NEOSPORA CANINUM AS CAUSATIVE-PATHOGEN OF ABORTION IN CATTLE
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ARTICLES
NEOSPORA CANINUM AS CAUSATIVE-PATHOGEN OF ABORTION IN CATTLE
Mazuz, l. M., Fish, l., Molad, t., Savitsky, i., Wolkomirsky, r., leibovitz, B. and Shkap, V. department of Parasitology, Kimron Veterinary institute, Bet dagan, israel. Corresponding author: Monica l. Mazuz, department of Parasitology, Kimron Veterinary institute, P.o.Box 12, 50250 Bet dagan, israel. Email: monica@int.gov.il
ABSTRACT
Neosporosis, caused by infection with the intracellular protozoan Neospora caninum, is recognized as one of the major causes of infectious abortion in cattle. Since abortion is the only clinical sign associated with N. caninum infection, diagnosis is complicated, however it is crucial to understand the epidemiology of the disease, and to undertake measures to reduce losses to the cattle industry. in israel the diagnosis of neosporosis is based on the detection of specific antibodies in aborted dams, or in the body fluids of aborted fetuses. the presence of antibodies in sera of aborted dams indicates exposure to N. caninum, but does not necessarily point to the causative agent of the abortion. in the present study sera from 7,951 aborted dams and body fluids from 446 fetuses were analyzed by the indirect fluorescent antibodies test (iFat). N. caninum seropositivity was found in 51.4% of the dams, and in 18.2% of the fetuses. in addition, primers were designed for the detection of N. caninum dNa, by a specific nested PCr (nPCr) test, in brain tissues from aborted fetuses. the primers do not react with other closely related apicomplexa species. the performance of both molecular and serological assays was evaluated in 97 samples of fetuses. Comparable results were observed in 57 negative and 20 positive samples, while 20 remaining samples showed positivity in only one of the assays. in addition, 26 paired dam and aborted fetuses were tested. of these, 15 samples obtained from dams or fetuses were found to be negative by both tests, 5 seropositive dams had positive fetuses in at least one of the assays, and 6 samples from seronegative dams were found to be positive in at least one of the fetus’ assays. the results obtained show high prevalence of N. caninum in cattle in israel and emphasize the necessity for the application of several methods for a reliable diagnosis of neosporosis as a cause of abortion.
Neosporosis is caused by the intracellular protozoan parasite Neospora caninum and is known as one of the major causes of infectious abortion, stillbirth and reproductive failure in cattle worldwide, including Israel (1, 2, 3, 4, 5). Cattle infected with N. caninum exhibit no clinical signs other than abortion. In the absence of typical clinical signs of infection, diagnosis and control of neosporosis is complicated. The mechanisms that cause abortion have not yet been fully elucidated (6). In Israel diagnosis of neosporosis is based on the detection of specific N. caninum antibodies on the aborting cow or in the body fluid of the aborted fetus. Presence of serum antibodies in dams indicates exposure to N. caninum, (7) but is not necessarily associated with the reproductive failure; on other hand, seronegative results do not exclude the presence of infection. (8). Non-aborting seropositive dams have been reported worldwide, including in Israel (5, 9). It is documented that only some of infected dams abort, while most of their calves are born infected but healthy (10). It has been widely reported that the level of abortion rate in N. caninum sero-positive dams is 3.5 to 7 times higher than in their seronegative counterparts (11, 12). Molecular methods allowing detection of specific N.caninum 14
INTRODUCTION
DNA in tissues of aborted fetuses have been applied for complementary diagnosis of neosporosis. The detection of N. caninum DNA provided evidence of intra-uterine transmission of parasites from dams to offspring (13, 14, 15, 16, 17). In the present study, aborting dams and aborted fetuses were examined using either serology or molecular methods in order to evaluate whether the two techniques complement each other thus allowing for a more reliable diagnosis of neosporosis.
