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Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil
Veterinary Parasitology 147 (2007) 61–66 www.elsevier.com/locate/vetpar
Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil H.F.J. Pena a,*, R.M. Soares a, A.M.A. Ragozo a, R.M. Monteiro a, L.E.O. Yai b, S.M. Nishi a, S.M. Gennari a a
Department of Preventive Veterinary Medicine and Animal Health, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-000 Sa˜o Paulo, Brazil b Centro de Controle de Zoonoses, R. Santa Eula´lia, 86, CEP 02031-020 Sa˜o Paulo, Brazil Received 9 February 2007; accepted 5 March 2007
Abstract Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70 g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400—Dog 1 and Sheep n. 342, IFAT 1:50—Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (103 oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus. # 2007 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Sheep; Dogs; Isolation; Oocyst shedding; Rodents; Sequence analysis; Brazil
1. Introduction Neospora caninum is a protozoan parasite that can cause paralysis and death in dogs and is a major cause of bovine abortion worldwide (Dubey, 2003). Dogs (Canis
* Corresponding author. Tel.: +55 11 3091 7701; fax: +55 11 3091 7928. E-mail address: [email protected] (H.F.J. Pena). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.03.002
familiaris) and coyotes (Canis latrans) are the only recognized definitive hosts for N. caninum, shedding oocysts in the faeces (McAllister et al., 1998; Gondim et al., 2004). Cattle and a wide range of other warmblooded animals can act as intermediate hosts (Dubey et al., 2006). It seems to occur less frequently in sheep. In Brazil, N. caninum seroprevalence value of 9.2% has been recently observed in sheep (Figliuolo et al., 2004). Ovine experimental neosporosis is clinically and histopathologically similar to that reported in cattle,
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as the occurrence of repetitive abortion (McAllister et al., 1996; Jolley et al., 1999). Natural infection in sheep is not common but can also be associated with abortion and weak pre-term offspring (Jolley et al., 1999; Ha¨ssig et al., 2003; West et al., 2006). N. caninum has been isolated from cattle, dogs, water buffaloes, white-tailed deer, as reviewed by Dubey et al. (2006), and there is a report of isolation from sheep in Japan (Koyama et al., 2001). The purposes of the present study were to isolate, for the first time, N. caninum from Brazilian naturally infected sheep, and to detect the parasite using molecular methods. 2. Materials and methods 2.1. Sheep Two 4-month-old sheep, with no clinical signs, tested for N. caninum by indirect fluorescent antibody test (IFAT) were seropositive considering 1:50 dilution as the cut-off. NC-1 strain of N. caninum maintained in Vero cell culture was used as the antigen. Sheep n. 302, from Ibitinga county, Sa˜o Paulo state, had IFAT 1:400 and Sheep n. 342, from Tieteˆ county, Sa˜o Paulo state, had IFAT 1:50. Both sheep had no antibodies to Toxoplasma gondii when tested by IFAT (<1:64). 2.2. Infection of dogs Brains from both seropositive sheep were collected at necropsy and separately homogenized. Five aliquots from each brain homogenate were stored in 2 ml microtubes at 70 8C for further DNA study. Approximately 70 g of each homogenate were offered to a different N. caninum and T. gondii seronegative dog (IFAT < 1:50 and < 1:16, respectively). Dogs were 3 months old. Dog 1 received brain from Sheep n. 302 and Dog 2 received brain from Sheep n. 342. Dog sera were tested weekly at 1:50 for IgG antibodies to N. caninum and at 1:16 for IgG antibodies to T. gondii (IFAT) from 0 to 70 days post-inoculation (PI). Dogs had never consumed uncooked meat and were fed commercial dry dog food during entire experiment. Faecal samples from each dog were examined daily for the detection of Neospora-like oocysts 0–40 days PI using a standard sucrose flotation technique. Each day, complete samples were weighed, homogenized and 1 g (5) was mixed with 12 ml concentrated sucrose (sp. gr. 1.205), sieved with a tea strainer and transferred to 15 ml tubes. After centrifugation (450 g, 10 min), two drops of the supernatant were recovered with a loop
from each tube, transferred to a slide and covered with a coverslip for microscopic examination. As daily concentration of oocysts was too low to count them in the Neubauer chamber, 1 g (3) of each positive faecal sample was examined using the sucrose flotation technique described above. When no more oocysts were observed and counted on the supernatant under microscopic examination, faecal suspension was resuspended and centrifuged again. Supernatant was reexamined until no more oocysts were found. The total number of oocysts produced was calculated from the weight of the dog’s faeces. A total of 100 unsporulated oocysts in concentrated sucrose solution were examined by light microscopy at a magnification of 1000 using an Olympus BX40 microscope connected to the Olympus DP70 microscope digital camera. Images were analysed using the program Image-Pro1 Plus Version 5.1 (Media Cybernetics, Inc., Silver Spring, MD). For sporulation, faecal samples containing