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Fall in antibody titer to small ruminant lentivirus in the periparturient period in goats
Small Ruminant Research 147 (2017) 37–40
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Fall in antibody titer to small ruminant lentivirus in the periparturient period in goats Michał Czopowicz a,∗ , Olga Szalu´s-Jordanow b , Marcin Mickiewicz a , Lucjan Witkowski a , c ´ Agata Moroz a , Iwona Markowska-Daniel a , Daria Reczynska , Emilia Bagnicka c , a Jarosław Kaba a Laboratory of Veterinary Epidemiology and Economics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland b Department of Small Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland c Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Post˛epu 36A, Jastrz˛ebiec, 05-552 Magdalenka, Poland
a r t i c l e
i n f o
Article history: Received 19 October 2016 Received in revised form 7 December 2016 Accepted 12 December 2016 Available online 15 December 2016 Keywords: Caprine arthritis-encephalitis CAE ELISA Gestation Pregnancy
a b s t r a c t The prospective study was carried out to document the change of antibody level to small ruminant lentivirus (SRLV) in chronically infected pregnant does. Thirteen dairy goats of Polish White Improved and Polish Fawn Improved breeds, asymptomatically infected with SRLV for at least a year, were enrolled. The goats were blood-sampled at mating, then four times during pregnancy, 2 weeks before kidding, at kidding and monthly for three months postpartum. Antibody titers to SRLV were determined by screening sera in increasing dilutions with three different commercial ELISAs: indirect whole-virus antigen (wELISA), indirect p28-transmembrane antigen (p28-TM-ELISA) and competitive gp135 (SU-ELISA). Then, the reciprocal of the greatest dilution at which a serum yielded the result greater than the cut-off of the test was considered the end-point antibody titer. Compared to the level at mating antibody titers significantly fell at kidding in all three tests. Significant decrease in antibody titer was observed for the longest time in SU-ELISA and for the shortest time in p28-TM-ELISA. At kidding false negative results were observed in two ELISAs (p28-TM-ELISA and SU-ELISA) and 3 of 13 goats became seronegative at kidding in at least one ELISA. At least four-fold fall in antibody titer between mating and kidding was observed in wELISA in 6 goats, in p28-TM-ELISA in 4 goats and in SU-ELISA in 5 goats. None of goats showed at least four-fold decrease in all three tests. Fall in antibody titer to SRLV in the periparturient period can interfere with the results of serological screening of pregnant goats. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Caprine arthritis-encephalitis (CAE) caused by a small ruminant lentivirus (SRLV) from the family Retroviridae, spreads insidiously in a herd with no symptoms apparent in infected goats for a long time following infection. Therefore, control of the disease requires early identification of infected individuals, which may only be achieved by laboratory testing. Even though PCR was first used for diagnosing SRLV infection over twenty years ago (HerrmannHoesing, 2010) and several highly accurate real-time PCRs for detection of SRLV have recently been developed (De Regge and Cay, 2013; Kuhar et al., 2013) none of them has so far been commercial-
∗ Corresponding author. E-mail address: [email protected] (M. Czopowicz). http://dx.doi.org/10.1016/j.smallrumres.2016.12.006 0921-4488/© 2016 Elsevier B.V. All rights reserved.
ized and they remain hardly available in routine veterinary practice. Hence, serological ELISA tests remain the mainstay of field CAE diagnostics, mostly owing to their high accuracy and wide commercial availability. Nowadays, various ELISA kits detecting antibodies to surface (SU), transmembrane (TM), capsid (CA) proteins as well as whole-virus antigen are available. Despite the variability of antibody types detected, the main pitfall of serological testing for SRLV results from a delayed seroconversion (Rimstad et al., 1983), fluctuations of antibody levels in chronically infected seropositive goats (Hanson et al., 1996), and genetic diversity of the virus (Lacerenza et al., 2006). One of the circumstances which may potentially interfere with humoral immune response is pregnancy (Herr et al., 2011) and antibody levels were shown to fall in the periparturient period in cows infected with another retrovirus – bovine leukemia virus (BLV) (Burridge et al., 1982). This has also been observed in cows infected with Bovine viral diarrhea virus (Bachofen et al., 2013) and
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M. Czopowicz et al. / Small Ruminant Research 147 (2017) 37–40
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Fig. 1. Antibody titers to small ruminant lentivirus throuhout the study determined using the ID Screen MVV-CAEV Indirect Screening test (ID.vet Innovative Diagnostics, France) (wELISA). Antibody titers presented on binary logarithm scale after dividing by two. * signifies statistical significance at ␣ = 0.05.