MATERIALS AND METHODS
Samples Sera from 7,951 aborting cows and thoracic fluid from 446 aborted fetuses, from farms with history of abortion, were submitted for Neospora caninum diagnosis between 20062009. Brain tissues from 98 aborted fetuses were tested by nested PCR (nPCR), and 26 paired samples from dams and their respective fetuses were examined by both nPCR and indirect fluorescent antibody test (IFAT). Indirect fluorescent antibody test (IFAT( Serum from dams and thoracic liquids from aborted fetuses were examined for the presence of antibodies to N. caninum Volume 66 (1) 2011
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ISRAEL JOURNAL OF VETERINARY MEDICINE by IFAT as previous described (18). The cut-off was 1:200 and 1:80 for dams and fetuses, respectively (19, 4). DNA extraction and purification from brain tissues Twenty milligrams of brain tissue collected from aborted fetuses were used for DNA purification. The ground tissues were digested with Proteinase K overnight at a temperature of 56º C, and then DNA purification was performed using Qiagen DNeasy Tissue Kit (QIAGEN, USA). Samples were frozen and stored at - 20ºC until use. For positive control N. caninum from the Israeli isolate NcIs491 were grown in Vero cells, and parasites were obtained after destruction of monolayer cells and subsequent release of parasites from the cells (5). The media containing parasites and cell debris were collected, parasites were separated from cells by differential centrifugation at 70x g for 5 minutes, and the supernatant containing parasites was passed to a new tube, centrifuged again at 1000x g for 20 minutes. The pelleted parasites were washed in PBS at least 3 times by centrifuge at 1000g for 20 minutes. The specificity of the nPCR test to N. caninum was evaluated using DNA from Toxoplasma gondii (RH strain) parasites and Besnoitia besnoiti, taxonomically related tissue cystforming coccidian parasite. Nested PCR (nPCR) amplification Two sets of primers were designed for nPCR, based on DNA sequence of N. caninum (GenBank Accession number X84238). The nPCR reaction was performed with an external set of primers: forward primer 5 ': CTG CTG ACG TGT CGT TGT TG-3' represent nucleotide (nt) position 476 of referred sequence and reverse primer 5 ': CAT CTA CCA GGC CGC TCT TC-3' reverse sequence of nt position 1014, and an internal set of primers: forward primer 5 ': GCG TCA GGG TGA GGA CAG TG-3' nt position 631 of referred sequence and reverse primer 5 ': CTC TCC GTT CGC CAG CAG TG -3' reverse sequence of nt position 910. The PCR reaction contained: 1µl of genomic DNA, each primer concentration 4ηg and 2x Readdy Mix (AB gene) containing: DNA Polymerase Thermoprime Plus (0.3125 units), 18.75 mM Tris-HCI (pH 8.8), 5mM (NH4)2SO4 0.375 mM MgCl, 0.0025% Tween 20 and 0.05mM dNTP mix in final volume of 50 μl. The cycling protocol, following preheating of 95ºC for 3 minutes were: 30 cycles of denaturizing at 94º C for 30 seconds, annealing for 30 seconds at 56º C for the external primers and 57º C for the internal primers, extension at 72ºC for 40 seconds and a final extension at 72ºC for 5 minutes. The nPCR was performed using 1μl of the first reaction as a template. After amplification the reaction products were visualized on 1.5% agarose gel. to 1:1600 in 22.7% (933 samples) and ≥1:3200 in 52% (2, 153 sample). The results obtained by the IFAT in 446 aborted fetuses were: 18.2% (81 samples) positive and 81.8% (365 samples) negative. As shown in Figure 1, the titer in seropositive fetuses ranged from 1:80 to 1:1280. Distribution of antibody titers in fetuses was 23.5% (19 samples) up to 1:160, 39.5 % (32 samples) from 1:320-1:640 and ≥1:1280 in 37% (30 samples).
Figure 1. distribution of N. caninum antibodies in 7951 dams and 446 aborted fetuses.
As shown in Figure 2, the primers designed for nPCR successfully detect N. caninum from aborted infected brain and no amplification was detected when DNA from T. gondii or B. besnoiti were applied. Ninety seven fetuses examined by IFAT and nPCR, showed comparable results. A complete agreement was detected in the assays in 20 positive and 57 negative samples. In 21 out of 98 samples tested, 11 samples were positive by the nPCR, but negative in IFAT, and 10 samples were found positive by IFAT, but negative by nPCR (Figure 3).
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RESULTS
Out of the 7,951 sera from aborted cows tested for N. caninum antibodies, 4,104 (51.4%) were seropositive with titers ranging from 1:200 to >1:3200, and 3,847 samples (48,4%) were seronegative (Figure 1). Among seropositive cows the titers found were: up to 1:400 in 24.8% (1,018 samples), between 1:800
Figure 2. Specificity of the nPCr. lanes: 1-Marker, 2 Negative control, 3- N. caninum – positive control, 4- N. caninum positive abortion brain, 5-7 Negative brains from aborted fetuses, 8Toxoplasma gondii and 9- Besnoitia besnoiti.
