Acta Tropica 130 (2014) 88–93
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Molecular identification of Neospora caninum from calf/foetal brain tissue and among oocysts recovered from faeces of naturally infected dogs in southern Ethiopia K. Asmare a,b,∗, E. Skjerve b, J. Bekele a, D. Sheferaw a, T. Stachurska-Hagen b, L.J. Robertson b a b
School of Veterinary Medicine, Hawassa University, P.O. Box 5 Hawassa, Ethiopia Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146, 0033 Oslo, Norway
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 21 October 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Neospora caninum Dog Calf ITS1 Oocyst
a b s t r a c t This study sought to confirm and investigate further recently published information regarding the occurrence of Neospora caninum in cattle in Ethiopia and investigate infection in dogs, the canine definitive host, in this region. Faecal samples from 383 dogs in Hawassa, Ethiopia were examined by microscopy for Neospora-like oocysts, and positive samples then analysed by a molecular approach (DNA isolation, PCR and sequencing at the ITS1 gene). Brain tissue samples from four late term aborted foetuses, one congenitally defective calf (hind leg arthrogryposis) and placental tissue from cattle in the same area were also examined by the same molecular approach. All foetal, calf and placental tissue were associated with Neospora seropositive dams. A high prevalence of Neospora-like oocysts (11.5 m ± 1.5 m diameter) was observed in faecal samples from dogs (37 positive samples; 9.7% prevalence), and in 17 of these the identification was confirmed by PCR, giving a prevalence of confirmed infection of 4.4%. N. caninum DNA was also detected in all foetal and calf brain tissue samples. Sequencing revealed only minor differences among all PCR products, whether from oocysts or from brain tissue samples. These data provide molecular evidence of the presence of N. caninum infection in both dog and cattle in this region of Ethiopia. Moreover these findings highlight the role of dogs in maintaining and spreading the infection horizontally in the study area. The high frequency of N. caninum infection in household dogs as well as farm dogs is worthy of further investigation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum is a tissue cyst forming Apicomplexan parasite in the family Sarcocystidae. The parasite has a facultative heteroxenous life cycle with canids as definitive hosts and a range of mammals as intermediate hosts (Bishop et al., 2010; ChavezVelasquez et al., 2004; Ferroglio et al., 2007), but is frequently associated with cattle (Canada et al., 2004; Hall et al., 2006; Fish et al., 2007). Vertical infection is generally considered to be the primary mode of transmission between cattle, but is insufficient to sustain the infection in a herd (Davison et al., 1999; French et al., 1999). Thus horizontal infection is considered complementary to vertical transmission and enables the introduction of new infections into naive herds (King et al., 2011).
∗ Corresponding author at: Centre for Epidemiology and Biostatistics, Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146, 0033 Oslo, Norway. Tel.: +47 22597369. E-mail addresses: [email protected], [email protected], [email protected] (K. Asmare). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.10.017
The primary clinical manifestation of infection in cattle is abortion, most commonly at 5–7 months of gestation. Serological surveys from several countries have identified neosporosis as a major cause of bovine reproductive failure, with up to 50% of abortions on farms caused by N. caninum (Brickell et al., 2010; Dubey et al., 2007; Kul et al., 2009). Some data suggest that this infection may also be of importance in reproductive failure in small ruminants (Moreno et al., 2012; Howe et al., 2012). The occurrence of N. caninum infection in cattle in Ethiopia, risk factors for infection, and association of N. caninum infections in exotic and crossbred cattle with abortion and stillbirth were reported recently (Asmare et al., 2013a,b). These data were based solely on serological studies in cattle, and thus demonstrate exposure rather than infection. Diagnosis based on identification of the parasite itself, or its DNA using molecular tools, provides conclusive evidence of infection with N. caninum (Pedraza-Diaz et al., 2009; Fish et al., 2007). Additionally, molecular investigations of N. caninum from both intermediate and definitive hosts can be instrumental in unravelling the epidemiology of this infection (Beck et al., 2009; Regidor-Cerrillo et al., 2006). Molecular tools can also be used to distinguish between different strains of parasite and to obtain
