this is considered to be a nonspecific reaction because the reaction between the serum of the uninfested animal in experiment 2 could not be removed by absorption. None of the cattle reacted to the extract of D. bovis. These results may be expected, as Fisher (unpubl. results) tested the sera of 63 goats and 99 cattle that were infested with Demodex spp. in agar-gel diffusion tests and found that none of them reacted to the extracts used. There were no prccipitins formed between the sera and the tissue extract which indicates that there was no reaction to host tissue proteins. The reaction between the blood extracts and the sera (26 of 65) is difficult to interpret. The reaction is probably not related to the P. ovis infestations because in the 1st experiment all of the infested and uninfested cattle sera reacted with the blood extract and secondly, absorption of the serum of a P. ovis-infested cow with the blood
lSI
Med. Ento_ol.
Vol. 14,
DO.
25 Nove_ber
2: 146-151
1977
PRECIPITATING ANTIBODIES IN CATTLE INFESTED BY PSOROPTES OVIS (ACARINA: PSOROPTIDAE)1,2 By WilliaDl F. Fisher3,4 and Grant I. Wilson3
Precipitating antibodies have been detected by agar-gel diffusion tests in the sera of animals infested with mites of the family Psoroptidae. Serum precipitins were found by Fox et al. (1967) in rabbits infested with Psoroptes cuniculi (Delafond), by Fisher (1972) in sheep infested with Psoroptes ovis (Hering), by Butler (1968) in cattle infested with Chorioptes bovis (Hering), and by Weisbroth et al. (1974) in cats infested with Otodectes 0Jnotis (Hering). Information on the antibody response to P. ovis in cattle and the immunochemical identification of those antibodies is needed. Such information may be useful in treatment and control of diseases caused by these mites. Because many cattle may be concomitantly parasitized by a number of arthropods (lice, grubs, and ticks), the antibody response to these parasites must be taken into account when they occur in animals being tested for antibodies to P. ovis. This paper presents the results of agar-gel diffusion tests on the sera of cattle infested with P. ovis, Hypoderma bovis (Linnaeus), Hypoderma lineatum (de Villiers), Otobius megnini (Duges), Haematopinus eurysternus (Denny), Linognathus vituli (Linnaeus), Bovicola bovis (Linnaeus), Demodex bovis (Stiles), and combinations of these parasites, against extracts of several of these arthropods. lMention of a trade name, a proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 'Address reprint requests to Fisher in Kerrville. ·Parasite Research Laboratory, Western Region, Agricultural Research Service, U.S. Department of Agriculture, P. O. Box 705, Albuquerque, New Mexico 87103, U.S.A. 'Present address: U.S. Livestock Insects Laboratory, USDA, ARS, P.O. Box 232. Kerrville, Texas 78028, U.S.A.
MATERIALS
AND METHODS
Experimental animals In the 1st experiment, sera were collected from 12 cattle (10 calves, 2 cows) with P. ovis infestations and 12 cattle (6 calves, 6 cows) without P. ovis infestations. The P. ovis infestations were from 1 to 8 months duration and were moderate to heavy at the time of sampling. Most of the infestations were the result of manually transferring mites to clean animals and allowing the infestations to develop until they became suitable subjects for the testing of experimental acaricides. In the 2nd experiment, sera were collected from 26 cattle (24 calves, 2 cows) with P. ovis infestations and 42 cattle (34 calves, 8 cows) without P. ovis infestations. The P. ovis infestations were from 2 to 9 months duration and were light to heavy at the time of sampling. The sera from 21 of the uninfested calves were the preinfestation samples of 21 of the P. ovis-infested calves. The sera from the 8 mature cows came from cattle in Beltsville, MD, U.S.A. Most of the cattle of both experiments were Hereford or Hereford-Angus crosses. The 8 mature cows of the 2nd experiment were dairy cows. Some of the cattle from both experiments (P. ovis-infested and uninfested) were parasitized with H. eurysternus (10 cows), L. vituli (12 calves), Hypoderma spp. (7 calves, 1 cow), O. megnini (17 calves, 1 cow), B. bovis (4 calves) and D. bovis (35 calves, 10 cows) at the time of bleeding. Thirty-three were found to be free of H. eurysternus, 21 free of L. vituli, 8 free of Hypoderma spp., 20 free of O. megnini, and 27 free of B. bovis at the time of sampling. All sera were stored at -18°C until needed.