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15
ARTICLES neosporosis have higher titers of those infected but non-aborting. The primers designed for nPCR in this study specifically detected N. caninum 251 bp fragment from DNA of aborted fetuses, and no DNA from T. gondii and B. besnoiti was amplified. The detection of N. caninum DNA in aborted fetuses by PCR and nPCR was described previously (14, 25, 29, 30). Examination of the aborted fetuses resulted in 79% agreement between nPCR and IFAT. The discrepancy found in negative IFA results but positive by nPCR, or on the other hand, positively tested by IFA, but negative in nPCR, was described in other studies when using various methods (15, 31, 32). There were several explanations for the differences observed using various assays. According to Dubey et al. (31), negative serological results in infected fetuses were influenced by the age of fetus, level of exposure and time between exposure and abortion. It has also been found that lack of antibodies in the fetuses might give inconclusive results concerning absence of infection, since the fetus might be infected in the late gestation, leaving insufficient time for antibody production (4). Negative results in nPCR in seropositive fetuses might result as the distribution pattern of parasites in the host tissue which is as yet unclear. Furthermore the distribution of parasites might vary between animals, and the parasite load might be influenced by the stage of gestation and age at which the fetus was aborted (33). In addition, the brain tissues although considered to be the most suitable for the detection of N. caninum DNA by PCR (14, 32), it is possible that examination of additional organs would lead to increased reliability of the diagnosis. On the other hand, as shown by Yao et al., (32), PCR applied to various tissues including brain, heart, lung, liver, spleen, kidney and skeletal muscle, resulted in a total of 15 positive samples tested from various tissues, while only 2 were negative from brain, but positive from kidney and gluteus. Examinations of the paired samples showed that all seropositive dams produced positive fetuses by at least one of the assays, while among seronegative samples from dams, positive fetuses were detected in 23 %. The results provided evidence of vertical transmission of N. caninum and emphasize the high rate of transmission of parasites from dams to their offspring. Studies with dams and asymptomatic progenies were summarized by Dubey et al., (4), with seropositivity in progeny ranging from 30.8% to 100% among seropositive dams, and from 0% to 23.5% in progenies from seronegative dams. Similar finding on seropositive fetuses born from seronegative dams were reported (12, 25, 34). It was shown that most cows that abort have N. caninum antibodies at the time of abortion, therefore seronegative dams are unlikely to be involved in the Neospora-induced abortion (34, 8). On the other hand, this possibility can not be ruled out since during pregnancy, antibody levels fluctuate (35), and their levels may fall below the threshold sensitivity of the test (36). Therefore it has been suggested that application of more than one diagnostic test would be more reliable to associate abortion to the N. caninum infection. The results obtained here showed that in addition to the conventional IFAT, nPCR is a complementary Volume 66 (1) 2011
Figure 3. results of nPCr from brain tissuses and iFat from thoracic fluids of 98 aborted fetuses. Results of paired samples from 26 dams and their respective fetuses are summarized in Table 1. Fifteen fetuses (57.7%) from serological negatives dams were found negative in both nPCR and IFAT tests. All seropositives dams and 6 out of 21 seronegative dams produced fetuses positive in at least one of the fetal assays. Table 1. Comparison between tests of the dams with respectively aborted fetuses aborted fetuses test iFat / nPCr positive / positive positive / negative negative / positive negative / negative iFat of dams Positive 1 3 1 0 Negative 3 0 3 15
In the present study high seroprevalence to N. caninum was observed in cattle from herds with history of abortions. From the dams tested 51.4% were seropositive to N. caninum indicating high exposure to the parasite. Similar seroprevalence levels has been reported in cattle worldwide (4). In Portugal, Korea and Argentina reports of seroprevalence of N. caninum antibodies among cattle suffering abortion were 46%, 48.7% and 64.5%, respectively (20, 21, 22). A highly efficient transmission rate (up to 95%) from dams to fetuses has been reported worldwide (8, 23). Although about 50% positivity was detected in cows, seroprevalence among fetuses was 18.2%. In Brazil infection in aborted fetuses reported was 39.1% (24), and in Switzerland about 21% (25). Among the seropositive cows examined, 52% exhibited N. caninum antibodies with titers ≥1:3200, these results indicate that N. caninum might be the causative pathogen of abortion. According to Dubey et al. (26); McAllister et al.(27); Schares et al. (23,28) dams aborting due to 16