Neospora caninum (Kyaw et al., 2005), and in buffalos with Toxocara vitulorum infection (Amerasinghe et al., 1994) as well as in sheep with Trichostrongylus colubriformis infection (Beasley et al., 2010). We carried out the prospective study to document the change of specific antibody levels in pregnant does chronically infected with SRLV. 2. Materials and methods 2.1. Animals The study was approved by the 3rd Local Ethical Committee in Warsaw (Approval No. 31/2013, 22 May 2013) and carried out in a dairy goat herd counting roughly 60 dairy does of Polish White Improved (PWI) and Polish Fawn Improved (PFI) breed. A SRLV infection was detected in this herd 20 years ago, first serologically and then by the virus isolation (Kaba et al., 2009). Subsequently, regular serological screening based on a whole-virus ELISA, has been performed twice a year until now and the within-herd seroprevalence ranged from 25% to 75% (Kaba et al., 2011). SRLV in this herd belonged to genotype A, which seems to predominate in Polish goat population (Olech et al., 2012). Initially, 21 SRLV-seropositive does aged 2 years or more were enrolled in the study at the beginning of a mating season. Eight of them were then excluded due to reproductive failures (5 goats), sudden death (1 goat) and testing negative in at least one of ELISA tests used in the study (2 goats). Finally, 8 PWI and 5 PFI goats, aged from 3 to 7 years with the median of 5 years and interquartile range between 4 and 6 years were included. They had become seropositive to SRLV 1–5 years (median of 2 years) before the onset of the study, however none of them had developed any clinical symptoms of CAE. 2.2. Sampling protocol All the goats were blood-sampled ten times in all: at mating, at the end of each month of gestation, 2 weeks before kidding, during the week preceding kidding and at the end of each of three months after parturition. Blood was collected to dry tubes and left at room
temperature overnight, then centrifuged, and the sera were stored at −20 ◦ C until testing. 2.3. Serological testing The sera were screened with three commercial ELISA tests – ® two indirect: ID Screen MVV-CAEV Indirect Screening test (ID.vet Innovative Diagnostics, France) containing whole-virus antigen (henceforth referred to as wELISA) and IDEXX MVV/CAEV p28 Ab Screening (IDEXX Laboratories, USA) based on recombinant TM and p28 antigen (p28-TM-ELISA) – and one competitive: Small Ruminant Lentivirus Antibody Test Kit, cELISA (VMRD, USA) coated with SU (gp135) antigen (SU-ELISA). They all were based on antigens from caprine isolates of SRLV and have proven highly accurate in diagnosing SRLV infection in goats (Brinkhof and van Maanen, 2007; Herrmann et al., 2003; Nowicka et al., 2014). Manufacturer’s cut-offs were used: wELISA – sample-to-positive control ratio percentage (S/P%) of 50%, p28-TM-ELISA – S/P% of 110%, SU-ELISA – percentage of inhibition (% I) of 35%. To determine the antibody titer each serum sample was tested in increasing dilutions: in wELISA and p28-TM-ELISA from 1:20 to 1:2560, whereas in SU-ELISA from 1:1 through 1:256. Then, the reciprocal of the greatest dilution at which a serum yielded the result (either S/P% or % I) greater than the cut-off of the test was considered the end-point antibody titer. For the needs of preparing plots antibody titers were logarithmically transformed using binary logarithm, either directly (SU-ELISA) or after division of the titer by 10 (wELISA and p28-TM-ELISA kits). 2.4. Statistical analysis Antibody titers were given in figures as median, interquartile range (25th–75th percentile) and raw data. Data were subjected to rank transformation of type RT-1 (Conover and Iman, 1984) and analyzed using repeated-measures analysis of variance. Given that a change in antibody titer should be at least 4-fold to be considered significant (Thrusfield, 2007), titers which differed from the titer at mating no more than 2-fold were assigned the same rank. A Dunnett’s post-hoc test was applied to compare antibody titers during pregnancy and three months after parturition with the basal level
M. Czopowicz et al. / Small Ruminant Research 147 (2017) 37–40
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Fig. 2. Antibody titers to small ruminant lentivirus throuhout the study determined using the IDEXX MVV/CAEV p28 Ab Screening (IDEXX Laboratories, USA) (p28-TM-ELISA). Antibody titers presented on binary logarithm scale after dividing by two. * signifies statistical significance at ␣ = 0.05.
