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Transmission of the caprine arthritis–encephalitis virus through artificial insemination
Small Ruminant Research 109 (2013) 193–198
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Transmission of the caprine arthritis–encephalitis virus through artificial insemination Kelma Costa de Souza a,b,∗ , Raymundo Rizaldo Pinheiro a , Diônes Oliveira Santos a , Roberta Lomonte Lemos de Brito a , Apoliana de Souza Rodrigues a , Lúcia Helena Sider a , Ney Rômulo Oliveira Paula c , Amanda Aragão Avila b , Janaina de Fátima Saraiva Cardoso c , Alice Andrioli a a b c
Brazilian Agricultural Research Center, Embrapa Goats and Sheep, Brazil Vale Acaraú University, Brazil Federal University of Piauí, Brazil
a r t i c l e
i n f o
Article history: Received 27 August 2010 Received in revised form 20 July 2012 Accepted 31 July 2012 Available online 13 September 2012 Keywords: CAEV Goats Viral load Artificial insemination Transmission
a b s t r a c t The objective of present study was to evaluate the transmissibility of the caprine arthritis–encephalitis virus (CAEV) through artificial insemination (AI), and to assess the influence of viral load on this probable transmission. It also aims to verify whether the inflammatory process caused by the use of intravaginal sponges would facilitate virus entry in the female reproductive tract. For this purpose, 30 undefined breed goats were used, all serologically negative for CAEV. One Anglo-Nubian buck, also seronegative, was used to artificially inseminate females in this study. His semen was contaminated with the standard CAEV-Cork virus strain, with two distinct infective titres, one 106 TCID50 /mL, for high viral load (HVL), and another of 102 TCID50 /mL, for low viral load (LVL). Females had estrus synchronised by using two protocols, intravaginal sponges in Group 1 (G1, N = 15) and auricular subcutaneous implants in Group 2 (G2, N = 15). For inseminations, the goats were divided into three groups of 10 animals each. One group was inseminated with HVL, another with LVL and the third with semen from the same virus-free buck, as a negative control. The experiment was conducted in accordance to the ethical principles for animal experimentation. Statistical analyses were performed by the chi-square test (P < 0.05). Thirty days after insemination, the experimental infection was confirmed, when 12 out of the 20 (60%) inseminated goats had seroconverted. Sixty days after insemination, all females from the HVL and LVL groups presented anti-CAEV antibodies. There was no statistical difference (P > 0.05) among groups regarding viral loads nor between the two estrus synchronisation protocols. Goats from the control group remained seronegative throughout the experiment (12 months). Concerning reproductive parameters, no difference was found between the control group and the infected groups. Based on these results, it is possible to conclude that the virus can be transmitted through artificial insemination with infected semen. Therefore, the venereal route is a potential route of infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Brazilian Agricultural Research Center, Embrapa Goats and Sheep, Brazil. Tel.: +55 88 9611 8072; fax: +55 88 3112 7455. E-mail address: kelma [email protected] (K.C.d. Souza). 0921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2012.07.031
Caprine arthritis–encephalitis (CAE) is an infectious disease caused by a virus from the Retroviridae family, Lentivirinae subfamily and Lentivirus genus, which infects goats at various stages of age development, regardless of
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sex and production system (Lara et al., 2005). The disease causes considerable economic losses in goat production, especially when breeders and matrices of high genetic value are affected, for they are removed from reproduction and often from a breeding programme. This may lead to loss of full potential of the animal concerning the transmission of their genetic characteristics for future generations. The main CAEV transmission route is through ingestion of colostrum or milk from infected females (Pisoni et al., 2007), although other routes are possible but not proven. This may include maternal–foetal and sexual transmission through semen (Andrioli et al., 2006; Ali Al Ahmad et al., 2008). Pinheiro et al. (2006) showed that the spread of CAEV among herds was mainly due to the introduction of infected bucks. In order to prove transmission through semen, the following steps are needed: detection of the etiologic agent in semen of infected animals; insemination of uninfected females with semen containing the virus, with subsequent confirmation of female infection through diagnostic tests (Peterson et al., 2008). The first step was proven by Andrioli et al. (1999), as they observed through the Nested polymerase chain reaction (Nested PCR) the presence of proviral CAEV DNA in semen of naturally infected goats. Nevertheless, bucks presented an intermittent of proviral CAEV DNA in semen (Andrioli et al., 2006; Paula et al., 2009; Peterson et al., 2008), attesting that the presence of the virus increases the possibility of transmission through sexual contact, which represents a potential risk of the disease spreading through natural mating (Rowe et al., 1991), and even more importantly, through artificial insemination (AI), when several females may be inseminated with a single ejaculate. In humans, transmission of human immunodeficiency virus (HIV) through semen depends on the stage of the disease, the patient nutritional or immune status and their association with other diseases (Alexander, 1990), as well as on virological factors such as infectious viral load. Moreover, the presence of mucosal inflammation and type of sexual relation interferes with the spread of HIV (Cohen, 2002). In goats, little is known about these factors. Moreover, this line of research needs to be strengthened for better control of chronic viral diseases. Therefore, the objective of the present study was to determine whether artificial insemination with contaminated semen could be a CAEV transmission route and to assess the influence of viral load in this probable transmission, and, finally, to verify whether the inflammatory process caused by the use of intravaginal sponges would facilitate virus entry in the female reproductive tract.
