DOI: 10.2478/s11686-014-0201-y © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(1), 1–4; ISSN 1230-2821
PCR detection of Neospora caninum in water buffalo foetal tissues Clementina Auriemma1*, Maria Gabriella Lucibelli1, Giorgia Borriello1, Esterina De Carlo2, Alessandra Martucciello2, Lorena Schiavo2, Amalia Gallo1, Francesca Bove1, Federica Corrado1, Santa Girardi1, Maria Grazia Amoroso1, Barbara Ďegli Uberti1 and Giorgio Galiero1 1 Department of Animal Health, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, 80055, Portici, NA, Italy; Centro di Referenza Nazionale sull’Igiene e le Tecnologie dell’Allevamento e delle Produzioni Bufaline, S.S. 18 Via delle Calabrie, 27-84131 Fuorni, SA, Italy
2
Abstract The seroprevalence of Neospora caninum was surveyed by an ELISA kit on two water buffalo herds of Southern Italy. Seropositive samples were detected in 47% and 59% of individuals, respectively, thus indicating high level of exposure to the parasite even if the possibility of vertical transmission cannot be excluded. Tissue samples collected from three aborted fetuses from the same herds were investigated for N. caninum presence by PCR assays targeting the 18S and the Nc5 DNA sequences, respectively. Both methods have shown the presence of N. caninum DNA in heart and brain. Sequencing of the Nc5 genomic DNA confirmed the presence of N. caninum in the samples; phylogenetic analysis of the obtained sequences showed high homology among the Neospora recovered from different samples. The present study suggests an important role of N. caninum as a possible abortive agent for water buffaloes.
Keywords Neospora caninum, water buffalo, abortion, PCR
Introduction Neospora caninum is a coccidian parasite affecting cattle and dogs worldwide (Dubey 2003). Evidence of Neospora infections has been reported in many other domestic species such as horses, goats, sheep and water buffaloes (Dubey et al. 1998). High prevalence values of anti-N. caninum antibodies have been observed in buffalo worldwide (Dubey et al. 1998, Guarino et al. 2000, Meenakshi Sandhu et al. 2007). The parasite has also been isolated from naturally infected water buffalo (Rodrigues et al. 2004), demonstrating that this animal species is an important natural intermediate host for N. caninum. Recently, experimental vertical transmission and abortion by this pathogen have been described in pregnant water buffalo (Konrad et al. 2012). Histological analysis showed the presence of Neospora-like tissue cysts in two water buffalo foetuses (Guarino et al. 2000). However, evident data on identification and molecular characterization of N. caninum as possible abortive agent for this animal species are still
missing. The aim of this study was to evaluate the role of N. caninum as a possible abortive agent in water buffalo farms characterized by abortions but negative to the major abortive agents.
Materials and Methods To assess the prevalence of N. caninum infection, sera from water buffaloes bred in two dairy farms in Campania region were analysed for the presence of antibodies to N. caninum using an ELISA kit (ID Screen® Neospora caninum Indirect, ID-Vet, France) according to the manufacturer’s instructions. The herds consisted of 588 and 321 animals, respectively. No cattle were present on the monitored farms and there was no evidence of any contact between the farms. During a four months surveillance period, three water buffalo foetuses aborted between the fourth and sixth month of gestation and collected from the same dairy farms included in
*Corresponding author: [email protected]
2