oocysts were mixed with 2% K2Cr2O7 in Petri dishes for 5 days at 25 8C, and then stored at 4 8C for further use. The dog that shed Neospora oocysts (Dog 1) was euthanized 70 days PI. Brain/cerebellum and spinal cord were collected at necropsy, washed in sterile antibiotic solution (10,000 UI penicillin, 20 mg streptomycin and 2.0 mg amphotericyn B per ml), separately homogenized with the antibiotic solution, passed through a 19-gauge needle and centrifuged twice using 50 ml tubes (3000 g, 10 min). Each homogenate was used to infect two flasks with Vero cell monolayer in RPMI-1640 supplemented with 2.5% foetal calf serum and antibiotics (200 UI penicillin and 0.2 mg streptomycin per ml). An aliquot from brain/cerebellum and from spinal cord homogenates, and also two aliquots from heart, liver, kidney, lung, spleen and thigh muscles homogenates were separately stored in 2 ml microtubes at 70 8C for further DNA study. 2.3. Infection of rodents After 2 months stored in 2% K2Cr2O7 at 4 8C, sporulated N. caninum oocysts in faecal material were sieved through a series of strainers (60, 100, 200 and 400 mesh) using tap water. After 24 h, the supernatant of the filtrate was discarded and aliquots (2 ml) of the sediment were transferred into 15 ml tubes, mixed with 10 ml of the concentrated sucrose and centrifuged (450 g, 10 min). Then, 1 ml of the top layer of flotation medium was carefully aspirated with a pipette and transferred to 50 ml tubes. The remaining flotation medium was remixed and the procedure was repeated.
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The collected oocysts were washed three times in distilled water by centrifugation (1000 g, 10 min), the supernatant was discarded and the sediment resuspended in distilled water. To count the oocysts, 1 ml (2) of the solution was examined in concentrated sucrose as described above. Two Swiss white outbred mice, two Mongolian gerbils (Meriones ungulatus), and two large vesper mice (Calomys callosus—Rodentia, Cricetidae) were orally inoculated with approximately 103 oocysts. Animals were euthanized 2 months PI and tissues were aseptically collected. Brain and heart of each gerbil were separately homogenized in sterile antibiotic solution, as described above, and centrifuged (3000 g, 10 min). Each gerbil brain homogenate was distributed to infect Vero cell monolayer in RPMI-1640 with 2.5% foetal calf serum and antibiotics, as described above, to be stored at 70 8C for further DNA study and for microscopic examination in fresh preparations. Gerbil heart suspensions were used the same way but the microscopic examination. A pool of brains and a pool of hearts from Calomys and from Swiss mice were used the same way as gerbil brains and hearts. 2.4. DNA extraction of tissues and oocysts
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using primers based on the Nc-5 gene (Yamage et al., 1996). Reactions consisted of 10 mM Tris–HCl, 50 mM KCl, 50 mM MgCl2, 25 pmol of each primer (Np4/Np7 or Np6/Np7), 0.2 mM of each dNTP, 1.5 U Taq DNA polymerase, and 5 ml target DNA for the first reaction or 1 ml of a 1/10 dilution of the product from the first amplification for the second reaction, in a total volume of 50 ml. Positive control (N. caninum tachyzoite DNA) and at least two negative (H2O) controls were included in each set of reactions. PCR amplifications were performed on Eppendorf MastercyclerTM gradient thermocycler. The conditions for both primary and secondary PCR reactions were an initial denaturation at 94 8C for 3 min, followed by 35 cycles of 95 8C for 30 s, 57 8C for 30 s and 72 8C for 60 s and a final extension at 72 8C for 10 min. The final amplified DNA was analysed by electrophoresis in 2% agarose gels and stained with ethidium bromide. Oocysts also have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid 18S and 5.8S coding genes and flanking the ITS1 region (ITS1-PCR). The amplification was performed in 50 ml final solution as previously described (Rodrigues et al., 2004) but using primers JS4 (Slapeta et al., 2002) and CT2b (50 TTG CGC GAG CCA AGA CAT C 30 , this study). The PCR products were run in 2% agarose gel and detected after staining with ethidium bromide.
DNA isolation from sheep brains, dog tissues and rodent tissues was based on protocols described by Ausubel et al. (1999). Briefly, 500 ml of each sample was washed in Tris–EDTA (TE) buffer (10 mM Tris– HCl, pH 8.0; 1 mM EDTA), and digested by overnight incubation at 37 8C in lysis buffer (10 mM Tris–HCl, pH 8.0; 100 mM NaCl; 25 mM EDTA; 1% SDS; 400 mg/ml proteinase K). DNA was extracted via phenol, phenol/chloroform and chloroform steps, and then was precipitated with two volumes of 100% ethanol. Following overnight incubation at 20 8C and centrifugation, each pellet was resuspended in 30 ml of TE buffer, incubated for 10 min at 56 8C and stored at 20 8C until amplification. To isolate DNA from oocysts, positive faecal floats were examined under light microscopy, slides were rinsed with TE and centrifuged twice at 12,000 g, 5 min. The pellet was resuspended with 20 ml of 10% SDS and 200 ml of TE, and submitted to five freeze (192 8C, 1 min)–thaw (65 8C, 2 min) cycles. Digestion and DNA extraction followed the steps described above.