DISCUSSION
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ISRAEL JOURNAL OF VETERINARY MEDICINE tool for an improved and confirmative diagnosis of N. caninum in fetuses. 1. E. and ortega-Mora, l.: Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain. Vet. Parasitol. 111, 143-152, 2003. Yamage,M., Flechtner,o. and Gottstein, B.: Neospora caninum: specific oligonucleotide primers for the detection of brain 'cyst' dNa of experimentally infected nude mice by the polymerase chain reaction (PCr). J. Parasitol. 82 (2):272-279, 1996. Pare, J., thurmond, M. and Hietal, S.: Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. res. 60: 133-139, 1996. Shkap., reske, a., Pipano, E., Fish, l. and Baszler, t.: immunological relationship between Neospora caninum and Besnoitia besnoiti. Vet. Parasitol., 106: 35-43, 2002. Hur, K., Kim H.J., Hwang, W. S., Hwang, E.K., Jean, Y. H., lee B.C., Bae, J.S., Kang Y.B., Yamane i. and Kim, d.Y.: Seroepidemiological study of Neospora caninum in Korean dairy cattle by indirect immunofluorescent antibody assay. Korean J. Vet. res. 38:859- 866, 1998. Venturini M., Venturini, C. l., Bacigalupe, d., Machuca M., Echaide i., Basso, W., Unzaga, J.M., di lorenzo, C., Guglielmone, a., Jenkins M. C. and dubey, J.P.: Neospora caninum infection in bovine fetuses and dairy cows with abortion in argentina int. J. Parasitol. 129: 253-257, 1999. Canada, N., Carvalheira , J., Meirele, C.S., Correia da Costa, J.M. and rocha, a.: Prevalence of Neospora caninum infection in dairy cows and its consequence for reproductive management. theriolog. 62:1229-1235, 2004. Schares, G., Peter, M., Wurm, r., Barwald a. and Conraths, F. G.: the efficiency of vertical transmission of Neospora caninum in dairy cattle analyzed by serological techniques. Vet. Parasitol. 80:87-98, 1998. Corbelini, l.G., driemeier, d., Cruz, C.F.E., Gondim, l.F.P. and Wald, V.: Neosporosis as a cause of abortion in dairy cattle in rio Grande do Sul, southern Brazil. Vet. Parasitol. 103: 195-202, 2002. Sager H., Fischer i. and Furrer, K.: Swiss case-control study to assess Neospora caninum- associated bovine abortions by PCr, histopathology and serology. Vet. Parasitol. 102: 1-15, 2001. dubey, J.P., Jenkins, M.C., adams d.S., Mcallister., M., anderson-Sprecher, r., Bazler, t. V., Knok, o.C.H., lally, N. C., Bjorkman, C. and Uggla, a.: antibodies responses of cows during an outbreak of neosporosis evaluated by indirect immunoabsorbent assays. J. Parasitol. 83:1063-1069, 1997. Mcallister, M., Huffman.E.m., Hietala, S.K., Conrad, P.a., anderson, M.l. and Salman, M.l.: Evidence suggesting g a point source exposure in an outbreak of bovine abortion due to neosporosis. J. Vet. diagn. invest. 8: 355-357, 1996. Schares, G., rauser, M., Zimmer, K., Peter, M., Wurm, r., dubey J.P., de Graaf, d.C., Edelhofer, r., Mertens, C., Hess, G. and Conraths, F. G.: Serological differences in Neospora caninum –associated epidemic and endemic abortion. J. Parasitol. 85:688-694, 1999. 17
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anderson, M., Blanchard, P., Barr, B., dubey, J., Hoffman, r. and Conrad, P.: Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. am. Vet. Med. assoc. 198: 241-244, 1991. dubey, J.P.: Neosporosis in cattle: biology and economic impact. J. am. Vet. Med. assoc. 214: 1160-1163, 1999. dubey, J.P., Shares, G. and ortega-Mora l.M..: Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbiol. rev. 20/2:323-67, 2007. Fish, l., Mazuz, M., Molad, t., Savitsky, i. and Shkap, V.: isolation of Neospora caninum from dairy zero grazing cattle in israel. Vet. Parasitol. 149, 167-171, 2007. dubey, J.P. and lindsay, d.: a review of Neospora caninum and neosporosis. Vet. Parasitol. 67:1-59, 1996. de Meerschman, F., Speybroeck, N., Berkvens, d., rettigner, C., Focant, C., leclipteux, t., Cassart, d. and losson, B.: Fetal infection with Neospora caninum in dairy and beef cattle in Belgium. theriogenol. 58: 933-945, 2002. dubey, J.P. and Shares, G.: diagnosis of neosporosis. Vet. Parasit.140: 1-34, 2006." to "dubey, J.P. and Schares, G.: diagnosis of bovine neosporosis. Vet. Parasit.140: 1-34, 2006. Pare, J., thurmond, M. and Hietal, S.: Neospora caninum antibodies in cows during pregnancy as a predictor of congenital infection and abortion. J. Parasitol. 83: 82-87, 1997. Guy C.S., Williams d.J.K., Kelly d.F., McGarry, J.W., Guy, F., Bjorkman, C., Smith r.F. and trees, a.J.: Neospora caninum in persistently infected, pregnant cows: spontaneous transplacental infection is associated with an increase in maternal antibody. Vet. rec. 149:443-449, 2001. Wouda, W., Moen, a. and Schukken, Y.: abortion risk in progeny of cows after a Neospora caninum epidemic. therogenol. 49:1311-1316, 1998. davison, H., otter, a. and trees, a.: Significance of Neospora caninum in British dairy cattle determined by estimation of seroprevalence in normally calving and aborting cattle. int. J. Parasitol., 29: 1189-1194, 1999. Ho. M., Barr, B., tarantal., l., lai, l., Hendricks, a., Marsh, a., Sverlow, K., Packham, a. and Conrad, P.: detection of Neospora from tissues of experimentally infected rhesus macaques by PCr and specific dNa probe hybridization. J. Clin. Microb. 35: 1740-1745, 1997. Baszler, t., Gay, l., long, M. and Mathison, B.: detection by PCr of Neospora caninum in fetal tissue from spontaneous bovine abortions. J. Clin. Microb. 37: 40594064, 1999. Gottstein, B., Hentrich, B., Wyss, r., thur, B., Busato, a., Stark, K. and Muller, N.: Molecular and immunodiagnostic investigations on bovine neoporosis in Switzerland. int. J. Parasitol., 28: 679-691, 1998. Pereira-Bueno, J., Quintanilla-Gozalo, a., Perez-Perez,V., Espi-Felgueroso, a., alverez-Garcia, G., Collantes-Fernandez,