the serological survey, were analysed for the presence of N. caninum by molecular method. Genomic DNA was extracted from brain and heart tissues using a commercial kit (QIAamp DNA mini kit, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. N. caninum was detected and identified by two PCR assays, targeting the 18S gene and the Nc5 genomic DNA sequence specific for N. caninum, respectively. The primers used were APIF (5’- AAGTATAAGCTTTTATACGGC-3’) and APIR (5’-CACTGCCACGGTAGTCCAA TA-3’) for the 18S gene (Magnino et al. 2000), and Np4 (5’CCTCCCAATGCGAACGAAA-3’), Np6 (5’-CAGTCAACCTACGTCTTCT-3’) and Np7 (5’-GGGTGAACCGAGGGA GTTG-3’) for the hemi-nested PCR assay targeting the Nc5 genomic DNA sequence (Yamage et al. 1996, Baszler et al. 1999). Positive (DNA from NC-1 strain for N. caninum and RH strain for T. gondii) and negative controls were included in each set of PCR reactions. In addition to N. caninum, the presence of pathogens typically associated with abortion (Brucella spp., Salmonella spp., Chlamydophila spp., Listeria spp., Campylobacter spp., Coxiella burnetii, Leptospira spp., Toxoplasma gondii, Bovine and Bubaline Herpesvirus – BoHV1, BuHV1 – and bovine viral diarrhoea virus – BVDV) was also analysed in specific tissues (kidney, spleen, liver, lung, placenta, heart, brain) by molecular assays as elsewhere described (Vilcek et al. 1994, Fuchs et al. 1999, Ossewaarde and Meijer 1999, Vemulapalli et al. 1999, Magnino et al. 2000, De Carlo et al. 2004, Parisi et al. 2006, Al Amri et al. 2007, Marianelli et al. 2007, OIE 2011). The Nc5 amplicons were purified and sequenced using the Big Dye Terminator cycle sequencing kit v.3.1 (Applied Biosystems) and the genetic analyser ABI3130 (Applied Biosystems) following the manufacturer’s instructions. Sequences were analysed by multiple alignment by CLUSTAL W version 1.5 software using neighbor joining tree-building method. Phylogenetic tree was inferred using Mega version 5.10 beta (Tamura et al. 2011). Complete necropsy was performed on the water buffalo foetuses, evaluating the presence of macroscopic lesions. Histological analysis was carried out on foetal tissues (brain, heart, liver, kidney, spleen, lung) as elsewhere described (Gonzales et al. 1999).
Results The results of the serological analysis carried out on 980 water buffalo serum samples revealed 51% of animals seropositive to N. caninum. Brain and heart tissues from the aborted foetuses were analysed by PCR targeting the 18S gene. This assay indicated that all the samples were positive to N. caninum (Fig. 1). The hemi-nested PCR targeting the Nc5 region of genomic DNA specific of N. caninum carried out on the same samples confirmed the results of the 18S PCR (Fig. 1).
Clementina Auriemma et al.
Fig. 1. Detection of N. caninum and T. gondii by PCR amplification of the Nc5 and 18S rRNA DNA sequences followed by BseDI restriction patterns of the amplified 18S rRNA fragments. Lanes: (M) QX size marker 50-800 bp (QIAgen); (1–3) samples of hearts positive to 18S rRNA; (4–6) samples of brains positive to 18S rRNA; (7) negative control; (8) positive control for T. gondii; (9) positive control for N. caninum; (10–12) restriction profile of heart samples positive to N. caninum; (13–15) restriction profile of brain samples positive to N. caninum; (16) negative control; (17–18) restriction profile of positive controls for T. gondii and N. caninum, respectively; (19–21): samples of hearts positive to Nc5; (22–24): samples of brains positive to Nc5; (25) negative control; (26) negative control for T. gondii; (27) positive control for N. caninum.
The foetal tissues included in this study, resulted negative to molecular detection of the major abortive agents mentioned above. During necropsy, no characteristic or consistent lesions were observed in the aborted foetuses, but haemorrhagic fluid was recovered from the abdominal cavity. Histological analysis displayed the presence of lesions characteristically associated with N. caninum infection (Gonzales et al. 1999) in all tissues from the infected foetuses. In particular focal necrosis, perivascular cuffing and glial nodules were observed in brain, with cuffs predominantly composed of lymphocytes, while myocardial lesions consisting of focal infiltration of mononuclear cells with minimal necrosis were observed in heart (data not shown).
N. caninum in water buffalo foetuses
3
Fig. 2. Phylogenetic relationships (neighbour joining tree-building method) among N. caninum as deduced from structure alignment of the Nc5 DNA. Samples isolated in the present study (Neospora caninum 1, 2 and 3) were compared to isolates selected from GenBank. Bovine Herpesvirus Canis lupus familiaris cytochrome b gene was used as out-group. Numbers at the nodes indicate the bootstrap confidence values obtained after 1000 replicates.