Preparation of extracts P. ovis mites were collected from cattle and P. cuniculi mites from rabbits, as described by Fisher (1972). P. ovis extract (PO) was prepared from mites that had been freshly killed by freezing and from others that had been stored frozen from 4 to 10 months. The mites were thawed, dried (a portion at 37°C for 5 hr and a portion at 39°C for 16 hr) and ground in a Ten Broeck tissue grinder for 5 min. in 1: 5000 merthiolated saline [20 ml of 1: 1000 thimerosal solution (MerthiolateiB> solution, Eli Lilly & Company, Indianapolis, Indiana 46206,
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Abstract: Precipitating antibodies, as demonstrated by agar-gel diffusion tests, were found in the sera of 23 of 38 Psoroptes ovis-infested cattle when tested against a P. ovis extract and in the sera of 9 of 17 cattle when tested against a P. cUlliculi extract. Sera from 5 of 54 P. ovis-uninfested cattle reacted to the P. ovis extract and 3 of 22 reacted to the P. cUlliculi extract. Analysis of the P. ovis and the P. CUlliculiextracts indicates that they share common antigens. The reaction between the sera of mite-infested cattle and the above antigens is a specific antigenantibody reaction. Extracts of Hypoderma bovis, Otobius megllilli, Haematopinus eurystemus, Demodex hovis, cattle tissue, and cattle blood were made and tested with some of the above sera. Only the H. bovis extract gave a specific reaction with the serum of a grub-infested calf. Reactions with the O. megllilli and blood extracts were considered nonspecific.
1977
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& Wilson:
Precipitating
TABLE
infested
SERUM NO. I
2 3 4 5 6 7 8 9 10 11 12 13-18 19-20 21 22 23 24
in P. ovis-infested J.
147
cattle
Agar-gel reactions in the sera of Psoroptes ovisand P. ovis-uninfested cattle with P. ovis (PO) and P. cuniculi (PC) extracts (Experiment I). DEGREE OF DURATION PARASITE INFESOF INFESTATION TATION· INFESTATION
P. ovis
5 5 5 5 4 4 4
3-4 3 3 t t
H. eurysternus Uninfested ttt ttt
I I
5 5
3 mo. 2 mo. 2 mo. 2 mo. 5 mo. 1.75 mo. 1.75 mo. 8 mo. 1.75 mo. 1.75 mo. 2-3 mo. 2-3 mo. -tt
EXTRACTS PO
PC
No. of bands
0 0 2 0
I··· I I·· 2 I
I···
I"
I··
0 0 2 0 0
I I
2
I
I
2 0
0 0 0 0 0
I
0 0 I
I
0
0
·5, heavy; 4, moderate to heavy; 3, moderate; 2, light to moderate; I, light infestation. • ·Positive on test; negative on retest or vice versa. ···Faint reaction. tConcurrent H. eurysternus infestation (heavy). ttField infestation; duration unknown. ttt Bovicola bovis present.
