Serologic study of anti-Neospora caninum antibodies in household dogs and dogs living in dairy and beef cattle farms in Tehran, Iran

A few studies have been done on the seroepidemiology of anti-Neospora caninum antibodies in dairy and beef cattle farms in Iran, which suggested the presence of N. caninum in these areas, but there is no published information directed on the presence or epidemiology of this organism in the dogs in Iran. To investigate anti-N. caninum antibodies in household dogs and dogs living in cattle farms, 100 blood samples were collected: 50 from dogs living in dairy and beef cattle farms and 50 from household dogs. Serum samples were screened for detection of anti-N. caninum IgG antibodies using indirect fluorescent antibody test (IFAT; ≥50). Antibodies were seen in 10 (20%) of 50 household dogs and in 23 (46%) of 50 farm dogs. There were significant statistical differences in seropositivity between these two groups (P = 0.005). The IFAT antibody titers were as follows: 1:50 in seven dogs, 1:100 in eight dogs, 1:200 in six dogs, 1:400 in seven dogs, 1:800 in three dogs, 1:1,600 in one dog, and 1:12,800 in one dog. There were no significant differences in seropositivity between males and females. The positive results were increasing with age, and positive results were significantly different in the age group of older than 2 years compared to the dogs of age group under 1 year (P = 0.000) and 1–2 years (P = 0.007). The results confirm the exposure of household and farm dogs to N. caninum in Tehran and the higher rate of exposure for the dogs of dairy and cattle farms around Tehran.     

Immunoanalysis of three litters born to a Doberman bitch infected with <Emphasis Type="Italic">Neospora caninum</Emphasis>

Neospora caninum is a tissue cyst-forming coccidium that may cause neuromuscular disorders in dogs. Infected bitches can transmit the parasite to their pups in utero. Vertical transmission may occur after primary infection during pregnancy and in subsequent pregnancies. The reason why only a few pups develop clinical neosporosis is unknown. We obtained sera from a Doberman bitch and its offspring delivered in three litters. The bitch had a titer of 1:640 in an indirect fluorescent antibody test (IFAT). At least three pups of litter A, one pup of litter B, and two pups of litter C were also seropositive for N. caninum. However, clinical neosporosis developed only in one pup of litter C, which had the highest IFAT titer (1:5,120) of all dogs examined. Western blots carried out after one-dimensional and two-dimensional separation of N. caninum tachyzoites revealed that the largest number of antigens was recognized by sera derived from the bitch. The lowest number of antigens was recognized by serum from the pup with clinical neosporosis. However, this pup uniquely recognized a major antigen with a molecular weight of about 17,000. The information collected in this study adds to our knowledge on why some pups develop clinical neosporosis and others do not.     

First report of <Emphasis Type="Italic">Neospora caninum</Emphasis>-associated bovine abortion in Mashhad area,Iran

Neospora caninum is an apicomplexan protozoan which causes abortion in cattle worldwide. The present study was designed to assess the importance of bovine neosporosis for causing abortion in Iranian cattle. Infection was primarily diagnosed by polymerase chain reaction (PCR), complemented with histopathology and immunohistochemistry (IHC). One hundred brains of aborted bovine fetuses were collected from Iranian dairy herd between 2003 and 2005 in Mashhad area. N. caninum was detected by PCR in the brains of 13 aborted fetuses. Lesions consistent with N. caninum infection were observed in 12 of fetal brains. Immunohistochemical examination of brain detected N. caninum organisms in three aborted fetuses, and a thick-walled (2 μm) cyst with 50 μm diameter was identified in one of the IHC-positive brain. The results indicated that neosporosis is an important cause of abortion in dairy cattle of Iran.     

Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus

In order to develop a vaccine against Neospora caninum in dogs and cattle, we constructed a recombinant vaccinia virus expressing the N. caninum surface protein, NcSRS2 (Nc-p43). Monoclonal antibodies to NcSRS2 and anti-N. caninum tachyzoite mouse serum recognized the NcSRS2 expressed by the recombinant vaccinia virus. In addition, recombinant NcSRS2 was transported to the cell surface. Mice infected with the recombinant virus predominantly produced IgG1 antibody (Ab) to N. caninum, rather than producing IgG2a Ab. Moreover, splenocytes from mice infected with the recombinant virus proliferated in the presence of the N. caninum antigen. Mice immunized with the recombinant virus gave rise to humoral and cellular immune responses to N. caninum tachyzoites. This study showed that a recombinant vaccinia virus expressing NcSRS2 might be useful for the production of a live vaccine against N. caninum infection. Received: 24 March 2000 / Accepted: 18 May 2000     

Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 10⁷ tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon‐γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite‐specific mucosal and circulating antibodies have a protective role against this parasitic infection.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in camels (Camelus dromedarius) in Mashhad, Iran

One hundred twenty camels were blood-sampled and used to evaluate serological screening for Neospora caninum and Toxoplasma gondii infection by indirect fluorescent antibody test (IFAT) in Mashhad, Iran, during years 2004–2005. Of the 120 camels, antibodies to N. caninum were found in three in titers of 1:20 and in four in titers of 1:40 using whole N. caninum tachyzoites as IFAT slide (VMRD Inc., Pullman, WA 99163, USA). Antibodies to T. gondii were found in three camels in titers 1:20 and in two camels in titers 1:40 using whole T. gondii tachyzoites as IFAT slide (BIOGENE, Iran).     

Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces,Thailand

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.     

Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene

Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarites and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis.     

Survey of <Emphasis Type="Italic">Neospora caninum</Emphasis> and bovine herpes virus 1 coinfection in cattle

A seroprevalence survey of Neospora caninum and bovine herpesvirus 1 (BHV-1) was conducted in cattle pasturing in an area of the southern Italian Apennines to investigate the coinfection of these two pathogens. Blood samples were collected from 948 pastured cattle raised on 81 farms. Sera were tested for antibodies to N. caninum and to BHV-1 using an ELISA assay and a neutralization test, respectively. Out of the 81 farms sampled, 63 (77.8%) were positive for N. caninum and 80 (98.8%) for BHV-1. Coinfection was found in 62 (76.5%) farms. Out of the 948 bovine sera samples, 303 (32.0%) had antibodies to N. caninum and 735 (77.5%) to BHV-1. The copresence of antibodies to N. caninum and BHV-1 was found in 256 (27.0%) cattle. The logistic regression results indicated that seropositivity for BHV-1 was a risk factor for N. caninum seropositivity and seropositivity for N. caninum was a risk factor for BHV-1 seropositivity.     

Prevalence of Neospora caninum infection in Sardinian dairy farms (Italy) detected by iscom ELISA on tank bulk milk

Neospora caninum is a heteroxenous cyst-forming coccidian closely related to Toxoplasma gondii and is considered one of the major causes of abortions in cattle worldwide. The present work aims to update the epidemiological trend of N. caninum of dairy cattle in Sardinia island, Western Mediterranean (Italy). For this reason, we used the newest enzyme-linked immunoassay (ELISA) methodology that exploits immune-stimulating complexes (iscoms) principle and allows us to point out the infection in the tank bulk milk too, besides the individual cattle. A total of 624 herds were sampled and tank bulk milk was submitted to iscom ELISA test. The analysis of the tank bulk milk samples revealed a total farm prevalence of 55% for N. caninum in Sardinia. In the provinces of Oristano and Cagliari the prevalences (64 and 65%, respectively) were significantly higher (p<0.01) than in Sassari and Nuoro (41 and 40%, respectively). The iscom Elisa test applied on tank bulk milk seems to be helpful and cost-effective for large epidemiological surveys, for monitoring control strategy plans for N. caninum, and for increasing the bio-safety level in dairy cattle farms.     