3. Results
2.5. PCR
3.1. Infection of dogs
The DNA isolated from animal tissues and oocysts was tested by hemi-nested PCR for N. caninum DNA
Dog 1 began shedding N. caninum oocysts 10 days PI, but they were detected again in faecal examinations
2.6. Molecular identification of oocysts After elution from agarose gel by using a clean-up system (GFX1, GE Healthcare, Buckinghamshire, UK), the amplicons of ITS1-PCR were sequenced in both directions using the ABI chemistry (ABI PRISM1, Foster City, CA) with sense and reverse primers. Each strand was sequenced at least four times to increase the reliability of the results. The sequences were assembled and the contig formed with the phred-base calling tool available on the website http://bioinformatica.ucb.br/. The final sequence were recovered with each residue score equal or greater than 20 and deposited in GenBank1 under accession number DQ832318. This sequence was compared with existing homologues in GenBank1.
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only 16 days PI and then until 25 days PI. During patent period, it was estimated a range of 1–419 oocysts per gram of faeces per day (mean 44) and a range of 1–11,890 considering total weight of faeces excreted per day (mean 1720). A total of approximately 27,600 oocysts were shed during 16 days of oocyst shedding. Dog 1 remained clinically healthy during the study. Dog 2 did not shed oocysts during the period of examination. None of the dogs developed IgG antibodies to N. caninum (IFAT < 1:50) or T. gondii (IFAT < 1:16) during the period of examination (7 weeks PI). The unsporulated oocysts observed in sucrose solution measured 9.56–10.84 (10.48 0.40) mm by 9.73–11.86 (10.80 0.44) mm. Most of the measured oocysts (63.5%) were <11.00 mm. The length–width ratios were 1–1.09 (mean 1.03), with 88.5% of the oocysts with length–width ratios 1.05. The two flasks cultured with brain/cerebellum and one flask cultured with spinal cord contaminated and were discarded few days after inoculation. The other flask cultured with spinal cord was examined during 2 months, but despite one parasite had been seen in the first day of inoculation, no culture was established. 3.2. Infection of rodents All rodents remained clinically normal. Thickwalled tissue cysts of N. caninum were seen only in brain squashes of the two gerbils (one cyst in one and three cysts in the other), measuring 24.47–47.07 mm by 25.33–53.91 mm (mean 39.03 40.52 mm). The cysts had 1.71–2.66 mm thick cyst walls. Parasites were not detected in cell cultures inoculated with rodent brains or hearts. Culture flasks were discarded 2 months after inoculation. 3.3. PCR The presence of N. caninum Nc-5 gene was confirmed in the five aliquots of brain homogenates from Sheep n. 302 and from Sheep n. 342. N. caninum Nc-5 gene was also detected in brain/cerebellum and spinal cord homogenates from Dog 1, but not in both aliquots from heart, liver, kidney, lung, spleen or thigh muscles homogenates. Oocysts proved positive by the PCR based on Nc-5 gene and ITS1 sequences. Fifty-four nucleotides of the 18S gene and 405 nucleotides of the ITS1 sequence from the ITS1-PCR products were sequenced. Such fragments had 100% nucleotide identity with homologous sequences from several N. caninum isolates available in GenBank1.
