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Neospora caninum is a cause of perinatal mortality in axis deer (Axis axis)
Veterinary Parasitology 199 (2014) 255–258
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Neospora caninum is a cause of perinatal mortality in axis deer (Axis axis) Walter Basso a,∗ , Gastón Moré b,c , Maria Alejandra Quiroga d , Diego Balducchi e , Gereon Schares f , Maria Cecilia Venturini b a
Institute of Parasitology, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 266a, 8057 Zurich, Switzerland Laboratory of Immunparasitology, Faculty of Veterinary Sciences, National University of La Plata, 60 y 118, 1900 La Plata, Argentina Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina d Department of Special Pathology, Faculty of Veterinary Sciences, National University of La Plata, 60 y 118, 1900 La Plata, Argentina e Zoo of La Plata, Paseo del Bosque, 1900 La Plata, Argentina f Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald, Insel Riems, Germany b c
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 18 October 2013 Accepted 27 October 2013 Keywords: Neospora caninum Axis axis Perinatal mortality Multilocus-microsatellite analysis
a b s t r a c t Neospora caninum is a worldwide distributed protozoan that may cause neuromuscular disease in dogs and reproductive failure in domestic and wild ruminants. One axis fawn (Axis axis) and four neonates from the same deer herd died at a zoo in Argentina within a four-month period. The fawn presented with dilatation of the anal sphincter at birth and incontinence, developed weakness and ataxia and died at 14 days of age. At necropsy, a mega formation of the distal large intestine was observed. Microscopically, non-suppurative encephalitis, suppurative bronchopneumonia, fibrin necrotic enteritis and degenerative changes in the liver were observed in hematoxilin and eosin-stained tissue sections, and thick-walled N. caninum-like cysts were observed in fresh brain samples. Serologic studies for N. caninum revealed an IFAT titer of 1:6400 in the fawn and 1:25, 1:400, 1:3200 and 1:6400 in the neonates. N. caninum DNA was detected in brain samples from the fawn and from one neonate by PCR, and the parasite was isolated in vitro from the fawn’ brain after passage through gerbils (Meriones unguiculatus) and gamma-interferon knock-out mice. N. caninum DNA obtained from the fawn, neonate and isolated parasites showed the same microsatellite pattern. This suggests a common infection source for both animals. The diagnosis of N. caninum infection was confirmed, suggesting its association with perinatal mortality in captive axis deer. To the best of our knowledge, this is the first report of clinical disease associated to N. caninum infection in axis deer and of isolation of the parasite from this wild ruminant species. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum infection is an important infectious cause of abortion, stillbirth and perinatal death in cattle and neuromuscular disease in dogs, and was also reported in other domestic and wild animal species. Vertical
∗ Corresponding author. Tel.: +41 44 635 8510; fax: +41 44 635 8907. E-mail address: [email protected] (W. Basso). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.10.020
transmission from an infected dam to the fetus, or postnatal transmission by ingestion of food or water contaminated with oocysts shed by definitive hosts may cause infection in ruminants (Dubey et al., 2007). So far, only domestic dogs, coyotes, Australian dingoes and gray wolves were confirmed as definitive hosts of N. caninum (Dubey and Schares, 2011). In cattle, N. caninum-associated abortion can have an endemic or an epidemic pattern. While endemic abortions have been associated with transplacental transmission of the parasite from chronically infected dams to the
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W. Basso et al. / Veterinary Parasitology 199 (2014) 255–258
Fig. 1. Neospora caninum tissue cyst in the brain of the axis fawn (fresh sample). Note the typical thick cyst wall.