setting-20 kc, Bronson Sonic Power, Div. Bronson Instruments, Inc., Mirybrook Rd, Danbury, Conn. 06810, U.S.A.) for 5 min. The mixture was placed at 4°C for 2 days and centrifuged at 900 g for 15 min. The supernatant was removed and frozen. The tissue extract was prepared from pieces of cattle hide that were dehaired with lime and sodium sulfide. Portions of the tissue were placed in 0.9% saline and some in 10% formalin. The specimens were removed from their respective solutions and dried at 37-38 DC for 24 hr, weighed (saline treated-635.3 mg; formalin treated-776.8 mg), and homogenized in phosphate-buffered saline (PBSpH 7.2) using a Virtis blender. An additional 8 ml of PBS was added to the mixture to obtain a better homogenization. The tissue suspension was further ground in a tissue grinder and centrifuged in a refrigerated centrifuge at 17,000 rpm for 30 min. The supernatant was removed, concentrated to 10 ml with an ultrafilter [Ami con Diaflow Ultrafilter (UM-2), 1000 mol.wt. cut off, Amicon Corporation, Lexington, Mass. 02173, U.S.A.] and frozen at -18°C until needed. A blood extract was prepared from whole heparinized cattle blood. The sample was taken from 2 cows that were heavily parasitized with H. eurysternus. The blood was then frozen at -18 DC; when
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U.S.A.) and 80 ml of 0.9% saline] at a ratio of 100 mg of dried mites per 1.5 ml of saline. The mixture was left standing, with periodic shaking, at 6 DC for 2 to 3 days. It was then centrifuged at 900 g for 20 min. The supernatant was removed and used as the test antigen. The P. cuniculi extract (PC) was prepared using the same method as for the PO extract. The Hypoderma bovis extract (HB) was prepared from 4 mature, 3rd-stage H. bovis larvae. The larvae were collected from the backs of several cattle, rinsed in saline and frozen at -18 DCfor 2 months. When needed, they were thawed and dried for 5 hr at 37°C, and prepared in the same manner as the PO extract except that the ratio of tissue to saline was 1 g larval tissue to 4 ml of saline. The O. megnini extract (OM-I) was prepared from 28 nymphal ear ticks that were collected from 1 cow. The ticks were kept at 22-24 DCfor 6 days and then frozen at -18 DCfor 9 months. When needed, they were thawed, dried at 37 DCfor 5 hr, and prepared in the same manner as the HB extract except that the ratio of the tick material to saline was 100 mg dried tick per 1 ml of saline. A 2nd extract (OM-2) was prepared in the same manner as the OM-l extract from 9 nymphal ear ticks obtained from goats' ears. The H. eurysternus extract (HE) was prepared from lice obtained from heavily parasitized cattle. The lice were separated from the hair and frozen at -18°C; when needed they were thawed and dried (a portion at 37°C for 5 hr and a portion at 39°C for 16 hr) and prepared in the same manner as OM-I. A D. bovis extract (DB) was prepared from mites obtained from cattle hides that had been chemically dehaired in a lime-sodium sulfide solution (Fisher 1973). The mites were removed from the nodules, rinsed in saline, transferred to a blender and homogenized at high speed for 60 sec. The suspension was passed through a strainer (4.65 mesh per cm2) to remove tissue debris and entrapped mites. The mites were sedimented by centrifugation and then frozen at -18 DC; when needed they were thawed, placed at 4°C overnight, and suspended in 0.9% saline the next day. The suspension was passed through a strainer and the entrapped mites and remaining tissue were collected in saline and placed at 4°C for several days. The suspension was then centrifuged and the mites resuspended in 32 ml of saline so that 1620 mites were contained in each ml of solution. Sixteen ml of the suspension was removed and the mites were disrupted with a sonicator (Bronson Sonicator, Model LS-75, #6
antibodies
J. Med.
148
needed it was thawed and used without any further treatment.
Serological methods
TABLE 2. Agar-gel reactions in the sera of Psoroptes ovis-infested and P. ovis-uninfested cattle with P. ovis (PO) and P. cuniculi
(PC) extracts (Experiment 2).
SERUM NO. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52-59 60 61 62-92
DEGREE EXTRACTS OF DURATION PO PC PARASITE INFESOF INFESTATIONTATION*INFESTATION No. of bands P.ovis
Uninfested·"
5 5 5 5 4 4 4 4 3-4 3-4 3 3 3 3 3 3 3 2-3 2 2 1 I 1 I 1 1 0 0 0 0 0
6-7 mo. 4mo. 3 mo. 3 mo. 9 mo. 6-7 mo. 4 mo. 2 mo. 4 mo. 2 mo. 6-7 mo. 4 mo. 2 mo. 2 mo. 2 mo. 2 mo. 2 mo. 4 mo. 4 mo. 2 mo. 4 mo. 4 mo. 4 mo. 4 mo. 2 mo. 9 mo.