Brain homogenates from each gerbil and brain homogenate from rodent C. callosus (pool) were positive for N. caninum Nc-5 gene, but there was no DNA detection in any of the heart homogenates from rodents. 4. Discussion This study demonstrates for the first time the behaviour of N. caninum in dogs fed tissue cysts from naturally infected sheep and it is the first isolation from a naturally infected sheep from Brazil. The identity of the isolate was based on biological and molecular characteristics and structure. Koyama et al. (2001) isolated N. caninum from the brain of a pregnant sheep by bioassay in mice. Tachyzoites were detected in the peritoneal exsudate of immunodeficient mice and maintained by continuous passage in nu/nu mice and cell cultures. In general, the results on this paper were very similar to that observed in different previous studies in which dogs consumed experimentally infected mouse carcasses or tissues directly from experimentally infected calves. In the present study, Dog 1 shed oocysts 10–25 days PI. This is comparable to previous reports of oocyst shedding by dogs from 13 to 23 days PI (McAllister et al., 1998) and from 10 to 28 days PI (Gondim et al., 2002). But a prepatent period as short as 5 days has also been observed in dogs (Lindsay et al., 1999; Gondim et al., 2002). Just one oocyst was seen in Dog 1 faeces on day 10 PI, despite faecal flotations from total faeces have been microscopically examined this day, and then oocysts were seen again on day 16 PI. This failure to detect oocysts by microscopy suggests that probably low numbers were being shed in the faeces. This is a feature of Neospora infection that has been noted by other authors (McAllister et al., 1998; Lindsay et al., 1999). The concentration of oocysts (approximately 27,600) during 16 days of oocyst shedding can be considered low. McAllister et al. (1998) pointed out that the number of cysts consumed by dogs is an important issue to Neospora oocyst shedding. Gondim et al. (2002) demonstrated that dogs shed great numbers of N. caninum oocysts after consuming tissues from infected calves (about 3 kg of different tissues per dog) than after consuming infected mouse carcasses (4–16 mice per dog) and Gondim et al. (2005) observed that dogs that consumed only brain and spinal cord shed less oocysts than dogs that consumed multiple tissues, so it is also possible that the type of tissue may influence oocyst production. At the present study, Dog 1 was only fed with sheep
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brain. But the distribution of N. caninum cysts in the tissues of different hosts should be better studied. In the present study, DNA detection in dog and rodents tissues was only observed in neural tissues. Dog 1 remained clinically healthy during the period of oocyst excretion. This was also observed in dogs examined by McAllister et al. (1998), Lindsay et al. (1999) and Gondim et al. (2002). IgG IFAT titres are generally 1:400 in dogs with confirmed clinical neosporosis (Barber and Trees, 1996), but the majority of dogs shedding N. caninum oocysts after experimental infection do not seroconvert in the N. caninum IFAT as observed by previous studies (McAllister et al., 1998; Lindsay et al., 1999; Gondim et al., 2002; Rodrigues et al., 2004). So, the fact that Dog 1 did not develop detectable IgG antibody titre within 7 weeks after infection is consistent with other reports. The number of oocysts shed seems not to be related with seroconvertion, because a puppy that shed 504,400 oocysts did not develop antibodies as observed by Gondim et al. (2005) and on the other hand, some dogs with high antibody titres had no detectable oocysts in the faeces (McAllister et al., 1998). The potential of N. caninum oocysts to infect sheep was experimentally demonstrated by O’Handley et al. (2002). The minimum infectious dose of N. caninum oocysts to infect sheep is not known, but it is proved to be low in calves, as few as 300 (Gondim et al., 2002). As dogs are frequently found in sheep stocks, oocysts shed by them can also be a natural source of N. caninum infection to sheep. Schares et al. (2005) study was the first demonstrating morphological differences by light microscopy between N. caninum isolates and other species, especially Hammondia heydorni. The results from the present paper are comparable because N. caninum unsporulated oocysts were also measured in concentrated sucrose solution using a computer image program. In that paper, the 75th percentiles of N. caninum oocyst isolates were 10.7 mm in length and 1.06 in length–width ratios. In Brazilian sheep isolate, from the present study, 63.5% of the oocysts were <11.00 mm in length, and most of the oocysts (88.5%) had a length–width ratio 1.05. Relatively to the rodents, N. caninum is nonpathogenic and rarely infective to Swiss white outbred mice (Lindsay and Dubey, 1989), and even immunosuppressed mice are resistant to oral feeding of oocysts (Lindsay et al., 1999), so it was expected that this group of rodents was not infected in the present experiment. Dubey and Lindsay (2000) demonstrate the high susceptibility of gerbils to oral infection with
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Fig. 1. Neospora caninum tissue cyst in the brain of a gerbil fed oocysts from dog experimentally infected with brain sheep. Fresh preparation.
N. caninum oocysts, but the pathogenicity has been variable. Gerbils became sick or died at 6–13 days after feeding 1000 oocysts (Dubey and Lindsay, 2000), or remained clinically normal (Basso et al., 2001; Schares et al., 2005). In the present study both gerbils became infected and did not develop disease. Basso et al. (2001) found tissue cysts measuring 26 mm 28 mm and 27 mm 32 mm in sections of brain of one gerbil. Gerbils were necropsied 63–75 days PI. Well-developed N. caninum tissue cysts have also been found in gerbils 60 days PI in the present study, the largest of them measuring 47.07 mm 53.91 mm in fresh preparation (Fig. 1). All four tissue cysts found in gerbil brains were thick walled (>1 mm). The large vesper mouse, C. callosus, is a rodent species from South America that has been used as a laboratory animal. It is highly susceptible to T. gondii infection, and has been used as an appropriate alternative model to experimental toxoplasmosis (Favoreto-Junior et al., 1998). In the present paper, the susceptibility of the wild mouse C. callosus to oral infection with N. caninum was demonstrated by molecular detection of N. caninum DNA in its brain, but no tissue cyst were observed by light microscopy, probably because the small amount of material examined. Unfortunately, the amount of oocysts was not enough to inoculate more animals. The use of this rodent as an alternative model to neosporosis should be better investigated. Attempts to isolate the parasite in cell cultures were not successful, probably because the tissues from Dog 1 and from rodents were just homogenized in a mortar and not digested. New attempts are being made to isolate the parasite in cell culture from gerbils orally inoculated with oocysts from the Brazilian sheep isolate.