fetuses after reactivation, epidemic abortion outbreaks are thought to occur after a primary oocyst-derived infection of pregnant dams (Dubey et al., 2007; Basso et al., 2010). Vertical transmission is considered the major route of transmission in cattle, whereas the epidemiological importance of horizontal transmission seems to vary regionally. In wild ruminants, the occurrence and importance of both ways of transmission are poorly understood. Most reports on N. caninum infection in wild ruminants consist of seroprevalence studies in asymptomatic animals, but there are only a few reports concerning clinical neosporosis in these species (Dubey et al., 2007; Dubey and Schares, 2011). The first observation on clinical neosporosis in a deer was made in a California black-tailed deer (Odocoileus hemionus columbianus) showing a generalized infection (Woods et al., 1994). Fatal cases were further reported in a full-term stillborn Eld deer (Cervus eldi siamensis) in a zoo in France (Dubey et al., 1996), in two full-term twin antelope calves (Tragelaphus imberbis) in a zoo in Germany (Peters et al., 2001) and in a captive-born 3-week-old fallow deer (Dama dama) in Switzerland (Soldati et al., 2004). In all cases, several organs were involved but the main changes consisted of non-suppurative encephalitis. In the United States, N. caninum infection seems to be prevalent in white-tailed deer (Odocoileus virginianus) as seroprevalences over 40% were observed (Dubey et al., 1999), and recently, congenital transmission was confirmed in this species (Dubey et al., 2013). It was assumed that there is an active sylvatic cycle between deer and wolves or coyotes in North America (Gondim, 2006; Dubey et al., 2011). Axis deer (Axis axis) inhabit wooden regions of India and Nepal and are bred in captivity in zoos and recreational hunting areas worldwide.
To the best of our knowledge, there are no reports of clinical or subclinical neosporosis in this deer species so far. 2. Case description One axis fawn (A. axis) at the zoo of La Plata, Argentina presented with dilatation of the anal sphincter at birth and incontinence. It developed weakness and ataxia during the first week of life and died at 14 days of age. A necropsy and histopathological examination were performed. Mega formation of distal part of the colon and rectum was observed, with fecal retention. Microscopically, non-suppurative encephalitis and gliosis, suppurative bronchopneumonia, fibrin-necrotic enteritis and degenerative changes in the liver were observed in Hematoxilin and Eosin (H&E) stained tissue sections, and thick-walled N. caninumlike cysts were observed in fresh brain samples (Fig. 1). Subsequently, serum samples collected from the heart during necropsy were serologically analyzed for antibodies against N. caninum and Toxoplasma gondii by indirect fluorescent antibody tests (IFAT) using a rabbit anti-bovine IgG fluorescein isothiocyanate conjugate, as described by Moré et al. (2009). A positive reaction for N. caninum (titer 1:6400) was observed, while antibodies against T. gondii were not detected (titer < 1:25). After DNA extraction from brain tissues using a commercial extraction kit (QIAmp DNA Mini Kit, QIAGEN GmbH, Hilden, Germany), a PCR for detection of N. caninum DNA was performed with the specific primers Np6+ and Np21+ (Müller et al., 1996) amplifying a 328 bp fragment. For parasite isolation, brain homogenate of the fawn in 0.9% saline solution was bioassayed in five gerbils (Meriones unguiculatus); N. caninum
W. Basso et al. / Veterinary Parasitology 199 (2014) 255–258
257
Table 1 Specific anti-N. caninum and anti-T. gondii antibody titers (IFAT) and PCR results for N. caninum in captive axis deer. Animal Nr. Adult animals 1 2 3 4 5 6 7 8 9 10 11 12 13
IFAT titer N. caninum
IFAT titer T. gondii
PCR for N. caninum
1:100 <1:25 1:800 1:200 1:800 1:800 1:800 1:800 ≥1:6400 1:800 1:800 1:200 1:400
<1:25 <1:25 1:25 1:50 <1:25 1:100 1:50 1:25 1:50 1:50 1:50 1:100 <1:25
– – – – – – – – – – – – –
Neonates 1 2 3 4
1:25a 1:400 1:3200a 1:6400a
<1:25a <1:25 1:50a <1:25a
Negative Negative Positive Negative
Fawn 1
1:6400
<1:25
Positive
a
Precolostral serum samples; –, not performed.