2 2 0 0 2 1 I 0 1 0 0 I I I 1 0
1** 0 0 1 0 0 0 2 I 0 I 0 0 I 0
2
3 0
Vo!. 14, no. 2
1% agar dissolved in glycine-buffered saline (Kagan & Norman 1970). The pH was adjusted to 7-8. Sixteen ml (large plates) or 8 ml (small plates) of melted agar was poured into the plates, allowed to harden for 0.5 hr at room temperature and placed in the refrigerator at 4°0 overnight. Wells, 5 mm in diam., were cut with a No.2 cork borer so that 6 peripheral wells were grouped around a central well. The center-to-center distance between the central well and each peripheral well was 10 mm. The wells were sealed with a drop of melted agar. The center well was either filled with test serum and the peripheral wells with the test antigens, or the central well was filled with the test antigen and the peripheral wells with the test sera. The plates were placed in a humid chamber at 22-24°0. The plates were usually set up in the afternoon and following incubation at 22-24 °0 overnight were refilled with the appropriate antigens or sera the next morning. In the case of 2 plates, the wells were filled in the morning and refilled in the late afternoon. In a few cases the plates were not refilled due to a shortage of a particular reagent. These 2 modifications in the procedure will be referred to in the results as the modified procedure. Observations of the results obtained were made over a period of 4 to 6 days with the aid of a 7 X hand lens over an illuminated dark background. Mter the final observation the plates were soaked in glycine buffer for 36 hr and then flooded with 0.002% amido black as described by Kagan & Norman (1970). Sera were usually tested against each extract once, but in several cases some of the sera were tested twice with certain of the extracts. The absorbed sera were treated in the same way as the unabsorbed sera.
0 0
RESULTS
The results of the 1st experiment are presented in Sera from 9 of the 12 mite-infested catde formed precipitin bands against the P. ovis extract and 7 against the P. cuniculi extract. Sera from 2 of the 8 cattle infested with H. eurystemus but not P. ovis and 1 of the 4 uninfested catde also reacted against the P. ovis extract; serum from the latter uninfested calf also reacted against the P. cuniculi extract. The sera from the 10 cattle infested with H. eurysternUJ did not react with the H. eurystemus extract nor did those of the 2 infested with B. bovis nor the 2 uninfested. The Hypoderma extract did not react with the sera of 5 grub-infested nor with those of 5 grub-free cattle. The serum from 1 of TABLE 1.
I 0 1
·5, heavyj 4, moderate to heavyj 3, moderate; 2, light to moderatej I,light infestation. • ·Positive on 1st test, negative on retest. ••• See text.
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Several sera were absorbed with some of the extracts in the following manner. Equal volumes of the serum were mixed with the test extract in a small test tube and placed at 4°0 for approximately 24 hr (some of the sera were absorbed for 2 days). Duplicate samples of the sera were mixed with saline instead of the extract and placed at 4 °0. Mter the prescribed time the mixture was centrifuged at 900 g for 10 min. and the supernatant removed. The saline-serum mixture provided a control reaction at the same dilution as the serum which had been absorbed with the extract. In experiments I and 2, agar-gel diffusion tests were performed in either 100 mm X 15 mm or 60 mm X 15 mm glass or plastic petri dishes with
Entomo!.
1977
Fisher & Wilson:
Precipitating antibodies in P. avis-infested cattle
treated serum. This same serum (31) reacted against the HB extract. The reaction was removed when the serum was absorbed with the HB extract but not when the serum was absorbed with the PO extract. Serum 8 (P. ovis-infested) showed similar results as serum 31, except that the absorbing of the
TABLE
3.
SERUM NO.