Nc5 amplicons from positive tissues were sequenced using the Np6-Np7 primers pair, and N. caninum was confirmed in the samples. For each foetus, the obtained sequences from heart and brain showed complete homology. Moreover, the sequences were compared with the existing homologues in GeneBank and differed only at eleven nucleotides from that deposited by Chryssafidis et al. (2011). In particular, the three fragments of the Nc5 genomic DNA region displayed homology levels of 96%, 97%, and 98%, respectively, with a Neospora caninum isolated from a naturally infected water buffalo foetus from Brazil (DQ059068.1). Phylogenetic trees obtained from CLUSTAL W alignment of the Nc5 sequence using neighbour joining method (Fig. 2) showed intraspecific variations among Neospora caninum isolated in the present study and other Neospora caninum deposited in GenBank.
Discussion Water buffalo industry is economically important for several Italian regions, and the reproductive failure due to N. caninum
infection has been poorly characterized. Our data indicate a seroprevalence of 51%, thus confirming the presence of this parasite in this animal species according to previously reported serological data (Guarino et al. 2000). In this study the presence of N. caninum in foetal brain and heart tissues from water buffalo abortions was detected by two different PCR assays, targeting the 18S and the Nc5 respectively, with comparable results. Both samples were positive to both PCR assays. The Nc5 genomic DNA sequences from the three foetuses differed at a total of eleven nucleotide sites, indicating the existence of different N. caninum strains within the tested herds. As expected, phylogenetic analysis showed high homology among the detected Neospora strains. Finally, the analysed foetuses resulted positive to N. caninum and negative to the other major pathogens associated with abortion and reproductive disorders in water buffalo. These data suggest an important role of N. caninum as a possible abortive agent for this animal species. Consequently, neosporosis should be included in the routine diagnosis of abortive agents in water buffalo, especially in herds characterized by fertility loss or pregnancy interruption events.
4
References Al Amri A., Senok A.C., Ismaeel A.Y., Al-Mahmeed A.E., Botta G.A. 2007. Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools. Journal of Medical Microbiology, 56, 1350–1355. DOI: 10.1099/jmm.0.47220-0. Baszler T.V., Gay L.J.C., Long M.T., Mathison B.A. 1999. Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions. Journal of Clinical Microbiology, 37, 4059–4064. Chryssafidis A.L., Soares R.M., Rodrigues A.A.R., Carvalho N.A.T., Gennari S.M. 2011. Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo (Bubalus bubalis) foetus from Brazil. Parasitology Research, 108, 741–743. DOI: 10.1007/s00436-010-2214-2. De Carlo E., Re G.N., Letteriello R., Del Vecchio V., Giordanelli M.P., Magnino S., Fabbi M. Bazzocchi C., Bandi C., Galiero G. 2004. Molecular characterisation of a field strain of bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological reactivation. Veterinary Record, 154, 171–174. DOI: 10.1136/vr.154.6.171. Dubey J.P., Romand S., Hilali M., Kwok O.C.H., Thullies P. 1998. Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt. International Journal of Parasitology, 28, 527–529. DOI: 10.1016/S0020-7519(97)00190-2. Dubey J.P. 2003. Review of Neospora caninum and neosporosis in animals. Korean Journal of Parasitology, 41, 1–16. DOI: 10.3347/kjp.2003.41.1.1. Fuchs M., Hübert P., Detterer J., Rziha H.J. 1999. Detection of bovine herpesvirus type 1 in blood from naturally infected cattle by using a sensitive PCR that discriminates between wild-type virus and virus lacking glycoprotein. European Journal of Clinical Microbiology, 37, 2498–2507. Gonzàlez L., Buxton D., Atxaerandio R., Aduriz G., Maley S., Marco J.C., Cuervo L.A. 1999. Bovine abortion associated with Neospora caninum in Northern Spain. Veterinary Record, 144, 145–150. DOI: 10.1136/vr.144.6.145. Guarino A., Fusco G., Savini G., Di Francesco G., Cringoli G. 2000. Neosporosis in water buffalo (Bubalus bubalis) in Southern Italy. Veterinary Parasitology, 91, 15–21. DOI: 10.1016/S0 304-4017(00)00239-9. Konrad J.L., Moore D.P., Crudeli G., Caspe S.G., Cano D.B., Leunda M.R., Lischinsky L., Regidor-Cerrillo J., Odeón A.C., OrtegaMora L.M., Echaide I., Campero C.M. 2012. Experimental inoculation of Neospora caninum in pregnant water buffalo. Veterinary Parasitology, 187, 72–78. DOI: 10.1016/j.vetpar. 2011.12.030. Magnino S., Vigo P.G., Bandi C., Rosignoli C., Boldini M., Vezzoli F., Alborali L., Cammi G., Foni E., Colombo N., Colombo
