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Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls
Theriogenology 62 (2004) 1329–1336
Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls Andrea Caetano-da-Silvaa,1, Ignacio Ferrea, Esther Collantes-Ferna´ndeza, Vanesa Navarroa, Gorka Adurizb, Carlos Ugarte-Garagalzac, Luis Miguel Ortega-Moraa,* a
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain b Instituto Vasco de Investigacio´n y Desarrollo Agrario (NEIKER), 48160 Derio, Spain c Aberekin Centro de Inseminacio´n, Unidades de Torrelavega y Derio, Parque Tecnolo´gico Ed. 600, 48160 Derio, Spain
Received 30 November 2003; received in revised form 23 January 2004; accepted 24 January 2004
Abstract Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low
* Corresponding author. Tel.: þ34-91-3944069; fax: þ34-91-3943908. E-mail address: [email protected] (L.M. Ortega-Mora). 1 Present address: Universidade Federal de Goia´s, Brasil.
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.01.010
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frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI. # 2004 Elsevier Inc. All rights reserved. Keywords: Neospora caninum; Bull; Frozen extended semen; PCR
1. Introduction Neospora caninum is a heteroxenous cyst-forming coccidian parasite closely related to Toxoplasma gondii which has emerged as an important cause of reproductive failure in cattle worldwide [1–3]. Neosporosis in bovines is a common cause of abortion and congenitally infected calves may show neural signs after birth [4,5]. In the Neospora life cycle, dogs are both intermediate and definitive hosts [6], and cattle and other animals are natural intermediate hosts [2]. N. caninum is mainly spread by transplacental transmission from cow to calf [7] but other sources of vertical transmission not yet proved in naturally infected cattle such as pooled colostrum or milk could also be possible [8]. Epidemiological evidence suggests that horizontal transmission also exists [7,9–11]. Calves [12] and pregnant cows [13] can be experimentally infected by Neospora oocysts when administered orally. In addition, the experimental life cycle of N. caninum by cyclical oral transmission between dog and cattle has been reproduced [14]. Recently, the presence of N. caninum DNA in semen from naturally infected bulls was reported [15]. The possibility of N. caninum transmission via contaminated semen could have important repercussions on cattle semen trade. Cryopreservation of semen increases the national as well as the international distribution of semen and the possibility of spreading different diseases among cattle population [16]. The aim of this work was to obtain more information on the relevance of frozen extended semen as a vehicle of N. caninum infection. To accomplish this, a retrospective study was designed and frozen extended straws from naturally infected bulls were studied for the presence of Neospora. Moreover, in positive straws, the parasite load and location were also investigated.
2. Materials and methods 2.1. Bulls and their N. caninum serological status A total of 25 bulls raised for reproductive purposes in a collaborating AI centre were selected for the study. Holstein–Friesian was the predominant breed of bulls and their age varied between 11 and 109 (mean of 38) months (Table 1). All animals were trained for semen collection and were located in individual pens and under strict nutritional, sanitary, and management control. Twenty bulls with antibody titres equal to or higher than 1:250 for N. caninum by an immunofluorescent antibody test (IFAT) [17] were defined as being naturally infected with Neospora (Table 1). Each bull involved in the study was sampled twice for serology.
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Table 1 Detection of N. caninum DNA by means of a nested-PCR in 230 frozen extended semen straws from 25 bulls Bull
Breed
Age (months)
IFAT titre
No. of straws assayed
No. of positive straws
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Limousine Holstein Holstein Holstein Holstein Holstein Holstein Jersey Holstein Holstein Holstein Limousine Limousine Limousine Limousine
20 17 22 14 54 61 58 54 24 60 109 77 36 11 20 24 31 11 52 Not recorded 94 24 26 24 26
1:1000 1:500 1:500 1:500 1:500 1:500 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 <1:100 <1:100 <1:100 <1:100 <1:100
10 10 10 10 21 10 10 10 7 10 3 10 10 3 10 5 10 7 10 4 10 10 10 10 10
3 0 0 0 3 0 1 3 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0
Semen straws analyzed from each bull were obtained at different time-points. Breed, age, and serum titre of specific antibodies to N. caninum by IFAT of the bulls studied are also shown.