Agar-gel reactions in selected sera after absorption with several of the test extracts. No. OF BANDS TEST ABSORBING Before After EXTRACT EXTRACT absorption absorption PO PO 1 0 PO PC 1 0* PO Saline 1 1 PO HB I 1 PO HE 1 1 PO OM-I 1 1 PO OM-2 1 1 PO Blood 1 1* PO Tissue 1 1* PO DB 1 1* HB HB I 0 HB PO 1 1** HB Saline 1 1*** PO PO 1* 1** PO PC 1* 0* PO HE 1* 1* PO OM-l 1* 1* PO OM-2 1* 1* PO HB 1* 1* PO Blood 1* 1* PO Tissue 1* 1* PO DB 1* 1* PO Saline 1* 1* PO PO 1* 0* PO PC 1* 0* PO Saline 1* 1* PO PO 1 0 PC PO 1 0 PO Saline 1 0 OM-I OM-I 1 1 OM-2 OM-2 1 I OM-l OM-2 1 1 OM-l OM-2 1 1
31 31 31 31 31 31 31 31 31 31 31 31 31 8 8 8 8 8 8 8 8 8 8 23 23 23 51 51 51 84 84 84 84 *Modified procedure-see text. **Very faint reaction. ***Extremely faint reaction.
serum with PO extract only removed part of the reaction. A faint band still was found when the absorbed serum was tested against the PO extract. Absorbing the serum with PC extract completely removed the reaction between the serum and the PO extract. Serum 23 (P. ovis-uninfested) reacted against the PO extract in the test proper. Absorbing this serum with the PO and PC extract removed the reaction between the serum and the PO extract. The saline-diluted serum still was reactive. Serum 51 (P. ovis-uninfested) reacted against the PO extract in the test proper. When the serum was absorbed with PO and PC extracts, no reaction occurred between the extract and the absorbed serum. The saline-treated serum also did not react. Serum 84 reacted against OM-l and OM-2 in the test proper. This animal was not infested with ear ticks. When this serum was absorbed with OM-l and OM-2 and retested with these extracts, the precipitin was
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the 8 cattle with tick-infested ears reacted with the O. megnini extract (OM-I). None of these 8 sera reacted with the OM-2 extract. The sera from the 4 cattle free of ear ticks did not react with either the OM-lor OM-2 extract. None of the sera from the 24 Demodex-infestedcattle reacted with the Demodex extract. None of the 24 sera (12 P. ovis-infested and 12 P. ovis-free cattle) reacted with the tissue extract but all 24 reacted against the blood extract. A thin precipitin line formed between the sera and blood extract. This line was not seen until after the plates had been treated with the stain. The results of the 2nd experiment are presented in TABLE 2. Sera from 14 of the 26 P. ovis-infested cattle formed precipitin bands against the P. ovis extract, and those from 2 of the 5 P. ovis-infested cattle reacted against the P. cuniculi extract. Sera from 2 of the P. ovis-uninfested cattle reacted with the P. ovis extract, and those from 2 of the 10 uninfested animals (51-60) reacted against the P. cuniculi extract. No precipitin bands Were formed between the H. eurysternus extract and the sera of 12 L. vituli-infested and 17 L. vituli-free cattle, nor between this extract and the sera of 2 B. bovisinfested and 25 B. bovis-uninfested cattle. The serum from 1 of the 3 grub-infested cattle formed a band against the Hypoderma extract; sera from the 3 grub-uninfested animals did not react. A precipitin band was formed between the serum of 1 of the 16 cattle without ear ticks with both the OM-l and OM-2 extracts. The sera from 10 animals infested with ear ticks did not react with the 1st extract but 1 reacted with the 2nd extract. None of the sera from 21 Demodex-infested cattle reacted against the Demodex extract, and no reaction occurred between 41 of the sera and the tissue extract. Only 2 of the 41 sera tested reacted against the blood extract. The results of the absorption tests are found in TABLE 3. The reaction that occurred with serum 31 (P. ovis-infested) and the PO extract was removed when the serum was absorbed with PO and PC extracts. Absorbing the serum with the other extracts did not remove the precipitin band that was formed between the serum and the PO extract. The serum was still reactive when diluted with the extracts as shown by the reaction of the saline-
149
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150