infection of these gerbils was serologically confirmed (IFAT titer ≥ 800) at 75 days after inoculation. Brain homogenate from one of the gerbils was reinoculated intraperitoneally into a gamma-interferon knock-out mouse (C.129S7 (B6)Ifngtm1Ts/J, The Jackson Laboratory, Bar Harbor, Maine, USA), and the parasites were successfully transferred to MARC 145 cell cultures using a homogenate of mouse brain and heart after 12 days. The isolate was named NC-Axis. The animal experiments were authorized by and performed in accordance to the guidelines of the FCV UNLP, Argentina and the Ministerium für Landwirtschaft, Umweltschutz und Raumordnung of the German Federal State of Brandenburg (see Table 1). Additionally, 13 asymptomatic adult axis deer that were housed in the same enclosure together with the infected fawn were serologically tested for antibodies against N. caninum and T. gondii. Twelve out of 13 animals had positive titers (1:200 to ≥1:6400) to N. caninum, and nine animals showed low antibody reactions against T. gondii antigens (IFAT titer 1:25–1:100) (Table 1). Four neonates (1–2 days old) that died within a period of two months before or after the death of the fawn in this same enclosure, tested serologically positive for N. caninum and negative for T. gondii infections (Table 1). N. caninum DNA was detected by PCR (Müller et al., 1996) in brain samples of one of the four neonates (Table 1). Unfortunately, vertical transmission could not be analyzed in more detail because a precise association of the offspring to the adult animals was not possible. N. caninum DNA from brain tissues of the fawn, the neonate and from the parasites isolated in vitro (isolate NC-Axis) were further characterized by multilocusmicrosatellite analysis based on the N. caninum microsatellites (MS) 1B, 2, 3, 5, 6A, 6B, 10, 12 and 21, using nested-PCR techniques followed by sequencing (MS2 and 10) or length determination (MS3, 5, 6A, 6B, 7, 12 and 21) of the amplified sequences as previously described (Basso et al., 2009,
2010). All nine N. caninum microsatellite markers could be amplified from the three DNA samples. The microsatellite pattern obtained was identical in all three samples (Table 2) but different from other reported N. caninum isolates so far. 3. Discussion The herd of axis deer entered the zoo 15 years ago and no new animals had been introduced during the past 11 years. No outbreaks of perinatal mortality were recorded in the past. Whether the mega formation of colon and rectum observed in the fawn in the present study were directly caused by N. caninum could not be confirmed, however, the presence of megaoesophagus was previously reported in dogs with clinical neosporosis (Barber and Trees, 1996; Basso et al., 2005). Axis deer have an epitheliochorial cotyledonary placenta (Hamilton et al., 1960) that prevents the passage of antibodies to the fetus. Therefore, the finding of antibodies against N. caninum in precolostral serum is indicative of a transplacental infection. The four neonates in this study were weak and died between one and two days of age, and only in Neonate nr. 2 was there evidence of ingestion of colostrum. Therefore, it can be assumed that the detection of antibodies in at least the remaining three neonates would strongly suggest intrauterine infections. This was confirmed by PCR in Neonate nr. 3. Isolation of viable N. caninum is considered difficult. So far, only a few successful in vitro isolations of the parasite have been performed worldwide (Dubey et al., 2007). N. caninum has been isolated so far from dogs, wolves, cattle, horses, sheep, water buffaloes, bisons and white tailed deer (Dubey and Schares, 2011). In this study, we could isolate N. caninum in vitro after passaging through gerbils and a gamma interferon knock-out mouse. These mice lack the gene to produce gamma-interferon and are especially susceptible to the infection with Apicomplexa parasites.