2.2. Experimental animals One six-year-old Anglo-Nubian buck and 30 undefined breed (UB) does aged between one and four years old were used. The animals were serologically evaluated before the experiment by Agarose Gel Immunodiffusion (AGID) and Western Blot (WB) tests. The tests showed no anti-CAEV antibodies, and thus, the animals were selected for their negative character. The experiment was conducted in accordance to the ethical principles for animal experimentation adopted by Brazilian College of Animal Experimentation (COBEA, 2008). 2.3. Viral inoculum The viral inoculum was prepared by concentration by dialysis (Plummer, 1978), using the standard CAEV-Cork1 virus strain. The titre was calculated by the method of Reed and Muench (1938) in order to obtain the infecting titres for high viral load (HVL) of 106 TCID50 /mL and low viral load (LVL) of 102 TCID50 /mL. 2.4. Semen extending Ejaculations of the seronegative buck for CAEV were collected through an artificial vagina coupled to a sterile tube (Falcon 15 mL), and then taken to the Semen Technology Laboratory at Embrapa Goats and Sheep to evaluate the volume, aspects, sperm motility and vitality. Semen that had spermatozoon in quality and number for fresh artificial insemination (120–500 × 106 spz/mL) was separated into three equal parts and then was extended in Minimum Essential Medium (MEM) with 2% foetal calf serum and glucose as energy source. One part did not contain CAEV, for the negative control (NC); in the second, the extended semen had 106 TCID50 /mL viral titre; and the third had 102 TCID50 /mL. Finally, the semen was put into 0.50 mL straws, sealed and placed in styrofoam with water at 37 ◦ C for fresh insemination. 2.5. Estrus synchronisation and artificial insemination with fixed time Before insemination, the females were subjected to hormonal synchronisation of estrus and then divided into two groups, G1 (n = 15) and G2 (n = 15). In G1, progesterone-releasing intravaginal sponges were used, with 60 mg of medroxyprogesterone acetate (MAP),2 which were inserted intravaginally. G2 received another method of progesterone application, by auricular subcutaneous implants, with 1.0 mg Norgestomet.3 This experiment aimed to verify whether the inflammatory process caused by sponges would facilitate virus entry in the female reproductive tract. Each protocol lasted 11 days, and day zero was considered the one on which the progesterone sources were introduced. On day 9, 50 g cloprostenol4 and 200 IU equine chorionic gonadotropin5 (eCG) intra-muscular applications were made, and on day 11, the progesterone source was removed. After removing the progesterone sources, signs of estrus in females were observed every 12 h, when they were exposed to the presence of the buck, but with no physical contact. Females from G1 (n = 15) treated with intravaginal sponges were divided into three subgroups (G1.1–1.3) with five animals each. The females in G2 group were divided similarly and they were synchronised with auricular subcutaneous implants (subgroups G2.1–2.3) (Table 1). All the females were inseminated transcervically on at a fixed time at 32 h and 48 h after removing the progesterone sources, including those which showed no signs of estrus. Females from subgroups G1.1 and G2.1 were inseminated with non-contaminated semen, corresponding to negative control (NC); subgroups G1.2 and G2.2 received contaminated semen with low viral load, becoming the group LVL, and subgroups G1.3 and G2.3 received contaminated semen with high viral load (HVL) (Table 1). At the time of AIs, the females were properly restrained and the cervix was located by using a vaginal speculum (duck-billed) and a light source and semen was applied with a 0.5 mL semen applicator as deeply as
2. Materials and methods 2.1. Location and period The study was conducted from October 2008 to October 2009 at Embrapa Goats and Sheep, located in Sobral, in the north of Ceará state, Brazil. The region lies at about 111 m altitude, 3◦ 45 0.5 latitude south and 40◦ 20 45.8 longitude west.
1 Laboratoire Associé de Recherches sur les Petits Ruminants, INRA, ENVL, France. 2 Progeston, SYNTEX S.A., Argentina. 3 Crestar, Intervet, Holanda. 4 Ciosin, Coopers, Brazil. 5 Novormon, SYNTEX S.A., Argentina.
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Table 1 Experimental groups and subgroups of goats subjected to hormonal synchronisation of estrus and artificial insemination with CAEV contaminated semen. Subgroups
Progesterone source
Negative control (virus-free) Low viral load titre (102 TCID50 /mL) High viral load titre (106 TCID50 /mL) * **
Intravaginal sponges* (G 1)
Auricular implants** (G2)
G1.1 (n = 5) G1.2 (n = 5) G1.3 (n = 5)
G2.1 (n = 5) G2.2 (n = 5) G2.3 (n = 5)
Intravaginal sponges = Progeston, SYNTEX S.A., Argentina. Auricular implants = Crestar, Intervet, Holanda.
possible. At this moment, the presence or absence of mucosal inflammation in the cervical region could be observed, characterised by redness and/or presence of foul smelling mucus, and the occurrence of bleeding during inseminations. Each female was inseminated with 1.0 mL semen (2 mL × 0.5 mL straw), in order to receive the previously established viral titre and the usually recommended method for AI.
12 months after insemination. The antigen used was produced in the Virology Laboratory at Embrapa Goats and Sheep, containing the p28 protein of CAEV (Pinheiro et al., 2006). The concentration obtained in the antigen was 4.25 mg/mL by the method of Bradford (1976). To perform WB, the methodology by Pinheiro et al. (2001) later adapted by Rodrigues et al. (2009) was used and AGID as reported by Gouveia et al. (2000).
2.6. Handling after artificial insemination and blood samples 2.8. Statistical analyses After inseminations, the negative control, LVL and HVL groups were kept in separate paddocks and isolated from each other by a double fence, keeping a distance of 1.5 m among paddocks. This distance was essential to prevent any physical contact among experimental animals from different experimental groups and with other near-by animals. However, horizontal transmission was not prevented within the groups due to technical limitations and because it would be very stressful for the animals to be kept isolated for a long time. Furthermore, each group was kept in an extensive system so that horizontal transmission was unlikely. Finally, the objective of the study was not to quantify how many females would be infected by semen, but whether this would be an infection route. Throughout the experiment, the animals were followed by WB diagnostic testing and all preventive measures were adopted to prevent transmission among the experimental groups, such as: each group had a management kit (thermometer, scissors, tweezers, stethoscope, etc.). Handling always started in the control group followed by LVL, and HVL. Needles for blood samples were disposable and no other animal was introduced in the flocks. Pregnancy was diagnosed 60 days after insemination by ultrasound examinations with Biosound-Esoat® equipment, Falco model, using a 5.0 MHz transrectal transducer. Blood samples were taken monthly up to four months after inseminations and, thereafter, every 60 days until 12 months after insemination. Samples were obtained by puncturing the jugular vein using the vacutainer® system, with 10 mL tubes without anticoagulant. The samples were centrifuged at 1500 g for 15 min to separate serum. These were stored in eppendorf® tubes labelled in duplicate and stored in a freezer at −20 ◦ C and then tested. 2.7. Serological test Agar Gel Immunodiffusion (AGID) and Western Blot (WB) were the diagnostic tests used in this research. They were conducted monthly until four months after insemination in order to detect the closest moment of seroconversion. Hereafter, the test was performed every 60 days until
Statistical analyses were performed by the Chi-square (2 ) test with established value of (P < 0.05) and the Yates correction, using the EPI-INFO version 6.0 statistical programme.
3. Results Reproductive parameters were compared between the control/seronegative and inoculated/seropositive groups. Pregnancy rates were 10% (1/10) in the control group and 35% (7/20) in the inoculated groups. Kidding rates were 10% (1/10) for the control group and 25% (5/20) for the seropositive groups. However, there were two abortions around 70 days of pregnancy in the seropositive group, emphasising that in one of these miscarriages, there were three foetuses, which probably contributed to the incident. No significant difference was observed between groups concerning any of the parameters studied (P ≥ 0.05). The experimental infection was confirmed 30 days after insemination with CAEV-Cork contaminated semen, when seroconversion was observed in 12 (60%) out of 20 inseminated goats with contaminated semen, of which ten (100%) belonged to the HVL group and two (20%) to the LVL group (Table 2). Sixty days after insemination, all the goats from both groups which had received contaminated semen presented anti-CAEV antibodies in WB, and remained so until 360 days after AIs. There was no statistical difference (P > 0.05) between groups, although seroconversion was detected earlier in the group inseminated with titre of 106 TCID50 /mL (HVL group). No goat from the control
Table 2 Percentage of female goats seropositive to CAEV in both intravaginal sponges or auricular implants for estrus synchronisation, with different viral loads, from 30 to 60 days after AI. Group
30 days
60 days *
HVL LVL
**
Intravaginal sponges
Auricular implants
Total
Intravaginal sponges*
Auricular implants**
Total
5/5 (100%) 0/5 (0%)
5/5 (100%) 2/5 (40%)
10/10 (100%) 2/10 (20%)
5/5 (100%) 5/5 (100%)
5/5 (100%) 5/5 (100%)
10/10 (100%) 10/10 (100%)
HVL = high viral load titre 106 TCID50 /mL. LVL = low viral load titre 102 TCID50 /mL. * Intravaginal sponges = Progeston, SYNTEX S.A., Argentina. ** Auricular implants = Crestar, Intervet, Holanda.