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still present in the absorbed serum. This test was repeated at a later date. The serum (unabsorbed) did not react to the O. megnini extracts the 2nd time. The results of the tests on cross-reactions (FIG. I) show (a) common antigens between P. ovis and P. cuniculi (FIG. Ia), and (b) no cross-reaction between H. bovis and P. ovis extracts (FIG. Ib), or P. cuniculi extracts (FIG. Ic). DISCUSSION
centrifuging the serum to remove the precipitated antigen-antibody complex. Only the P. ovis and P. cuniculi extracts could remove the reaction that occurs between the serum and the PO extract. The other extracts tested failed to remove the reaction. In 1 case (Serum 8, TABLE 3), the PO extract only partially removed the reaction but the PC extract removed the reaction completely. The difference between this serum and Serum 31 is that the 2 sera were tested with 2 different batches of PO antigen. In preparation of the extracts, the 2nd batch did not seem to be as dryas the 1st batch and may have resulted in a slightly different dilution of the antigen. The absorption tests were carried out by using equal amounts of serum and antigen. In the 1st example (Serum 31) enough antigen may have been present and all the antibody precipitated out, whereas in the 2nd example (Serum 8) there may not have been enough antigen to completely remove the reaction. The PC extracts were in sufficient strengths to remove the reaction completely. The reactions between the sera of the mite-free cattle and the P. ovis and P. cuniculi extracts are difficult to interpret. Two of the animals (51 & 61) were from herds with no known history of P. ovis infestation. The other cattle were bought from cattle traders and were free of the infestation when purchased but they may have been exposed previously. The reaction between serum 51 and the P. ovis extract could be removed by absorption, indicating that it was an antigen-antibody reaction. Perhaps the animal had been exposed to a parasite that was antigenically related to P. ovis. Only 1 of the 8 cattle with grub cysts in their backs reacted to the 3rd-stage larval H. bovis extract. These results agree with those of Beesley (1970)
abc FIG. I. Agar-gel diffusion reaction between selected cattle sera and P. avis, P. cuniculi, H. eurysternus, O. megnini, and H. bovis extracts. (a) Reaction between extracts and serum showing relationship between P. avis and P. cuniculi. (b) Reaction between serum and extracts showing no relationship between H. bovis and P. Dvis extracts. (c) Reaction between extracts and serum showing no relationship between H. bovis and P. cuniculi
extracts.
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The present results indicate that precIpItating antibodies are present in the sera of P. ovis-infested cattle and that these antibodies can be detected by the use of either P. ovis or P. cuniculi extract. Some of the sera only reacted to one or the other of the extracts while others reacted to both extracts. Common antigens are shared by these 2 mites as shown in FIG. I. These results confirm previous studies (Fisher 1972) on common antigens between P. ovis and P. cuniculi. At least 2 antigens are present in the P. ovis extract and 3 in the P. cuniculi extract. A possible reason that only 23 of the 38 P. ovisinfested cattle reacted to the P. ovis extract and 9 of the 17 reacted to the P. cuniculi extract is that most of the animals were less than I year old and some of them may not have been able to respond to the same degree as others. These animals may have produced antibodies but at a low level which was not detectable by agar-gel diffusion tests. In previous tests with sheep (Fisher 1972) infestations were from older animals, over 1 year old. The reaction between the P. ovis extract and the sera of the P. ovis-infested cattle is considered an antigen-antibody reaction, as the reaction correlates well with the infestation and can be removed by absorbing the serum with the test extract, and
Vol. 14, no. 2
1977
Fisher & Wilson:
Precipitating antibodies in P. ouis-infested cattle