K. Asmare et al. / Acta Tropica 130 (2014) 88–93
data on establishment and spread of infection, as well as transmission routes. The molecular diagnostic methods used are often based on those described for the related parasite Toxoplasma gondii. These include analysis of the potentially polymorphic markers such as the ribosomal DNA sequences from the 18S subunit, surface antigen and dense granule protein genes and the internal transcribed spacer 1 (ITS1) region of rDNA (Pedraza-Diaz et al., 2009). In the study described here, analysis at the ITS1 gene of rDNA was used to examine tissues from potentially infected calves/foetuses and from oocysts isolated from dog faecal samples. This gene has been used extensively in phylogenetic studies in several species including N. caninum (Beck et al., 2009; Gondim et al., 2004). Furthermore, it has also been used to differentiate oocysts of N. caninum from closely related parasite Hammondia heydorni that has morphologically identical oocysts (Slapeta et al., 2002). 2. Materials and methods 2.1. Study design and sampling 2.1.1. Dog samples In collaboration with the district veterinary department, a list of dairy farms in Hawassa, Ethiopia was prepared, most of which were known to own dogs, and these were requested to provide dog faecal samples. Dog owners in residential areas were also contacted with the same request. The sample size was estimated based on 13.3% expected prevalence (Asmare et al., 2013b), 95% confidence level and 5% desired precision. Accordingly, samples from a minimum of 178 dogs had to be collected. However, a total of 383 faecal samples were collected from 165 farm dogs and 218 household dogs. The dogs were owned by 137 individuals, of whom 55 owned a dairy farm. Dog owners participating in the study were briefed on the study objectives and provided with bottles for collection of faecal samples at defecation. Samples were transported to the School of Veterinary Medicine at Hawassa University in a cold box, and kept at 4 ◦ C until analysis (a maximum of 4 days following collection). Dog owners were also interviewed to obtain information on type of dog (farm dog or household), age of dog, sex, and level of control (partial or fully free). Dogs with partial control are chained during the day and released during the night. 2.1.2. Bovine tissue samples Farm owners with herds identified as having seropositive cattle were advised via the district veterinary department to contact the research team if abortion or congenital abnormality was encountered at full-term parturition, or if any of their calves were born with physical abnormalities. On the basis of this advice, we were able to collect clinical material from four late-term aborted foetuses (two from one farm) and a congenitally defective calf (hind leg arthrogryposis). In addition, placental tissue from a cow with normal parturition was collected. All the dams were seropositive for N. caninum exposure. As brain is the most consistently affected organ (Dubey et al., 2006), foetal samples were collected from the cerebellum, cerebrum and midbrain. 2.2. Laboratory analyses 2.2.1. Oocyst recovery from dog faeces At the parasitology laboratory, School of Veterinary Medicine, Hawassa University, faecal samples were processed by a modified flotation technique, as described briefly below. Approximately 12 g faecal sample was suspended in 25 ml tap water and mixed thoroughly. The suspension was sieved through a strainer (0.5–0.8 mm mesh aperture) and the filtrate collected in centrifuge tubes and centrifuged at 3000 rpm for five min. The supernatant was decanted
89