Fig. 3. Antibody titers to small ruminant lentivirus throuhout the study determined using the Small Ruminant Lentivirus Antibody Test Kit, cELISA (VMRD, USA) (SU-ELISA). Antibody titers presented on binary logarithm scale. * signifies statistical significance at ␣ = 0.05.
at mating. A significance level (␣) was set at 0.05. Analyses and plots were performed in Statistica 12 (StatSoft Inc.). 3. Results Compared to the basal level at mating antibody titer significantly fell at kidding in all three ELISA kits (repeated-measures ANOVA p < 0.001 in all three kits; antibody titer at kidding vs. at mating in Dunnett’s test: wELISA – p = 0.002, p28-TM-ELISA – p = 0.005, SU-ELISA – p < 0.001; Figs. 1–3). Significant decrease in antibody titer was observed for the longest time in SU-ELISA and for the shortest time in p28-TM-ELISA. Two goats turned seronegative at kidding in p28-TM-ELISA and so did two goats in SU-ELISA, whereas none in wELISA. As one of the aforementioned goats was
seronegative at kidding simultaneously in p28-TM-ELISA and SUELISA, three of 13 goats became seronegative at kidding in at least one ELISA. At least four-fold fall in antibody titer between mating and kidding was observed in wELISA in 6 goats, in p28-TM-ELISA in 4 goats and in SU-ELISA in 5 goats. None of the goats showed at least four-fold decrease simultaneously in all three ELISA kits. 4. Discussion Our study shows that antibody titers to all SRLV antigens tend to fall around parturition. This observation is similar to the one of Burridge et al. (1982) in bovine viral leukemia. Given that caprine colostral immunoglobulins mainly come from circulating blood (Larson, 1958) and passive immunity of goat kids is
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based on colostrum intake (Guedes et al., 2010; Pisarska et al., 2002), the fall of antibodies may partly result from the transfer of immunoglobulins from serum to colostrum. Another likely explanation may be the periparturient relaxation of immunity – a phenomenon described so far mainly in gastrointestinal nematode infestations, and associated with impairment of both humoral and cell-dependent immunity in the periparturient period (Beasley et al., 2010). The analogy between the fall in antibody titer observed in our study and the periparturient relaxation of immunity is likely to exist since the link between the onset of lactation and increasing viral load in SRLV infected goats was shown (Morin et al., 2003). From the three ELISA kits used in the study wELISA was the only test which did not miss any of pregnant does at kidding. This probably results from the broader antigen spectrum it covers compared to the remaining two kits. Two goats which became seronegative in a p28-TM-ELISA at kidding (one of them also in SU-ELISA) had the lowest antibody titers at mating and for the entire pregnancy in all ELISA kits. This indicates that turning seronegative was a simple consequence of the low initial antibody level. Although the second goat which tested negative in SU-ELISA at kidding similarly had very low antibody titer in this ELISA kit before, its antibody titers in p28-TM-ELISA were high. This could be explained by an impaired capability of producing neutralizing antibodies to viral envelope antigens observed in some chronically infected goats (Narayan et al., 1984), or by diversity of surface glycoprotein antigens affecting the affinity of the circulating antibodies to the antigen used in SU-ELISA (Herrmann-Hoesing, 2010). Higher antibody titers to capsid than to surface antigen of BLV were also observed by Burridge et al. (1982). The main