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Table 2 Results of the multilocus-microsatellite analysis from N. caninum DNA derived from brain tissues of the axis fawn, brain of neonate (nr. 3) and from in vitro cultured tachyzoites isolated from the fawn by bioassay (all 3 samples displayed the same microsatellite pattern). N. caninum microsatellites 1B
2
3
5
6A
6B
7
10
12
21
Sequence
(AT)8 AC (AT)3
(AT)6 TTGT ATC(AT)11 GT(AT)2
(AT)11
(TA)15 TGTA
(TA)13
(AT)11
(TA)11
(ACT)6 (AGA)15 (TGA)7
(GT)15
(TACA)9
Length (bp)
24
47
22
34
26
22
22
84
30
36
The direct isolation (without bioassay) into cell culture is often unsuccessful (Dubey and Schares, 2006). The use of microsatellite-DNA sequence analysis has proved to be a valuable tool to study genetic diversity in N. caninum (Dubey and Schares, 2011). Recently, it was shown that a common N. caninum microsatellite pattern prevailed in all fetuses from individual epidemic abortion outbreaks in cattle, and that this pattern was unique for each herd, supporting the hypothesis of an infection from a common point source in each outbreak (Basso et al., 2010). In this study, transplacental N. caninum infections in several animals of one herd were recorded during a short period of time, and the finding that N. caninum isolated from both the fawn and from the Neonate nr. 3 displayed the same microsatellite pattern are highly suggestive of a recent infection from a common source. So far, the only demonstrated postnatal mode of infection in ruminants under natural conditions is the ingestion of sporulated oocysts from the environment (Dubey et al., 2007). Among the wild canid species kept at the zoo at that moment (i.e. wolves (Canis lupus), maned wolves (Chrysocyon brachyurus) and Pampas foxes (Lycalopex gymnocercus), only wolves have been confirmed as definitive hosts for N. caninum so far (Dubey et al., 2011). It is also possible that oocysts had been introduced with contaminated grass or hay that was used to feed the deer. However, there was also some evidence of stray dogs entering the zoo by night that could have accounted for environmental contamination with N. caninum oocysts. The high seroprevalence for N. caninum observed in the adult deer (92%) and detection of transplacental infections, suggest an elevated level of transmission in the herd. To the best of our knowledge, this is the first report of clinical disease associated to N. caninum infection in axis deer and in vitro isolation of N. caninum from this wild ruminant species. Acknowledgements The authors would like to thank Lais Pardini, Alejandra Larsen and Pavlo Maksimov for their help with the experiments, M. del Carmen Villanueva for her support at the zoo, and Susann Schares, Andrea Bärwald and Isidoro Ercoli for their excellent technical assistance. References Barber, J.S., Trees, A.J., 1996. Clinical aspects of 27 cases of neosporosis in dogs. Vet. Rec. 139, 439–443.
Basso, W., Venturini, M.C., Bacigalupe, D., Kienast, M., Unzaga, J.M., Larsen, A., Machuca, M., Venturini, L., 2005. Confirmed clinical Neospora caninum infection in a boxer puppy from Argentina. Vet. Parasitol. 131, 299–303. Basso, W., Schares, S., Bärwald, A., Herrmann, D.C., Conraths, F.J., Pantchev, N., Vrhovec, M.G., Schares, G., 2009. Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culturederived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers. Vet. Parasitol. 160, 43–50. Basso, W., Schares, S., Minke, L., Bärwald, A., Maksimov, A., Peters, M., Schulze, C., Müller, M., Conraths, F.J., Schares, G., 2010. Microsatellite typing and avidity analysis suggest a common source of infection in herds with epidemic Neospora caninum-associated bovine abortion. Vet. Parasitol. 173, 24–31. Dubey, J.P., Hollis, K., Romand, S., Thulliez, P., Kwok, O.C., Hungerford, L., Anchor, C., Etter, D., 1999. High prevalence of antibodies to Neospora caninum in white-tailed deer (Odocoileus virginianus). Int. J. Parasitol. 