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.     

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.     

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.     

Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo (Bubalus bubalis) fetus from Brazil

The aim of this study was to determine the congenital infection by Neospora caninum in the water buffalo (Bubalus bubalis), a natural intermediate host. Nine pregnant water buffalos, raised under free-grazing condition, were slaughtered, and their fetuses were collected. Samples of brain and thoracic fluid were obtained from those fetuses, with gestational ages ranging from 2 to 5 months. The DNA of N. caninum was detected and identified in the brain of one of those fetuses, using two PCR assays, one directed to the Nc5 gene and the other, to the common toxoplasmatiid ITS1 sequence. The DNA fragments produced on PCR were sequenced, and N. caninum was confirmed in the samples. No antibodies to N. caninum were detected on any sample of thoracic fluid by immunofluorescent antibody test (IFAT < 25). This is the first confirmation of congenital transmission of N. caninum in water buffalos.     

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.     

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.     

Epidemiology of neosporosis in dairy cattle in Galicia (NW Spain)

This comprehensive study of neosporosis in dairy cattle in Galicia (NW Spain) included: (1) a comparative study of three serological techniques for detection of Neospora caninum antibodies (direct agglutination, enzyme-linked immunosorbent assay and indirect immunofluorescence); (2) a cross-sectional serological survey in which 276 herds and 5,196 animals were tested; (3) a study of N. caninum antibody dynamics; (4) the isolation of viable tachyzoites of N. caninum. Data were analysed to determine the risk factors associated with the infection. A total of 219 herds (79.3%) and 816 heads of cattle (15.7%) were found to be seropositive. Seropositivity was higher on farms with dogs than on farms without dogs, and there was a negative correlation between the size of the herds and seroprevalence. Co-infection with Toxoplasma gondii increased the risk of seropositivity. Cows infected with N. caninum were 5.3 times more likely to abort than non-infected cows. The dynamics study showed an increase in anti-N. caninum antibody titres during the third trimester of pregnancy. Viable tachyzoites were isolated from brain samples. These results indicate that the economic impact of N. caninum is high in Galicia, and therefore, the inclusion of control measures for neosporosis in the official control health programmes is strongly recommended.     

Comparative study for the detection of antibodies to Neospora caninum in milk and sera in dairy cattle in southern Romania

The study aimed to assess the within-herd Neospora caninum exposure in dairy cattle in southern Romania, based on the detection of specific antibodies in milk and serum. A total of 104 paired samples of milk and serum were collected from four dairy farms. Individual samples were analyzed for N. caninum antibodies by ELISA: IDEXX Neospora Ab (Idx) (three farms: A, B, C; n = 60) and ID-VET Lab (Idv) (farm D; n = 44). Additionally, four pooled milk samples, one per each farm (A, B, C) and a composed one (A+B+C), were analyzed with Idx ELISA. Optimized cut-off values for milk samples were determined by receiver operating characteristic (ROC) analysis, with serum results considered as true status. The agreement was expressed by K values. The overall seroprevalence of N. caninum infection was 45% in the farms tested by Idx ELISA and 56.8% in the farm tested by Idv ELISA. A good agreement between serum and milk was obtained for both ELISA kits (K = 0.72 and 0.77, respectively). The specificity and sensitivity at optimized cut-off of S/P>0.704 for Idx and S/P%>7.966% for Idv were 100% and 70.37% for Idx and 89.47% and 88% for Idv. Testing pooled milk samples, there were identified as N. caninum positive the dairy farms with a 15% or higher within-herd seroprevalence at the cut-off value of S/P>0.51. This is the first study in Romania in which milk samples were tested to determine the N. caninum infection status in dairy farms, providing a base for further researches.     

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).     

First identification of Neospora caninum by PCR in aborted bovine foetuses in Romania

The first identification of Neospora caninum infection in aborted bovine foetuses in Romania is reported. Nine aborted foetuses were collected from a dairy farm. The foetal age of foetuses was between 3 and 7 months. A 7-month-old foetus was mummified. N. caninum DNA Nc-5 region was amplified from samples extracted from brain tissues of three (33.3%) aborted foetuses. The foetal ages of PCR positive foetuses were 3, 4 and 7 months (mummified). No specific lesions or tissue cysts were found to the histological examination, because of advanced autolysis of brains.     