and 2 g of sediment re-suspended in 10 ml of saturated salt solution (specific gravity, 1.2) and vortexed for 1 min. The suspension was overlaid with 5 ml tap water and centrifuged at 3000 rpm for 10 min. The interface was collected into two separate clean tubes and tap water was added to the top of the tubes and vortexed for 5 s. The mixture was centrifuged for 10 min at 3000 rpm. The last step was repeated once to wash out the salt. Aliquots of the sediment were examined for protozoan oocysts at 400× magnification. Oocysts with a diameter of 11.5 ± 1.5 m with morphological similarity to sporulated or non-sporulated N. caninum oocysts were noted as positive for Neospora-like oocysts (NLO). Samples considered positive or presumptive for NLO were transferred to 2 ml microcentrifuge tubes to which 1.5 ml of 75% ethanol was added. The samples were couriered to the Norwegian School of Veterinary Science (NSVS), Oslo, Norway. At the NSVS Parasitology laboratory, the samples were centrifuged at 3000 rpm for 5 min. After decanting the supernatant, the sediment was re-suspended in distilled water, vortexed and centrifuged for a second time. The procedure was repeated once again to wash out the alcohol and the sediment was examined at 400× magnification for NLO. All samples considered positive for NLO at this second examination were subjected to molecular investigation. 2.2.2. Preservation of tissue samples Following aseptic opening of the skull of the aborted foetuses and defective calf, samples weighing 25–50 g were taken from the cerebral cortex, cerebellum and midbrain. Haemorrhagic parts or any visible gross lesions of the tissue were deliberately incorporated in to the samples. The same aseptic procedure was followed for placental tissue. The tissue samples were transferred into 10 ml tubes and 75% ethanol was added until the tissue samples were completely covered. These samples were couriered to NSVS, along with the positive faecal samples described above. 2.2.3. DNA extraction from oocysts Samples that had been selected for molecular investigation were subjected to DNA extraction based on the method used for Cryptosporidium oocysts at NSVS using a QIAamp DNA mini kit (QIAGEN GmbH, Germany). The protocol involved a pre-incubation step in which the oocysts were resuspended in AL buffer and heated to 100 ◦ C in a heating block for an hour. 2.2.4. DNA extraction from tissue samples The tissue samples were cut into small pieces, and homogenised by hand using a pestle and mortar. From the homogenate, 25 mg were transferred into microcentrifuge tubes and DNA extracted using a QIAamp DNA mini kit (QIAGEN GmbH, Germany). The control DNA extraction was performed in a similar approach. Liver samples from cattle were spiked with N. caninum cell culture tachyzoites stock (1 × 107 tachyzoites/ml) (NC-Liverpool strain, kindly provided by Dr. Siv Klevar, Norwegian Veterinary Institute, and processed according to the protocol described here. 2.2.5. Polymerase chain reaction (PCR) PCR targeting the ITS1 gene in the ribosomal DNA was run on DNA extracts both from tissue and oocysts using the primer pair PN3 [5 -TACTACTCCCTGTGAGTTG-3 ] and PN4 [5 TCTCTTCCCTCAAACGCTA-3 ] (Holmdahl and Mattsson, 1996) using 2 l template DNA for each reaction. DNA extracted from N. caninum tachyzoites was used as positive control and molecular grade water as negative control, which followed each sample batch from DNA extraction onwards. Following a 2.5 min hot start at 94 ◦ C, the PCR was carried out with 50 cycles of 94 ◦ C for 40 s, 54 ◦ C for 1 min, and 72 ◦ C for 30 s. The PCR was completed with 3 min annealing at 72 ◦ C. PCR products were electrophoresed on 2% agarose and stained with SYBR Green. In order to increase the sensitivity, DNA
90
K. Asmare et al. / Acta Tropica 130 (2014) 88–93
Fig. 2. PCR products targeting the Internal Transcribed Spacer 1 (ITS1) gene of N. caninum in foetal and calf brain tissue. Fig. 1. PCR products targeting the Internal Transcribed Spacer 1 (ITS1) gene of Neospora caninum oocysts recovered from naturally infected dogs.
extraction and PCR was run repeatedly on the same samples for a minimum of 10 and maximum of 20 times, until the first positive result was detected. If none of these replicates resulted in a band of the correct size, the sample was considered negative. 2.2.6. PCR product purification and sequencing PCR products of positive samples at the ITS1 gene were purified (High Pure PCR product purification kit; Roche Diagnostics GmbH). Sequencing was performed in both directions by a commercial company (Eurofins MWG Operon, Germany). 2.2.7. Data analysis Sequences were edited, manually checked and assembled using DNAStar software (www.dnastar.com), and compared with sequences of N. caninum available in GenBank by a BLAST search. Six representative new sequences were deposited in GenBank with accession numbers from KC461931 to KC461936. Sequences were aligned using CLUSTALW in BioEdit. Descriptive statistics with 95% confidence interval estimates were conducted on questionnaire and coprological data using STATA 11 (SE for Windows, StataCorp, College Station, TX).