29, 1709–1711. Dubey, J.P., Jenkins, M.C., Kwok, O.C.H., Ferreira, L.R., Choudhary, S., Verma, S.K., Villena, I., Butler, E., Carstensen, M., 2013. Congenital transmission of Neospora caninum in white-tailed deer (Odocoileus virginianus). Vet. Parasitol., http://dx.doi.org/10.1016/j.vetpar.2013.03. 004. Dubey, J.P., Jenkins, M.C., Rajendran, C., Miska, K., Ferreira, L.R., Martins, J., Kwok, O.C., Choudhary, S., 2011. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet. Parasitol. 181, 382–387. Dubey, J.P., Rigoulet, J., Lagourette, P., George, C., Longeart, L., LeNet, J.L., 1996. Fatal transplacental neosporosis in a deer (Cervus eldi siamensis). J. Parasitol. 82, 338–339. Dubey, J.P., Schares, G., 2006. Diagnosis of bovine neosporosis. Vet. Parasitol. 140, 1–34. Dubey, J.P., Schares, G., 2011. Neosporosis in animals – the last five years. Vet. Parasitol. 180, 90–108. Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbiol. Rev. 20, 323–367. Gondim, L.F., 2006. Neospora caninum in wildlife. Trends Parasitol. 22, 247–252. Hamilton, W.J., Harrison, R.J., Young, B.A., 1960. Aspects of placentation in certain Cervidae. J. Anat. 94, 1–33. Moré, G., Bacigalupe, D., Basso, W., Rambeaud, M., Beltrame, F., Ramirez, B., Venturini, M.C., Venturini, L., 2009. Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle. Vet. Parasitol. 160, 51–54. Müller, N., Zimmermann, V., Hentrich, B., Gottstein, B., 1996. Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay. J. Clin. Microbiol. 34, 2850–2852. Peters, M., Wohlsein, P., Knieriem, A., Schares, G., 2001. Neospora caninum infection associated with stillbirths in captive antelopes (Tragelaphus imberbis). Vet. Parasitol. 97, 153–157. Soldati, S., Kiupel, M., Wise, A., Maes, R., Botteron, C., Robert, N., 2004. Meningoencephalomyelitis caused by Neospora caninum in a juvenile fallow deer (Dama dama). J. Vet. Med. 51, 280–283. Woods, L.W., Anderson, M.L., Swift, P.K., Sverlow, K.W., 1994. Systemic neosporosis in a California black-tailed deer (Odocoileus hemionus columbianus). J. Vet. Diagn. Invest. 6, 508–510.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Neospora caninum is a cause of perinatal mortality in axis deer (Axis axis) Walter Basso a,∗ , Gastón Moré b,c , Maria Alejandra Quiroga d , Diego Balducchi e , Gereon Schares f , Maria Cecilia Venturini b a
Institute of Parasitology, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 266a, 8057 Zurich, Switzerland Laboratory of Immunparasitology, Faculty of Veterinary Sciences, National University of La Plata, 60 y 118, 1900 La Plata, Argentina Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina d Department of Special Pathology, Faculty of Veterinary Sciences, National University of La Plata, 60 y 118, 1900 La Plata, Argentina e Zoo of La Plata, Paseo del Bosque, 1900 La Plata, Argentina f Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald, Insel Riems, Germany b c
a r t i c l e
i n f o
Article history: Received 29 July 2013 Received in revised form 18 October 2013 Accepted 27 October 2013 Keywords: Neospora caninum Axis axis Perinatal mortality Multilocus-microsatellite analysis
a b s t r a c t Neospora caninum is a worldwide distributed protozoan that may cause neuromuscular disease in dogs and reproductive failure in domestic and wild ruminants. One axis fawn (Axis axis) and four neonates from the same deer herd died at a zoo in Argentina within a four-month period. The fawn presented with dilatation of the anal sphincter at birth and incontinence, developed weakness and ataxia and died at 14 days of age. At necropsy, a mega formation of the distal large intestine was observed. Microscopically, non-suppurative encephalitis, suppurative bronchopneumonia, fibrin necrotic enteritis and degenerative changes in the liver were observed in hematoxilin and eosin-stained tissue sections, and thick-walled N. caninum-like cysts were observed