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Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil H.F.J. Pena a,*, R.M. Soares a, A.M.A. Ragozo a, R.M. Monteiro a, L.E.O. Yai b, S.M. Nishi a, S.M. Gennari a a
Department of Preventive Veterinary Medicine and Animal Health, Faculdade de Medicina Veterina´ria e Zootecnia, Universidade de Sa˜o Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-000 Sa˜o Paulo, Brazil b Centro de Controle de Zoonoses, R. Santa Eula´lia, 86, CEP 02031-020 Sa˜o Paulo, Brazil Received 9 February 2007; accepted 5 March 2007
Abstract Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70 g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400—Dog 1 and Sheep n. 342, IFAT 1:50—Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (103 oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus. # 2007 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Sheep; Dogs; Isolation; Oocyst shedding; Rodents; Sequence analysis; Brazil
1. Introduction Neospora caninum is a protozoan parasite that can cause paralysis and death in dogs and is a major cause of bovine abortion worldwide (Dubey, 2003). Dogs (Canis
* Corresponding author. Tel.: +55 11 3091 7701; fax: +55 11 3091 7928. E-mail address: [email protected] (H.F.J. Pena). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.03.002
familiaris) and coyotes (Canis latrans) are the only recognized definitive hosts for N. caninum, shedding oocysts in the faeces (McAllister et al., 1998; Gondim et al., 2004). Cattle and a wide range of other warmblooded animals can act as intermediate hosts (Dubey et al., 2006). It seems to occur less frequently in sheep. In Brazil, N. caninum seroprevalence value of 9.2% has been recently observed in sheep (Figliuolo et al., 2004). Ovine experimental neosporosis is clinically and histopathologically similar to that reported in cattle,
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as the occurrence of repetitive abortion (McAllister et al., 1996; Jolley et al., 1999). Natural infection in sheep is not common but can also be associated with abortion and weak pre-term offspring (Jolley et al., 1999; Ha¨ssig et al., 2003; West et al., 2006). N. caninum has been isolated from cattle, dogs, water buffaloes, white-tailed deer, as reviewed by Dubey et al. (2006), and there is a report of isolation from sheep in Japan (Koyama et al., 2001). The purposes of the present study were to isolate, for the first time, N. caninum from Brazilian naturally infected sheep, and to detect the parasite using molecular methods. 2. Materials and methods 2.1. Sheep Two 4-month-old sheep, with no clinical signs, tested for N. caninum by indirect fluorescent antibody test (IFAT) were seropositive considering 1:50 dilution as the cut-off. NC-1 strain of N. caninum maintained in Vero cell culture was used as the antigen. Sheep n. 302, from Ibitinga county, Sa˜o Paulo state, had IFAT 1:400 and Sheep n. 342, from Tieteˆ county, Sa˜o Paulo state, had IFAT 1:50. Both sheep had no antibodies to Toxoplasma gondii when tested by IFAT (<1:64). 2.2. Infection of dogs Brains from both seropositive sheep were collected at necropsy and separately homogenized. Five aliquots from each brain homogenate were stored in 2 ml microtubes at 70 8C for further DNA study. Approximately 70 g of each homogenate were offered to a different N. caninum and T. gondii seronegative dog (IFAT < 1:50 and < 1:16, respectively). Dogs were 3 months old. Dog 1 received brain from Sheep n. 302 and Dog 2 received brain from Sheep n. 342. Dog sera were tested weekly at 1:50 for IgG antibodies to N. caninum and at 1:16 for IgG antibodies to T. gondii (IFAT) from 0 to 70 days post-inoculation (PI). Dogs had never consumed uncooked meat and were fed commercial dry dog food during entire experiment. Faecal samples from each dog were examined daily for the detection of Neospora-like oocysts 0–40 days PI using a standard sucrose flotation technique. Each day, complete samples were weighed, homogenized and 1 g (5) was mixed with 12 ml concentrated sucrose (sp. gr. 1.205), sieved with a tea strainer and transferred to 15 ml tubes. After centrifugation (450 g, 10 min), two drops of the supernatant were recovered with a loop
from each tube, transferred to a slide and covered with a coverslip for microscopic examination. As daily concentration of oocysts was too low to count them in the Neubauer chamber, 1 g (3) of each positive faecal sample was examined using the sucrose flotation technique described above. When no more oocysts were observed and counted on the supernatant under microscopic examination, faecal suspension was resuspended and centrifuged again. Supernatant was reexamined until no more oocysts were found. The total number of oocysts produced was calculated from the weight of the dog’s faeces. A total of 100 unsporulated oocysts in concentrated sucrose solution were examined by light microscopy at a magnification of 1000 using an Olympus BX40 microscope connected to the Olympus DP70 microscope digital camera. Images were analysed using the program Image-Pro1 Plus Version 5.1 (Media Cybernetics, Inc., Silver Spring, MD). For sporulation, faecal samples containing oocysts were mixed with 