Infection was also confirmed by showing the reactivity of sera with at least one immunodominant tachyzoite antigen by immunoblotting (IB) [18]. The other five bulls showed no serum specific antibodies to N. caninum by these techniques and acted as noninfected controls (Table 1). 2.2. Experimental design and semen samples A retrospective study was designed to investigate the presence of N. caninum by a nested-PCR in frozen extended semen straws of bulls with naturally acquired neosporosis. Ten extended semen straws from each bull obtained at different time-points (and therefore, different batches) during previous 2 years (2000–2002) were obtained from the semen bank collection of the AI centre. All semen samples were originally collected from bulls at the collaborating AI centre with the help of an artificial vagina (inside temperature of 43 8C). Fresh semen was diluted in a tris-buffered-fructose-glycerol-yolk extender to 30 million spermatozoa per straw according to standard procedures and stored in liquid nitrogen. All of the bulls produced
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high quality semen with normal sperm morphology and good fertility rates (data not shown). 2.3. Detection and quantification of N. caninum DNA in semen samples A protocol developed to purify N. caninum DNA from bovine semen and their PCR detection has been described previously by Ortega-Mora et al. [15]. Before DNA extraction, each semen straw was thawed for 60 s at 37 8C in a water bath, cut with a sterile scalpel, and passed through a Sephacryl S400 (Amersham Biosciences, Uppsala, Sweden) chromatography column as described by Santurde et al. [19] to avoid potential inhibitory effects of extender in the PCR assay. Afterwards, each semen sample (250 mL) was separated into a seminal fluid and a cellular (nonsperm and sperm cells, respectively) fraction by centrifugation at 12,000 g for 3 min. Total DNA was extracted from seminal fluid and nonsperm cells of semen by using the Genomic-Prep cell and tissue DNA isolation kit (Amersham Biosciences Limited, UK) as described previously [15]. An amount of 5 mL from 50 mL DNA solution obtained (equivalent to 20 mL of extended semen sample) was used for PCR amplification. Samples of seminal fluid and cellular fraction were analyzed separately using a nestedPCR on the internal transcribed spacer (ITS1) region of N. caninum [20] which was carried out with four oligonucleotides as described by Buxton et al. [21]. Briefly, DNA amplification was performed in 25 mL total volume and after the primary amplification, 2 mL of PCR product was added to the secondary amplification reaction mixture. Secondary amplification product was visualized as a 213 bp band by 1% agarose gel electrophoresis and ethidium bromide staining. In each amplification, a DNA extraction and PCR controls equivalent to 102 tachyzoites, respectively, were employed as positive controls. To identify false-positive results, negative control reactions (reactions without template or reactions with DNA of N. caninum-negative semen) were added to each set of PCRs. Quantification of Neospora DNA from nested-PCR positive samples was performed by a real-time PCR test based on the Nc-5 sequence using the double-stranded DNA-binding (dsDNA) dye SYBR Green I. Amplification, data acquisition, and data analysis of reactions for Neospora Nc5 sequence were carried out in the ABI 7700 Prism Sequence Detector machine (Applied Biosystems) as described previously [22]. The number of N. caninum organisms was quantified by interpolation of the corresponding Ct values (cycle threshold: the fractional cycle number reflecting a positive PCR result) in a standard curve from DNA equivalent to 101 to 104 tachyzoites. A sequence Detection System Software v.1.6 (Applied Biosystems) was used to analyze the data, and the number of parasites in semen samples (parasite load) was expressed as parasite number per milliliter of semen.
3. Results Table 1 shows the age, breed, and serological status to Neospora of the 25 bulls included in the study. A total of 230 frozen semen straws were supplied by the collaborating AI centre (Table 1). For the seropositive bulls, the total number of semen straws analyzed (180) was little lower than expected (200) because less than 10 straws were available from
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6 bulls, although 21 straws were available from 1 bull. Eight of the seropositive bulls (40%) investigated showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws sampled (7.8%) were found to be positive (Table 1). In all positive samples, we consistently detected specific DNA in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw analyzed was under the detection level of the real-time PCR and parasite load could not be estimated. On the other hand, 10 extended semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained (Table 1). In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws of the same batch of each positive straw from bulls number 1, 5, and 8 (Table 1) were requested from the collaborating AI centre and analyzed (total 27 semen straws) but N. caninum DNA was only detected in one straw from one bull (bull number 5 in Table 1).