in fresh brain samples. Serologic studies for N. caninum revealed an IFAT titer of 1:6400 in the fawn and 1:25, 1:400, 1:3200 and 1:6400 in the neonates. N. caninum DNA was detected in brain samples from the fawn and from one neonate by PCR, and the parasite was isolated in vitro from the fawn’ brain after passage through gerbils (Meriones unguiculatus) and gamma-interferon knock-out mice. N. caninum DNA obtained from the fawn, neonate and isolated parasites showed the same microsatellite pattern. This suggests a common infection source for both animals. The diagnosis of N. caninum infection was confirmed, suggesting its association with perinatal mortality in captive axis deer. To the best of our knowledge, this is the first report of clinical disease associated to N. caninum infection in axis deer and of isolation of the parasite from this wild ruminant species. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum infection is an important infectious cause of abortion, stillbirth and perinatal death in cattle and neuromuscular disease in dogs, and was also reported in other domestic and wild animal species. Vertical
∗ Corresponding author. Tel.: +41 44 635 8510; fax: +41 44 635 8907. E-mail address: [email protected] (W. Basso). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.10.020
transmission from an infected dam to the fetus, or postnatal transmission by ingestion of food or water contaminated with oocysts shed by definitive hosts may cause infection in ruminants (Dubey et al., 2007). So far, only domestic dogs, coyotes, Australian dingoes and gray wolves were confirmed as definitive hosts of N. caninum (Dubey and Schares, 2011). In cattle, N. caninum-associated abortion can have an endemic or an epidemic pattern. While endemic abortions have been associated with transplacental transmission of the parasite from chronically infected dams to the
256
W. Basso et al. / Veterinary Parasitology 199 (2014) 255–258
Fig. 1. Neospora caninum tissue cyst in the brain of the axis fawn (fresh sample). Note the typical thick cyst wall.
fetuses after reactivation, epidemic abortion outbreaks are thought to occur after a primary oocyst-derived infection of pregnant dams (Dubey et al., 2007; Basso et al., 2010). Vertical transmission is considered the major route of transmission in cattle, whereas the epidemiological importance of horizontal transmission seems to vary regionally. In wild ruminants, the occurrence and importance of both ways of transmission are poorly understood. Most reports on N. caninum infection in wild ruminants consist of seroprevalence studies in asymptomatic animals, but there are only a few reports concerning clinical neosporosis in these species (Dubey et al., 2007; Dubey and Schares, 2011). The first observation on clinical neosporosis in a deer was made in a California black-tailed deer (Odocoileus hemionus columbianus) showing a generalized infection (Woods et al., 1994). Fatal cases were further reported in a full-term stillborn Eld deer (Cervus eldi siamensis) in a zoo in France (Dubey et al., 1996), in two full-term twin antelope calves (Tragelaphus imberbis) in a zoo in Germany (Peters et al., 2001) and in a captive-born 3-week-old fallow deer (Dama dama) in Switzerland (Soldati et al., 2004). In all cases, several organs were involved but the main changes consisted of non-suppurative encephalitis. In the United States, N. caninum infection seems to be prevalent in white-tailed deer (Odocoileus virginianus) as seroprevalences over 40% were observed (Dubey et al., 1999), and recently, congenital transmission was confirmed in this species (Dubey et al., 2013). It was assumed that there is an active sylvatic cycle between deer and wolves or coyotes in North America (Gondim, 2006; Dubey et al., 2011). Axis deer (Axis axis) inhabit wooden regions of India and Nepal and are bred in captivity in zoos and recreational hunting areas worldwide.