Received: September 21, 2012 Revised: October 22, 2013 Accepted for publication: December 3, 2013
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M., Bergami C., Mellini A., Fabbi M., Genchi C. 2000. Neosporosi bovina in Italia: un biennio di attività diagnostica. Selezione Veterinaria, S15–S23. Marianelli C., Tarantino M., Astarita S., Martucciello A., Capuano F., Galiero G. 2007. Molecular detection of Leptospira species in aborted foetuses of water buffalo. Veterinary Record, 161, 310–312. Meenakshi Sandhu K.S., Ball M.S., Kumar H., Sharma S., Sidhu P.K., Sreekumar C., Dubey J.P. 2007. Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India. Journal of Parasitology, 93, 1374–1377. DOI: 10.1645/ GE-1317.1. OIE 2011. Chapter 2.9.7 “Listeria monocytogenes”, Chapter 2.9.9 ”Salmonellosis”, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Ossewaarde J.M., Meijer A. 1999. Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology, 145, 411–417. DOI: 10.1099/13500872-1452-411. Parisi A., Fraccalvieri R., Cafiero M., Miccolupo A., Padalino I., Montagna C., Capuano F., Sottili R. 2006. Diagnosis of Coxiella burnetii-related abortion in Italian domestic ruminants using single-tube nested PCR. Veterinary Microbiology, 118, 101–106. DOI: 10.1016/j.vetmic.2006.06.023. Rodrigues A. A., R. Gennari S.M., Aguiar D.M., Sreekumar C., Hill D.E., Miska K., Vianna M.C.B., Dubey J.P. 2004. Shedding of Neospora caninum oocysts by dogs fed tissues from naturally infected water buffaloes (Bubalus bubalis) from Brazil. Veterinary Parasitology, 124, 139–150. DOI: 10.1016/j.vetpar.2004.07.007. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011. Mega5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731–2739. DOI: 10.1093/molbev/msr121. Vemulapalli R., Mcquiston J.R., Schurig G.G., Sriranganathan N., Halling S.M., Boyle S.M. 1999. Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains. Clinical and Diagnostic Laboratory Immunology, 6, 760 –764. Vilcek S., Herring A.J., Herring J.A., Nettleton P.F., Lowings J.P., Paton D.J. 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Archives of Virology 136, 309–323. Yamage M., Flechtner O., Gottstein B. 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain “cyst” DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). Journal of Parasitology, 82, 272–279.
PCR detection of Neospora caninum in water buffalo foetal tissues Clementina Auriemma1*, Maria Gabriella Lucibelli1, Giorgia Borriello1, Esterina De Carlo2, Alessandra Martucciello2, Lorena Schiavo2, Amalia Gallo1, Francesca Bove1, Federica Corrado1, Santa Girardi1, Maria Grazia Amoroso1, Barbara Ďegli Uberti1 and Giorgio Galiero1 1 Department of Animal Health, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute, 2, 80055, Portici, NA, Italy; Centro di Referenza Nazionale sull’Igiene e le Tecnologie dell’Allevamento e delle Produzioni Bufaline, S.S. 18 Via delle Calabrie, 27-84131 Fuorni, SA, Italy
2
Abstract The seroprevalence of Neospora caninum was surveyed by an ELISA kit on two water buffalo herds of Southern Italy. Seropositive samples were detected in 47% and 59% of individuals, respectively, thus indicating high level of exposure to the parasite even if the possibility of vertical transmission cannot be excluded. Tissue samples collected from three aborted fetuses from the same herds were investigated for N. caninum presence by PCR assays targeting the 18S and the Nc5 DNA sequences, respectively. Both methods have shown the presence of N. caninum DNA in heart and brain. Sequencing of the Nc5 genomic DNA confirmed the presence of N. caninum in the samples; phylogenetic analysis of the obtained sequences showed high homology among the Neospora recovered from different samples. The present study suggests an important role of N. caninum as a possible abortive agent for water buffaloes.