4. Discussion In the present study, we looked for Neospora DNA in a panel of 180 extended semen straws from 20 naturally infected bulls raised for reproductive purposes in an AI centre. Results obtained show that it was possible to detect N. caninum DNA using a nested-PCR in some extended semen straws from the seropositive bulls. Although the N. caninum DNA detection rate compared to the total of semen straws analyzed was low, an important percentage of seropositive bulls investigated showed at least one contaminated semen straw. The sporadic detection of N. caninum in semen was confirmed when unexpectedly, only one out of 27 semen straws was found to be positive when additional semen straws of nine semen batches considered as positive from three different bulls were investigated. On the other hand, no significant correlation was found between the detection of positive straws by PCR of the protozoa and serum IFAT titres since the number of bulls with higher serum antibody titres was low. The sporadic detection of Neospora DNA in semen straws of the serologically positive bulls investigated may be explained, at least in part, by the low parasite numbers of parasite template in semen which made it undetectable by the technique employed. Semen samples were collected from bulls in the chronic stages of the infection [23] where parasites are located as dormant forms in tissue cysts in the central nervous system (CNS) and probably in muscles [1,5] and sporadic detection could be compatible with intermittent shedding of the parasite from CNS following parasitaemia. Moreover, the standard dilution used to prepare the extended semen straws may make these more insensitive to parasite detection. The nested-PCR technique used to detect N. caninum in semen samples has shown a high degree of sensitivity [15]. However, all positive extended semen samples analyzed by quantitative-PCR to determine the parasite load showed that the parasite number was under the detection level of the technique. Discrepant results between quantitative-PCR and nested-PCR may be due to very low parasite numbers in the sample detectable only by nested-PCR. Considering the extremely high degree of sensitivity, one could consider that by testing extended semen samples with very low target molecule concentrations, it is likely that Neospora DNA will not always be pipetted into each reaction tube, thus yielding a negative PCR result. Simultaneous tests of several extended semen samples, theoretically
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with one tachyzoite in each sample, may yield both positive and negative result. On the other hand, although we used a protocol of DNA extraction [15] that reduced PCR inhibitors present in semen, the persistence of these inhibitors in some samples could reduce, at least in part, the sensitivity of the PCR techniques. Although these factors cannot be ruled out, it must be emphasized that all PCR studies were carried out with meticulous care to prevent cross-contamination of the PCR assay. In addition, exhaustive controls were included during the extraction of Neospora DNA and PCR protocols. The fact that N. caninum was not always detected in repeated reactions with samples from the same batch indicates the presence of very low parasite numbers. Moreover, the finding that PCR analyses performed on different semen straws from the same batch thought to be positive were not always positive could indicate that parasites are not uniformly distributed in semen. Similar results have been reported when N. caninum detection is carried out by PCR in other fluids and tissues [4,13,24]. The difficulty to detect N. caninum for both naturally or experimentally infected adult cattle probably reflects the limited amount of parasite DNA present in tissues. Also, the existence of infected seropositive cattle giving negative PCR results in semen examination is reported for other infectious agents such as Brucella melitensis [25] or bovine herpesvirus type 1 [26]. The observation that most N. caninum DNA is detected in the cell fraction and virtually no specific DNA is present in the seminal fluid suggests that parasites can be associated with some types of cells. It is likely that immune cells such as mononuclear phagocytic cells or PMNL would be the cells responsible for transporting the parasite in blood, since circulating antibodies and complement would probably kill extracellular tachyzoites during reactivation. Trafficking of leukocytes to disseminate intracellular parasites via a Trojan horse-type mechanism has been postulated for other apicomplexan parasites such as T. gondii [27] but this phenomenon needs to be investigated for N. caninum. At present, binding of N. caninum antiserum to phagocytes [28,29] and the ability to resist lysis in the macrophage by the related coccidian T. gondii [30] have been reported. The parasite stage excreted in semen is more likely to be tachyzoites rather than bradyzoites as the former are the invasive stage and may infect many cell types and tissues [1]. On the contrary, tissue cysts containing bradyzoites are confined mainly to the CNS [5]. The presence of N. caninum in frozen semen straws suggests the possibility of parasite transmission by AI. From our results, the frequency of N. caninum DNA detection seems to be low suggesting that the risk of sexual transmission is also probably low. In addition, the parasite number seems also to be low. It is perhaps relevant that the close-related coccidian T. gondii has been shown in semen of experimentally infected rams [31,32], goats [33], and bulls [34] but venereal transmission has not been demonstrated. In the case of N. caninum, we do not know if processing the extended semen straws by addition of the extender and/or by freezing and thawing of the insemination straws could have adversely affected or prevented the infectivity of the protozoa. Since AI is being used for introducing new genetics in bovine herds and also for better control of product quality in relation to transmission of diseases, it is of interest to determine whether the semen of infected bulls has the potential to introduce N. caninum into a herd. Further studies are needed to determine the effect of using N. caninum infected semen to inseminate heifers or cows. Until then, the use of spermatozoa from bulls persistently infected with N. caninum constitutes a theoretical risk of infection for
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inseminated cows. Although detection of N. caninum in semen does not necessarily imply the presence of infectious parasitic stages, our results indicate the necessity for the reinforcement of precautionary measures for bulls infected with N. caninum in AI centres until the risks are further evaluated.