To the best of our knowledge, there are no reports of clinical or subclinical neosporosis in this deer species so far. 2. Case description One axis fawn (A. axis) at the zoo of La Plata, Argentina presented with dilatation of the anal sphincter at birth and incontinence. It developed weakness and ataxia during the first week of life and died at 14 days of age. A necropsy and histopathological examination were performed. Mega formation of distal part of the colon and rectum was observed, with fecal retention. Microscopically, non-suppurative encephalitis and gliosis, suppurative bronchopneumonia, fibrin-necrotic enteritis and degenerative changes in the liver were observed in Hematoxilin and Eosin (H&E) stained tissue sections, and thick-walled N. caninumlike cysts were observed in fresh brain samples (Fig. 1). Subsequently, serum samples collected from the heart during necropsy were serologically analyzed for antibodies against N. caninum and Toxoplasma gondii by indirect fluorescent antibody tests (IFAT) using a rabbit anti-bovine IgG fluorescein isothiocyanate conjugate, as described by Moré et al. (2009). A positive reaction for N. caninum (titer 1:6400) was observed, while antibodies against T. gondii were not detected (titer < 1:25). After DNA extraction from brain tissues using a commercial extraction kit (QIAmp DNA Mini Kit, QIAGEN GmbH, Hilden, Germany), a PCR for detection of N. caninum DNA was performed with the specific primers Np6+ and Np21+ (Müller et al., 1996) amplifying a 328 bp fragment. For parasite isolation, brain homogenate of the fawn in 0.9% saline solution was bioassayed in five gerbils (Meriones unguiculatus); N. caninum
W. Basso et al. / Veterinary Parasitology 199 (2014) 255–258
257
Table 1 Specific anti-N. caninum and anti-T. gondii antibody titers (IFAT) and PCR results for N. caninum in captive axis deer. Animal Nr. Adult animals 1 2 3 4 5 6 7 8 9 10 11 12 13
IFAT titer N. caninum
IFAT titer T. gondii
PCR for N. caninum
1:100 <1:25 1:800 1:200 1:800 1:800 1:800 1:800 ≥1:6400 1:800 1:800 1:200 1:400
<1:25 <1:25 1:25 1:50 <1:25 1:100 1:50 1:25 1:50 1:50 1:50 1:100 <1:25
– – – – – – – – – – – – –
Neonates 1 2 3 4
1:25a 1:400 1:3200a 1:6400a
<1:25a <1:25 1:50a <1:25a
Negative Negative Positive Negative
Fawn 1
1:6400
<1:25
Positive
a
Precolostral serum samples; –, not performed.
infection of these gerbils was serologically confirmed (IFAT titer ≥ 800) at 75 days after inoculation. Brain homogenate from one of the gerbils was reinoculated intraperitoneally into a gamma-interferon knock-out mouse (C.129S7 (B6)Ifngtm1Ts/J, The Jackson Laboratory, Bar Harbor, Maine, USA), and the parasites were successfully transferred to MARC 145 cell cultures using a homogenate of mouse brain and heart after 12 days. The isolate was named NC-Axis. The animal experiments were authorized by and performed in accordance to the guidelines of the FCV UNLP, Argentina and the Ministerium für Landwirtschaft, Umweltschutz und Raumordnung of the German Federal State of Brandenburg (see Table 1). Additionally, 13 asymptomatic adult axis deer that were housed in the same enclosure together with the infected fawn were serologically tested for antibodies against N. caninum and T. gondii. Twelve out of 13 animals had positive titers (1:200 to ≥1:6400) to N. caninum, and nine animals showed low antibody reactions against T. gondii antigens (IFAT titer 1:25–1:100) (Table 1). Four neonates (1–2 days old) that died within a period of two months before or after the death of the fawn in this same enclosure, tested serologically positive for N. caninum and negative for T. gondii infections (Table 1). N. caninum DNA was detected by PCR (Müller et al., 1996) in brain samples of one of the four neonates (Table 1). Unfortunately, vertical transmission could not be analyzed in more detail because a precise association of the offspring to the adult animals was not possible. N. caninum DNA from brain tissues of the fawn, the neonate and from the parasites isolated in vitro (isolate NC-Axis) were further characterized by multilocusmicrosatellite analysis based on the N. caninum microsatellites (MS) 1B, 2, 3, 5, 6A, 6B, 10, 12 and 21, using nested-PCR techniques followed by sequencing (MS2 and 10) or length determination (MS3, 5, 6A, 6B, 7, 12 and 21) of the amplified sequences as previously described (Basso et al., 2009,
2010). All nine N. caninum microsatellite markers could be amplified from the three DNA samples. The microsatellite pattern obtained was identical in all three samples (Table 2) but different from other reported N. caninum isolates so far. 3. Discussion The herd of axis deer entered the zoo 15 years ago and no new animals had been introduced during the past 11 years. No outbreaks of perinatal mortality were recorded in the past. Whether the mega formation of colon and rectum observed in the fawn in the present study were directly caused by N. caninum could not be confirmed, however, the presence of megaoesophagus was previously reported in dogs with clinical neosporosis (Barber and Trees, 1996; Basso et al., 2005). Axis deer have an epitheliochorial cotyledonary placenta (Hamilton et al., 1960) that prevents the passage of antibodies to the fetus. Therefore, the finding of antibodies against N. caninum in precolostral serum is indicative of a transplacental infection. The four neonates in this study were weak and died between one and two days of age, and only in Neonate nr. 2 was there evidence of ingestion of colostrum. Therefore, it can be assumed that the detection of antibodies in at least the remaining three neonates would strongly suggest intrauterine infections. This was confirmed by PCR in Neonate nr. 3. Isolation of viable N. caninum is considered difficult. So far, only a few successful in vitro isolations of the parasite have been performed worldwide (Dubey et al., 2007). N. caninum has been isolated so far from dogs, wolves, cattle, horses, sheep, water buffaloes, bisons and white tailed deer (Dubey and Schares, 2011). In this study, we could isolate N. caninum in vitro after passaging through gerbils and a gamma interferon knock-out mouse. These mice lack the gene to produce gamma-interferon and are especially susceptible to the infection with Apicomplexa parasites.