2% K2Cr2O7 in Petri dishes for 5 days at 25 8C, and then stored at 4 8C for further use. The dog that shed Neospora oocysts (Dog 1) was euthanized 70 days PI. Brain/cerebellum and spinal cord were collected at necropsy, washed in sterile antibiotic solution (10,000 UI penicillin, 20 mg streptomycin and 2.0 mg amphotericyn B per ml), separately homogenized with the antibiotic solution, passed through a 19-gauge needle and centrifuged twice using 50 ml tubes (3000 g, 10 min). Each homogenate was used to infect two flasks with Vero cell monolayer in RPMI-1640 supplemented with 2.5% foetal calf serum and antibiotics (200 UI penicillin and 0.2 mg streptomycin per ml). An aliquot from brain/cerebellum and from spinal cord homogenates, and also two aliquots from heart, liver, kidney, lung, spleen and thigh muscles homogenates were separately stored in 2 ml microtubes at 70 8C for further DNA study. 2.3. Infection of rodents After 2 months stored in 2% K2Cr2O7 at 4 8C, sporulated N. caninum oocysts in faecal material were sieved through a series of strainers (60, 100, 200 and 400 mesh) using tap water. After 24 h, the supernatant of the filtrate was discarded and aliquots (2 ml) of the sediment were transferred into 15 ml tubes, mixed with 10 ml of the concentrated sucrose and centrifuged (450 g, 10 min). Then, 1 ml of the top layer of flotation medium was carefully aspirated with a pipette and transferred to 50 ml tubes. The remaining flotation medium was remixed and the procedure was repeated.
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The collected oocysts were washed three times in distilled water by centrifugation (1000 g, 10 min), the supernatant was discarded and the sediment resuspended in distilled water. To count the oocysts, 1 ml (2) of the solution was examined in concentrated sucrose as described above. Two Swiss white outbred mice, two Mongolian gerbils (Meriones ungulatus), and two large vesper mice (Calomys callosus—Rodentia, Cricetidae) were orally inoculated with approximately 103 oocysts. Animals were euthanized 2 months PI and tissues were aseptically collected. Brain and heart of each gerbil were separately homogenized in sterile antibiotic solution, as described above, and centrifuged (3000 g, 10 min). Each gerbil brain homogenate was distributed to infect Vero cell monolayer in RPMI-1640 with 2.5% foetal calf serum and antibiotics, as described above, to be stored at 70 8C for further DNA study and for microscopic examination in fresh preparations. Gerbil heart suspensions were used the same way but the microscopic examination. A pool of brains and a pool of hearts from Calomys and from Swiss mice were used the same way as gerbil brains and hearts. 2.4. DNA extraction of tissues and oocysts
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using primers based on the Nc-5 gene (Yamage et al., 1996). Reactions consisted of 10 mM Tris–HCl, 50 mM KCl, 50 mM MgCl2, 25 pmol of each primer (Np4/Np7 or Np6/Np7), 0.2 mM of each dNTP, 1.5 U Taq DNA polymerase, and 5 ml target DNA for the first reaction or 1 ml of a 1/10 dilution of the product from the first amplification for the second reaction, in a total volume of 50 ml. Positive control (N. caninum tachyzoite DNA) and at least two negative (H2O) controls were included in each set of reactions. PCR amplifications were performed on Eppendorf MastercyclerTM gradient thermocycler. The conditions for both primary and secondary PCR reactions were an initial denaturation at 94 8C for 3 min, followed by 35 cycles of 95 8C for 30 s, 57 8C for 30 s and 72 8C for 60 s and a final extension at 72 8C for 10 min. The final amplified DNA was analysed by electrophoresis in 2% agarose gels and stained with ethidium bromide. Oocysts also have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid 18S and 5.8S coding genes and flanking the ITS1 region (ITS1-PCR). The amplification was performed in 50 ml final solution as previously described (Rodrigues et al., 2004) but using primers JS4 (Slapeta et al., 2002) and CT2b (50 TTG CGC GAG CCA AGA CAT C 30 , this study). The PCR products were run in 2% agarose gel and detected after staining with ethidium bromide.
DNA isolation from sheep brains, dog tissues and rodent tissues was based on protocols described by Ausubel et al. (1999). Briefly, 500 ml of each sample was washed in Tris–EDTA (TE) buffer (10 mM Tris– HCl, pH 8.0; 1 mM EDTA), and digested by overnight incubation at 37 8C in lysis buffer (10 mM Tris–HCl, pH 8.0; 100 mM NaCl; 25 mM EDTA; 1% SDS; 400 mg/ml proteinase K). DNA was extracted via phenol, phenol/chloroform and chloroform steps, and then was precipitated with two volumes of 100% ethanol. Following overnight incubation at 20 8C and centrifugation, each pellet was resuspended in 30 ml of TE buffer, incubated for 10 min at 56 8C and stored at 20 8C until amplification. To isolate DNA from oocysts, positive faecal floats were examined under light microscopy, slides were rinsed with TE and centrifuged twice at 12,000 g, 5 min. The pellet was resuspended with 20 ml of 10% SDS and 200 ml of TE, and submitted to five freeze (192 8C, 1 min)–thaw (65 8C, 2 min) cycles. Digestion and DNA extraction followed the steps described above.