Keywords Neospora caninum, water buffalo, abortion, PCR
Introduction Neospora caninum is a coccidian parasite affecting cattle and dogs worldwide (Dubey 2003). Evidence of Neospora infections has been reported in many other domestic species such as horses, goats, sheep and water buffaloes (Dubey et al. 1998). High prevalence values of anti-N. caninum antibodies have been observed in buffalo worldwide (Dubey et al. 1998, Guarino et al. 2000, Meenakshi Sandhu et al. 2007). The parasite has also been isolated from naturally infected water buffalo (Rodrigues et al. 2004), demonstrating that this animal species is an important natural intermediate host for N. caninum. Recently, experimental vertical transmission and abortion by this pathogen have been described in pregnant water buffalo (Konrad et al. 2012). Histological analysis showed the presence of Neospora-like tissue cysts in two water buffalo foetuses (Guarino et al. 2000). However, evident data on identification and molecular characterization of N. caninum as possible abortive agent for this animal species are still
missing. The aim of this study was to evaluate the role of N. caninum as a possible abortive agent in water buffalo farms characterized by abortions but negative to the major abortive agents.
Materials and Methods To assess the prevalence of N. caninum infection, sera from water buffaloes bred in two dairy farms in Campania region were analysed for the presence of antibodies to N. caninum using an ELISA kit (ID Screen® Neospora caninum Indirect, ID-Vet, France) according to the manufacturer’s instructions. The herds consisted of 588 and 321 animals, respectively. No cattle were present on the monitored farms and there was no evidence of any contact between the farms. During a four months surveillance period, three water buffalo foetuses aborted between the fourth and sixth month of gestation and collected from the same dairy farms included in
*Corresponding author: [email protected]
2
the serological survey, were analysed for the presence of N. caninum by molecular method. Genomic DNA was extracted from brain and heart tissues using a commercial kit (QIAamp DNA mini kit, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. N. caninum was detected and identified by two PCR assays, targeting the 18S gene and the Nc5 genomic DNA sequence specific for N. caninum, respectively. The primers used were APIF (5’- AAGTATAAGCTTTTATACGGC-3’) and APIR (5’-CACTGCCACGGTAGTCCAA TA-3’) for the 18S gene (Magnino et al. 2000), and Np4 (5’CCTCCCAATGCGAACGAAA-3’), Np6 (5’-CAGTCAACCTACGTCTTCT-3’) and Np7 (5’-GGGTGAACCGAGGGA GTTG-3’) for the hemi-nested PCR assay targeting the Nc5 genomic DNA sequence (Yamage et al. 1996, Baszler et al. 1999). Positive (DNA from NC-1 strain for N. caninum and RH strain for T. gondii) and negative controls were included in each set of PCR reactions. In addition to N. caninum, the presence of pathogens typically associated with abortion (Brucella spp., Salmonella spp., Chlamydophila spp., Listeria spp., Campylobacter spp., Coxiella burnetii, Leptospira spp., Toxoplasma gondii, Bovine and Bubaline Herpesvirus – BoHV1, BuHV1 – and bovine viral diarrhoea virus – BVDV) was also analysed in specific tissues (kidney, spleen, liver, lung, placenta, heart, brain) by molecular assays as elsewhere described (Vilcek et al. 1994, Fuchs et al. 1999, Ossewaarde and Meijer 1999, Vemulapalli et al. 1999, Magnino et al. 2000, De Carlo et al. 2004, Parisi et al. 2006, Al Amri et al. 2007, Marianelli et al. 2007, OIE 2011). The Nc5 amplicons were purified and sequenced using the Big Dye Terminator cycle sequencing kit v.3.1 (Applied Biosystems) and the genetic analyser ABI3130 (Applied Biosystems) following the manufacturer’s instructions. Sequences were analysed by multiple alignment by CLUSTAL W version 1.5 software using neighbor joining tree-building method. Phylogenetic tree was inferred using Mega version 5.10 beta (Tamura et al. 2011). Complete necropsy was performed on the water buffalo foetuses, evaluating the presence of macroscopic lesions. Histological analysis was carried out on foetal tissues (brain, heart, liver, kidney, spleen, lung) as elsewhere described (Gonzales et al. 1999).
