Detection of Neospora caninum in semen of bulls

Veterinary Parasitology 117 (2003) 301–308

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Detection of Neospora caninum in semen of bulls Luis Miguel Ortega-Mora a,∗ , Ignacio Ferre a , Itziar del-Pozo b , Andrea Caetano-da-Silva a,1 , Esther Collantes-Fernández a , Javier Regidor-Cerrillo a , Carlos Ugarte-Garagalza c , Gorka Aduriz b a

c

Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain b Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER), 48160 Derio, Spain Aberekin Centro de Inseminación, Unidades de Torrelavega y Derio, Parque Tecnológico Ed. 600, 48160 Derio, Spain Received 29 July 2003; accepted 25 September 2003

Abstract In cattle, transplacental infection is the main route of Neospora caninum transmission, but postnatal transmission by the oral uptake of sporozoite-containing oocysts shed by dogs may also be possible. Other routes of horizontal transmission, such as the venereal route, have not been investigated. In this study, we evaluated the presence of N. caninum DNA by a nested-PCR in fresh non-extended semen and frozen extended semen straws of five Holstein–Friesian bulls with naturally-acquired neosporosis. The infection status was assessed by an immunofluorescent antibody test (IFAT) and confirmed by immunoblotting (IB). Because of inhibitory components of semen, a protocol was developed to purify N. caninum DNA from bovine semen. Sporadically, N. caninum DNA was detected in non-extended fresh semen samples and frozen extended semen straws of the five seropositive bulls. In all positive samples, specific DNA was consistently found in the cell fraction of semen and not in seminal plasma. The parasite mean load in positive fresh semen samples determined by a real-time PCR was low oscillating between 1 and 2.8 parasites/ml of semen (maximum parasite load detected in one sample was 7.5 parasites/ml of semen). In parallel, another three similar but uninfected bulls acted as controls and no N. caninum DNA was amplified in any of their fresh and straw semen samples assayed. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined. © 2003 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Bull; Semen; PCR; Venereal transmission

∗ Corresponding author. Tel.: +34-91-3944069; fax: +34-91-3943908. E-mail address: [email protected] (L.M. Ortega-Mora). 1 Present address: Universidade Federal de Goi´ as, Brasil.

0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.09.015

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1. Introduction Neospora caninum is a heteroxenous cyst-forming coccidian closely related to Toxoplasma gondii (Dubey et al., 2002). In cattle, neosporosis is manifested by reproductive failure, which includes abortion and neural signs in neonatal calves (Dubey and Lindsay, 1996; Dubey, 1999). Due to its high prevalence in cattle, N. caninum has emerged as an important cause of bovine abortion (Anderson et al., 2000), and neosporosis has been recognized as an economically important disease with a considerable impact on the livestock industry (Trees et al., 1999). Advances concerning the Neospora life cycle have proved dogs to be both intermediate and definitive hosts (McAllister et al., 1998), and cattle and other animals to be its natural intermediate hosts (Dubey, 1999). Routes of Neospora transmission include transplacental infection through tachyzoites, ingestion of tissues harbouring cysts and oral uptake of sporozoite-containing oocysts. Transplacental transmission seems to be very efficient for N. caninum in naturally infected cattle and plays a major role in the maintenance and spread of the disease (Davison et al., 1999). Other sources of vertical transmission, such as cow to calf transmission via pooled colostrum or milk could also be possible (Uggla et al., 1998), but until now this has not been proven in naturally infected cattle. Mathematical models indicate that vertical transmission alone is not sufficient to sustain the infection in cattle herds (French et al., 1999) and epidemiological evidence suggests that horizontal transmission exists (Davison et al., 1999; Dijkstra et al., 2001, 2002). Calves (De Marez et al., 1999) and pregnant cows (Trees et al., 2002) can be experimentally infected by Neospora oocysts when administered orally, and recently, the life cycle of N. caninum between dog and cattle has been experimentally reproduced (Gondim et al., 2002). However, some routes of postnatal transmission of N. caninum in cattle, e.g. venereal transmission, must be investigated. Recently, the seroprevalence of N. caninum infection in breeder bulls was shown to be moderate (Caetano-da-Silva et al., 2004), and the possibility of bulls playing some role in the horizontal transmission of N. caninum has been suggested (Moore et al., 2003). The close-related coccidian T. gondii has been detected in semen of experimentally infected rams (Blewett et al., 1982; Teale et al., 1982), goats (Dubey and Sharma, 1980), and bulls (Scarpelli et al., 2001). However, at least in ovine toxoplasmosis, the possibility of venereal transmission is questionable (Blewett et al., 1982). At present, the presence of N. caninum in bull semen and its possible venereal transmission has not been reported. The possibility of N. caninum transmission via semen could imply profound repercussions on cattle semen trade. Artificial insemination (AI) is an important procedure for the improvement of cattle production and millions of doses of frozen bovine semen are exchanged annually around the world, increasing the possibility of spreading different diseases among cattle populations (Philpott, 1994). This is the case of some important bovine pathogens such as the bovine herpesvirus-1 (BHV-1), the causal agent of infectious bovine rhinotracheitis (Afshar and Eaglesome, 1990). BHV-1 is the most common bovine viral pathogen found in semen and its detection is of great importance because infected semen may lead to clinical signs in inseminated cows or heifers. To prevent the transmission of BHV-1 by AI, only semen that is free of BHV-1 should be used. Many tests have been used to detect infectious agents in bovine semen. Polymerase chain reaction (PCR) has been shown to be a useful tool to identify some pathogens, like BHV-1, present in semen (Van