tissue samples spiked with 5 l N. caninum Liverpool strain tachyzoites gave positive bands at 1:10, 1:50, 1:100 and 1:1000 dilutions; the 1:50 dilution was used as a positive control throughout the PCR investigation. 3.3. Sequence analysis Sequences obtained from the ITS1 gene PCR were aligned with standard sequences obtained from GenBank as follows (Accession numbers provided in parentheses): NC-Liverpool (EU564166), NC-Bahia (AY259043), NC-Illinois (AY259041) and NC-Nowra (AF338411). Alignment with our sequences provides confirmation that both dogs and cattle were infected with N. caninum. With the exception of three DNA isolates obtained from oocysts isolated from dog faecal samples (NCD162, NCD335 and NCD380), the sequences obtained from all the other positive samples, whether acquired from oocysts in dog faeces or from foetal or calf brain tissue, were identical to one another. It should be noted that for all our samples, clear double peaks were seen on the electropherograms at positions 247 and 249 (Fig. 3). The three dog-derived samples that differed from the other samples demonstrated only minor SNPs or double peaks. 4. Discussion
3. Results 3.1. Detection of Neospora in dog faeces Among the 383 dog faecal samples (218 household and 165 farm dogs) from in and around Hawassa, Neospora-like oocysts (NLO) were detected in 37 (9.7%) by coproscopy (Table 1). Of those 37 samples considered positive by microscopy, 17 (46%) were confirmed to be positive for N. caninum by PCR at the ITS1 gene (Fig. 1), indicating a minimum of 4.4% prevalence. Most of the positive samples (by both microscopy and PCR) were from dogs less than one year old (P < 0.05). N. caninum oocysts were detected in faecal samples from both farm dogs and household dogs, and no significant difference in prevalence was observed between these two groups (P > 0.05). Similarly there was no significant difference in prevalence between sex and control categories. 3.2. Detection of Neospora DNA in tissue samples Six tissue samples (brain tissue from 4 late term aborted foetuses and from a 2 month old congenitally defective calf, and a placental tissue sample following normal parturition), all from dams with positive serological status for N. caninum antibody, were examined by PCR. Neospora caninum DNA was detected at least once in all the brain tissue samples (Fig. 2), but not in the placental tissue. The liver
A high prevalence of N. caninum infection, along with a strong association with reproductive disorders, has recently been reported in Ethiopian dairy and breeding cattle (Asmare et al., 2013a,b). Additionally, Neospora infection in cattle was shown to be associated with an extensive presence of farm dogs (Asmare et al., 2013b). However, in the previous studies, pathological or molecular evidence of infection with N. caninum in cattle was not provided, and nor was any evidence of infection in dogs. The current study is built upon these results, and not only provides molecular evidence of N. caninum infection in aborted foetuses and a congenitally deformed calf, but also demonstrates excretion of N. caninum oocysts in dog faeces, with initial identification by microscopy and confirmation by molecular methods. Although the seroprevalence of N. caninum infection in dogs can be relatively high (Bruhn et al., 2012; King et al., 2012; Ghalmi et al., 2009; Mitrea et al., 2013), the prevalence of Neospora excretion in faeces (as detected by coproscopy or molecular techniques) tends to be lower, with previous studies reporting prevalences ranging from 0.03% in Germany (Schares et al., 2005) to 1.15% in Iran (Razmi, 2009) and 1.5% in Australia (King et al., 2012). The relatively high prevalence of PCR-confirmed Neospora oocyst shedding in dogs found in our study (4.4%), may reflect the scavenging nature, or in some cases entire survival, of dogs on untreated offal in this region, and is more similar to the oocyst excretion prevalence reported
K. Asmare et al. / Acta Tropica 130 (2014) 88–93
91
Table 1 Prevalence of Neospora-like oocysts (NLO) in dog faeces from Hawassa, Southern Ethiopia: results from coproscopy and PCR (confirmed N. caninum). Variables
Category
Number examined
Coproscopy (NLO)
PCR (confirmed N. caninum)
Type of ownership
Farm dog Household dog