Prevalence of antibodies against Neospora caninum in dogs from urban areas in Central Poland

Neospora caninum is a protozoan parasite which causes abortion in cattle as well as reproduction problems and neurological disorders in dogs. To assess the prevalence of the parasite in urban dogs in the Mazovian Voivodeship, Central Poland, serum samples from 257 dogs were analyzed for the presence of specific IgG antibodies. The examined dogs visited three private veterinary clinics located in Warsaw due to control tests, vaccinations, or other reasons not directly connected with neosporosis. Using ELISA and Western blot, antibodies against the parasite were detected in 56 out of 257 dogs, giving a prevalence of 21.7%. A greater prevalence was observed in female dogs than in males, 28% and 17.3%, respectively, and the differences were statistically significant (p < 0.05). There were no significant differences in seroprevalence of Neospora infection within the age groups (p > 0.05). This study indicates the presence of N. caninum in the Mazovian Voivodeship, in dogs which live in urban areas and exposure of these dogs to the parasite. The fact that seropositive dogs had no contact with cattle confirms the important role of dogs in the parasite’s epidemiology.     

On the Efficacy and Safety of Vaccination with Live Tachyzoites of Neospora caninum for Prevention of Neospora-Associated Fetal Loss in Cattle

Infection of cattle with Neospora caninum may result in abortion or the birth of a congenitally infected calf. Vaccination with live N. caninum protects against experimental infection of cattle and mice, and the naturally attenuated Nc-Nowra strain of N. caninum is of particular interest as a potential vaccine candidate. Vaccination of heifers prior to breeding with live Nc-Nowra tachyzoites by either the subcutaneous or the intravenous route reduced the rate of abortion and the presence of the parasite in calves as determined by PCR and serology after infection of cows with a virulent isolate. Protected fractions were 55.6% to 85.2% depending on the route of vaccination and growth conditions of the vaccine strain, with cryopreserved Nc-Nowra tachyzoites being less effective, with a 25.9% protected fraction. Vaccination appeared to reduce the rate of pregnancy after artificial insemination in some groups compared to nonvaccinated, nonchallenged controls. One animal that was vaccinated but not challenged experienced an abortion, but Nc-Nowra could not be detected in any of the cows in this group or their progeny. This study confirms that live vaccination can be an effective method of preventing neosporosis in cattle and yet highlights the technical hurdle of preservation of live parasites that must be overcome for a vaccine to be commercially successful.     

Serologic profiles for Sarcocystis sp. and Neospora caninum and productive performance in naturally infected beef calves

Sarcocystis sp. and Neospora caninum infections affect cattle worldwide causing important economic losses. The objective of the present study was to trace serologic profiles for Sarcocystis sp. and N. caninum in naturally infected beef calves and analyze their relationship with transmission routes and productive performance. Samples were collected in two cow-calf operations located in Buenos Aires province, Argentina. In farm 1, 43 calves were bled and weighed three times. In farm 2, 69 calves were bled and weighed six times. Sarcocystis sp. and N. caninum immunofluorescence antibody test (IFAT) titers were averaged for each sampling point in order to trace serologic profiles for each infection. Categories were created to evaluate differences in daily weight gain. For S. cruzi antigen, animals were separated in a low-titer (≤200) and high-titer group (>200); for N. caninum, animals were grouped as infected and uninfected. Sarcocystis sp. antibody titer as well as the number of infected animals increased gradually over time in both farms. In farm 2 the low-titer group had significantly higher daily weight gain than the high-titer group. For N. caninum 44% (farm 1) and 65% (farm 2) of calves were considered infected, and the serological profile was horizontal or decreasing over time. However, seroprevalence increased in both farms and vertical and horizontal transmission frequency were estimated between 18.5%–29% and 22–25.5%, respectively. No differences were detected in daily weight gain between N. caninum groups from both farms. This is the first report of serological profiles for Sarcocystis sp. and N. caninum by IFAT in naturally infected beef calves and their relationship to different transmission routes and productive performance.     