shortcoming of the study is a small sample size. We hoped to follow up roughly 20 goats, however unfortunate coincidences eliminated as many as 8 of 21 initially enrolled individuals. Small sample size does not allow to formulate firm recommendations regarding diagnostic procedures in the periparturient period. However, it seems probable that wELISAs is less likely to yield false negative results due to falling antibody titers. Therefore, if only one ELISA has to be used for screening pregnant goats wELISA seems to suit best. Given that no goat showed at least four-fold antibody titer decrease simultaneously in all three ELISA kits, the parallel use of them appears to increase sensitivity, if valuable goats need to be screened in the periparturient period. Perhaps, parallel use of ELISA and a real-time PCR would ensure the highest accuracy, however limited availability of PCR in routine field practice renders this recommendation difficult to follow. Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgement This work was financially supported by the National Science Center (grant number 2013/09/B/NZ6/03514). References Amerasinghe, P.H., Vasanthathilake, V.W., Lloyd, S., Fernando, S.T., 1994. Periparturient reduction in buffalo of mitogen-induced lymphocyte proliferation and antibody to Toxocara vitulorum. Trop. Anim. Health Prod. 26, 109–116.
Bachofen, C., Bollinger, B., Peterhans, E., Stalder, H., Schweizer, M., 2013. Diagnostic gap in Bovine viral diarrhea virus serology during the periparturient period in cattle. J. Vet. Diagn. Invest. 25, 655–656. Beasley, A.M., Kahn, L.P., Windon, R.G., 2010. The periparturient relaxation of immunity in Merino ewes infected with Trichostrongylus colubriformis: parasitological and immunological responses. Vet. Parasitol. 168, 60–70. Brinkhof, J., van Maanen, C., 2007. Evaluation of five enzyme-linked immunosorbent assays and an agar gel immunodiffusion test for detection of antibodies to small ruminant lentiviruses. Clin. Vaccine Immunol. 14, 1210–1214. Burridge, M.J., Thurmond, M.C., Miller, J.M., Schmerr, M.J., Van Der Maaten, M.J., 1982. Fall in antibody titer to bovine leukemia virus in the periparturient period. Can. J. Comp. Med. 46, 270–271. Conover, W.J., Iman, R.L., 1984. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35, 124–129. De Regge, N., Cay, B., 2013. Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses. J. Virol. Methods 194, 250–257. Guedes, M.T., Zacharias, F., Couto, R.D., Portela, R.W., Santos, L.C., Santos, S.C., Pedroza, K.C., Peixoto, A.P., López, J.A., Mendonc¸a-Lima, F.W., 2010. Maternal transference of passive humoral immunity to Haemonchus contortus in goats. Vet. Immunol. Immunopathol. 136, 138–143. Hanson, J., Hydbring, E., Olsson, K., 1996. A long term study of goats naturally infected with caprine arthritis-encephalitis virus. Acta vet. Scand. 37, 31–39. Herr, M., Bostedt, H., Failing, K., 2011. IgG and IgM levels in dairy cows during the periparturient period. Theriogenology 75, 377–385. Herrmann, L.M., Cheevers, W.P., McGuire, T.C., Adams, D.S., Hutton, M.M., Gavin, W.G., Knowles, D.P., 2003. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication. Clin. Diagn. Lab. Immunol. 10, 267–271. Herrmann-Hoesing, L.M., 2010. Diagnostic assays used to control small ruminant lentiviruses. J. Vet. Diagn. Invest. 22, 843–855. ´ J., Nowicki, M., 2009. Isolation and Kaba, J., Rola, M., Materniak, M., Kuzmak, characterization of caprine arthritis encephalitis virus in goats from Poland. Pol. J. Vet. Sci. 12, 183–188. Kaba, J., Bagnicka, E., Czopowicz, M., Nowicki, M., Witkowski, L., Szalu´s-Jordanow, O., 2011. Long-term study on the spread of caprine arthritis-encephalitis in a goat herd. Centr. Eur. J. Immunol. 