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Molecular identification of Neospora caninum from calf/foetal brain tissue and among oocysts recovered from faeces of naturally infected dogs in southern Ethiopia K. Asmare a,b,∗, E. Skjerve b, J. Bekele a, D. Sheferaw a, T. Stachurska-Hagen b, L.J. Robertson b a b
School of Veterinary Medicine, Hawassa University, P.O. Box 5 Hawassa, Ethiopia Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146, 0033 Oslo, Norway
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 21 October 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Neospora caninum Dog Calf ITS1 Oocyst
a b s t r a c t This study sought to confirm and investigate further recently published information regarding the occurrence of Neospora caninum in cattle in Ethiopia and investigate infection in dogs, the canine definitive host, in this region. Faecal samples from 383 dogs in Hawassa, Ethiopia were examined by microscopy for Neospora-like oocysts, and positive samples then analysed by a molecular approach (DNA isolation, PCR and sequencing at the ITS1 gene). Brain tissue samples from four late term aborted foetuses, one congenitally defective calf (hind leg arthrogryposis) and placental tissue from cattle in the same area were also examined by the same molecular approach. All foetal, calf and placental tissue were associated with Neospora seropositive dams. A high prevalence of Neospora-like oocysts (11.5 m ± 1.5 m diameter) was observed in faecal samples from dogs (37 positive samples; 9.7% prevalence), and in 17 of these the identification was confirmed by PCR, giving a prevalence of confirmed infection of 4.4%. N. caninum DNA was also detected in all foetal and calf brain tissue samples. Sequencing revealed only minor differences among all PCR products, whether from oocysts or from brain tissue samples. These data provide molecular evidence of the presence of N. caninum infection in both dog and cattle in this region of Ethiopia. Moreover these findings highlight the role of dogs in maintaining and spreading the infection horizontally in the study area. The high frequency of N. caninum infection in household dogs as well as farm dogs is worthy of further investigation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum is a tissue cyst forming Apicomplexan parasite in the family Sarcocystidae. The parasite has a facultative heteroxenous life cycle with canids as definitive hosts and a range of mammals as intermediate hosts (Bishop et al., 2010; ChavezVelasquez et al., 2004; Ferroglio et al., 2007), but is frequently associated with cattle (Canada et al., 2004; Hall et al., 2006; Fish et al., 2007). Vertical infection is generally considered to be the primary mode of transmission between cattle, but is insufficient to sustain the infection in a herd (Davison et al., 1999; French et al., 1999). Thus horizontal infection is considered complementary to vertical transmission and enables the introduction of new infections into naive herds (King et al., 2011).
∗ Corresponding author at: Centre for Epidemiology and Biostatistics, Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146, 0033 Oslo, Norway. Tel.: +47 22597369. E-mail addresses: [email protected], [email protected], [email protected] (K. Asmare). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.10.017
The primary clinical manifestation of infection in cattle is abortion, most commonly at 5–7 months of gestation. Serological surveys from several countries have identified neosporosis as a major cause of bovine reproductive failure, with up to 50% of abortions on farms caused by N. caninum (Brickell et al., 2010; Dubey et al., 2007; Kul et al., 2009). Some data suggest that this infection may also be of importance in reproductive failure in small ruminants (Moreno et al., 2012; Howe et al., 2012). The occurrence of N. caninum infection in cattle in Ethiopia, risk factors for infection, and association of N. caninum infections in exotic and crossbred cattle with abortion and stillbirth were reported recently (Asmare et al., 2013a,b). These data were based solely on serological studies in cattle, and thus demonstrate exposure rather than infection. Diagnosis based on identification of the parasite itself, or its DNA using molecular tools, provides conclusive evidence of infection with N. caninum (Pedraza-Diaz et al., 2009; Fish et al., 2007). Additionally, molecular investigations of N. caninum from both intermediate and definitive hosts can be instrumental in unravelling the epidemiology of this infection (Beck et al., 2009; Regidor-Cerrillo et al., 2006). Molecular tools can also be used to distinguish between different strains of parasite and to obtain