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.     

Molecular detection of Neospora caninum in house sparrows (Passer domesticus) in Iran

Neospora caninum is an intracellular protozoan parasite with a wide range of intermediate bird hosts. There is little information describing the prevalence and genetic characterization of N. caninum in bird hosts worldwide and in Iran. In this study, a total of 217 brain samples of house sparrow (Passer domesticus) were examined for N. caninum presence by nested polymerase chain reaction targeting the Nc-5 gene. N. caninum DNA was detected in 3.68% (8/217) of sparrows. Sequencing of the Nc5 genomic DNA revealed 97–99% of similarity with N. caninum sequences deposited in Genbank. To our knowledge, this study is the first molecular evidence of N. caninum DNA in bird hosts in Iran. The results of this study highlight the role of the house sparrow (Passer domesticus) in maintaining and spreading N. caninum infection to canines in the feral and domestic environment.     

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).     

Investigation of Neospora caninum, Hammondia sp., and Toxoplasma gondii in tissues from slaughtered beef cattle in Bahia, Brazil

Neospora caninum, Hammondia sp., and Toxoplasma gondii are parasites with morphological and genetic similarities. N. caninum and T. gondii are important abortive agents of cattle and sheep, respectively, and may infect numerous animal species. Hammondia sp. is not known to induce disease in animals, but may cause confusion in the identification of closely related coccidia. The aim of this study was to investigate infection rates caused by N. caninum, Hammondia sp., and T. gondii in beef cattle using a nested PCR for Toxoplasmatinae rDNA, followed by sequencing of the PCR products. Antibodies to N. caninum and T. gondii were also investigated in the tested animals. Brains and hearts were obtained from 100 beef cattle in a slaughterhouse in Bahia. Seven samples from brain tested positive for Toxoplasmatinae DNA. No positive reactions were found in heart tissues. After sequencing of the PCR products from all positive tissues, five sequences matched with N. caninum and two matched with T. gondii. Antibodies to N. caninum and T. gondii were found in 20% and 26% of the animals, respectively. The confirmation of N. caninum and the absence of Hammondia heydorni in the tested animals is suggestive that cattle are not efficient intermediate hosts of H. heydorni; however further studies need to be performed using a greater variety of tissues and a higher sample size. The detection of T. gondii DNA in bovine tissues reinforces the potential risk of transmission of this parasite to humans and other animals through the consumption of bovine meat.     

PCR detection of Neospora caninum in water buffalo foetal tissues

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.     

Prevalence of Cryptosporidium spp. infection in some dairy herds of Mashhad (Iran) and its association with diarrhea in newborn calves

A 1:1 matched case-control study of calves less than 1 month of age was carried out by weekly visits to some dairy farms in Mashhad, Iran. Fecal samples were collected over a 6-month period from a total of 112 calves with clinical signs of diarrhea and from 112 matched animals without clinical signs of diarrhea as assessed by a scoring system. The samples were investigated for the presence of Cryptosporidium spp. oocysts by modified Ziehl–Neelson staining method. Cryptosporidium spp. oocysts were present in 51.8% of calves with diarrhea and in 21.4% of the controls. Among diarrheic calves, the highest prevalence of Cryptosporidium oocyst shedding was in age group 8–14 days (74.5%), and the lowest prevalence of Cryptosporidium oocyst shedding was in age group 22–30 days (23.8%). The McNemar test in all sampled calves showed significant differences in odds ratio for the presence of Cryptosporidium spp. in fecal samples, and the excretion of parasite in the feces of scouring calves was significantly higher (odds ratio 6.1) than in healthy calves. Differences between animals with and without diarrhea were statistically significant for age groups 1–7 and also 8–14 day-old calves. These results indicate that in these industrial dairy farms in Mashhad, infections by Cryptosporidium should be considered as a potential cause for newborn calf.     

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).     