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der Engelenburg et al., 1993; Santurde et al., 1996; Rocha et al., 1998; Moore et al., 2000). In neosporosis, recent research has focused on the development of fast and sensitive PCR systems to detect N. caninum in cattle tissues (Ferre et al., 2003), but not in semen. In the present study, we evaluated fresh non-extended semen and frozen extended semen of bulls with naturally-acquired neosporosis for the presence of N. caninum by means of a nested-PCR. In addition, the parasite load in each positive semen sample was determined by a real-time PCR. 2. Materials and methods 2.1. Bulls and serological status to neosporosis Eight bulls (Holstein–Friesian, 1–6 years old) raised for reproductive purposes in an AI centre and trained for semen collection were used in this study. All animals were located in individual pens and under strict nutritional, sanitary and management control. Five of the eight bulls were naturally infected with N. caninum and infection was assessed by the detection of serum specific antibodies by an immunofluorescent antibody test (IFAT). Sera from bulls with titers equal to or higher than 1:250 were considered to be positive for antibodies specific for N. caninum (Álvarez-Garc´ıa et al., 2003). In addition, the infection was confirmed showing the reactivity of sera with at least one immunodominant tachyzoite antigen by immunoblotting (IB). The other three bulls showed no serum specific antibodies to N. caninum by any of these techniques and acted as non-infected controls. Each bull involved in the study was sampled twice for serology. The IFAT and IB procedures have been described before (Álvarez-Garc´ıa et al., 2002, 2003). 2.2. Semen collection All semen samples were collected from Holstein bulls at the AI centre with the help of an artificial vagina (inside temperature of 43 ◦ C). All of the bulls produced high quality semen with normal sperm morphology and good fertility rates (data not shown). Fresh non-extended semen samples were obtained once weekly, on the same day for all animals, over 10 consecutive weeks from the eight bulls. A seropositive bull was retired at week 6 of the experiment because of an arthritic leg problem. Thus, a total of 76 semen samples were collected, transported and stored in our laboratory at 4 ◦ C, and processed within 24 h of collection. Ten extended semen straws from each bull were also included in the study. These samples (a total of 80 semen straws) were originally collected over the 2 years before the experiment started. Fresh semen was diluted in a Tris-buffered-fructose–glycerol–yolk extender to 30 million of spermatozoa per straw according to standard procedures at the AI centre, and stored in liquid nitrogen. 2.3. Semen samples preparation A protocol was developed to purify N. caninum DNA from bovine semen because direct PCR detection of N. caninum in semen was impossible due to the inhibitory components