36, 170–173. Kuhar, U., Barliˇc-Maganja, D., Grom, J., 2013. Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype A and B small ruminant lentivirus strains. BMC Vet. Res. 9, 172. Kyaw, T., Suwimonteerabutr, J., Virakul, P., Lohachit, C., Kalpravidh, W., 2005. Seronegative conversion in four Neospora caninum–infected cows, with a low rate of transplacental transmission. Vet. Parasitol. 131, 145–150. Lacerenza, D., Giammarioli, M., Grego, E., Marini, C., Profiti, M., Rutili, D., Rosati, S., 2006. Antibody response in sheep experimentally infected with different small ruminant lentivirus genotypes. Vet. Immunol. Immunopathol. 112, 264–271. Larson, B.L., 1958. Transfer of specific blood serum proteins to lacteal secretions near parturition. J. Dairy Sci. 16, 1033–1044. Morin, T., Grézel, D., Bouzar, B., Villet, S., Greenland, T., Guiguen, F., Fornazéro, C., Mornex, J.F., Chebloune, Y., 2003. Experimental model of lentivirus reactivation for virological and immunological studies in caprine arthritis-encephalitis virus (CAEV) infected goats. Bull. Vet. Inst. Pulawy 47, 275–286. Narayan, O., Sheffer, D., Griffin, D.E., Clements, J., Hess, J., 1984. Lack of neutralizing antibodies to caprine arthritis-encephalitis lentivirus in persistently infected goats can be overcome by immunization with inactivated Mycobacterium tuberculosis. J. Virol. 49, 349–355. Nowicka, D., Czopowicz, M., Mickiewicz, M., Szalu´s-Jordanow, O., Witkowski, L., Bagnicka, E., Kaba, J., 2014. Diagnostic performance of ID screen MVV-CAEV Indirect Screening ELISA in identifying small ruminant lentiviruses-infected goats. Pol. J. Vet. Sci. 17, 501–506. ´ Olech, M., Rachid, A., Croisé, B., Kuzmak, J., Valas, S., 2012. Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland. Virus Res. 163, 528–536. ´ Pisarska, A., Stefaniak, T., Popławski, M., Przewozny, M., Ratajski, R., Polak, A., Nowacki, W., 2002. Transfer of maternal passive immunity to kids in goat herd. Pol. J. Vet. Sci. 5, 251–255. Rimstad, E., East, N.E., Torten, M., Higgins, J., DeRock, E., Pedersen, N.C., 1983. Delayed seroconversion following naturally acquired caprine arthritis-encephalitis virus infection in goats. Am. J. Vet. Res. 54, 1858–1862. Thrusfield, M., 2007. Veterinary Epidemiology, third edition. Wiley-Blackwell, USA (p. 330).
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Fall in antibody titer to small ruminant lentivirus in the periparturient period in goats Michał Czopowicz a,∗ , Olga Szalu´s-Jordanow b , Marcin Mickiewicz a , Lucjan Witkowski a , c ´ Agata Moroz a , Iwona Markowska-Daniel a , Daria Reczynska , Emilia Bagnicka c , a Jarosław Kaba a Laboratory of Veterinary Epidemiology and Economics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland b Department of Small Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159c, 02-776 Warsaw, Poland c Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Post˛epu 36A, Jastrz˛ebiec, 05-552 Magdalenka, Poland
a r t i c l e
i n f o
Article history: Received 19 October 2016 Received in revised form 7 December 2016 Accepted 12 December 2016 Available online 15 December 2016 Keywords: Caprine arthritis-encephalitis CAE ELISA Gestation Pregnancy
