Introduction

Neospora caninum is a protozoan parasite that is a primary abortifacient in cattle, and antibodies to N. caninum has been identified from many regions of the world (Dubey and Schares 2006) including Iran (Sadrebazzaz et al. 2004; Razmi et al. 2006). Cattle with antibodies to N. caninum are more to abort than seronegative cows. Foetus may die in utero, resorbed, mummified, autolyzed or stillborn or born alive with or without clinical signs (Dubey 2003).

Bovine neosporosis can be diagnosed in cattle using indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay and in foetuses using methods such as histopathology (HP), immunohistochemistry (IHC) and polymerase chain reaction (PCR; Dubey and Schares 2006). In Iran, results based on seroprevalence of N. caninum in aborted dairy cattle showed that neosporosis should be regarded as a cause of economic loss in dairy cattle (Sadrebazzaz et al. 2004; Razmi et al. 2006). The objective of the study was to analyse through detection N. caninum as a cause of bovine abortion in dairy cows by IFAT, semi-nested PCR and HP in Mashhad, Iran.

Materials and methods

Study site

The study was carried out in Mashhad, center of Khorasan-Razavi province, Iran, particularly in dairy cattle.

Sampling

The study was made during 2003–2005. Twelve aborted foetuses (with 4 to 9 months of age) were obtained. Foetuses and dams’ fluids and blood were sampled and centrifuged at 1,000×g for 10 min to eliminate cellular debris and stored at −20°C until tested. Brain tissue samples from aborted foetuses were obtained. Tissue samples were sized from 0.5 to 2 cm3 and fixed in 10% buffered formalin (pH 7.2).

Serology

A total of 12 foetal sera and fluids and 12 dams’ sera were tested in Razi Serum and Vaccine Research Institute (Mashhad branch) for antibodies to N. caninum using the IFAT slides, in which whole N. caninum tachyzoites (VMRD, Pullman, 99163 WA, USA) and Toxoplasma gondii tachyzoites (Biogen®, Iran) was used as antigens and a fluorescent-labelled antisera to IgG1 and IgG2 as conjugate. A cut off ratio of 1:200 for dams’ sera and 1:20 for foetal fluids and sera were defined as positive.

Histopathology

Brain tissue samples from aborted foetuses fixed in buffer formalin (10%) were processed by histological preparation including final embedding in paraffin wax. Two sections in each samples (5 μm) were cut for a total of 12 foetuses’ brains. All sections were stained with haematoxylin and eosin. Diagnosis of N. caninum was made on the observation of the non-suppurative encephalomyelitis characterized by micro-focal non-suppurative infiltration, perivascular cuffing and glial proliferation.

Semi-nested PCR

Brain samples, a total of six foetuses seropositive for N. caninum, were analysed using a semi-nested PCR. The dams of these foetuses had been also tested seropositive.

DNA was extracted from 50 mg of thawed brain tissues by using proteinase K digestion phenol–chloroform purification followed by ethanol precipitation (Sambrook et al. 1989). As positive and negative controls, DNA of N. caninum, T. gondii, Babesia spp. and Theileria spp. were also extracted (50 μl contained 0.1–1.0 μg of target DNA). Amplification of the internal transcribed spacer 1 (ITS1) region and 18S rRNA sequence (GenBank accession no. AY463245) of N. caninum were designed to amplify a 357-bp DNA fragment with oligonucleotide primers. The N. caninum Nc1 forward primer spans nucleotides 111 to 129 (5′-AGC GTG ATA TAC TAC TCC C-3′), Nc2 reverse primer spans nucleotides 446 to 467 (5′-CGA GCC AAG ACA TCC ATT GCT G-3′) and Nc3 semi-nested PCR primer spans nucleotides 209 to 227 (5′-GTG TGT GCA TAT ATC CGG G-3′). The PCR mixture of 50 μl contained 0.1–1.0 μg of target DNA, 2 mM MgCl2, 10× reaction buffer (50 mM KCl, 10 mM Tris–HCl [pH 8.3]), 10 pmol of each PCR primer, 200 μM of each deoxyribonucleotide triphosphate and 1 U of TaqDNA polymerase (Cinagen®, Iran). PCRs were performed in a thermocycler (Techgene, Techne, Germany) for 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s and extension at 72°C for 60 s. For semi-nested PCR, second-round primers Nc2 and Nc3 used 2 μl of amplicon solution from first-round Nc1–Nc2 PCR amplification as target DNA with the same PCR mixture subjected to 35 cycle of denaturation at 94°C for 30 s, annealing at 55°C for 45 s and extension at 72°C for 60 s. Amplicons were resolved on a 2% agarose gel stained with ethidium bromide and photographed under UV light. Positive (N. caninum DNA) and negative controls (no DNA) as well as irrelevant templates (50 μl contained 0.1–1.0 μg of target DNA of T. gondii, Babesia spp. and Theileria spp.) were included in each PCR run. Positive samples were tested at least three times for showing reproducibility of the specific PCR. Amplification products were analysed by electrophoresis through a 2% agarose gel for the specific N. caninum PCR. The PCR products were digested with endonuclease HinfI (Fermentas, Germany) according to the manufacturer’s instruction.