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Volume 66 (1) 2011
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ARTICLES 28. Muller, N., Zimmermann, V. and Gottstein, B.: diagnosis of Neospora caninum and Toxoplasma gondii infection by PCr and dNa hybridization immunoassay. J. Clin. Microbiol. 34: 2850-2852, 1996. 29. Ellis, J.t., McMillan, d., ryce, C., Payne, S., atkinson, r. and Harper, P.a.W.: development of a single tube bested polymerase chain reaction assay for detection of Neospora caninum dNa. int. J. Parasitol. 29:1589-1596, 1999. 30. dubey, J.P.: review of Neospora caninum and neosporosis in animals. Korean J. Parasitol., 41: 1, 1-16, 2003. 31. Yao l., Yang N., liu Q., Wang M., Zhang W., Qian W.F., Hu Y. F. and ding, J.: detection of Neospora caninum in aborted bovine fetuses and dam and blood samples by nested PCr and Elisa and seroprevalence in Beijing and tianjin, China. Parasitol. 136:1251-1256, 2009. 32. Collantes-Fernandes, E., lopez-Perez, i., alvarez-Garcia, G. and ortega-Mora, l.M.: temporal distribution and parasite load kinetics in blood and tissues during Neospora caninum infection in mice. infect. immun. 74:2491-2494, 2006. 33. otter, a., Jeffrey, M., Chiles, S.F.E., Helmick, B., Wilesmith, J.W. and tress, a.J.: Comparison of histology with maternal and fetal serology for diagnostic of abortion due to bovine neosporosis. Vet. rec. 141:487-489, 1997. 34. Conrad, P.a., Sverlow, K.W., anderson, M., rowel J., Bondurant , r., tuter,G., Breitmeyer, r., Palmer C., thurmond, M., ardans, a., dubey J.P., duhamel, G. and Barr, B.: detection of serum antibody response in cattle with natural or experimental Neospora infection. J. Vet. invest. 5: 572-578, 1993. 35. Wouda, W., Brinkhof, J., van Maanen,C., de Gee, a.l. and Moen, a.r.: Serodiagnosis of neosporosis in individual cows and dairy herds: a comparative study in three enzyme –linked immunsorbent assays. Clin. diagn. lab. immunol. 5, 711-716, 1998.
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Volume 66 (1) 2011
ARTICLES
NEOSPORA CANINUM AS CAUSATIVE-PATHOGEN OF ABORTION IN CATTLE
Mazuz, l. M., Fish, l., Molad, t., Savitsky, i., Wolkomirsky, r., leibovitz, B. and Shkap, V. department of Parasitology, Kimron Veterinary institute, Bet dagan, israel. Corresponding author: Monica l. Mazuz, department of Parasitology, Kimron Veterinary institute, P.o.Box 12, 50250 Bet dagan, israel. Email: monica@int.gov.il
ABSTRACT
Neosporosis, caused by infection with the intracellular protozoan Neospora caninum, is recognized as one of the major causes of infectious abortion in cattle. Since abortion is the only clinical sign associated with N. caninum infection, diagnosis is complicated, however it is crucial to understand the epidemiology of the disease, and to undertake measures to reduce losses to the cattle industry. in israel the diagnosis of neosporosis is based on the detection of specific antibodies in aborted dams, or in the body fluids of aborted fetuses. the presence of antibodies in sera of aborted dams indicates exposure to N. caninum, but does not necessarily point to the causative agent of the abortion. in the present study sera from 7,951 aborted dams and body fluids from 446 fetuses were analyzed by the indirect fluorescent antibodies test (iFat). N. caninum seropositivity was found in 51.4% of the dams, and in 18.2% of the fetuses. in addition, primers were designed for the detection of N. caninum dNa, by a specific nested PCr (nPCr) test, in brain tissues from aborted fetuses. the primers do not react with other closely related apicomplexa species. the performance of both molecular and serological assays was evaluated in 97 samples of fetuses. Comparable results were observed in 57 negative and 20 positive samples, while 20 remaining samples showed positivity in only one of the assays. in addition, 26 paired dam and aborted fetuses were tested. of these, 15 samples obtained from dams or fetuses were found to be negative by both tests, 5 seropositive dams had positive fetuses in at least one of the assays, and 6 samples from seronegative dams were found to be positive in at least one of the fetus’ assays. the results obtained show high prevalence of N. caninum in cattle in israel and emphasize the necessity for the application of several methods for a reliable diagnosis of neosporosis as a cause of abortion.