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of semen (Von Beroldingen et al., 1990). Before DNA extraction, each extended semen straw was thawed for 60 s at 37 ◦ C 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. (1996) in order to avoid potential inhibitory effects of extender in PCR assay. Each semen sample (400 ␮l of fresh non-extended semen and 200–250 ␮l of extended semen, respectively) was separated into a seminal fluid and a cellular (nonsperm and sperm cells) fraction by centrifugation at 12,000 × g for 3 min. Total DNA was extracted from seminal fluid and cellular fraction of semen by using the Genomic-Prep cell and tissue DNA isolation kit (Amersham Biosciences Limited, UK). Briefly, seminal fluid and the cellular fraction of each semen sample were treated with a cell lysis solution with proteinase K (200 ␮g/ml) and RNAse A (20 ␮g/ml), and the samples were incubated at 60 ◦ C for 1 h. In the cellular fraction sample, nonsperm cells and spermatozoan tails were lysed, but sperm heads remained intact (Von Beroldingen et al., 1990). Sperm heads contain most of the DNA present in semen and cause inhibitory effects on PCR assay (Von Beroldingen et al., 1990; Van der Engelenburg et al., 1993) and we decided not to obtain DNA from that fraction. The lysates obtained before were deproteinated with a protein precipitation solution. The precipitates were removed by centrifugation, and the DNA-containing supernatant was pipetted into a new 1.5 ml centrifuge tube. The DNA was then precipitated with isopropanol and resuspended in 50 ␮l of DNA hydration solution. In both fractions from extended semen samples, before adding the supernatant from the protein precipitation step, 1 ␮l glycogen (20 mg/ml) was added to isopropanol to improve DNA yield. An amount of 5 ␮l (equivalent to 40 ␮l of fresh semen and 20 ␮l of extended semen samples, respectively) was used for PCR amplification. 2.4. PCR procedures Seminal fluid and cellular fraction were separately analyzed using a nested-PCR on the internal transcribed spacer (ITS1) region of N. caninum (Payne and Ellis, 1996) which was carried out with four oligonucleotides as described by Buxton et al. (1998). Briefly, DNA amplification was performed in 25 ␮l total volume and after the primary amplification, 2 ␮l of PCR product was added to the secondary amplification reaction mixture. Secondary amplification product was visualized by 1.5% agarose gel electrophoresis and ethidium bromide staining. To avoid false positive reactions, DNA extraction, PCR sample preparation and electrophoresis were performed in separate rooms with different sets of instruments and aerosol barrier tips and disposable gloves were employed. 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. To determine the sensitivity of PCR amplifications on seminal samples, N. caninum tachyzoites of the Nc-1 isolate were serially 10-fold diluted in 250 ␮l N. caninum free non-extended and extended bull semen samples, respectively. The final concentrations of N. caninum ranged from 104 to 1 tachyzoites per sample. Neospora DNA were extracted from each dilution as described above and 5 ␮l from 50 ␮l DNA solution obtained was analyzed by PCR. The expected 213 bp band was observed from the cellular fraction sample

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containing a DNA equivalent of 1 tachyzoite (0.1 tachyzoites per PCR reaction), in seminal fluid sample the detection limit of N. caninum varied between 1 and 10 tachyzoites. 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 Nc-5 sequence were carried out in the ABI 7700 Prism Sequence Detector machine (Applied Biosystems) as described previously (Collantes-Fernández et al., 2002). 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 10−1 –104 tachyzoites. A sequence Detection System Software v.1.6 (Applied Biosystems) was used to analyse the data, and the number of parasites in semen samples (parasite load) was expressed as parasite number/ml of semen.

3. Results N. caninum DNA was detected in semen samples of the five seropositive bulls (Table 1). In fresh non-extended semen samples, N. caninum DNA was detected in four of the five seropositive bulls. One positive sample was detected in each of two bulls and the other two bulls showed three positive samples, respectively. In the frozen extended semen, Neospora DNA was detected in three straws corresponding to three different bulls. No N. caninum DNA was amplified in fresh and frozen semen samples from the seronegative bulls. The total frequency of occurrence of Neospora DNA in all semen samples analyzed was low (11 positives out of 156 semen samples). N. caninum DNA was detected in 8 out of 76 fresh non-extended semen samples, while only 3 out of 80 extended semen straws were positive. In all positive samples, we consistently found specific DNA in the cell fraction and not in seminal plasma. The mean parasite load in positive fresh semen samples oscillated between 1 and 2.8 parasites/ml of semen (Table 1). The maximum parasite load detected was 7.5