Acknowledgements Funding for this work was provided by a research grant from the Spanish government (AGL2000-0112-P4-03). A. Caetano-da-Silva and I. Ferre were financed by CAPESBRASIL and the Ramo´ n y Cajal Spanish Scientific Programme, respectively. This work was also part of the EU research collaboration COST-854. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
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Dubey JP, Lindsay DS. A review of Neospora caninum and neosporosis. Vet Parasitol 1996;67:1–59. Dubey JP. Recent advances in Neospora and neosporosis. Vet Parasitol 1999;84:349–67. Dubey JP. Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 2003;41:1–16. Anderson ML, Andrianarivo AG, Conrad PA. Neosporosis in cattle. Anim Reprod Sci 2000;60:417–31. Dubey JP. Neosporosis in cattle. J Parasitol 2003;89:S42–56. McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are definitive hosts of Neospora caninum. Int J Parasitol 1998;28:1473–8. Davison HC, Otter A, Trees AJ. Estimation of vertical and horizontal transmission parameters of Neospora caninum infections in dairy cattle. Int J Parasitol 1999;29:1683–9. Uggla A, Stenlund S, Holmdahl OJM, Thebo P, Kindahl H, Bjo¨ rkman C. Oral Neospora caninum inoculation of neonatal calves. Int J Parasitol 1998;28:1467–72. McAllister MM, Bjo¨ rkman C, Anderson-Sprecher R, Rogers DG. Evidence of point-source exposure to Neospora caninum and protective immunity in a herd of beef cows. J Am Vet Med Assoc 2000;217:881–7. Dijkstra Th, Barkema HW, Eysker M, Wouda W. Evidence of post-natal transmission of Neospora caninum in Dutch dairy herds. Int J Parasitol 2001;31:209–15. Dijkstra Th, Barkema HW, Bjo¨ rkman C, Wouda W. A high rate of seroconversion for Neospora caninum in a dairy herd without an obvious increased incidence of abortions. Vet Parasitol 2002;109:203–11. De-Marez T, Liddell S, Dubey JP, Jenkins MC, Gasbarre L. Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses. Int J Parasitol 1999;29:1647–57. Trees AJ, McAllister MM, Guy CS, McGarry JW, Smith RF, Williams DJL. Neospora caninum: oocyst challenge of pregnant cows. Vet Parasitol 2002;109:147–54. Gondim LM, Gao L, McAllister MM. Improved production of Neospora caninum oocysts, cyclical oral transmission between dogs and cattle, and in vitro isolation from oocysts. J Parasitol 2002;88:1159–63. Ortega-Mora LM, Ferre I, del-Pozo I, Caetano-da-Silva A, Collantes-Ferna´ ndez E, Regidor-Cerrillo J, et al. Detection of Neospora caninum in semen of bulls. Vet Parasitol 2003;117:301–8. Philpott M. The dangers of disease transmission by artificial insemination and embryo transfer. Br Vet J 1994;149:339–69. ´ lvarez-Garcı´a G, Collantes-Ferna´ ndez E, Costas E, Rebordosa X, Ortega-Mora LM. Influence of age and A purpose for testing on the cut-off selection of serological methods in bovine neosporosis. Vet Res 2003;34:341–52. ´ lvarez-Garcı´a G, Pereira-Bueno J, Go´ mez-Bautista M, Ortega-Mora LM. Pattern of recognition of A Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses. Vet Parasitol 2002;107:15–27. Santurde G, Da-Silva N, Villares R, Tabare´ s E, Solana A, Bautista JM, et al. Rapid and high sensitivity test for direct detection of bovine herpesvirus-1 genome in clinical samples. Vet Microbiol 1996;49:81–92.