Neosporosis is caused by the intracellular protozoan parasite Neospora caninum and is known as one of the major causes of infectious abortion, stillbirth and reproductive failure in cattle worldwide, including Israel (1, 2, 3, 4, 5). Cattle infected with N. caninum exhibit no clinical signs other than abortion. In the absence of typical clinical signs of infection, diagnosis and control of neosporosis is complicated. The mechanisms that cause abortion have not yet been fully elucidated (6). In Israel diagnosis of neosporosis is based on the detection of specific N. caninum antibodies on the aborting cow or in the body fluid of the aborted fetus. Presence of serum antibodies in dams indicates exposure to N. caninum, (7) but is not necessarily associated with the reproductive failure; on other hand, seronegative results do not exclude the presence of infection. (8). Non-aborting seropositive dams have been reported worldwide, including in Israel (5, 9). It is documented that only some of infected dams abort, while most of their calves are born infected but healthy (10). It has been widely reported that the level of abortion rate in N. caninum sero-positive dams is 3.5 to 7 times higher than in their seronegative counterparts (11, 12). Molecular methods allowing detection of specific N.caninum 14
INTRODUCTION
DNA in tissues of aborted fetuses have been applied for complementary diagnosis of neosporosis. The detection of N. caninum DNA provided evidence of intra-uterine transmission of parasites from dams to offspring (13, 14, 15, 16, 17). In the present study, aborting dams and aborted fetuses were examined using either serology or molecular methods in order to evaluate whether the two techniques complement each other thus allowing for a more reliable diagnosis of neosporosis.
MATERIALS AND METHODS
Samples Sera from 7,951 aborting cows and thoracic fluid from 446 aborted fetuses, from farms with history of abortion, were submitted for Neospora caninum diagnosis between 20062009. Brain tissues from 98 aborted fetuses were tested by nested PCR (nPCR), and 26 paired samples from dams and their respective fetuses were examined by both nPCR and indirect fluorescent antibody test (IFAT). Indirect fluorescent antibody test (IFAT( Serum from dams and thoracic liquids from aborted fetuses were examined for the presence of antibodies to N. caninum Volume 66 (1) 2011
website: www.isrvma.org
ISRAEL JOURNAL OF VETERINARY MEDICINE by IFAT as previous described (18). The cut-off was 1:200 and 1:80 for dams and fetuses, respectively (19, 4). DNA extraction and purification from brain tissues Twenty milligrams of brain tissue collected from aborted fetuses were used for DNA purification. The ground tissues were digested with Proteinase K overnight at a temperature of 56º C, and then DNA purification was performed using Qiagen DNeasy Tissue Kit (QIAGEN, USA). Samples were frozen and stored at - 20ºC until use. For positive control N. caninum from the Israeli isolate NcIs491 were grown in Vero cells, and parasites were obtained after destruction of monolayer cells and subsequent release of parasites from the cells (5). The media containing parasites and cell debris were collected, parasites were separated from cells by differential centrifugation at 70x g for 5 minutes, and the supernatant containing parasites was passed to a new tube, centrifuged again at 1000x g for 20 minutes. The pelleted parasites were washed in PBS at least 3 times by centrifuge at 1000g for 20 minutes. The specificity of the nPCR test to N. caninum was evaluated using DNA from Toxoplasma gondii (RH strain) parasites and Besnoitia besnoiti, taxonomically related tissue cystforming coccidian parasite. Nested PCR (nPCR) amplification Two sets of primers were designed for nPCR, based on DNA sequence of N. caninum (GenBank Accession number X84238). The nPCR reaction was performed with an external set of primers: forward primer 5 ': CTG CTG ACG TGT CGT TGT TG-3' represent nucleotide (nt) position 476 of referred sequence and reverse primer 5 ': CAT CTA CCA GGC CGC TCT TC-3' reverse sequence of nt position 1014, and an internal set of primers: forward primer 5 ': GCG TCA GGG TGA GGA CAG TG-3' nt position 631 of referred sequence and reverse primer 5 ': CTC TCC GTT CGC CAG CAG TG -3' reverse sequence of nt position 910. The PCR reaction contained: 1µl of genomic DNA, each primer concentration 4ηg and 2x Readdy Mix (AB gene) containing: DNA Polymerase Thermoprime Plus (0.3125 units), 18.75 mM Tris-HCI (pH 8.8), 5mM (NH4)2SO4 0.375 mM MgCl, 0.0025% Tween 20 and 0.05mM dNTP mix in final volume of 50 μl. The cycling protocol, following preheating of 95ºC for 3 minutes were: 30 cycles of denaturizing at 94º C for 30 seconds, annealing for 30 seconds at 56º C for the external primers and 57º C for the internal primers, extension at 72ºC for 40 seconds and a final extension at 72ºC for 5 minutes. The nPCR was performed using 1μl of the first reaction as a template. After amplification the reaction products were visualized on 1.5% agarose gel. to 1:1600 in 22.7% (933 samples) and ≥1:3200 in 52% (2, 153 sample). The results obtained by the IFAT in 446 aborted fetuses were: 18.2% (81 samples) positive and 81.8% (365 samples) negative. As shown in Figure 1, the titer in seropositive fetuses ranged from 1:80 to 1:1280. Distribution of antibody titers in fetuses was 23.5% (19 samples) up to 1:160, 39.5 % (32 samples) from 1:320-1:640 and ≥1:1280 in 37% (30 samples).