Results The results of the serological analysis carried out on 980 water buffalo serum samples revealed 51% of animals seropositive to N. caninum. Brain and heart tissues from the aborted foetuses were analysed by PCR targeting the 18S gene. This assay indicated that all the samples were positive to N. caninum (Fig. 1). The hemi-nested PCR targeting the Nc5 region of genomic DNA specific of N. caninum carried out on the same samples confirmed the results of the 18S PCR (Fig. 1).
Clementina Auriemma et al.
Fig. 1. Detection of N. caninum and T. gondii by PCR amplification of the Nc5 and 18S rRNA DNA sequences followed by BseDI restriction patterns of the amplified 18S rRNA fragments. Lanes: (M) QX size marker 50-800 bp (QIAgen); (1–3) samples of hearts positive to 18S rRNA; (4–6) samples of brains positive to 18S rRNA; (7) negative control; (8) positive control for T. gondii; (9) positive control for N. caninum; (10–12) restriction profile of heart samples positive to N. caninum; (13–15) restriction profile of brain samples positive to N. caninum; (16) negative control; (17–18) restriction profile of positive controls for T. gondii and N. caninum, respectively; (19–21): samples of hearts positive to Nc5; (22–24): samples of brains positive to Nc5; (25) negative control; (26) negative control for T. gondii; (27) positive control for N. caninum.
The foetal tissues included in this study, resulted negative to molecular detection of the major abortive agents mentioned above. During necropsy, no characteristic or consistent lesions were observed in the aborted foetuses, but haemorrhagic fluid was recovered from the abdominal cavity. Histological analysis displayed the presence of lesions characteristically associated with N. caninum infection (Gonzales et al. 1999) in all tissues from the infected foetuses. In particular focal necrosis, perivascular cuffing and glial nodules were observed in brain, with cuffs predominantly composed of lymphocytes, while myocardial lesions consisting of focal infiltration of mononuclear cells with minimal necrosis were observed in heart (data not shown).
N. caninum in water buffalo foetuses
3
Fig. 2. Phylogenetic relationships (neighbour joining tree-building method) among N. caninum as deduced from structure alignment of the Nc5 DNA. Samples isolated in the present study (Neospora caninum 1, 2 and 3) were compared to isolates selected from GenBank. Bovine Herpesvirus Canis lupus familiaris cytochrome b gene was used as out-group. Numbers at the nodes indicate the bootstrap confidence values obtained after 1000 replicates.
Nc5 amplicons from positive tissues were sequenced using the Np6-Np7 primers pair, and N. caninum was confirmed in the samples. For each foetus, the obtained sequences from heart and brain showed complete homology. Moreover, the sequences were compared with the existing homologues in GeneBank and differed only at eleven nucleotides from that deposited by Chryssafidis et al. (2011). In particular, the three fragments of the Nc5 genomic DNA region displayed homology levels of 96%, 97%, and 98%, respectively, with a Neospora caninum isolated from a naturally infected water buffalo foetus from Brazil (DQ059068.1). Phylogenetic trees obtained from CLUSTAL W alignment of the Nc5 sequence using neighbour joining method (Fig. 2) showed intraspecific variations among Neospora caninum isolated in the present study and other Neospora caninum deposited in GenBank.