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[20] Payne S, Ellis J. Detection of Neospora caninum DNA by the polymerase chain reaction. Int J Parasitol 1996;26:347–51. [21] Buxton D, Maley SW, Wright S, Thomson KM, Rae AG, Innes EA. The pathogenesis of experimental neosporosis in pregnant sheep. J Comp Pathol 1998;118:267–79. ´ lvarez-Garcı´a G, Ortega-Mora LM. Quantitative detection of [22] Collantes-Ferna´ ndez E, Zaballos A, A Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR. J Clin Microbiol 2002;40:1194–8. ´ lvarez-Garcı´a G, del-Pozo I, Atxaerandio R, et al. Neospora [23] Caetano-da-Silva A, Ferre I, Aduriz G, A caninum infection in breeder bulls: seroprevalence and comparison of serological methods used for diagnosis. Vet Parasitol, submitted. [24] Williams DJL, Guy CS, McGarry JW, Guy F, Tasker L, Smith RF, et al. Neospora caninum-associated abortion in cattle: the time of experimentally-induced parasitaemia during gestation determines foetal survival. Parasitology 2000;121:347–58. [25] Amin AS, Hamdy MER, Ibrahim AK. Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Vet Microbiol 2001;83:37–44. [26] Rocha MA, Barbosa EF, Guimaraes SEF, Dias-Neto E, Gouveia AMG. A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls. Vet Microbiol 1998;63:1–11. [27] Barragan A, Sibley LD. Migration of Toxoplasma gondii across biological barriers. Trends Microbiol 2003;11:426–30. [28] Boger LA, Hattel AL. Additional evaluation of undiagnosed bovine abortion cases may reveal fetal neosporosis. Vet Parasitol 2003;113:1–6. [29] Wouda W, Moen AR, Visser IJR, van Knapen F. Bovine fetal neosporosis: a comparison of epizootic and sporadic abortion cases and different age classes with regard to lesion severity and identification of organisms in brain, heart, and liver. J Vet Diagn Invest 1997;9:180–5. [30] McLeod R, Mack D, Brown C. T. gondii: new advances in cellular and molecular biology. Exp Parasitol 1991;72:109–21. [31] Blewett DA, Teale AJ, Miller JK, Scott GR, Buxton D. Toxoplasmosis in rams: possible significance of venereal transmission. Vet Rec 1982;111:73–5. [32] Teale AJ, Blewett DA, Miller JK. Experimentally induced toxoplasmosis in young rams: the clinical syndrome and semen secretion of toxoplasma. Vet Rec 1982;111:53–5. [33] Dubey JP, Sharma SP. Prolonged excretion of Toxoplasma gondii in semen of goats. Am J Vet Res 1980;41:794–5. [34] Scarpelli LC, Costa AJ, Migani MB, Varandas NP, Ferro MIT, Esper CR. Venereal transmission viability of Toxoplasma gondii in bovines. In: Proceedings of the XVIII International Conference of the World Association for the Advancement of Veterinary Parasitology; 2001. p. 22.
Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls Andrea Caetano-da-Silvaa,1, Ignacio Ferrea, Esther Collantes-Ferna´ndeza, Vanesa Navarroa, Gorka Adurizb, Carlos Ugarte-Garagalzac, Luis Miguel Ortega-Moraa,* a
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain b Instituto Vasco de Investigacio´n y Desarrollo Agrario (NEIKER), 48160 Derio, Spain c Aberekin Centro de Inseminacio´n, Unidades de Torrelavega y Derio, Parque Tecnolo´gico Ed. 600, 48160 Derio, Spain
Received 30 November 2003; received in revised form 23 January 2004; accepted 24 January 2004
Abstract Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low
* Corresponding author. Tel.: þ34-91-3944069; fax: þ34-91-3943908. E-mail address: [email protected] (L.M. Ortega-Mora). 1 Present address: Universidade Federal de Goia´s, Brasil.