Sequences and primers were analysed using the “GeneRunner” programme, and homology studies were performed by “blastn” (blastn, nucleotide–nucleotide basic local alignment search tool) online programme.

Results

Serology

Of the 12 aborted foetuses and their dam sampled, six (50%) of the dams were seropositive, and five (41%) of their aborted foetuses were seropositive for antibodies to N. caninum. There were five cases with a positive serology in both the dams and its foetuses. All of the 12 dams and foetuses were seronegative for antibodies to T. gondii.

Histopathology

In six foetuses aborted by seropositive dams, the lesions consistent with N. caninum were observed in three foetuses. Lesions included neural oedematous, congestion, perivascular oedematous, mild status spongiosis, focal gliosis, perivascular cuffing and granulomatose foci surrounded by mononuclear inflammatory cells (lymphocytes and hystocytes). In two foetuses’ brains, N. caninum-like tachyzoites were observed.

Semi-nested PCR

DNA was successfully extracted from frozen brains of aborted foetuses using proteinase K digestion and further phenol–chloroform purification method. Homology analysis of the selected primers using “blastn” demonstrated specificity of the designed primers. The primers Nc1 and Nc2 were used to amplify 357 bp of the above sequences. Moreover, Nc2 and Nc3 amplified 259 bp of the same regions. The semi-nested PCR demonstrated that four of six aborted foetal brain samples were infected by N. caninum. ITS1 and 5.8S rRNA genomic sequence of N. caninum contain a unique restriction cut site for endonuclease HinfI at nucleotide 243. Digestion of PCR products with this enzyme produced two fragments of 225 and 132 bp in length, which confirms specificity of the PCR.

Discussion

In the 12 aborted bovine foetuses analysed by Neospora, diagnostic procedures include serology, HP and PCR techniques. Presence of N. caninum antibodies in aborted and healthy dairy cattle was detected (Sadrebazzaz et al. 2004; Razmi et al. 2006), but there was no information about bovine abortion associated with N. caninum by complex techniques in Iran. In our study, 6 of the 12 dams were diagnosed as seropositive by N. caninum using IFAT, and four (33%) foetuses of six infected dams were considered as positive by IFAT. In this study, 33% of N. caninum foetal infection cases were diagnosed by semi-nested PCR (Habibi et al. 2005); this value was the same as reported in Mexico, 34% diagnosed by HP (Morales et al. 2001). Nested PCR were developed and used in studies on pathogenesis of bovine neosporosis. This PCR was used to examine aborted foetuses in other countries, and it was used for first time in Iran. The specifically designed PCR analyses showed that nested PCR procedure (to increase sensitivity and specificity) was necessary to detect N. caninum infected foetuses. There is an up to 1,000 times increased efficiency at generating second-round amplicons (Jackson et al. 1992).

Our study are consistent with previous studies showing tissue parasites detected most frequently in brain by PCR (reviewed in Dubey and Schares 2006). In our study, 25% of aborted foetuses showed suggestive lesions of neosporosis by HP, which was lower than our PCR results (33%), in Morales et al. (2001) and in Sager et al. (2001) by HP and PCR. In HP, neural and perivascular oedematous, perivascular cuffing, focal gliosis, granulomatose foci surrounded by mononuclear inflammatory cells and N. caninum-like tachyzoites were considered as indicative of infection by N. caninum. (Dubey et al. 2006; Dubey and Schares 2006). PCR methods generally had a higher sensitivity than IHC methods, and also a high specificity (Van Maanen et al. 2004).

In the present study, good agreement between HP, IFAT and PCR was observed, and in our study, PCR was used as the sensitive technique. Because of among the ITS1 regions of T. gondii, N. caninum, Babesia spp. and Theileria spp., there are number of sequence differences that allow the establishment of specific PCRs. In our study, the aborted foetal brains have been shown as a reliable tissue for PCR analysis, and a nested PCR procedure may be used to increase sensitivity and specificity to detect N. caninum infected foetuses. In conclusion, the presence of N. caninum infection in aborted foetuses in dairies studied was demonstrated using IFAT, HP and semi-nested PCR in Iran, which was similar to those recorded in previous studies in Mexico (Morales et al. 2001) and the higher percentages of foetal infection in UK (10–13%; Otter et al. 1995; Schock et al. 2000).

However, it is necessary to carry out further studies especially in definitive hosts to determine vertical or horizontal transmission modes in the study area to develop adequate epidemiology and preventive management.