Discussion Water buffalo industry is economically important for several Italian regions, and the reproductive failure due to N. caninum
infection has been poorly characterized. Our data indicate a seroprevalence of 51%, thus confirming the presence of this parasite in this animal species according to previously reported serological data (Guarino et al. 2000). In this study the presence of N. caninum in foetal brain and heart tissues from water buffalo abortions was detected by two different PCR assays, targeting the 18S and the Nc5 respectively, with comparable results. Both samples were positive to both PCR assays. The Nc5 genomic DNA sequences from the three foetuses differed at a total of eleven nucleotide sites, indicating the existence of different N. caninum strains within the tested herds. As expected, phylogenetic analysis showed high homology among the detected Neospora strains. Finally, the analysed foetuses resulted positive to N. caninum and negative to the other major pathogens associated with abortion and reproductive disorders in water buffalo. These data suggest an important role of N. caninum as a possible abortive agent for this animal species. Consequently, neosporosis should be included in the routine diagnosis of abortive agents in water buffalo, especially in herds characterized by fertility loss or pregnancy interruption events.
4
References Al Amri A., Senok A.C., Ismaeel A.Y., Al-Mahmeed A.E., Botta G.A. 2007. Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools. Journal of Medical Microbiology, 56, 1350–1355. DOI: 10.1099/jmm.0.47220-0. Baszler T.V., Gay L.J.C., Long M.T., Mathison B.A. 1999. Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions. Journal of Clinical Microbiology, 37, 4059–4064. Chryssafidis A.L., Soares R.M., Rodrigues A.A.R., Carvalho N.A.T., Gennari S.M. 2011. Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo (Bubalus bubalis) foetus from Brazil. Parasitology Research, 108, 741–743. DOI: 10.1007/s00436-010-2214-2. De Carlo E., Re G.N., Letteriello R., Del Vecchio V., Giordanelli M.P., Magnino S., Fabbi M. Bazzocchi C., Bandi C., Galiero G. 2004. Molecular characterisation of a field strain of bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological reactivation. Veterinary Record, 154, 171–174. DOI: 10.1136/vr.154.6.171. Dubey J.P., Romand S., Hilali M., Kwok O.C.H., Thullies P. 1998. Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt. International Journal of Parasitology, 28, 527–529. DOI: 10.1016/S0020-7519(97)00190-2. Dubey J.P. 2003. Review of Neospora caninum and neosporosis in animals. Korean Journal of Parasitology, 41, 1–16. DOI: 10.3347/kjp.2003.41.1.1. Fuchs M., Hübert P., Detterer J., Rziha H.J. 1999. Detection of bovine herpesvirus type 1 in blood from naturally infected cattle by using a sensitive PCR that discriminates between wild-type virus and virus lacking glycoprotein. European Journal of Clinical Microbiology, 37, 2498–2507. Gonzàlez L., Buxton D., Atxaerandio R., Aduriz G., Maley S., Marco J.C., Cuervo L.A. 1999. Bovine abortion associated with Neospora caninum in Northern Spain. Veterinary Record, 144, 145–150. DOI: 10.1136/vr.144.6.145. Guarino A., Fusco G., Savini G., Di Francesco G., Cringoli G. 2000. Neosporosis in water buffalo (Bubalus bubalis) in Southern Italy. Veterinary Parasitology, 91, 15–21. DOI: 10.1016/S0 304-4017(00)00239-9. Konrad J.L., Moore D.P., Crudeli G., Caspe S.G., Cano D.B., Leunda M.R., Lischinsky L., Regidor-Cerrillo J., Odeón A.C., OrtegaMora L.M., Echaide I., Campero C.M. 2012. Experimental inoculation of Neospora caninum in pregnant water buffalo. Veterinary Parasitology, 187, 72–78. DOI: 10.1016/j.vetpar. 2011.12.030. Magnino S., Vigo P.G., Bandi C., Rosignoli C., Boldini M., Vezzoli F., Alborali L., Cammi G., Foni E., Colombo N., Colombo
Received: September 21, 2012 Revised: October 22, 2013 Accepted for publication: December 3, 2013
Clementina Auriemma et al.
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