Figure 1. distribution of N. caninum antibodies in 7951 dams and 446 aborted fetuses.
As shown in Figure 2, the primers designed for nPCR successfully detect N. caninum from aborted infected brain and no amplification was detected when DNA from T. gondii or B. besnoiti were applied. Ninety seven fetuses examined by IFAT and nPCR, showed comparable results. A complete agreement was detected in the assays in 20 positive and 57 negative samples. In 21 out of 98 samples tested, 11 samples were positive by the nPCR, but negative in IFAT, and 10 samples were found positive by IFAT, but negative by nPCR (Figure 3).
299bp
1
2
3
4
5
6
7
8
9
RESULTS
Out of the 7,951 sera from aborted cows tested for N. caninum antibodies, 4,104 (51.4%) were seropositive with titers ranging from 1:200 to >1:3200, and 3,847 samples (48,4%) were seronegative (Figure 1). Among seropositive cows the titers found were: up to 1:400 in 24.8% (1,018 samples), between 1:800
Figure 2. Specificity of the nPCr. lanes: 1-Marker, 2 Negative control, 3- N. caninum – positive control, 4- N. caninum positive abortion brain, 5-7 Negative brains from aborted fetuses, 8Toxoplasma gondii and 9- Besnoitia besnoiti.
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ARTICLES neosporosis have higher titers of those infected but non-aborting. The primers designed for nPCR in this study specifically detected N. caninum 251 bp fragment from DNA of aborted fetuses, and no DNA from T. gondii and B. besnoiti was amplified. The detection of N. caninum DNA in aborted fetuses by PCR and nPCR was described previously (14, 25, 29, 30). Examination of the aborted fetuses resulted in 79% agreement between nPCR and IFAT. The discrepancy found in negative IFA results but positive by nPCR, or on the other hand, positively tested by IFA, but negative in nPCR, was described in other studies when using various methods (15, 31, 32). There were several explanations for the differences observed using various assays. According to Dubey et al. (31), negative serological results in infected fetuses were influenced by the age of fetus, level of exposure and time between exposure and abortion. It has also been found that lack of antibodies in the fetuses might give inconclusive results concerning absence of infection, since the fetus might be infected in the late gestation, leaving insufficient time for antibody production (4). Negative results in nPCR in seropositive fetuses might result as the distribution pattern of parasites in the host tissue which is as yet unclear. Furthermore the distribution of parasites might vary between animals, and the parasite load might be influenced by the stage of gestation and age at which the fetus was aborted (33). In addition, the brain tissues although considered to be the most suitable for the detection of N. caninum DNA by PCR (14, 32), it is possible that examination of additional organs would lead to increased reliability of the diagnosis. On the other hand, as shown by Yao et al., (32), PCR applied to various tissues including brain, heart, lung, liver, spleen, kidney and skeletal muscle, resulted in a total of 15 positive samples tested from various tissues, while only 2 were negative from brain, but positive from kidney and gluteus. Examinations of the paired samples showed that all seropositive dams produced positive fetuses by at least one of the assays, while among seronegative samples from dams, positive fetuses were detected in 23 %. The results provided evidence of vertical transmission of N. caninum and emphasize the high rate of transmission of parasites from dams to their offspring. Studies with dams and asymptomatic progenies were summarized by Dubey et al., (4), with seropositivity in progeny ranging from 30.8% to 100% among seropositive dams, and from 0% to 23.5% in progenies from seronegative dams. Similar finding on seropositive fetuses born from seronegative dams were reported (12, 25, 34). It was shown that most cows that abort have N. caninum antibodies at the time of abortion, therefore seronegative dams are unlikely to be involved in the Neospora-induced abortion (34, 8). On the other hand, this possibility can not be ruled out since during pregnancy, antibody levels fluctuate (35), and their levels may fall below the threshold sensitivity of the test (36). Therefore it has been suggested that application of more than one diagnostic test would be more reliable to associate abortion to the N. caninum infection. The results obtained here showed that in addition to the conventional IFAT, nPCR is a complementary Volume 66 (1) 2011