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.01.010
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frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI. # 2004 Elsevier Inc. All rights reserved. Keywords: Neospora caninum; Bull; Frozen extended semen; PCR
1. Introduction Neospora caninum is a heteroxenous cyst-forming coccidian parasite closely related to Toxoplasma gondii which has emerged as an important cause of reproductive failure in cattle worldwide [1–3]. Neosporosis in bovines is a common cause of abortion and congenitally infected calves may show neural signs after birth [4,5]. In the Neospora life cycle, dogs are both intermediate and definitive hosts [6], and cattle and other animals are natural intermediate hosts [2]. N. caninum is mainly spread by transplacental transmission from cow to calf [7] but other sources of vertical transmission not yet proved in naturally infected cattle such as pooled colostrum or milk could also be possible [8]. Epidemiological evidence suggests that horizontal transmission also exists [7,9–11]. Calves [12] and pregnant cows [13] can be experimentally infected by Neospora oocysts when administered orally. In addition, the experimental life cycle of N. caninum by cyclical oral transmission between dog and cattle has been reproduced [14]. Recently, the presence of N. caninum DNA in semen from naturally infected bulls was reported [15]. The possibility of N. caninum transmission via contaminated semen could have important repercussions on cattle semen trade. Cryopreservation of semen increases the national as well as the international distribution of semen and the possibility of spreading different diseases among cattle population [16]. The aim of this work was to obtain more information on the relevance of frozen extended semen as a vehicle of N. caninum infection. To accomplish this, a retrospective study was designed and frozen extended straws from naturally infected bulls were studied for the presence of Neospora. Moreover, in positive straws, the parasite load and location were also investigated.
2. Materials and methods 2.1. Bulls and their N. caninum serological status A total of 25 bulls raised for reproductive purposes in a collaborating AI centre were selected for the study. Holstein–Friesian was the predominant breed of bulls and their age varied between 11 and 109 (mean of 38) months (Table 1). All animals were trained for semen collection and were located in individual pens and under strict nutritional, sanitary, and management control. Twenty bulls with antibody titres equal to or higher than 1:250 for N. caninum by an immunofluorescent antibody test (IFAT) [17] were defined as being naturally infected with Neospora (Table 1). Each bull involved in the study was sampled twice for serology.
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Table 1 Detection of N. caninum DNA by means of a nested-PCR in 230 frozen extended semen straws from 25 bulls Bull
Breed
Age (months)
IFAT titre
No. of straws assayed
No. of positive straws
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Limousine Holstein Holstein Holstein Holstein Holstein Holstein Jersey Holstein Holstein Holstein Limousine Limousine Limousine Limousine
20 17 22 14 54 61 58 54 24 60 109 77 36 11 20 24 31 11 52 Not recorded 94 24 26 24 26
1:1000 1:500 1:500 1:500 1:500 1:500 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 1:250 <1:100 <1:100 <1:100 <1:100 <1:100
10 10 10 10 21 10 10 10 7 10 3 10 10 3 10 5 10 7 10 4 10 10 10 10 10
3 0 0 0 3 0 1 3 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0
Semen straws analyzed from each bull were obtained at different time-points. Breed, age, and serum titre of specific antibodies to N. caninum by IFAT of the bulls studied are also shown.
Infection was also confirmed by showing the reactivity of sera with at least one immunodominant tachyzoite antigen by immunoblotting (IB) [18]. The other five bulls showed no serum specific antibodies to N. caninum by these techniques and acted as noninfected controls (Table 1). 2.2. Experimental design and semen samples A retrospective study was designed to investigate the presence of N. caninum by a nested-PCR in frozen extended semen straws of bulls with naturally acquired neosporosis. Ten extended semen straws from each bull obtained at different time-points (and therefore, different batches) during previous 2 years (2000–2002) were obtained from the semen bank collection of the AI centre. All semen samples were originally collected from bulls at the collaborating AI centre with the help of an artificial vagina (inside temperature of 43 8C). Fresh semen was diluted in a tris-buffered-fructose-glycerol-yolk extender to 30 million spermatozoa per straw according to standard procedures and stored in liquid nitrogen. All of the bulls produced
