First isolation of Neospora caninum from the faeces of a dog from Portugal

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of γ-interferon knockout mice with 10² sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.     

Pigeons (Columba livia) are a suitable experimental model for Neospora caninum infection in birds

Neospora caninum infections in chickens have been recently described by epidemiological and experimental approaches, and these birds may be considered natural intermediate hosts of the parasite. It has been postulated that other bird species might perform this role in wildlife as well. To better understand the sylvatic life cycle of N. caninum, further studies are required. In that sense, this work aimed to observe infection kinetics in pigeons experimentally infected with N. caninum. Experimental infections were conducted in parallel with a related protozoan, Toxoplasma gondii, which has been already described as able to infect pigeons in nature. Our results demonstrated that N. caninum disseminated through various tissues of this host and induced parasite-specific IgG seroconversion. Infection parameters were similar to that observed in the T. gondii infected group, although N. caninum-infected pigeons presented lower IgG titers during acute phase. The results herein described demonstrate that pigeons are a suitable model for N. caninum infection, considering that these data are in agreement with those observed in chickens experimentally infected with this parasite. As pigeons may be revealed as important reservoirs for N. caninum infection in nature, future studies are necessary to determine the real prevalence of this parasite in this and other birds in wildlife.     

Occurrence of Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta cow-calf operations

Mycobacterium avium subsp. paratuberculosis (MAP) and Neospora caninum (NC) are two pathogens causing important production limiting diseases in the cattle industry. Significant impacts of MAP and NC have been reported on dairy cattle herds, but little is known about the importance, risk factors and transmission patterns in western Canadian cow-calf herds. In this cross-sectional study, the prevalence of MAP and NC infection in southwest Alberta cow-calf herds was estimated, risk factors for NC were identified, and the reproductive impacts of the two pathogens were assessed.     

Neospora caninum, potential cause of abortions in dairy cows: The current serological follow-up in Slovakia

Neosporosis is considered to be a contributing risk factor for abortions in dairy cows and other farm animals and has negative economic impact on their breeding. In respect of the rapid spread of neosporosis in herds throughout the world, our aim was to detect the prevalence of anti-Neospora antibodies in cows post-abortion (PA) (PA Group n = 716) and in cows without any reproduction problems (Control Group n = 247) on large breeding farms from Eastern Slovakia. The overall mean seropositivity in PA Group (20.1%) was significantly higher (p < 0.0001) in comparison with the Control Group (2.3%), and this demonstrates the causal dependency of abortions on neosporosis. On farms from south-east region, 15.6% of cows were seropositive on average ranging from 7.8% to 25.8% in different districts. In the northern region, significantly (p = 0.0002) higher 26.2% mean seropositivity was detected. The seroprevalence in different districts ranged from 2.9% to 39.4%. In the PA Group a high seropositivity to infectious bovine rhinotracheitis (41.5%) and sporadic occurrence of Q-fever, leptospirosis and chlamydiosis was also detected. From 11 Toxoplasma-seropositive cows, only one animal was simultaneously positive to Neospora. The relatively high prevalence rate in cows warrants the attention and a need of surveillance in Slovak herds. A better knowledge of epidemiology of this etiological agent and the mechanisms of its transmission may help in the introduction of more effective preventive and control measures.     

Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy

The Nc-Spain 1H isolate of Neospora caninum, which was newly obtained from the brain of a congenitally asymptomatic infected calf, demonstrated a reduced in vitro tachyzoite yield and viability rate, as well as low virulence in mouse models. The objective of the present study was to determine the ability of this isolate to induce foetal death in a pregnant bovine model. For this purpose, 13 naïve pregnant heifers were divided into three groups and were experimentally challenged with either 10⁷ tachyzoites of Nc-1 (group 1, n = 5), Nc-Spain 1H (group 2, n = 5) isolates or phosphate-buffered saline (group 3, n = 3) intravenously at 70 days of gestation. After inoculation, pregnancy was monitored and dams were sacrificed when foetal death was detected. The remaining animals were slaughtered at 45 days post-infection. Maternal and foetal samples were collected for examination by histology and parasite DNA detection. Parasitaemia, specific anti-N. caninum IgG and interferon γ responses were also studied. At 3–4 weeks after infection, foetal death was detected in 3 out of 5 Nc-1-infected dams. However, no evidence of foetal death was observed in either Nc-Spain 1H-infected or control groups during the period studied. The most severe histopathological lesions were observed in the placenta and foetal organs from Nc-1-infected cattle that exhibited foetal death. It was in these animals that N. caninum DNA was more frequently detected. Parasitaemia was observed in all Nc-1-infected dams and in only 3 out of 5 Nc-Spain 1H-infected animals. The magnitude of the immune response was significantly higher in the Nc-1-inoculated group than in the group inoculated with the Nc-Spain 1H isolate. These data reveal the reduced virulence of the Nc-Spain 1H isolate in cattle.     