Figure 3. results of nPCr from brain tissuses and iFat from thoracic fluids of 98 aborted fetuses. Results of paired samples from 26 dams and their respective fetuses are summarized in Table 1. Fifteen fetuses (57.7%) from serological negatives dams were found negative in both nPCR and IFAT tests. All seropositives dams and 6 out of 21 seronegative dams produced fetuses positive in at least one of the fetal assays. Table 1. Comparison between tests of the dams with respectively aborted fetuses aborted fetuses test iFat / nPCr positive / positive positive / negative negative / positive negative / negative iFat of dams Positive 1 3 1 0 Negative 3 0 3 15
In the present study high seroprevalence to N. caninum was observed in cattle from herds with history of abortions. From the dams tested 51.4% were seropositive to N. caninum indicating high exposure to the parasite. Similar seroprevalence levels has been reported in cattle worldwide (4). In Portugal, Korea and Argentina reports of seroprevalence of N. caninum antibodies among cattle suffering abortion were 46%, 48.7% and 64.5%, respectively (20, 21, 22). A highly efficient transmission rate (up to 95%) from dams to fetuses has been reported worldwide (8, 23). Although about 50% positivity was detected in cows, seroprevalence among fetuses was 18.2%. In Brazil infection in aborted fetuses reported was 39.1% (24), and in Switzerland about 21% (25). Among the seropositive cows examined, 52% exhibited N. caninum antibodies with titers ≥1:3200, these results indicate that N. caninum might be the causative pathogen of abortion. According to Dubey et al. (26); McAllister et al.(27); Schares et al. (23,28) dams aborting due to 16
DISCUSSION
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ISRAEL JOURNAL OF VETERINARY MEDICINE tool for an improved and confirmative diagnosis of N. caninum in fetuses. 1. E. and ortega-Mora, l.: Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain. Vet. Parasitol. 111, 143-152, 2003. Yamage,M., Flechtner,o. and Gottstein, B.: Neospora caninum: specific oligonucleotide primers for the detection of brain 'cyst' dNa of experimentally infected nude mice by the polymerase chain reaction (PCr). J. Parasitol. 82 (2):272-279, 1996. Pare, J., thurmond, M. and Hietal, S.: Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. res. 60: 133-139, 1996. Shkap., reske, a., Pipano, E., Fish, l. and Baszler, t.: immunological relationship between Neospora caninum and Besnoitia besnoiti. Vet. Parasitol., 106: 35-43, 2002. Hur, K., Kim H.J., Hwang, W. S., Hwang, E.K., Jean, Y. H., lee B.C., Bae, J.S., Kang Y.B., Yamane i. and Kim, d.Y.: Seroepidemiological study of Neospora caninum in Korean dairy cattle by indirect immunofluorescent antibody assay. Korean J. Vet. res. 38:859- 866, 1998. Venturini M., Venturini, C. l., Bacigalupe, d., Machuca M., Echaide i., Basso, W., Unzaga, J.M., di lorenzo, C., Guglielmone, a., Jenkins M. C. and dubey, J.P.: Neospora caninum infection in bovine fetuses and dairy cows with abortion in argentina int. J. Parasitol. 129: 253-257, 1999. Canada, N., Carvalheira , J., Meirele, C.S., Correia da Costa, J.M. and rocha, a.: Prevalence of Neospora caninum infection in dairy cows and its consequence for reproductive management. theriolog. 62:1229-1235, 2004. Schares, G., Peter, M., Wurm, r., Barwald a. and Conraths, F. G.: the efficiency of vertical transmission of Neospora caninum in dairy cattle analyzed by serological techniques. Vet. Parasitol. 80:87-98, 1998. Corbelini, l.G., driemeier, d., Cruz, C.F.E., Gondim, l.F.P. and Wald, V.: Neosporosis as a cause of abortion in dairy cattle in rio Grande do Sul, southern Brazil. Vet. Parasitol. 103: 195-202, 2002. Sager H., Fischer i. and Furrer, K.: Swiss case-control study to assess Neospora caninum- associated bovine abortions by PCr, histopathology and serology. Vet. Parasitol. 102: 1-15, 2001. dubey, J.P., Jenkins, M.C., adams d.S., Mcallister., M., anderson-Sprecher, r., Bazler, t. V., Knok, o.C.H., lally, N. C., Bjorkman, C. and Uggla, a.: antibodies responses of cows during an outbreak of neosporosis evaluated by indirect immunoabsorbent assays. J. Parasitol. 83:1063-1069, 1997. Mcallister, M., Huffman.E.m., Hietala, S.K., Conrad, P.a., anderson, M.l. and Salman, M.l.: Evidence suggesting g a point source exposure in an outbreak of bovine abortion due to neosporosis. J. Vet. diagn. invest. 8: 355-357, 1996. Schares, G., rauser, M., Zimmer, K., Peter, M., Wurm, r., dubey J.P., de Graaf, d.C., Edelhofer, r., Mertens, C., Hess, G. and Conraths, F. G.: Serological differences in Neospora caninum –associated epidemic and endemic abortion. J. Parasitol. 85:688-694, 1999. 17
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