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high quality semen with normal sperm morphology and good fertility rates (data not shown). 2.3. Detection and quantification of N. caninum DNA in semen samples A protocol developed to purify N. caninum DNA from bovine semen and their PCR detection has been described previously by Ortega-Mora et al. [15]. Before DNA extraction, each semen straw was thawed for 60 s at 37 8C in a water bath, cut with a sterile scalpel, and passed through a Sephacryl S400 (Amersham Biosciences, Uppsala, Sweden) chromatography column as described by Santurde et al. [19] to avoid potential inhibitory effects of extender in the PCR assay. Afterwards, each semen sample (250 mL) was separated into a seminal fluid and a cellular (nonsperm and sperm cells, respectively) fraction by centrifugation at 12,000 g for 3 min. Total DNA was extracted from seminal fluid and nonsperm cells of semen by using the Genomic-Prep cell and tissue DNA isolation kit (Amersham Biosciences Limited, UK) as described previously [15]. An amount of 5 mL from 50 mL DNA solution obtained (equivalent to 20 mL of extended semen sample) was used for PCR amplification. Samples of seminal fluid and cellular fraction were analyzed separately using a nestedPCR on the internal transcribed spacer (ITS1) region of N. caninum [20] which was carried out with four oligonucleotides as described by Buxton et al. [21]. Briefly, DNA amplification was performed in 25 mL total volume and after the primary amplification, 2 mL of PCR product was added to the secondary amplification reaction mixture. Secondary amplification product was visualized as a 213 bp band by 1% agarose gel electrophoresis and ethidium bromide staining. In each amplification, a DNA extraction and PCR controls equivalent to 102 tachyzoites, respectively, were employed as positive controls. To identify false-positive results, negative control reactions (reactions without template or reactions with DNA of N. caninum-negative semen) were added to each set of PCRs. Quantification of Neospora DNA from nested-PCR positive samples was performed by a real-time PCR test based on the Nc-5 sequence using the double-stranded DNA-binding (dsDNA) dye SYBR Green I. Amplification, data acquisition, and data analysis of reactions for Neospora Nc5 sequence were carried out in the ABI 7700 Prism Sequence Detector machine (Applied Biosystems) as described previously [22]. The number of N. caninum organisms was quantified by interpolation of the corresponding Ct values (cycle threshold: the fractional cycle number reflecting a positive PCR result) in a standard curve from DNA equivalent to 101 to 104 tachyzoites. A sequence Detection System Software v.1.6 (Applied Biosystems) was used to analyze the data, and the number of parasites in semen samples (parasite load) was expressed as parasite number per milliliter of semen.
3. Results Table 1 shows the age, breed, and serological status to Neospora of the 25 bulls included in the study. A total of 230 frozen semen straws were supplied by the collaborating AI centre (Table 1). For the seropositive bulls, the total number of semen straws analyzed (180) was little lower than expected (200) because less than 10 straws were available from
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6 bulls, although 21 straws were available from 1 bull. Eight of the seropositive bulls (40%) investigated showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws sampled (7.8%) were found to be positive (Table 1). In all positive samples, we consistently detected specific DNA in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw analyzed was under the detection level of the real-time PCR and parasite load could not be estimated. On the other hand, 10 extended semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained (Table 1). In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws of the same batch of each positive straw from bulls number 1, 5, and 8 (Table 1) were requested from the collaborating AI centre and analyzed (total 27 semen straws) but N. caninum DNA was only detected in one straw from one bull (bull number 5 in Table 1).