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Table 2 Results of the multilocus-microsatellite analysis from N. caninum DNA derived from brain tissues of the axis fawn, brain of neonate (nr. 3) and from in vitro cultured tachyzoites isolated from the fawn by bioassay (all 3 samples displayed the same microsatellite pattern). N. caninum microsatellites 1B
2
3
5
6A
6B
7
10
12
21
Sequence
(AT)8 AC (AT)3
(AT)6 TTGT ATC(AT)11 GT(AT)2
(AT)11
(TA)15 TGTA
(TA)13
(AT)11
(TA)11
(ACT)6 (AGA)15 (TGA)7
(GT)15
(TACA)9
Length (bp)
24
47
22
34
26
22
22
84
30
36
The direct isolation (without bioassay) into cell culture is often unsuccessful (Dubey and Schares, 2006). The use of microsatellite-DNA sequence analysis has proved to be a valuable tool to study genetic diversity in N. caninum (Dubey and Schares, 2011). Recently, it was shown that a common N. caninum microsatellite pattern prevailed in all fetuses from individual epidemic abortion outbreaks in cattle, and that this pattern was unique for each herd, supporting the hypothesis of an infection from a common point source in each outbreak (Basso et al., 2010). In this study, transplacental N. caninum infections in several animals of one herd were recorded during a short period of time, and the finding that N. caninum isolated from both the fawn and from the Neonate nr. 3 displayed the same microsatellite pattern are highly suggestive of a recent infection from a common source. So far, the only demonstrated postnatal mode of infection in ruminants under natural conditions is the ingestion of sporulated oocysts from the environment (Dubey et al., 2007). Among the wild canid species kept at the zoo at that moment (i.e. wolves (Canis lupus), maned wolves (Chrysocyon brachyurus) and Pampas foxes (Lycalopex gymnocercus), only wolves have been confirmed as definitive hosts for N. caninum so far (Dubey et al., 2011). It is also possible that oocysts had been introduced with contaminated grass or hay that was used to feed the deer. However, there was also some evidence of stray dogs entering the zoo by night that could have accounted for environmental contamination with N. caninum oocysts. The high seroprevalence for N. caninum observed in the adult deer (92%) and detection of transplacental infections, suggest an elevated level of transmission in the herd. To the best of our knowledge, this is the first report of clinical disease associated to N. caninum infection in axis deer and in vitro isolation of N. caninum from this wild ruminant species. Acknowledgements The authors would like to thank Lais Pardini, Alejandra Larsen and Pavlo Maksimov for their help with the experiments, M. del Carmen Villanueva for her support at the zoo, and Susann Schares, Andrea Bärwald and Isidoro Ercoli for their excellent technical assistance. References Barber, J.S., Trees, A.J., 1996. Clinical aspects of 27 cases of neosporosis in dogs. Vet. Rec. 139, 439–443.
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