3. Results
2.5. PCR
3.1. Infection of dogs
The DNA isolated from animal tissues and oocysts was tested by hemi-nested PCR for N. caninum DNA
Dog 1 began shedding N. caninum oocysts 10 days PI, but they were detected again in faecal examinations
2.6. Molecular identification of oocysts After elution from agarose gel by using a clean-up system (GFX1, GE Healthcare, Buckinghamshire, UK), the amplicons of ITS1-PCR were sequenced in both directions using the ABI chemistry (ABI PRISM1, Foster City, CA) with sense and reverse primers. Each strand was sequenced at least four times to increase the reliability of the results. The sequences were assembled and the contig formed with the phred-base calling tool available on the website http://bioinformatica.ucb.br/. The final sequence were recovered with each residue score equal or greater than 20 and deposited in GenBank1 under accession number DQ832318. This sequence was compared with existing homologues in GenBank1.
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only 16 days PI and then until 25 days PI. During patent period, it was estimated a range of 1–419 oocysts per gram of faeces per day (mean 44) and a range of 1–11,890 considering total weight of faeces excreted per day (mean 1720). A total of approximately 27,600 oocysts were shed during 16 days of oocyst shedding. Dog 1 remained clinically healthy during the study. Dog 2 did not shed oocysts during the period of examination. None of the dogs developed IgG antibodies to N. caninum (IFAT < 1:50) or T. gondii (IFAT < 1:16) during the period of examination (7 weeks PI). The unsporulated oocysts observed in sucrose solution measured 9.56–10.84 (10.48 0.40) mm by 9.73–11.86 (10.80 0.44) mm. Most of the measured oocysts (63.5%) were <11.00 mm. The length–width ratios were 1–1.09 (mean 1.03), with 88.5% of the oocysts with length–width ratios 1.05. The two flasks cultured with brain/cerebellum and one flask cultured with spinal cord contaminated and were discarded few days after inoculation. The other flask cultured with spinal cord was examined during 2 months, but despite one parasite had been seen in the first day of inoculation, no culture was established. 3.2. Infection of rodents All rodents remained clinically normal. Thickwalled tissue cysts of N. caninum were seen only in brain squashes of the two gerbils (one cyst in one and three cysts in the other), measuring 24.47–47.07 mm by 25.33–53.91 mm (mean 39.03 40.52 mm). The cysts had 1.71–2.66 mm thick cyst walls. Parasites were not detected in cell cultures inoculated with rodent brains or hearts. Culture flasks were discarded 2 months after inoculation. 3.3. PCR The presence of N. caninum Nc-5 gene was confirmed in the five aliquots of brain homogenates from Sheep n. 302 and from Sheep n. 342. N. caninum Nc-5 gene was also detected in brain/cerebellum and spinal cord homogenates from Dog 1, but not in both aliquots from heart, liver, kidney, lung, spleen or thigh muscles homogenates. Oocysts proved positive by the PCR based on Nc-5 gene and ITS1 sequences. Fifty-four nucleotides of the 18S gene and 405 nucleotides of the ITS1 sequence from the ITS1-PCR products were sequenced. Such fragments had 100% nucleotide identity with homologous sequences from several N. caninum isolates available in GenBank1.