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Fig. 1. Western Blot results of female goats inseminated with CAEV contaminated semen using different viral loads at 60 days after insemination (p28, protein band; C+, positive control; C−, negative control; S+, positive sample; S−, negative sample).
group showed antibodies to CAEV in testing throughout the 12 months after insemination. Fig. 1 shows the results of WB. In this study, the method of estrus synchronising did not influence the virus transmission (Table 2) and genital tract inflammation was not observed at the time of AI in any of the females studied. Regarding the reproductive characteristics of the 30 females undergoing treatment for estrus synchronisation, in G1, 11 (73.3%) showed estrus between 24 and 48 h after removing the progesterone sources and in G2, 12 (80%) showed estrus, and three (20%) did not present any sign. The estrus percentage showed the efficiency of the two procedures. Pregnancy was diagnosed after 60 days, when all goats inseminated with contaminated semen had already seroconverted. 4. Discussion Evidence of CAEV transmission through AI corroborates studies by Andrioli et al. (1999, 2006), Cruz et al. (2009), Paula et al. (2009) and Ricarte et al. (2010), who suggested that the semen from infected goats contained the virus and thus could transmit CAEV. Likewise, Andrioli et al. (2006) emphasised the risk of spreading the infection through artificial insemination, which was found in this study, as it proved CAEV transmission by experimentally infected semen, and also that a single ejaculate can spread the virus in up to 100% of the artificially inseminated females. Rowe et al. (1991) observed that some goats seroconverted between 100 and 140 days after being subjected to natural mating with seropositive males and suspected that the transmission occurred through copulation. The authors did not confirm this route, probably because the females had direct contact with the infected male. Unlike the approach described above, in the present study the contaminated semen was introduced directly into the female reproductive tract through AI, as suggested by Peterson et al. (2008), so there was no contact between female goats and the buck, which was proven seronegative for CAEV in order to provide more safety for the study. Thus, sexual
CAEV transmission reinforces the importance of buck reproductive health and the need to test them regularly before using as semen donors in artificial insemination programmes and genetic selection (Ali Al Ahmad et al., 2008). Other lentiviruses, also responsible for infections in cattle, cats, primates and humans, have also been detected in semen, presenting potential transmission (Jordan et al., 1995; Mermin et al., 1991; Nash et al., 1995). The feline immunodeficiency virus (FIV) had sexual transmission proven when Holly et al. (1998) inseminated six females with fresh semen from males infected by FIV and three of them seroconverted. In the case of human lentivirus, the sexual route is the most responsible for the spread of human immunodeficiency virus (HIV), which is responsible for AIDS. In Africa, for example, 60% of new infections are in women who have had sexual relations with men positive for HIV (Swaminathan, 2009). Studies on the influence of viral load in CAEV transmission, especially through sexual contact, are still incipient. In the present experiment, even though all the goats were contaminated regardless of the viral load, the ones which received the highest titre seroconverted earlier. There are no data in the literature on quantities of potentially harmful viral loads which are present in semen or that are capable of transmitting infection. Some studies demonstrate factors which may influence CAEV presence in semen, such as stress caused by increased sexual activity in the breeding season and during times of high environmental temperature (Andrioli et al., 2006; Paula et al., 2009; Peterson et al., 2008). Andrioli et al. (2006) found that, in males infected with CAEV, the percentage of positive semen by PCR before and after testicular injury was 21.4% and 50%, respectively, demonstrating that the inflammatory process increases the flow of defense cells and increases the viral load. Some authors evaluated the profile of anti-CAEV antibodies in blood by AGID and proviral DNA of CAEV in blood and semen by PCR, from infected bucks. They found no correlation between them (Ali Al Ahmad et al., 2008; Paula et al., 2009), and that in both blood and semen the presence of the CAEV was intermittent (Andrioli et al., 2006; Paula et al., 2009). In addition, Paula et al. (2009) found that CAEV
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was detected in semen (PCR) from experimentally infected bucks before the diagnosis by AGID, demonstrating that males with recent infection represent important infection sources and that the absence of virus in the bloodstream did not exclude the excretion of the pathogenic agent in semen, or even a possible transmission. The use of intravaginal sponges to synchronise estrus in goats may cause inflammation, although in the present study there was no inflammation in the vaginal canal of the goats at insemination. What may have contributed to the infection of inseminated females was the fact that the monocyte and macrophage population (which are target cells) responsible for protecting the vagina are similar to those found in the mucosal surfaces exposed to environmental antigens, such as found in the gastrointestinal tract and upper respiratory tract (Hafez and Hafez, 2004). The latter are proven to be infection sources for CAEV with the human lentivirus. Lazzarin et al. (1991) suggested that the use of intrauterine devices was potentially associated with a higher possibility of HIV sexual transmission in women. The fertility data in the present experiment were low, when compared to other studies with fresh semen, perhaps because the insemination had fixed time with hormonally synchronised estrus, since the literature is somewhat conflicted about the issue, and Machado and Simplicio (2001) showed that inseminations performed earlier or later led to reduction in fertility rate. 5. Conclusions As a result of this study, it can be concluded that CAEV transmission by experimentally infected semen is a distinct possibility. The two viral loads used in this experiment were able to infect females, and seroconversion tended to be faster in goats which received the highest viral loads. The CAEV transmission was not influenced by the treatment method of estrus synchronisation and ovulation induction. MEM with 2% foetal calf serum and glucose can be used as goat semen extender as well as for semen contamination in experiments on pathogen transmission. CAEV probably has no negative influence on the fertility of females, at least in early infection. Acknowledgements The authors thank the Ceará Foundation for Support to Scientific and Technological Development – FUNCAP, Banco do Nordeste – BNB, State Government of Ceara – for financial support and the Brazilian Centre for Goats and Sheep Research (Centro Nacional de Pesquisa de Caprinos e Ovinos – EMBRAPA-CNPC) for use of the structure and technical and financial support. The Vale do Acaraú University. References Alexander, N.J., 1990. Sexual transmission of human immunodeficiency virus: virus entry into the male and female genital tract. Fertil. Steril. (54), 1–18.