K. Asmare et al. / Acta Tropica 130 (2014) 88–93
data on establishment and spread of infection, as well as transmission routes. The molecular diagnostic methods used are often based on those described for the related parasite Toxoplasma gondii. These include analysis of the potentially polymorphic markers such as the ribosomal DNA sequences from the 18S subunit, surface antigen and dense granule protein genes and the internal transcribed spacer 1 (ITS1) region of rDNA (Pedraza-Diaz et al., 2009). In the study described here, analysis at the ITS1 gene of rDNA was used to examine tissues from potentially infected calves/foetuses and from oocysts isolated from dog faecal samples. This gene has been used extensively in phylogenetic studies in several species including N. caninum (Beck et al., 2009; Gondim et al., 2004). Furthermore, it has also been used to differentiate oocysts of N. caninum from closely related parasite Hammondia heydorni that has morphologically identical oocysts (Slapeta et al., 2002). 2. Materials and methods 2.1. Study design and sampling 2.1.1. Dog samples In collaboration with the district veterinary department, a list of dairy farms in Hawassa, Ethiopia was prepared, most of which were known to own dogs, and these were requested to provide dog faecal samples. Dog owners in residential areas were also contacted with the same request. The sample size was estimated based on 13.3% expected prevalence (Asmare et al., 2013b), 95% confidence level and 5% desired precision. Accordingly, samples from a minimum of 178 dogs had to be collected. However, a total of 383 faecal samples were collected from 165 farm dogs and 218 household dogs. The dogs were owned by 137 individuals, of whom 55 owned a dairy farm. Dog owners participating in the study were briefed on the study objectives and provided with bottles for collection of faecal samples at defecation. Samples were transported to the School of Veterinary Medicine at Hawassa University in a cold box, and kept at 4 ◦ C until analysis (a maximum of 4 days following collection). Dog owners were also interviewed to obtain information on type of dog (farm dog or household), age of dog, sex, and level of control (partial or fully free). Dogs with partial control are chained during the day and released during the night. 2.1.2. Bovine tissue samples Farm owners with herds identified as having seropositive cattle were advised via the district veterinary department to contact the research team if abortion or congenital abnormality was encountered at full-term parturition, or if any of their calves were born with physical abnormalities. On the basis of this advice, we were able to collect clinical material from four late-term aborted foetuses (two from one farm) and a congenitally defective calf (hind leg arthrogryposis). In addition, placental tissue from a cow with normal parturition was collected. All the dams were seropositive for N. caninum exposure. As brain is the most consistently affected organ (Dubey et al., 2006), foetal samples were collected from the cerebellum, cerebrum and midbrain. 2.2. Laboratory analyses 2.2.1. Oocyst recovery from dog faeces At the parasitology laboratory, School of Veterinary Medicine, Hawassa University, faecal samples were processed by a modified flotation technique, as described briefly below. Approximately 12 g faecal sample was suspended in 25 ml tap water and mixed thoroughly. The suspension was sieved through a strainer (0.5–0.8 mm mesh aperture) and the filtrate collected in centrifuge tubes and centrifuged at 3000 rpm for five min. The supernatant was decanted
89
and 2 g of sediment re-suspended in 10 ml of saturated salt solution (specific gravity, 1.2) and vortexed for 1 min. The suspension was overlaid with 5 ml tap water and centrifuged at 3000 rpm for 10 min. The interface was collected into two separate clean tubes and tap water was added to the top of the tubes and vortexed for 5 s. The mixture was centrifuged for 10 min at 3000 rpm. The last step was repeated once to wash out the salt. Aliquots of the sediment were examined for protozoan oocysts at 400× magnification. Oocysts with a diameter of 11.5 ± 1.5 m with morphological similarity to sporulated or non-sporulated N. caninum oocysts were noted as positive for Neospora-like oocysts (NLO). Samples