a b s t r a c t The prospective study was carried out to document the change of antibody level to small ruminant lentivirus (SRLV) in chronically infected pregnant does. Thirteen dairy goats of Polish White Improved and Polish Fawn Improved breeds, asymptomatically infected with SRLV for at least a year, were enrolled. The goats were blood-sampled at mating, then four times during pregnancy, 2 weeks before kidding, at kidding and monthly for three months postpartum. Antibody titers to SRLV were determined by screening sera in increasing dilutions with three different commercial ELISAs: indirect whole-virus antigen (wELISA), indirect p28-transmembrane antigen (p28-TM-ELISA) and competitive gp135 (SU-ELISA). Then, the reciprocal of the greatest dilution at which a serum yielded the result greater than the cut-off of the test was considered the end-point antibody titer. Compared to the level at mating antibody titers significantly fell at kidding in all three tests. Significant decrease in antibody titer was observed for the longest time in SU-ELISA and for the shortest time in p28-TM-ELISA. At kidding false negative results were observed in two ELISAs (p28-TM-ELISA and SU-ELISA) and 3 of 13 goats became seronegative at kidding in at least one ELISA. At least four-fold fall in antibody titer between mating and kidding was observed in wELISA in 6 goats, in p28-TM-ELISA in 4 goats and in SU-ELISA in 5 goats. None of goats showed at least four-fold decrease in all three tests. Fall in antibody titer to SRLV in the periparturient period can interfere with the results of serological screening of pregnant goats. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Caprine arthritis-encephalitis (CAE) caused by a small ruminant lentivirus (SRLV) from the family Retroviridae, spreads insidiously in a herd with no symptoms apparent in infected goats for a long time following infection. Therefore, control of the disease requires early identification of infected individuals, which may only be achieved by laboratory testing. Even though PCR was first used for diagnosing SRLV infection over twenty years ago (HerrmannHoesing, 2010) and several highly accurate real-time PCRs for detection of SRLV have recently been developed (De Regge and Cay, 2013; Kuhar et al., 2013) none of them has so far been commercial-
∗ Corresponding author. E-mail address: [email protected] (M. Czopowicz). http://dx.doi.org/10.1016/j.smallrumres.2016.12.006 0921-4488/© 2016 Elsevier B.V. All rights reserved.
ized and they remain hardly available in routine veterinary practice. Hence, serological ELISA tests remain the mainstay of field CAE diagnostics, mostly owing to their high accuracy and wide commercial availability. Nowadays, various ELISA kits detecting antibodies to surface (SU), transmembrane (TM), capsid (CA) proteins as well as whole-virus antigen are available. Despite the variability of antibody types detected, the main pitfall of serological testing for SRLV results from a delayed seroconversion (Rimstad et al., 1983), fluctuations of antibody levels in chronically infected seropositive goats (Hanson et al., 1996), and genetic diversity of the virus (Lacerenza et al., 2006). One of the circumstances which may potentially interfere with humoral immune response is pregnancy (Herr et al., 2011) and antibody levels were shown to fall in the periparturient period in cows infected with another retrovirus – bovine leukemia virus (BLV) (Burridge et al., 1982). This has also been observed in cows infected with Bovine viral diarrhea virus (Bachofen et al., 2013) and
38
M. Czopowicz et al. / Small Ruminant Research 147 (2017) 37–40
®
Fig. 1. Antibody titers to small ruminant lentivirus throuhout the study determined using the ID Screen MVV-CAEV Indirect Screening test (ID.vet Innovative Diagnostics, France) (wELISA). Antibody titers presented on binary logarithm scale after dividing by two. * signifies statistical significance at ␣ = 0.05.