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Ali Al Ahmad, M.Z., Fieni, F., Pellerin, J.L., Guiguen, F., Cherel, Y., Chatagnon, G., Bouzar, A.B., Chebloune, Y., 2008. Detection of viral genomes of caprine arthritis–encephalitis virus (CAEV) in semen and in genital tract tissues of male goat. Theriogenology (69), 473–480. Andrioli, A., Gouveia, A.M.G., Pinheiro, R.R., Rocha, M.A., Martins, A., Santos, D.O., 1999. Detecc¸ão do DNA pro-viral do lentivírus caprino em sêmen de bodes naturalmente infectados (Detection of caprine lentivirus proviral DNA in semen of naturally infected bucks). Rev. Bras. Rep. Anim. (23), 420–421. Andrioli, A., Gouveia, A.M.G., Martins, A.S., Pinheiro, R.R., Santos, D.O., 2006. Fatores de risco na transmissão do lentivírus caprino pelo sêmen (Risk factors in caprine lentivirus transmission through semen). Pesq. Agropec. Bras. (41), 1313–1319. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (72), 248–254. COBEA – Colégio Brasileiro de Experimentac¸ão Animal. Available at: http://www.cobea.org.br/index.php (accessed 08.09.08). Cohen, M.S., 2002. HAART and prevention of HIV transmission. Conference reports. Medscape HIV/AIDS, Medscape. Available at: http://www.medscape.com/viewarticle/437545 (accessed 27.01.10). Cruz, J.C.M., Gouveia, A.M.G., Souza, K.C., Braz, G.F., Teixeira, B.M., Heinemann, M.B., Leite, R.C., Reis, J.K.P., Pinheiro, R.R., Andrioli, A., 2009. Caprine arthritis–encephalitis virus (CAEV) detection in semen of endangered goat breeds by nested polymerase chain reaction. Small Rum. Res. (85), 149–152. Gouveia, A.M.G., Melo, L.M., Pires, L.L., Pinheiro, R.R., 2000. Microimunodifusão em gel de ágar para o diagnóstico sorológico de infecc¸ão por lentivírus de pequenos ruminantes (Agar gel microimmunodiffusion for sorologic diagnosis of small ruminants lentivirus infection). Congresso Brasileiro de Med. Veterinária (27), 33. Hafez, B., Hafez, E.S.E., 2004. Reproduc¸ão Animal, seventh ed. Manole, São Paulo. Holly, L., Gayle Howard, Jordan J.O., Bucci, Joseph G., Butterworth, Jennifer L., English, Robert, Suzanne, Kennedy-Stoskopf, Tompkins, Mary B., Tompkins, Wayne A., 1998. Horizontal transmission of feline immunodeficiency virus with semen from seropositive cats. J. Reprod. Immunol. (41), 341–357. Jordan, H.L., Howard, J., Tompkins, W.A., Kennedystoskopf, S., 1995. Detection of feline immunodeficiency virus in semen from seropositive domestic cats (Felis catus). J. Virol. (69), 7328–7333. Lara, M.C.C.S.H., Birgel Junior, E.H., Birgel, E.H., 2005. Possibility of vertical transmission of Caprine Arthritis–Encephalitis Virus in neonate kids. Arq. Bras. Med. Vet. Zootec. (57), 553–555. Lazzarin, A., Saracco, A., Musicco, A., Nicolosi, A., 1991. Man-to-woman sexual transmission of the human immunodeficiency virus. Risk factors related to sexual behavior, man’s infectiousness, and woman’s susceptibility. Italian Study Group on HIV Heterosexual Transmission. Arch. Intern. Med. (151), 2411–2416. Machado, R., Simplicio, A.A., 2001. Avaliac¸ão de programas hormonais para a induc¸ão e sincronizac¸ão do estro em caprinos. Pesq. agropec. Bras. (36), 171–178. Mermin, J.H., Holodniy, M., Katzenstein, D.A., Merigan, T.C., 1991. Detection of human immunodeficiency virus DNA and RNA in semen by the polymerase chain reaction. J. Infect. Dis. (164), 769–772. Nash, J.W., Hanson, L.A., Coats, K.C., 1995. Bovine immunodeficiency virus in stud bull semen. Am. J. Vet. Res. (56), 760–763. Paula, N.R.O., Andrioli, A., Cardoso, J.F.S., Pinheiro, R.R., Sousa, F.M.L., Souza, K.C., Alves, F.S.F., Campello, C.C., Ricarte, A.R.F., Teixeira, M.F.S., 2009. Profile of the Caprine arthritis–encephalitis virus (CAEV) in blood, semen from bucks naturally and experimentally infected in the semiarid region of Brazil. Small Rum. Res. (85), 27–33. Peterson, K., Brinkhof, J., Houwers, D., Colenbrander, B., Gadella, B., 2008. Presence of pro-lentiviral DNA in male sexual organs and ejaculates of small ruminants. Theriogenology (69), 433–442. Pinheiro, R.R., Gouvea, A.M.G., Alves, F.S.F., 2001. Prevalência da infecc¸ão pelo vírus da artrite encefalite no Estado do Ceará. Brasil. Ciênc. Rural (31), 449–454. Pinheiro, R.R., Olortegui, C.D.C., Gouvea, A.M.G., Araujo, S.C., Andrioli, A., 2006. Desenvolvimento de dot-blot para detecc¸ão de anticorpos para o vírus da Artrite-Encefalite Caprina em caprinos. Rev. Port. Ciên. Vet. (101), 51–56. Pisoni, G., Moroni, P., Turin, L., Bertoni, G., 2007. Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats. Virology (369), 119–130. Plummer, D.J., 1978. An Introduction to Pratical Biochemistry, second ed. McGraw-Hill, London. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. (27), 493–497.
198
K.C.d. Souza et al. / Small Ruminant Research 109 (2013) 193–198
Ricarte, A.R.F., Andrioli, A., Pinheiro, R.R., Báo, S.N., Silva, J.S., Braz, S.V., Name, K.P.O., Lima-Verde, I.B., Brito, I.F., Dias, R.P., Freitas Aguiar, T.D., Dantas, T.V.M., Araújo, S.A.C., Cavalcanti, D.M.L.P., Paula, N.R.O., Teixeira, M.F.S., 2010. Avaliac¸ão imunohistoquímica e ultraestrutural de gametas e embriões caprinos infectados com o CAEV (Immunohistochemical and ultrastructural evaluation of gametes and embryos of goats infected with CAEV). Arq. Inst. Biol. (77), 217–223. Rodrigues, A.S., Brito, R.L.L., Santos, V.W.S., Dias, R.P., Brito, I.F., Anrioli, A., Pinheiro, R.R., 2009. Comparac¸ão de dois testes sorológicos na
evoluc¸ão natural de caprinos leiteiros com o vírus da Artrite-Encefalite Caprina-dados preliminares. Sincorte. João Pessoa (4). Rowe, J.D., Esast, N.E., Franti, C.E., Thurmond, M.C., Pederson, N.C., Theilen, G.H., 1991. Risk factores associated with the incidence of seroconversion to caprine arthritis–encephalitis virus in goats on California dairies. Am. J. Vet. Res. (53), 2396–2430. Swaminathan, N. Sêmen torna o HIV mais potente. Available at: www2.uol.com.br/./semen torna o hiv mais potente.html Accessed: 21 Dez 2009.