considered positive or presumptive for NLO were transferred to 2 ml microcentrifuge tubes to which 1.5 ml of 75% ethanol was added. The samples were couriered to the Norwegian School of Veterinary Science (NSVS), Oslo, Norway. At the NSVS Parasitology laboratory, the samples were centrifuged at 3000 rpm for 5 min. After decanting the supernatant, the sediment was re-suspended in distilled water, vortexed and centrifuged for a second time. The procedure was repeated once again to wash out the alcohol and the sediment was examined at 400× magnification for NLO. All samples considered positive for NLO at this second examination were subjected to molecular investigation. 2.2.2. Preservation of tissue samples Following aseptic opening of the skull of the aborted foetuses and defective calf, samples weighing 25–50 g were taken from the cerebral cortex, cerebellum and midbrain. Haemorrhagic parts or any visible gross lesions of the tissue were deliberately incorporated in to the samples. The same aseptic procedure was followed for placental tissue. The tissue samples were transferred into 10 ml tubes and 75% ethanol was added until the tissue samples were completely covered. These samples were couriered to NSVS, along with the positive faecal samples described above. 2.2.3. DNA extraction from oocysts Samples that had been selected for molecular investigation were subjected to DNA extraction based on the method used for Cryptosporidium oocysts at NSVS using a QIAamp DNA mini kit (QIAGEN GmbH, Germany). The protocol involved a pre-incubation step in which the oocysts were resuspended in AL buffer and heated to 100 ◦ C in a heating block for an hour. 2.2.4. DNA extraction from tissue samples The tissue samples were cut into small pieces, and homogenised by hand using a pestle and mortar. From the homogenate, 25 mg were transferred into microcentrifuge tubes and DNA extracted using a QIAamp DNA mini kit (QIAGEN GmbH, Germany). The control DNA extraction was performed in a similar approach. Liver samples from cattle were spiked with N. caninum cell culture tachyzoites stock (1 × 107 tachyzoites/ml) (NC-Liverpool strain, kindly provided by Dr. Siv Klevar, Norwegian Veterinary Institute, and processed according to the protocol described here. 2.2.5. Polymerase chain reaction (PCR) PCR targeting the ITS1 gene in the ribosomal DNA was run on DNA extracts both from tissue and oocysts using the primer pair PN3 [5 -TACTACTCCCTGTGAGTTG-3 ] and PN4 [5 TCTCTTCCCTCAAACGCTA-3 ] (Holmdahl and Mattsson, 1996) using 2 l template DNA for each reaction. DNA extracted from N. caninum tachyzoites was used as positive control and molecular grade water as negative control, which followed each sample batch from DNA extraction onwards. Following a 2.5 min hot start at 94 ◦ C, the PCR was carried out with 50 cycles of 94 ◦ C for 40 s, 54 ◦ C for 1 min, and 72 ◦ C for 30 s. The PCR was completed with 3 min annealing at 72 ◦ C. PCR products were electrophoresed on 2% agarose and stained with SYBR Green. In order to increase the sensitivity, DNA
90
K. Asmare et al. / Acta Tropica 130 (2014) 88–93
Fig. 2. PCR products targeting the Internal Transcribed Spacer 1 (ITS1) gene of N. caninum in foetal and calf brain tissue. Fig. 1. PCR products targeting the Internal Transcribed Spacer 1 (ITS1) gene of Neospora caninum oocysts recovered from naturally infected dogs.
extraction and PCR was run repeatedly on the same samples for a minimum of 10 and maximum of 20 times, until the first positive result was detected. If none of these replicates resulted in a band of the correct size, the sample was considered negative. 2.2.6. PCR product purification and sequencing PCR products of positive samples at the ITS1 gene were purified (High Pure PCR product purification kit; Roche Diagnostics GmbH). Sequencing was performed in both directions by a commercial company (Eurofins MWG Operon, Germany). 2.2.7. Data analysis Sequences were edited, manually checked and assembled using DNAStar software (www.dnastar.com), and compared with sequences of N. caninum available in GenBank by a BLAST search. Six representative new sequences were deposited in GenBank with accession numbers from KC461931 to KC461936. Sequences were aligned using CLUSTALW in BioEdit. Descriptive statistics with 95% confidence interval estimates were conducted on questionnaire and coprological data using STATA 11 (SE for Windows, StataCorp, College Station, TX).