Neospora caninum (Kyaw et al., 2005), and in buffalos with Toxocara vitulorum infection (Amerasinghe et al., 1994) as well as in sheep with Trichostrongylus colubriformis infection (Beasley et al., 2010). We carried out the prospective study to document the change of specific antibody levels in pregnant does chronically infected with SRLV. 2. Materials and methods 2.1. Animals The study was approved by the 3rd Local Ethical Committee in Warsaw (Approval No. 31/2013, 22 May 2013) and carried out in a dairy goat herd counting roughly 60 dairy does of Polish White Improved (PWI) and Polish Fawn Improved (PFI) breed. A SRLV infection was detected in this herd 20 years ago, first serologically and then by the virus isolation (Kaba et al., 2009). Subsequently, regular serological screening based on a whole-virus ELISA, has been performed twice a year until now and the within-herd seroprevalence ranged from 25% to 75% (Kaba et al., 2011). SRLV in this herd belonged to genotype A, which seems to predominate in Polish goat population (Olech et al., 2012). Initially, 21 SRLV-seropositive does aged 2 years or more were enrolled in the study at the beginning of a mating season. Eight of them were then excluded due to reproductive failures (5 goats), sudden death (1 goat) and testing negative in at least one of ELISA tests used in the study (2 goats). Finally, 8 PWI and 5 PFI goats, aged from 3 to 7 years with the median of 5 years and interquartile range between 4 and 6 years were included. They had become seropositive to SRLV 1–5 years (median of 2 years) before the onset of the study, however none of them had developed any clinical symptoms of CAE. 2.2. Sampling protocol All the goats were blood-sampled ten times in all: at mating, at the end of each month of gestation, 2 weeks before kidding, during the week preceding kidding and at the end of each of three months after parturition. Blood was collected to dry tubes and left at room
temperature overnight, then centrifuged, and the sera were stored at −20 ◦ C until testing. 2.3. Serological testing The sera were screened with three commercial ELISA tests – ® two indirect: ID Screen MVV-CAEV Indirect Screening test (ID.vet Innovative Diagnostics, France) containing whole-virus antigen (henceforth referred to as wELISA) and IDEXX MVV/CAEV p28 Ab Screening (IDEXX Laboratories, USA) based on recombinant TM and p28 antigen (p28-TM-ELISA) – and one competitive: Small Ruminant Lentivirus Antibody Test Kit, cELISA (VMRD, USA) coated with SU (gp135) antigen (SU-ELISA). They all were based on antigens from caprine isolates of SRLV and have proven highly accurate in diagnosing SRLV infection in goats (Brinkhof and van Maanen, 2007; Herrmann et al., 2003; Nowicka et al., 2014). Manufacturer’s cut-offs were used: wELISA – sample-to-positive control ratio percentage (S/P%) of 50%, p28-TM-ELISA – S/P% of 110%, SU-ELISA – percentage of inhibition (% I) of 35%. To determine the antibody titer each serum sample was tested in increasing dilutions: in wELISA and p28-TM-ELISA from 1:20 to 1:2560, whereas in SU-ELISA from 1:1 through 1:256. Then, the reciprocal of the greatest dilution at which a serum yielded the result (either S/P% or % I) greater than the cut-off of the test was considered the end-point antibody titer. For the needs of preparing plots antibody titers were logarithmically transformed using binary logarithm, either directly (SU-ELISA) or after division of the titer by 10 (wELISA and p28-TM-ELISA kits). 2.4. Statistical analysis Antibody titers were given in figures as median, interquartile range (25th–75th percentile) and raw data. Data were subjected to rank transformation of type RT-1 (Conover and Iman, 1984) and analyzed using repeated-measures analysis of variance. Given that a change in antibody titer should be at least 4-fold to be considered significant (Thrusfield, 2007), titers which differed from the titer at mating no more than 2-fold were assigned the same rank. A Dunnett’s post-hoc test was applied to compare antibody titers during pregnancy and three months after parturition with the basal level
M. Czopowicz et al. / Small Ruminant Research 147 (2017) 37–40
39
Fig. 2. Antibody titers to small ruminant lentivirus throuhout the study determined using the IDEXX MVV/CAEV p28 Ab Screening (IDEXX Laboratories, USA) (p28-TM-ELISA). Antibody titers presented on binary logarithm scale after dividing by two. * signifies statistical significance at ␣ = 0.05.