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Transmission of the caprine arthritis–encephalitis virus through artificial insemination Kelma Costa de Souza a,b,∗ , Raymundo Rizaldo Pinheiro a , Diônes Oliveira Santos a , Roberta Lomonte Lemos de Brito a , Apoliana de Souza Rodrigues a , Lúcia Helena Sider a , Ney Rômulo Oliveira Paula c , Amanda Aragão Avila b , Janaina de Fátima Saraiva Cardoso c , Alice Andrioli a a b c
Brazilian Agricultural Research Center, Embrapa Goats and Sheep, Brazil Vale Acaraú University, Brazil Federal University of Piauí, Brazil
a r t i c l e
i n f o
Article history: Received 27 August 2010 Received in revised form 20 July 2012 Accepted 31 July 2012 Available online 13 September 2012 Keywords: CAEV Goats Viral load Artificial insemination Transmission
a b s t r a c t The objective of present study was to evaluate the transmissibility of the caprine arthritis–encephalitis virus (CAEV) through artificial insemination (AI), and to assess the influence of viral load on this probable transmission. It also aims to verify whether the inflammatory process caused by the use of intravaginal sponges would facilitate virus entry in the female reproductive tract. For this purpose, 30 undefined breed goats were used, all serologically negative for CAEV. One Anglo-Nubian buck, also seronegative, was used to artificially inseminate females in this study. His semen was contaminated with the standard CAEV-Cork virus strain, with two distinct infective titres, one 106 TCID50 /mL, for high viral load (HVL), and another of 102 TCID50 /mL, for low viral load (LVL). Females had estrus synchronised by using two protocols, intravaginal sponges in Group 1 (G1, N = 15) and auricular subcutaneous implants in Group 2 (G2, N = 15). For inseminations, the goats were divided into three groups of 10 animals each. One group was inseminated with HVL, another with LVL and the third with semen from the same virus-free buck, as a negative control. The experiment was conducted in accordance to the ethical principles for animal experimentation. Statistical analyses were performed by the chi-square test (P < 0.05). Thirty days after insemination, the experimental infection was confirmed, when 12 out of the 20 (60%) inseminated goats had seroconverted. Sixty days after insemination, all females from the HVL and LVL groups presented anti-CAEV antibodies. There was no statistical difference (P > 0.05) among groups regarding viral loads nor between the two estrus synchronisation protocols. Goats from the control group remained seronegative throughout the experiment (12 months). Concerning reproductive parameters, no difference was found between the control group and the infected groups. Based on these results, it is possible to conclude that the virus can be transmitted through artificial insemination with infected semen. Therefore, the venereal route is a potential route of infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Brazilian Agricultural Research Center, Embrapa Goats and Sheep, Brazil. Tel.: +55 88 9611 8072; fax: +55 88 3112 7455. E-mail address: kelma [email protected] (K.C.d. Souza). 0921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.smallrumres.2012.07.031
Caprine arthritis–encephalitis (CAE) is an infectious disease caused by a virus from the Retroviridae family, Lentivirinae subfamily and Lentivirus genus, which infects goats at various stages of age development, regardless of
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sex and production system (Lara et al., 2005). The disease causes considerable economic losses in goat production, especially when breeders and matrices of high genetic value are affected, for they are removed from reproduction and often from a breeding programme. This may lead to loss of full potential of the animal concerning the transmission of their genetic characteristics for future generations. The main CAEV transmission route is through ingestion of colostrum or milk from infected females (Pisoni et al., 2007), although other routes are possible but not proven. This may include maternal–foetal and sexual transmission through semen (Andrioli et al., 2006; Ali Al Ahmad et al., 2008). Pinheiro et al. (2006) showed that the spread of CAEV among herds was mainly due to the introduction of infected bucks. In order to prove transmission through semen, the following steps are needed: detection of the etiologic agent in semen of infected animals; insemination of uninfected females with semen containing the virus, with subsequent confirmation of female infection through diagnostic tests (Peterson et al., 2008). The first step was proven by Andrioli et al. (1999), as they observed through the Nested polymerase chain reaction (Nested PCR) the presence of proviral CAEV DNA in semen of naturally infected goats. Nevertheless, bucks presented an intermittent of proviral CAEV DNA in semen (Andrioli et al., 2006; Paula et al., 2009; Peterson et al., 2008), attesting that the presence of the virus increases the possibility of transmission through sexual contact, which represents a potential risk of the disease spreading through natural mating (Rowe et al., 1991), and even more importantly, through artificial insemination (AI), when several females may be inseminated with a single ejaculate. In humans, transmission of human immunodeficiency virus (HIV) through semen depends on the stage of the disease, the patient nutritional or immune status and their association with other diseases (Alexander, 1990), as well as on virological factors such as infectious viral load. Moreover, the presence of mucosal inflammation and type of sexual relation interferes with the spread of HIV (Cohen, 2002). In goats, little is known about these factors. Moreover, this line of research needs to be strengthened for better control of chronic viral diseases. Therefore, the objective of the present study was to determine whether artificial insemination with contaminated semen could be a CAEV transmission route and to assess the influence of viral load in this probable transmission, and, finally, to verify whether the inflammatory process caused by the use of intravaginal sponges would facilitate virus entry in the female reproductive tract.