tissue samples spiked with 5 l N. caninum Liverpool strain tachyzoites gave positive bands at 1:10, 1:50, 1:100 and 1:1000 dilutions; the 1:50 dilution was used as a positive control throughout the PCR investigation. 3.3. Sequence analysis Sequences obtained from the ITS1 gene PCR were aligned with standard sequences obtained from GenBank as follows (Accession numbers provided in parentheses): NC-Liverpool (EU564166), NC-Bahia (AY259043), NC-Illinois (AY259041) and NC-Nowra (AF338411). Alignment with our sequences provides confirmation that both dogs and cattle were infected with N. caninum. With the exception of three DNA isolates obtained from oocysts isolated from dog faecal samples (NCD162, NCD335 and NCD380), the sequences obtained from all the other positive samples, whether acquired from oocysts in dog faeces or from foetal or calf brain tissue, were identical to one another. It should be noted that for all our samples, clear double peaks were seen on the electropherograms at positions 247 and 249 (Fig. 3). The three dog-derived samples that differed from the other samples demonstrated only minor SNPs or double peaks. 4. Discussion
3. Results 3.1. Detection of Neospora in dog faeces Among the 383 dog faecal samples (218 household and 165 farm dogs) from in and around Hawassa, Neospora-like oocysts (NLO) were detected in 37 (9.7%) by coproscopy (Table 1). Of those 37 samples considered positive by microscopy, 17 (46%) were confirmed to be positive for N. caninum by PCR at the ITS1 gene (Fig. 1), indicating a minimum of 4.4% prevalence. Most of the positive samples (by both microscopy and PCR) were from dogs less than one year old (P < 0.05). N. caninum oocysts were detected in faecal samples from both farm dogs and household dogs, and no significant difference in prevalence was observed between these two groups (P > 0.05). Similarly there was no significant difference in prevalence between sex and control categories. 3.2. Detection of Neospora DNA in tissue samples Six tissue samples (brain tissue from 4 late term aborted foetuses and from a 2 month old congenitally defective calf, and a placental tissue sample following normal parturition), all from dams with positive serological status for N. caninum antibody, were examined by PCR. Neospora caninum DNA was detected at least once in all the brain tissue samples (Fig. 2), but not in the placental tissue. The liver
A high prevalence of N. caninum infection, along with a strong association with reproductive disorders, has recently been reported in Ethiopian dairy and breeding cattle (Asmare et al., 2013a,b). Additionally, Neospora infection in cattle was shown to be associated with an extensive presence of farm dogs (Asmare et al., 2013b). However, in the previous studies, pathological or molecular evidence of infection with N. caninum in cattle was not provided, and nor was any evidence of infection in dogs. The current study is built upon these results, and not only provides molecular evidence of N. caninum infection in aborted foetuses and a congenitally deformed calf, but also demonstrates excretion of N. caninum oocysts in dog faeces, with initial identification by microscopy and confirmation by molecular methods. Although the seroprevalence of N. caninum infection in dogs can be relatively high (Bruhn et al., 2012; King et al., 2012; Ghalmi et al., 2009; Mitrea et al., 2013), the prevalence of Neospora excretion in faeces (as detected by coproscopy or molecular techniques) tends to be lower, with previous studies reporting prevalences ranging from 0.03% in Germany (Schares et al., 2005) to 1.15% in Iran (Razmi, 2009) and 1.5% in Australia (King et al., 2012). The relatively high prevalence of PCR-confirmed Neospora oocyst shedding in dogs found in our study (4.4%), may reflect the scavenging nature, or in some cases entire survival, of dogs on untreated offal in this region, and is more similar to the oocyst excretion prevalence reported
K. Asmare et al. / Acta Tropica 130 (2014) 88–93
91
Table 1 Prevalence of Neospora-like oocysts (NLO) in dog faeces from Hawassa, Southern Ethiopia: results from coproscopy and PCR (confirmed N. caninum). Variables
Category
Number examined
Coproscopy (NLO)
PCR (confirmed N. caninum)
Type of ownership
Farm dog Household dog