Brain homogenates from each gerbil and brain homogenate from rodent C. callosus (pool) were positive for N. caninum Nc-5 gene, but there was no DNA detection in any of the heart homogenates from rodents. 4. Discussion This study demonstrates for the first time the behaviour of N. caninum in dogs fed tissue cysts from naturally infected sheep and it is the first isolation from a naturally infected sheep from Brazil. The identity of the isolate was based on biological and molecular characteristics and structure. Koyama et al. (2001) isolated N. caninum from the brain of a pregnant sheep by bioassay in mice. Tachyzoites were detected in the peritoneal exsudate of immunodeficient mice and maintained by continuous passage in nu/nu mice and cell cultures. In general, the results on this paper were very similar to that observed in different previous studies in which dogs consumed experimentally infected mouse carcasses or tissues directly from experimentally infected calves. In the present study, Dog 1 shed oocysts 10–25 days PI. This is comparable to previous reports of oocyst shedding by dogs from 13 to 23 days PI (McAllister et al., 1998) and from 10 to 28 days PI (Gondim et al., 2002). But a prepatent period as short as 5 days has also been observed in dogs (Lindsay et al., 1999; Gondim et al., 2002). Just one oocyst was seen in Dog 1 faeces on day 10 PI, despite faecal flotations from total faeces have been microscopically examined this day, and then oocysts were seen again on day 16 PI. This failure to detect oocysts by microscopy suggests that probably low numbers were being shed in the faeces. This is a feature of Neospora infection that has been noted by other authors (McAllister et al., 1998; Lindsay et al., 1999). The concentration of oocysts (approximately 27,600) during 16 days of oocyst shedding can be considered low. McAllister et al. (1998) pointed out that the number of cysts consumed by dogs is an important issue to Neospora oocyst shedding. Gondim et al. (2002) demonstrated that dogs shed great numbers of N. caninum oocysts after consuming tissues from infected calves (about 3 kg of different tissues per dog) than after consuming infected mouse carcasses (4–16 mice per dog) and Gondim et al. (2005) observed that dogs that consumed only brain and spinal cord shed less oocysts than dogs that consumed multiple tissues, so it is also possible that the type of tissue may influence oocyst production. At the present study, Dog 1 was only fed with sheep
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brain. But the distribution of N. caninum cysts in the tissues of different hosts should be better studied. In the present study, DNA detection in dog and rodents tissues was only observed in neural tissues. Dog 1 remained clinically healthy during the period of oocyst excretion. This was also observed in dogs examined by McAllister et al. (1998), Lindsay et al. (1999) and Gondim et al. (2002). IgG IFAT titres are generally 1:400 in dogs with confirmed clinical neosporosis (Barber and Trees, 1996), but the majority of dogs shedding N. caninum oocysts after experimental infection do not seroconvert in the N. caninum IFAT as observed by previous studies (McAllister et al., 1998; Lindsay et al., 1999; Gondim et al., 2002; Rodrigues et al., 2004). So, the fact that Dog 1 did not develop detectable IgG antibody titre within 7 weeks after infection is consistent with other reports. The number of oocysts shed seems not to be related with seroconvertion, because a puppy that shed 504,400 oocysts did not develop antibodies as observed by Gondim et al. (2005) and on the other hand, some dogs with high antibody titres had no detectable oocysts in the faeces (McAllister et al., 1998). The potential of N. caninum oocysts to infect sheep was experimentally demonstrated by O’Handley et al. (2002). The minimum infectious dose of N. caninum oocysts to infect sheep is not known, but it is proved to be low in calves, as few as 300 (Gondim et al., 2002). As dogs are frequently found in sheep stocks, oocysts shed by them can also be a natural source of N. caninum infection to sheep. Schares et al. (2005) study was the first demonstrating morphological differences by light microscopy between N. caninum isolates and other species, especially Hammondia heydorni. The results from the present paper are comparable because N. caninum unsporulated oocysts were also measured in concentrated sucrose solution using a computer image program. In that paper, the 75th percentiles of N. caninum oocyst isolates were 10.7 mm in length and 1.06 in length–width ratios. In Brazilian sheep isolate, from the present study, 63.5% of the oocysts were <11.00 mm in length, and most of the oocysts (88.5%) had a length–width ratio 1.05. Relatively to the rodents, N. caninum is nonpathogenic and rarely infective to Swiss white outbred mice (Lindsay and Dubey, 1989), and even immunosuppressed mice are resistant to oral feeding of oocysts (Lindsay et al., 1999), so it was expected that this group of rodents was not infected in the present experiment. Dubey and Lindsay (2000) demonstrate the high susceptibility of gerbils to oral infection with
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Fig. 1. Neospora caninum tissue cyst in the brain of a gerbil fed oocysts from dog experimentally infected with brain sheep. Fresh preparation.
N. caninum oocysts, but the pathogenicity has been variable. Gerbils became sick or died at 6–13 days after feeding 1000 oocysts (Dubey and Lindsay, 2000), or remained clinically normal (Basso et al., 2001; Schares et al., 2005). In the present study both gerbils became infected and did not develop disease. Basso et al. (2001) found tissue cysts measuring 26 mm 28 mm and 27 mm 32 mm in sections of brain of one gerbil. Gerbils were necropsied 63–75 days PI. Well-developed N. caninum tissue cysts have also been found in gerbils 60 days PI in the present study, the largest of them measuring 47.07 mm 53.91 mm in fresh preparation (Fig. 1). All four tissue cysts found in gerbil brains were thick walled (>1 mm). The large vesper mouse, C. callosus, is a rodent species from South America that has been used as a laboratory animal. It is highly susceptible to T. gondii infection, and has been used as an appropriate alternative model to experimental toxoplasmosis (Favoreto-Junior et al., 1998). In the present paper, the susceptibility of the wild mouse C. callosus to oral infection with N. caninum was demonstrated by molecular detection of N. caninum DNA in its brain, but no tissue cyst were observed by light microscopy, probably because the small amount of material examined. Unfortunately, the amount of oocysts was not enough to inoculate more animals. The use of this rodent as an alternative model to neosporosis should be better investigated. Attempts to isolate the parasite in cell cultures were not successful, probably because the tissues from Dog 1 and from rodents were just homogenized in a mortar and not digested. New attempts are being made to isolate the parasite in cell culture from gerbils orally inoculated with oocysts from the Brazilian sheep isolate.
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