2.2. Experimental animals One six-year-old Anglo-Nubian buck and 30 undefined breed (UB) does aged between one and four years old were used. The animals were serologically evaluated before the experiment by Agarose Gel Immunodiffusion (AGID) and Western Blot (WB) tests. The tests showed no anti-CAEV antibodies, and thus, the animals were selected for their negative character. The experiment was conducted in accordance to the ethical principles for animal experimentation adopted by Brazilian College of Animal Experimentation (COBEA, 2008). 2.3. Viral inoculum The viral inoculum was prepared by concentration by dialysis (Plummer, 1978), using the standard CAEV-Cork1 virus strain. The titre was calculated by the method of Reed and Muench (1938) in order to obtain the infecting titres for high viral load (HVL) of 106 TCID50 /mL and low viral load (LVL) of 102 TCID50 /mL. 2.4. Semen extending Ejaculations of the seronegative buck for CAEV were collected through an artificial vagina coupled to a sterile tube (Falcon 15 mL), and then taken to the Semen Technology Laboratory at Embrapa Goats and Sheep to evaluate the volume, aspects, sperm motility and vitality. Semen that had spermatozoon in quality and number for fresh artificial insemination (120–500 × 106 spz/mL) was separated into three equal parts and then was extended in Minimum Essential Medium (MEM) with 2% foetal calf serum and glucose as energy source. One part did not contain CAEV, for the negative control (NC); in the second, the extended semen had 106 TCID50 /mL viral titre; and the third had 102 TCID50 /mL. Finally, the semen was put into 0.50 mL straws, sealed and placed in styrofoam with water at 37 ◦ C for fresh insemination. 2.5. Estrus synchronisation and artificial insemination with fixed time Before insemination, the females were subjected to hormonal synchronisation of estrus and then divided into two groups, G1 (n = 15) and G2 (n = 15). In G1, progesterone-releasing intravaginal sponges were used, with 60 mg of medroxyprogesterone acetate (MAP),2 which were inserted intravaginally. G2 received another method of progesterone application, by auricular subcutaneous implants, with 1.0 mg Norgestomet.3 This experiment aimed to verify whether the inflammatory process caused by sponges would facilitate virus entry in the female reproductive tract. Each protocol lasted 11 days, and day zero was considered the one on which the progesterone sources were introduced. On day 9, 50 g cloprostenol4 and 200 IU equine chorionic gonadotropin5 (eCG) intra-muscular applications were made, and on day 11, the progesterone source was removed. After removing the progesterone sources, signs of estrus in females were observed every 12 h, when they were exposed to the presence of the buck, but with no physical contact. Females from G1 (n = 15) treated with intravaginal sponges were divided into three subgroups (G1.1–1.3) with five animals each. The females in G2 group were divided similarly and they were synchronised with auricular subcutaneous implants (subgroups G2.1–2.3) (Table 1). All the females were inseminated transcervically on at a fixed time at 32 h and 48 h after removing the progesterone sources, including those which showed no signs of estrus. Females from subgroups G1.1 and G2.1 were inseminated with non-contaminated semen, corresponding to negative control (NC); subgroups G1.2 and G2.2 received contaminated semen with low viral load, becoming the group LVL, and subgroups G1.3 and G2.3 received contaminated semen with high viral load (HVL) (Table 1). At the time of AIs, the females were properly restrained and the cervix was located by using a vaginal speculum (duck-billed) and a light source and semen was applied with a 0.5 mL semen applicator as deeply as
2. Materials and methods 2.1. Location and period The study was conducted from October 2008 to October 2009 at Embrapa Goats and Sheep, located in Sobral, in the north of Ceará state, Brazil. The region lies at about 111 m altitude, 3◦ 45 0.5 latitude south and 40◦ 20 45.8 longitude west.
1 Laboratoire Associé de Recherches sur les Petits Ruminants, INRA, ENVL, France. 2 Progeston, SYNTEX S.A., Argentina. 3 Crestar, Intervet, Holanda. 4 Ciosin, Coopers, Brazil. 5 Novormon, SYNTEX S.A., Argentina.
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Table 1 Experimental groups and subgroups of goats subjected to hormonal synchronisation of estrus and artificial insemination with CAEV contaminated semen. Subgroups
Progesterone source
Negative control (virus-free) Low viral load titre (102 TCID50 /mL) High viral load titre (106 TCID50 /mL) * **
Intravaginal sponges* (G 1)
Auricular implants** (G2)
G1.1 (n = 5) G1.2 (n = 5) G1.3 (n = 5)
G2.1 (n = 5) G2.2 (n = 5) G2.3 (n = 5)
Intravaginal sponges = Progeston, SYNTEX S.A., Argentina. Auricular implants = Crestar, Intervet, Holanda.
possible. At this moment, the presence or absence of mucosal inflammation in the cervical region could be observed, characterised by redness and/or presence of foul smelling mucus, and the occurrence of bleeding during inseminations. Each female was inseminated with 1.0 mL semen (2 mL × 0.5 mL straw), in order to receive the previously established viral titre and the usually recommended method for AI.
12 months after insemination. The antigen used was produced in the Virology Laboratory at Embrapa Goats and Sheep, containing the p28 protein of CAEV (Pinheiro et al., 2006). The concentration obtained in the antigen was 4.25 mg/mL by the method of Bradford (1976). To perform WB, the methodology by Pinheiro et al. (2001) later adapted by Rodrigues et al. (2009) was used and AGID as reported by Gouveia et al. (2000).
2.6. Handling after artificial insemination and blood samples 2.8. Statistical analyses After inseminations, the negative control, LVL and HVL groups were kept in separate paddocks and isolated from each other by a double fence, keeping a distance of 1.5 m among paddocks. This distance was essential to prevent any physical contact among experimental animals from different experimental groups and with other near-by animals. However, horizontal transmission was not prevented within the groups due to technical limitations and because it would be very stressful for the animals to be kept isolated for a long time. Furthermore, each group was kept in an extensive system so that horizontal transmission was unlikely. Finally, the objective of the study was not to quantify how many females would be infected by semen, but whether this would be an infection route. Throughout the experiment, the animals were followed by WB diagnostic testing and all preventive measures were adopted to prevent transmission among the experimental groups, such as: each group had a management kit (thermometer, scissors, tweezers, stethoscope, etc.). Handling always started in the control group followed by LVL, and HVL. Needles for blood samples were disposable and no other animal was introduced in the flocks. Pregnancy was diagnosed 60 days after insemination by ultrasound examinations with Biosound-Esoat® equipment, Falco model, using a 5.0 MHz transrectal transducer. Blood samples were taken monthly up to four months after inseminations and, thereafter, every 60 days until 12 months after insemination. Samples were obtained by puncturing the jugular vein using the vacutainer® system, with 10 mL tubes without anticoagulant. The samples were centrifuged at 1500 g for 15 min to separate serum. These were stored in eppendorf® tubes labelled in duplicate and stored in a freezer at −20 ◦ C and then tested. 2.7. Serological test Agar Gel Immunodiffusion (AGID) and Western Blot (WB) were the diagnostic tests used in this research. They were conducted monthly until four months after insemination in order to detect the closest moment of seroconversion. Hereafter, the test was performed every 60 days until
Statistical analyses were performed by the Chi-square (2 ) test with established value of (P < 0.05) and the Yates correction, using the EPI-INFO version 6.0 statistical programme.
3. Results Reproductive parameters were compared between the control/seronegative and inoculated/seropositive groups. Pregnancy rates were 10% (1/10) in the control group and 35% (7/20) in the inoculated groups. Kidding rates were 10% (1/10) for the control group and 25% (5/20) for the seropositive groups. However, there were two abortions around 70 days of pregnancy in the seropositive group, emphasising that in one of these miscarriages, there were three foetuses, which probably contributed to the incident. No significant difference was observed between groups concerning any of the parameters studied (P ≥ 0.05). The experimental infection was confirmed 30 days after insemination with CAEV-Cork contaminated semen, when seroconversion was observed in 12 (60%) out of 20 inseminated goats with contaminated semen, of which ten (100%) belonged to the HVL group and two (20%) to the LVL group (Table 2). Sixty days after insemination, all the goats from both groups which had received contaminated semen presented anti-CAEV antibodies in WB, and remained so until 360 days after AIs. There was no statistical difference (P > 0.05) between groups, although seroconversion was detected earlier in the group inseminated with titre of 106 TCID50 /mL (HVL group). No goat from the control
Table 2 Percentage of female goats seropositive to CAEV in both intravaginal sponges or auricular implants for estrus synchronisation, with different viral loads, from 30 to 60 days after AI. Group
30 days
60 days *
HVL LVL
**
Intravaginal sponges
Auricular implants
Total
Intravaginal sponges*
Auricular implants**
Total
5/5 (100%) 0/5 (0%)
5/5 (100%) 2/5 (40%)
10/10 (100%) 2/10 (20%)
5/5 (100%) 5/5 (100%)
5/5 (100%) 5/5 (100%)
10/10 (100%) 10/10 (100%)
HVL = high viral load titre 106 TCID50 /mL. LVL = low viral load titre 102 TCID50 /mL. * Intravaginal sponges = Progeston, SYNTEX S.A., Argentina. ** Auricular implants = Crestar, Intervet, Holanda.