Fig. 3. Antibody titers to small ruminant lentivirus throuhout the study determined using the Small Ruminant Lentivirus Antibody Test Kit, cELISA (VMRD, USA) (SU-ELISA). Antibody titers presented on binary logarithm scale. * signifies statistical significance at ␣ = 0.05.
at mating. A significance level (␣) was set at 0.05. Analyses and plots were performed in Statistica 12 (StatSoft Inc.). 3. Results Compared to the basal level at mating antibody titer significantly fell at kidding in all three ELISA kits (repeated-measures ANOVA p < 0.001 in all three kits; antibody titer at kidding vs. at mating in Dunnett’s test: wELISA – p = 0.002, p28-TM-ELISA – p = 0.005, SU-ELISA – p < 0.001; Figs. 1–3). Significant decrease in antibody titer was observed for the longest time in SU-ELISA and for the shortest time in p28-TM-ELISA. Two goats turned seronegative at kidding in p28-TM-ELISA and so did two goats in SU-ELISA, whereas none in wELISA. As one of the aforementioned goats was
seronegative at kidding simultaneously in p28-TM-ELISA and SUELISA, three of 13 goats became seronegative at kidding in at least one ELISA. At least four-fold fall in antibody titer between mating and kidding was observed in wELISA in 6 goats, in p28-TM-ELISA in 4 goats and in SU-ELISA in 5 goats. None of the goats showed at least four-fold decrease simultaneously in all three ELISA kits. 4. Discussion Our study shows that antibody titers to all SRLV antigens tend to fall around parturition. This observation is similar to the one of Burridge et al. (1982) in bovine viral leukemia. Given that caprine colostral immunoglobulins mainly come from circulating blood (Larson, 1958) and passive immunity of goat kids is
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based on colostrum intake (Guedes et al., 2010; Pisarska et al., 2002), the fall of antibodies may partly result from the transfer of immunoglobulins from serum to colostrum. Another likely explanation may be the periparturient relaxation of immunity – a phenomenon described so far mainly in gastrointestinal nematode infestations, and associated with impairment of both humoral and cell-dependent immunity in the periparturient period (Beasley et al., 2010). The analogy between the fall in antibody titer observed in our study and the periparturient relaxation of immunity is likely to exist since the link between the onset of lactation and increasing viral load in SRLV infected goats was shown (Morin et al., 2003). From the three ELISA kits used in the study wELISA was the only test which did not miss any of pregnant does at kidding. This probably results from the broader antigen spectrum it covers compared to the remaining two kits. Two goats which became seronegative in a p28-TM-ELISA at kidding (one of them also in SU-ELISA) had the lowest antibody titers at mating and for the entire pregnancy in all ELISA kits. This indicates that turning seronegative was a simple consequence of the low initial antibody level. Although the second goat which tested negative in SU-ELISA at kidding similarly had very low antibody titer in this ELISA kit before, its antibody titers in p28-TM-ELISA were high. This could be explained by an impaired capability of producing neutralizing antibodies to viral envelope antigens observed in some chronically infected goats (Narayan et al., 1984), or by diversity of surface glycoprotein antigens affecting the affinity of the circulating antibodies to the antigen used in SU-ELISA (Herrmann-Hoesing, 2010). Higher antibody titers to capsid than to surface antigen of BLV were also observed by Burridge et al. (1982). The main shortcoming of the study is a small sample size. We hoped to follow up roughly 20 goats, however unfortunate coincidences eliminated as many as 8 of 21 initially enrolled individuals. Small sample size does not allow to formulate firm recommendations regarding diagnostic procedures in the periparturient period. However, it seems probable that wELISAs is less likely to yield false negative results due to falling antibody titers. Therefore, if only one ELISA has to be used for screening pregnant goats wELISA seems to suit best. Given that no goat showed at least four-fold antibody titer decrease simultaneously in all three ELISA kits, the parallel use of them appears to increase sensitivity, if valuable goats need to be screened in the periparturient period. Perhaps, parallel use of ELISA and a real-time PCR would ensure the highest accuracy, however limited availability of PCR in routine field practice renders this recommendation difficult to follow. Conflict of interest statement None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgement This work was financially supported by the National Science Center (grant number 2013/09/B/NZ6/03514). References Amerasinghe, P.H., Vasanthathilake, V.W., Lloyd, S., Fernando, S.T., 1994. Periparturient reduction in buffalo of mitogen-induced lymphocyte proliferation and antibody to Toxocara vitulorum. Trop. Anim. Health Prod. 26, 109–116.
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