Evaluation of bovine abortion associated with <Emphasis Type="Italic">Neospora caninum</Emphasis> by different diagnostic techniques in Mashhad,Iran

Twelve aborted foetuses (gestational ranged from 4–9 months) and dams from dairies cattle farms in (Mashhad) Iran were analysed to investigate the participation of Neospora caninum in abortion. Diagnosis of the infection was determined by histopathology, serology (indirect fluorescent antibody test [IFAT]) and semi-nested polymerase chain reaction (PCR). A total 33% of bovine foetuses were considered to be infected by PCR technique. Microscopic lesions consistent with N. caninum infection in foetal brains were observed in 25% of the samples, whereas 33% were positive using IFAT (with a cut-off titre of 1:20). This study confirms the importance of N. caninum as an important cause of abortion in Iran.     

Seroprevalence of Neospora caninum infection in dogs in Chaharmahal-va-Bakhtiari Province, Iran

The aim of this study was to determine seroprevalence of Neospora caninum infection in dogs in Chaharmahal-va-Bakhtiari province in Iran. One hundred serum samples were collected from dogs of different sex, age, and breed. Collected serum samples were examined using an indirect fluorescent antibody test. Thirty-two of the examined samples showed detectable IgG antibodies in titers of 1:50. Dogs older than one year old and stray dogs had a higher seroprevalence than other dogs. No sex or breed predispositions to N. caninum infection were detected during this serological assay (P ≤ 0.05).     

The role of B- and T-cell immunity in toltrazuril-treated C57BL/6 WT, µMT and nude mice experimentally infected with<Emphasis Type="Italic"> Neospora caninum</Emphasis>

Neospora caninum is a protozoan parasite predominantly known for causing abortion in cattle and neuromuscular disease in dogs. So far, no efficient metaphylactic chemotherapy has been developed. In preliminary studies, toltrazuril had been successfully used against experimental neosporosis in mice and calves. In the present study, we used immunocompetent and immunodeficient mouse strains to address the role of immunity in supporting the chemotherapy of experimental N. caninum infection. WT, µMT and athymic nude mice were intraperitoneally inoculated with 1×10⁶ Nc-1 tachyzoites. The drug was administered in the drinking water for 6 consecutive days so as to obtain a daily dose of approximately 20 mg toltrazuril/kg body weight. The course of infection was monitored by clinical, histological and immunohistochemical means, as well as by the search for parasite DNA using PCR-analyses of various organs. In immunocompetent WT mice, treatment proved to be of high efficacy by abrogation of any lesion formation or PCR-positivity in medicated C57BL/6 mice and a significant reduction of lesion formation or PCR-positivity in BALB/c animals. Similarly, treated µMT mice exhibited a significant reduction in cerebral lesion formation as well as in parasite DNA detectability by PCR when compared to untreated animals. Athymic nude mice, however, did not respond to treatment in that only a delay of the parasite dissemination was achieved, and nude mice still showed the neosporosis disease symptoms, although later than untreated animals. We conclude that treatment with toltrazuril appears to act parasitostatically rather than parasitocidically. This is supported by the fact that: (1) although the lack of B-cells did not impair the effect of toltrazuril, (2) the lack of T-cells did not allow for a full efficacy of treatment. Therefore, chemotherapy with toltrazuril against experimental infections with N. caninum requires the support of T-cell immunity in order to be successful.An erratum to this article can be found at     

Characterization of the B-cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite‐evasion immune‐mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69‐expressing peritoneal and splenic B cells. This early B‐cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin‐secreting splenic B cells and a rise in serum levels of N. caninum‐specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum‐infected mice. The B‐cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with γ‐irradiated parasites. Contrasting with the peripheral B‐cell expansion, a depletion of B‐lineage cells was observed in the bone‐marrow of the N. caninum‐infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite‐specific antibodies nevertheless impaired interferon‐γ (IFN‐γ) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non‐lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti‐N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite‐specific, non‐polyclonal, B‐cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN‐γ in the host protective immune mechanisms against this parasite.     