extract did not affect the reaction between the serum and the P. ovis extract. Acknowledgments: We wish to thank Dr S. M. Gaafar, Purdue University, Lafayette, Indiana, U.S.A., and Dr 1. Fox, University of Puerto Rico, San Juan, Puerto Rico, for kindly reviewing this manuscript. We also thank Dr S. Tokuda, University of New Mexico, Albuquerque, New Mexico, U.S.A., for technical advice during these experiments; Mr C. James, University of New Mexico, Albuquerque, New Mexico, U.S.A., for disrupting the demodectic mites; Dr R. W. Miller, Agricultural Environmental Quality Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville,MD, U.S.A., for supplying sera from 8 dairy cows; and Dr R. D. Romanowski, Animal Parasitology Institute, Agricultural Research Service, for preparation of the tissue extract. LITERATURE CITED
Beesley, W. N. 1970. Observations on the biology of the ox warble, Hypoderma (Diptera: Oestridae). IV. Some serological reactions against the parasite in natural and artificial hosts. Ann. Trop. Med. Parasitol. 64: 277-81. Boulard, C. 1970. Etude pri:liminare d'une collagenase brute extraite de la larve de premier stade d'Hypoderma lineatum (de Villers). C. R. Acad. Sci., Paris 270: 1349-51. Buder, J. F. 1968. Population dynamics of Chorioptes bouis (Hering): As affected by seasonal conditions in the microclimate and host-parasite interactions. PhD thesis, Cornell University, Ithaca, New York, U.S.A. 218 p. Fisher, W. F. 1972. Precipitating antibodies in sheep infested with Psoroptes ouis (Acarina: Psoroptidae), the sheep scab mite. j. Parasitol. 58: 1218-19. 1973. Natural transmission of Demodex bouis Stiles in cattle. J. Parasitol. 59: 223-24. Fox, I., I. G. Bayona, C. C. Umpierre & J. M. Morris. 1967. Circulating precipitating antibodies in the rabbit from mite infection as shown by agar-gel tests. J. Parasitol. 53: 4Q2-05.
Kagan, I. G. & L. Norman. 1970. Serodiagnosisof parasitic diseases.p. 453-86. In: Blair,J. E., E. H. Lennette &J. P. Truant, eds., Manual of clinical microbiology, 1sted. American Society for Microbiology, Bethesda, MD, U.S.A. Weisbroth, S. H., M. B. Powell, L. Roth & S. Scher. 1974. Immunopathology of naturally occurring otodectic otoacariasis in the domestic cat. j. Am. Vet. Med. Assoc. 165: 1088-93.
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who found that only 2 of 15 naturally infested cattle reacted with a Hypoderma antigen in his agar-gel diffusion tests. Boulard (1970), in similar experiments, did not find any precipitins in cattle sera by the immunoelectrophoresis technique. The reaction between the serum of an animal infested with both grubs and P. ovis and the HB extract was considered a specific reaction, as it could be removed by absorption (TABLE 3) and there was no evidence of cross-reaction between the grub extract and the P. ovis extract (FIG. I). No reactions occurred between the sera of H. eurysternus- or L. vituli-infested cattle and the H. eurysternus extract, although some of these cattle carried heavy burdens oflice. Under the conditions of this test, precipitins were not present in the sera of cattle infested with these species of lice. Zaugg (pel's. commun.), similarly, did not find precipitins in cattle infested with sucking lice. Several sera reacted to the O. megnini extracts, but this is considered to be a nonspecific reaction because the reaction between the serum of the uninfested animal in experiment 2 could not be removed by absorption. None of the cattle reacted to the extract of D. bovis. These results may be expected, as Fisher (unpubl. results) tested the sera of 63 goats and 99 cattle that were infested with Demodex spp. in agar-gel diffusion tests and found that none of them reacted to the extracts used. There were no prccipitins formed between the sera and the tissue extract which indicates that there was no reaction to host tissue proteins. The reaction between the blood extracts and the sera (26 of 65) is difficult to interpret. The reaction is probably not related to the P. ovis infestations because in the 1st experiment all of the infested and uninfested cattle sera reacted with the blood extract and secondly, absorption of the serum of a P. ovis-infested cow with the blood
lSI