Toltrazuril treatment of congenitally acquired <Emphasis Type="Italic">Neospora caninum</Emphasis> infection in newborn mice

C57BL/6 mice were infected with Neospora caninum tachyzoites during pregnancy, yielding a transplacental infection of developing fetuses. Subsequently, congenitally infected newborn mice were treated either once or three times with toltrazuril (or placebo) at a concentration of 31.25 mg compound per kg body weight. Both toltrazuril and placebo treatment had no negative effect on newborns, as noninfected treated pups developed normally without differences in mortality and morbidity to matching nontreated control animals. Already one application of toltrazuril was significantly (p < 0.01) able to delay the outbreak of neosporosis in newborn mice, when compared to placebo-treated infected controls. We found significantly higher proportion of surviving newborns in one-time-toltrazuril-treated and three-time-toltrazuril-treated groups (34% and 54%, respectively) when compared to one-time-placebo-treated and three-time-placebo-treated groups (14% and 30%, respectively). There was no significant difference (p = 0.2) in the proportion of surviving pups between one-time-toltrazuril and three-time-toltrazuril treatment. However, the number of diseased and Neospora-positive pups (46% and 47%, respectively) was markedly reduced after three-time-toltrazuril treatment compared to all other groups. Three-time-treatment also resulted in the highest antibody (IgG, IgG2a) response. Pharmacokinetic analyses using individual serum samples revealed that, although toltrazuril was absorbed and metabolized to toltrazuril sulfone by newborn mice, medicated animals exhibited an unexpected rapid turn-over (half-life time) of the compound. Toltrazuril and the metabolite were also found in brain tissues, indicating that passage of the blood–brain barrier occurred. In conclusion, we could show that three times treatment with toltrazuril had a high impact on the course of infection in congenitally N. caninum-infected newborn mice.     

First <Emphasis Type="Italic">in vitro</Emphasis> isolation of <Emphasis Type="Italic">Neospora caninum</Emphasis> from a naturally infected adult dairy cow in Slovakia

The impact of in vitro isolation and molecular characterisation of Neospora caninum as well as sequence analyses was studied. The brain homogenate of a naturally Neospora-infected dairy cow (positive in ELISA and Western blot) was intraperitoneally inoculated into Mongolian gerbils (Meriones unguiculatus). The brain of gerbils on day 60 post-inoculation was homogenized, and, after trypsin-digestion, cultured on Vero cells. Neospora-like tachyzoites were first observed after 77 days of cultivation. The parasite was confirmed by polymerase chain reaction (PCR) using Neospora-specific primers Np21 and Np6. The PCR product of the first Slovak isolate (NC-SKB1) was subsequently sequenced and published in GenBank under accession number GU300774. Sequencing and BLAST search identified the isolate as N. caninum.     

Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions

The routine diagnosis of Neospora caninum abortion is based upon histopathologic changes in fetal tissues and identification of tissue parasites by immunohistochemistry. Confirmation of N. caninum infection by immunohistochemistry has low sensitivity. In the present study, we examined the utility of PCR in detecting N. caninum infection in fetal tissues from spontaneous bovine abortion. DNA was obtained from fresh and formalin-fixed tissues from 61 bovine fetuses submitted for abortion diagnosis. Histopathology and immunohistochemistry determined the true status of N. caninum infection in each fetus. In formalin-fixed paraffin-embedded tissues, PCR detected N. caninum DNA in 13 of 13 true-positive fetuses (100%) and in 1 of 16 true-negative fetuses (6%). In fresh or frozen tissues, PCR detected N. caninum DNA in 10 of 13 true-positive fetuses (77%) and 0 of 11 true-negative fetuses (0%). PCR also detected N. caninum DNA in 6 of 8 fetuses that had typical lesions of N. caninum but were immunohistochemistry negative, indicating a higher sensitivity of PCR in comparison to that of immunohistochemistry. N. caninum DNA was amplified most consistently from brain tissue. PCR detection of N. caninum DNA in formalin-fixed, paraffin-embedded tissues was superior to that in fresh tissues, presumably because of the increased accuracy of sample selection inherent in histologic specimens.