Serological study of Neospora caninum infection in dogs and cattle from west of Iran

The aim of this study was to determine the seroprevalence of Neospora caninum infection in dogs and cattle from Hamedan province (West of Iran). Blood samples were collected from 1,046 cattle and 270 dogs in this area. Cattle and dog samples were tested and analyzed using ELISA and IFAT, respectively. IgG-antibodies to N. caninum were found in 27 and 17.4 % of dogs and cattle samples, respectively. In cattle study, The association between infection and type of cattle was statistically significant (P?=?0.004). Also, significant statistical differences were observed regarding to stray canids presence in farm (P?P?P?=?0.195) and breed (P?=?0.077). In dog study, there was statistical differences among age groups (P?P?P?=?0.112). This study is the first report of N. caninum infection in dogs and cattle from west of Iran. There is both horizontal and vertical transmission of N. caninum in this area, and the presence of stray dogs may be a risk factor for N. caninum infection in cattle. N. caninum is an important factor in the economic losses of the cattle breeding in Hamedan province. Therefore, further investigations and designing control strategies for improving management in cattle farms is highly recommended.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in dogs from Korea

Toxoplasma gondii and Neospora caninum are closely related protozoan parasites, they share many common hosts, and can cause neurological diseases in dogs. Dogs can have close contacts with humans and livestock and therefore they can act as reservoirs of these parasites. The aim of this study was to survey the seroprevalence of antibodies against T. gondii and N. caninum and their co-infection rate in dogs in Korea. In total, sera from 553 domestic dogs were collected from different breeds, sexes, and ages of dogs from nine provinces across the country of Korea during 2006 and 2007. The presence of antibodies against T. gondii and N. caninum was analyzed using the latex agglutination test (LAT) with a cut-off value of 1:32, and the indirect fluorescent antibody test (IFAT) using a serum titer of 1:100. In the total dog population, 71 (12.8%) dogs were positive for anti-T. gondii antibodies and only 20 (3.6%) were positive for anti-N. caninum antibodies. Relatively higher seropositive frequencies of antibodies against T. gondii (20.1%) and N. caninum (4.9%) were detected in the dog population from the Gyeonggi. A higher proportion of animals seropositive for anti-T. gondii antibodies was found in stray dog populations as compared to household dog populations: 18.5% (59/319) vs 5.1% (12/234), respectively. The Chi-square tests revealed significant differences in the seropositive frequencies of antibodies against T. gondii between stray and household dogs in the total population (p<0.0001), and in dogs from the Gyeonggi (p<0.01). No significant differences were observed for the presence of antibodies against T. gondii or N. caninum when compared across the sex or age (p>0.05). The first serological survey on antibodies against both T. gondii and N. caninum parasites across the entire country showed that co-infection was not common in these canine populations with a seropositive level of 0.72%. The significantly higher positive frequency of T. gondii antibodies in stray dogs in both, Gyeonggi and in the total dog populations suggests that further investigation on the seroprevalence of parasites should focus on stray dogs.     

A survey of Neospora caninum-associated bovine abortion in large dairy farms of Mashhad, Iran

Neospora caninum is an apicomplexan protozoon causing abortion in cattle worldwide. The present study was designed to assess the importance of this parasite for causing abortion in dairy farms in the Mashhad area of Iran. Of the aborted bovine fetuses, 151 were collected from dairy farms between 2006 and 2008. First, brain samples were examined by polymerase chain reaction (PCR) for the presence of N. caninum DNA, diagnosis was complemented with immunohistochemistry (IHC) and fetal serology (ELISA). Twenty-two (14.5%) of bovine fetuses were considered to be infected with N. caninum with at least one diagnostic technique being positive. PCR yielded 18 (11.9%) positive out of 151 brain samples. Only 52 brain samples were suitable for IHC examination, and N. caninum organism was detected in six (11.5%) of these 52 brain samples. Fetal fluids (n = 151) were assessed with a N. caninum-ELISA, resulting in 15 (9.9%) seropositive fetal fluids samples. In the present study, a good agreement was observed between PCR and ELISA, and a fair agreement between PCR and IHC. The results indicated that abortion due to N. caninum infection is prevalent among large-size dairy farms in the Mashhad area of Iran, and that different complementary diagnostic techniques should be used to increase the chance to detect N. caninum.