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Fig. 1. Western Blot results of female goats inseminated with CAEV contaminated semen using different viral loads at 60 days after insemination (p28, protein band; C+, positive control; C−, negative control; S+, positive sample; S−, negative sample).
group showed antibodies to CAEV in testing throughout the 12 months after insemination. Fig. 1 shows the results of WB. In this study, the method of estrus synchronising did not influence the virus transmission (Table 2) and genital tract inflammation was not observed at the time of AI in any of the females studied. Regarding the reproductive characteristics of the 30 females undergoing treatment for estrus synchronisation, in G1, 11 (73.3%) showed estrus between 24 and 48 h after removing the progesterone sources and in G2, 12 (80%) showed estrus, and three (20%) did not present any sign. The estrus percentage showed the efficiency of the two procedures. Pregnancy was diagnosed after 60 days, when all goats inseminated with contaminated semen had already seroconverted. 4. Discussion Evidence of CAEV transmission through AI corroborates studies by Andrioli et al. (1999, 2006), Cruz et al. (2009), Paula et al. (2009) and Ricarte et al. (2010), who suggested that the semen from infected goats contained the virus and thus could transmit CAEV. Likewise, Andrioli et al. (2006) emphasised the risk of spreading the infection through artificial insemination, which was found in this study, as it proved CAEV transmission by experimentally infected semen, and also that a single ejaculate can spread the virus in up to 100% of the artificially inseminated females. Rowe et al. (1991) observed that some goats seroconverted between 100 and 140 days after being subjected to natural mating with seropositive males and suspected that the transmission occurred through copulation. The authors did not confirm this route, probably because the females had direct contact with the infected male. Unlike the approach described above, in the present study the contaminated semen was introduced directly into the female reproductive tract through AI, as suggested by Peterson et al. (2008), so there was no contact between female goats and the buck, which was proven seronegative for CAEV in order to provide more safety for the study. Thus, sexual
CAEV transmission reinforces the importance of buck reproductive health and the need to test them regularly before using as semen donors in artificial insemination programmes and genetic selection (Ali Al Ahmad et al., 2008). Other lentiviruses, also responsible for infections in cattle, cats, primates and humans, have also been detected in semen, presenting potential transmission (Jordan et al., 1995; Mermin et al., 1991; Nash et al., 1995). The feline immunodeficiency virus (FIV) had sexual transmission proven when Holly et al. (1998) inseminated six females with fresh semen from males infected by FIV and three of them seroconverted. In the case of human lentivirus, the sexual route is the most responsible for the spread of human immunodeficiency virus (HIV), which is responsible for AIDS. In Africa, for example, 60% of new infections are in women who have had sexual relations with men positive for HIV (Swaminathan, 2009). Studies on the influence of viral load in CAEV transmission, especially through sexual contact, are still incipient. In the present experiment, even though all the goats were contaminated regardless of the viral load, the ones which received the highest titre seroconverted earlier. There are no data in the literature on quantities of potentially harmful viral loads which are present in semen or that are capable of transmitting infection. Some studies demonstrate factors which may influence CAEV presence in semen, such as stress caused by increased sexual activity in the breeding season and during times of high environmental temperature (Andrioli et al., 2006; Paula et al., 2009; Peterson et al., 2008). Andrioli et al. (2006) found that, in males infected with CAEV, the percentage of positive semen by PCR before and after testicular injury was 21.4% and 50%, respectively, demonstrating that the inflammatory process increases the flow of defense cells and increases the viral load. Some authors evaluated the profile of anti-CAEV antibodies in blood by AGID and proviral DNA of CAEV in blood and semen by PCR, from infected bucks. They found no correlation between them (Ali Al Ahmad et al., 2008; Paula et al., 2009), and that in both blood and semen the presence of the CAEV was intermittent (Andrioli et al., 2006; Paula et al., 2009). In addition, Paula et al. (2009) found that CAEV
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was detected in semen (PCR) from experimentally infected bucks before the diagnosis by AGID, demonstrating that males with recent infection represent important infection sources and that the absence of virus in the bloodstream did not exclude the excretion of the pathogenic agent in semen, or even a possible transmission. The use of intravaginal sponges to synchronise estrus in goats may cause inflammation, although in the present study there was no inflammation in the vaginal canal of the goats at insemination. What may have contributed to the infection of inseminated females was the fact that the monocyte and macrophage population (which are target cells) responsible for protecting the vagina are similar to those found in the mucosal surfaces exposed to environmental antigens, such as found in the gastrointestinal tract and upper respiratory tract (Hafez and Hafez, 2004). The latter are proven to be infection sources for CAEV with the human lentivirus. Lazzarin et al. (1991) suggested that the use of intrauterine devices was potentially associated with a higher possibility of HIV sexual transmission in women. The fertility data in the present experiment were low, when compared to other studies with fresh semen, perhaps because the insemination had fixed time with hormonally synchronised estrus, since the literature is somewhat conflicted about the issue, and Machado and Simplicio (2001) showed that inseminations performed earlier or later led to reduction in fertility rate. 5. Conclusions As a result of this study, it can be concluded that CAEV transmission by experimentally infected semen is a distinct possibility. The two viral loads used in this experiment were able to infect females, and seroconversion tended to be faster in goats which received the highest viral loads. The CAEV transmission was not influenced by the treatment method of estrus synchronisation and ovulation induction. MEM with 2% foetal calf serum and glucose can be used as goat semen extender as well as for semen contamination in experiments on pathogen transmission. CAEV probably has no negative influence on the fertility of females, at least in early infection. Acknowledgements The authors thank the Ceará Foundation for Support to Scientific and Technological Development – FUNCAP, Banco do Nordeste – BNB, State Government of Ceara – for financial support and the Brazilian Centre for Goats and Sheep Research (Centro Nacional de Pesquisa de Caprinos e Ovinos – EMBRAPA-CNPC) for use of the structure and technical and financial support. The Vale do Acaraú University. References Alexander, N.J., 1990. Sexual transmission of human immunodeficiency virus: virus entry into the male and female genital tract. Fertil. Steril. (54), 1–18.