4. Discussion In the present study, we looked for Neospora DNA in a panel of 180 extended semen straws from 20 naturally infected bulls raised for reproductive purposes in an AI centre. Results obtained show that it was possible to detect N. caninum DNA using a nested-PCR in some extended semen straws from the seropositive bulls. Although the N. caninum DNA detection rate compared to the total of semen straws analyzed was low, an important percentage of seropositive bulls investigated showed at least one contaminated semen straw. The sporadic detection of N. caninum in semen was confirmed when unexpectedly, only one out of 27 semen straws was found to be positive when additional semen straws of nine semen batches considered as positive from three different bulls were investigated. On the other hand, no significant correlation was found between the detection of positive straws by PCR of the protozoa and serum IFAT titres since the number of bulls with higher serum antibody titres was low. The sporadic detection of Neospora DNA in semen straws of the serologically positive bulls investigated may be explained, at least in part, by the low parasite numbers of parasite template in semen which made it undetectable by the technique employed. Semen samples were collected from bulls in the chronic stages of the infection [23] where parasites are located as dormant forms in tissue cysts in the central nervous system (CNS) and probably in muscles [1,5] and sporadic detection could be compatible with intermittent shedding of the parasite from CNS following parasitaemia. Moreover, the standard dilution used to prepare the extended semen straws may make these more insensitive to parasite detection. The nested-PCR technique used to detect N. caninum in semen samples has shown a high degree of sensitivity [15]. However, all positive extended semen samples analyzed by quantitative-PCR to determine the parasite load showed that the parasite number was under the detection level of the technique. Discrepant results between quantitative-PCR and nested-PCR may be due to very low parasite numbers in the sample detectable only by nested-PCR. Considering the extremely high degree of sensitivity, one could consider that by testing extended semen samples with very low target molecule concentrations, it is likely that Neospora DNA will not always be pipetted into each reaction tube, thus yielding a negative PCR result. Simultaneous tests of several extended semen samples, theoretically
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with one tachyzoite in each sample, may yield both positive and negative result. On the other hand, although we used a protocol of DNA extraction [15] that reduced PCR inhibitors present in semen, the persistence of these inhibitors in some samples could reduce, at least in part, the sensitivity of the PCR techniques. Although these factors cannot be ruled out, it must be emphasized that all PCR studies were carried out with meticulous care to prevent cross-contamination of the PCR assay. In addition, exhaustive controls were included during the extraction of Neospora DNA and PCR protocols. The fact that N. caninum was not always detected in repeated reactions with samples from the same batch indicates the presence of very low parasite numbers. Moreover, the finding that PCR analyses performed on different semen straws from the same batch thought to be positive were not always positive could indicate that parasites are not uniformly distributed in semen. Similar results have been reported when N. caninum detection is carried out by PCR in other fluids and tissues [4,13,24]. The difficulty to detect N. caninum for both naturally or experimentally infected adult cattle probably reflects the limited amount of parasite DNA present in tissues. Also, the existence of infected seropositive cattle giving negative PCR results in semen examination is reported for other infectious agents such as Brucella melitensis [25] or bovine herpesvirus type 1 [26]. The observation that most N. caninum DNA is detected in the cell fraction and virtually no specific DNA is present in the seminal fluid suggests that parasites can be associated with some types of cells. It is likely that immune cells such as mononuclear phagocytic cells or PMNL would be the cells responsible for transporting the parasite in blood, since circulating antibodies and complement would probably kill extracellular tachyzoites during reactivation. Trafficking of leukocytes to disseminate intracellular parasites via a Trojan horse-type mechanism has been postulated for other apicomplexan parasites such as T. gondii [27] but this phenomenon needs to be investigated for N. caninum. At present, binding of N. caninum antiserum to phagocytes [28,29] and the ability to resist lysis in the macrophage by the related coccidian T. gondii [30] have been reported. The parasite stage excreted in semen is more likely to be tachyzoites rather than bradyzoites as the former are the invasive stage and may infect many cell types and tissues [1]. On the contrary, tissue cysts containing bradyzoites are confined mainly to the CNS [5]. The presence of N. caninum in frozen semen straws suggests the possibility of parasite transmission by AI. From our results, the frequency of N. caninum DNA detection seems to be low suggesting that the risk of sexual transmission is also probably low. In addition, the parasite number seems also to be low. It is perhaps relevant that the close-related coccidian T. gondii has been shown in semen of experimentally infected rams [31,32], goats [33], and bulls [34] but venereal transmission has not been demonstrated. In the case of N. caninum, we do not know if processing the extended semen straws by addition of the extender and/or by freezing and thawing of the insemination straws could have adversely affected or prevented the infectivity of the protozoa. Since AI is being used for introducing new genetics in bovine herds and also for better control of product quality in relation to transmission of diseases, it is of interest to determine whether the semen of infected bulls has the potential to introduce N. caninum into a herd. Further studies are needed to determine the effect of using N. caninum infected semen to inseminate heifers or cows. Until then, the use of spermatozoa from bulls persistently infected with N. caninum constitutes a theoretical risk of infection for
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inseminated cows. Although detection of N. caninum in semen does not necessarily imply the presence of infectious parasitic stages, our results indicate the necessity for the reinforcement of precautionary measures for bulls infected with N. caninum in AI centres until the risks are further evaluated.
Acknowledgements Funding for this work was provided by a research grant from the Spanish government (AGL2000-0112-P4-03). A. Caetano-da-Silva and I. Ferre were financed by CAPESBRASIL and the Ramo´ n y Cajal Spanish Scientific Programme, respectively. This work was also part of the EU research collaboration COST-854. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
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