Table 1 Detection of N. caninum DNA by a nested-PCR in fresh non-extended semen and frozen extended semen straws of eight bulls Bull

A B C D E F G H

IFAT titre

Fresh non-extended semen

Frozen extended semen

Number of semen samples assayed

Positive number

Mean number of parasites/ml of semen

Number of semen straws assayed

Positive number

1:250 1:250 1:250 1:250 1:250 <1:100 <1:100 <1:100

6 10 10 10 10 10 10 10

1 3 0 3 1 0 0 0

1 1.5 – 2.8 1 – – –

10 10 10 10 10 10 10 10

1 0 1 0 1 0 0 0

The mean parasite load determined by a real-time PCR in positive fresh semen samples of four seropositive bulls is also shown.

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parasites/ml of fresh semen. In all positive extended semen samples the parasite numbers were under the detection level of real-time PCR (<0.1 tachyzoites per reaction).

4. Discussion Results obtained show that all seropositive bulls shed N. caninum DNA in fresh nonextended and frozen extended semen sporadically. It cannot be concluded that infectious stages of N. caninum were present in the semen since no parasite isolation was performed and PCR only detects specific DNA. However, the stage excreted in semen is more likely to be tachyzoite than cyst including bradyzoites. Whereas tachyzoites are found in many cell types and tissues, cysts have only been reported in the central and peripheral nervous system of cattle, especially in brain (Dubey and Lindsay, 1996), and recently in the skeletal muscle of two congenitally infected calves (Peters et al., 2001). There may be several reasons for the sporadic presence of Neospora DNA in semen of serologically positive bulls. These samples were collected from animals in the chronic stages of infection (Caetano-da-Silva et al., 2004) and tachyzoites may be shed intermittently. On the other hand, N. caninum may be excreted continuously, but not detected in certain weeks because of their low presence. The different detection limits between fresh and frozen semen samples can, at least in part, be explained by the dilution that was used to prepare the extended semen straws, whereas fresh semen samples were not diluted before testing. In addition, the volume of frozen semen sample assayed by PCR was lower than that used in fresh samples. The relative sensitivity of the nested-PCR assay, determined by amplification of the N. caninum DNA products of 10-fold dilutions of bull semen, detected 1 tachyzoite/250 ␮l of artificially contaminated semen. However, since in the PCR the usable portion of the sample used for detection of N. caninum DNA was very small, positive results can be considered as confirmatory, but negative results cannot be considered as a true negative. It is not known whether N. caninum circulates in semen extracellularly or intracellularly. 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 tachyzoites were associated to any type of cell. It was not possible, using the detection methods described in this study, to determine the cell type associated to N. caninum in semen, but it is likely that macrophages were the cells which vehicle N. caninum tachyzoites. The dissemination of tachyzoites in infected macrophages via blood could explain the presence of N. caninum in tissues and organs. However, the presence and possible multiplication of N. caninum in the male genital tract remain unclear. The presence of N. caninum DNA in semen is a strong argument in favor of venereal transmission, but the frequency of N. caninum DNA detection was low suggesting that the risk of sexual transmission is also probably low. The parasite load detected in positive fresh semen samples confirmed this. The maximum number of parasites found was 7.5 parasites/ml of semen, which corresponded to an ejaculate containing hardly 100 parasites. In addition, we do not know if processing of the extended semen straws (by addition of the extender or/and by freezing and thawing of the insemination straws) may have decreased or prevented the infectivity of the protozoa. Further studies are needed to determine the effect

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of using N. caninum infected semen to inseminate heifers or cows. However, these results should be considered in the broader perspective of safety in AI centers and for management of seropositive bulls. Since artificial insemination is being used to introduce new genetics in bovine herds and also for better control of product quality with regard to transmission of diseases, it would be interesting to determine whether the semen of infected bulls represents a potential source of N. caninum introduction in the herd. At present, we report the detection of N. caninum DNA in the semen of naturally infected bulls for the first time. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined, since N. caninum is one of the major causes of abortion in cattle.

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