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Ali Al Ahmad, M.Z., Fieni, F., Pellerin, J.L., Guiguen, F., Cherel, Y., Chatagnon, G., Bouzar, A.B., Chebloune, Y., 2008. Detection of viral genomes of caprine arthritis–encephalitis virus (CAEV) in semen and in genital tract tissues of male goat. Theriogenology (69), 473–480. Andrioli, A., Gouveia, A.M.G., Pinheiro, R.R., Rocha, M.A., Martins, A., Santos, D.O., 1999. Detecc¸ão do DNA pro-viral do lentivírus caprino em sêmen de bodes naturalmente infectados (Detection of caprine lentivirus proviral DNA in semen of naturally infected bucks). Rev. Bras. Rep. Anim. (23), 420–421. Andrioli, A., Gouveia, A.M.G., Martins, A.S., Pinheiro, R.R., Santos, D.O., 2006. Fatores de risco na transmissão do lentivírus caprino pelo sêmen (Risk factors in caprine lentivirus transmission through semen). Pesq. Agropec. Bras. (41), 1313–1319. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (72), 248–254. COBEA – Colégio Brasileiro de Experimentac¸ão Animal. Available at: http://www.cobea.org.br/index.php (accessed 08.09.08). Cohen, M.S., 2002. HAART and prevention of HIV transmission. Conference reports. Medscape HIV/AIDS, Medscape. Available at: http://www.medscape.com/viewarticle/437545 (accessed 27.01.10). Cruz, J.C.M., Gouveia, A.M.G., Souza, K.C., Braz, G.F., Teixeira, B.M., Heinemann, M.B., Leite, R.C., Reis, J.K.P., Pinheiro, R.R., Andrioli, A., 2009. Caprine arthritis–encephalitis virus (CAEV) detection in semen of endangered goat breeds by nested polymerase chain reaction. Small Rum. Res. (85), 149–152. Gouveia, A.M.G., Melo, L.M., Pires, L.L., Pinheiro, R.R., 2000. Microimunodifusão em gel de ágar para o diagnóstico sorológico de infecc¸ão por lentivírus de pequenos ruminantes (Agar gel microimmunodiffusion for sorologic diagnosis of small ruminants lentivirus infection). Congresso Brasileiro de Med. Veterinária (27), 33. Hafez, B., Hafez, E.S.E., 2004. Reproduc¸ão Animal, seventh ed. Manole, São Paulo. Holly, L., Gayle Howard, Jordan J.O., Bucci, Joseph G., Butterworth, Jennifer L., English, Robert, Suzanne, Kennedy-Stoskopf, Tompkins, Mary B., Tompkins, Wayne A., 1998. Horizontal transmission of feline immunodeficiency virus with semen from seropositive cats. J. Reprod. Immunol. (41), 341–357. Jordan, H.L., Howard, J., Tompkins, W.A., Kennedystoskopf, S., 1995. Detection of feline immunodeficiency virus in semen from seropositive domestic cats (Felis catus). J. Virol. (69), 7328–7333. Lara, M.C.C.S.H., Birgel Junior, E.H., Birgel, E.H., 2005. Possibility of vertical transmission of Caprine Arthritis–Encephalitis Virus in neonate kids. Arq. Bras. Med. Vet. Zootec. (57), 553–555. Lazzarin, A., Saracco, A., Musicco, A., Nicolosi, A., 1991. Man-to-woman sexual transmission of the human immunodeficiency virus. Risk factors related to sexual behavior, man’s infectiousness, and woman’s susceptibility. Italian Study Group on HIV Heterosexual Transmission. Arch. Intern. Med. (151), 2411–2416. Machado, R., Simplicio, A.A., 2001. Avaliac¸ão de programas hormonais para a induc¸ão e sincronizac¸ão do estro em caprinos. Pesq. agropec. Bras. (36), 171–178. Mermin, J.H., Holodniy, M., Katzenstein, D.A., Merigan, T.C., 1991. Detection of human immunodeficiency virus DNA and RNA in semen by the polymerase chain reaction. J. Infect. Dis. (164), 769–772. Nash, J.W., Hanson, L.A., Coats, K.C., 1995. Bovine immunodeficiency virus in stud bull semen. Am. J. Vet. Res. (56), 760–763. Paula, N.R.O., Andrioli, A., Cardoso, J.F.S., Pinheiro, R.R., Sousa, F.M.L., Souza, K.C., Alves, F.S.F., Campello, C.C., Ricarte, A.R.F., Teixeira, M.F.S., 2009. Profile of the Caprine arthritis–encephalitis virus (CAEV) in blood, semen from bucks naturally and experimentally infected in the semiarid region of Brazil. Small Rum. Res. (85), 27–33. Peterson, K., Brinkhof, J., Houwers, D., Colenbrander, B., Gadella, B., 2008. Presence of pro-lentiviral DNA in male sexual organs and ejaculates of small ruminants. Theriogenology (69), 433–442. Pinheiro, R.R., Gouvea, A.M.G., Alves, F.S.F., 2001. Prevalência da infecc¸ão pelo vírus da artrite encefalite no Estado do Ceará. Brasil. Ciênc. Rural (31), 449–454. Pinheiro, R.R., Olortegui, C.D.C., Gouvea, A.M.G., Araujo, S.C., Andrioli, A., 2006. Desenvolvimento de dot-blot para detecc¸ão de anticorpos para o vírus da Artrite-Encefalite Caprina em caprinos. Rev. Port. Ciên. Vet. (101), 51–56. Pisoni, G., Moroni, P., Turin, L., Bertoni, G., 2007. Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats. Virology (369), 119–130. Plummer, D.J., 1978. An Introduction to Pratical Biochemistry, second ed. McGraw-Hill, London. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. (27), 493–497.
198
K.C.d. Souza et al. / Small Ruminant Research 109 (2013) 193–198
Ricarte, A.R.F., Andrioli, A., Pinheiro, R.R., Báo, S.N., Silva, J.S., Braz, S.V., Name, K.P.O., Lima-Verde, I.B., Brito, I.F., Dias, R.P., Freitas Aguiar, T.D., Dantas, T.V.M., Araújo, S.A.C., Cavalcanti, D.M.L.P., Paula, N.R.O., Teixeira, M.F.S., 2010. Avaliac¸ão imunohistoquímica e ultraestrutural de gametas e embriões caprinos infectados com o CAEV (Immunohistochemical and ultrastructural evaluation of gametes and embryos of goats infected with CAEV). Arq. Inst. Biol. (77), 217–223. Rodrigues, A.S., Brito, R.L.L., Santos, V.W.S., Dias, R.P., Brito, I.F., Anrioli, A., Pinheiro, R.R., 2009. Comparac¸ão de dois testes sorológicos na
evoluc¸ão natural de caprinos leiteiros com o vírus da Artrite-Encefalite Caprina-dados preliminares. Sincorte. João Pessoa (4). Rowe, J.D., Esast, N.E., Franti, C.E., Thurmond, M.C., Pederson, N.C., Theilen, G.H., 1991. Risk factores associated with the incidence of seroconversion to caprine arthritis–encephalitis virus in goats on California dairies. Am. J. Vet. Res. (53), 2396–2430. Swaminathan, N. Sêmen torna o HIV mais potente. Available at: www2.uol.com.br/./semen torna o hiv mais potente.html Accessed: 21 Dez 2009.