Comparison of two PCR protocols for the detection of Neospora caninum DNA in rodents

The objective of the present study was, due to increasing interest in the epidemiological role of small mammals as potential reservoir of Neospora caninum, to compare two different PCR protocols for the diagnosis of N. caninum in rodents. We tested tissue samples from 50 house mice (Mus musculus), 50 rats (Rattus norvegicus) and 35 field mice (Apodemus sylvaticus). Two different PCR protocols based on primer pairs, Np4-Np7 and Np6plus-Np21plus, were used for diagnosis on these samples. While there were not mismatches between the results of both PCR from rats or field mice, 49 out of 50 samples from house mice gave positive results with Np4-Np7 primer set. However after cloning and sequencing the PCR products, only six of these were confirmed to be N. caninum, while all the other 43 amplicons matched with house mice DNA sequence from clone RP23-14F5 on chromosome 11 sequence. Our results evidence that Np4-Np7 PCR could not be reliable in diagnosis of N. caninum in rodents.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain)

The prevalence of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and cELISA, respectively, in 160 roe deer (Capreolus capreolus), 177 sheep and 178 cattle sharing pastures in Galicia (Northwest Spain). The seroprevalence for T. gondii was 13.7% in roe deer, 57% in sheep and 7.3% in cattle. The seroprevalence for N. canimum was 6.8%, 10.1% and 24.1% in roe deer, sheep and cattle, respectively. Statistically significant differences were observed between sheep and the other species for T. gondii and between cattle and the other ruminants for N. caninum. Only 19/515 animals were positive for both, T. gondii and N. caninum. Statistically significant differences were observed among different geographical areas for T. gondii but not for Neospora, seroprevalence being higher in the coastal area lower than in other areas. This study reveals a widespread exposure to T. gondii in Galician ruminants, and therefore, those species, particularly sheep, should be regarded as a potential source of infection for humans.     

Neospora caninum associated with epidemic abortions in dairy cattle: The first clinical neosporosis report in Turkey

Neospora caninum, a protozoan parasite, has been considered as one of the most important etiological agents responsible for abortion in dairy cattle throughout the world since it was first identified in dogs in 1988. In this report, characteristics of neosporosis, detected in a dairy cow ranch having epidemic abortions as high as 18.4%, were described. Blood samples were collected from 25 infertile or aborted dairy cattle, 6 calves born in 2006 and 40 heifers that were born in 2005 and raised in the same ranch. Necropsy was conducted in a 20-day-old Simmental calf that exhibited neurological signs including incoordination, head shaking, hyperextension in forelimbs and hindlimbs and tremor. The seroprevalance in aborted or infertile dairy cattle, heifers, and calves was 60%, 40%, and 33.3%, respectively. The mothers of seropositive two calves including clinically affected calf and its dam were N. caninum seropositive. In immunoperoxidase examinations, N. caninum antigen immunopositivity was observed in the degenerative and necrotic neurons in the brain, cerebellum as well as neurons in dorsal root ganglia of the cervical and thoracic regions of the spinal cord. In the heart, myocytes and Purkinje cells exhibited granular and linear patterns of immunoreactivity. Striated myofibers around the eyeball also showed immunolocalization for N. caninum antigen. Ultrastructurally, tachyzoites with typical apical complex, rhoptries and double-layered parasitic membrane were detected in the brain and heart sections. In conclusion, this report described clinical neosporosis for the first time in Turkey with tissue localization of the causative agents. This scientific communication also discusses the possible impact of cattle neosporosis by clinical, serologic and pathologic evidences collected from the survey of calves born in two successive generations in a ranch.     

Associations of Neospora caninum seropositivity with gestation number and pregnancy outcome in Danish dairy herds

The prevalence and distribution of seropositivity towards the protozoan parasite Neospora caninum were studied in single blood samples from 1561 cows from 31 Danish dairy herds. Blood samples were analysed by an indirect enzyme-linked immunoassay and an indirect fluorescent-antibody test. Seroprevalence in 15 herds with previous abortions assigned to neosporosis ranged from 1% to 58%, with a mean frequency of 22%. In eight out of 16 herds without a history of N. caninum related abortions, no seroreactors were found. In the remaining eight herds, the seroprevalence ranged from 6% to 59%. The prevalence and distribution of seropositivity, gestation number prior to sampling, and breed were related to abortions and perinatal deaths using a random-effects logistic-regression model. Abortion risk was significantly increased in seropositive animals (OR=3) and in 2nd-gestation cows (OR=3). Perinatal death was significantly influenced by gestation number and breed, but not by serostatus. Reproductive performance and culling risk of cows were not affected by serostatus. Seropositivity increased with “age” (i.e. gestation number) (P=0.02). In open cows, seropositivity tended to decrease with distance from calving (P=0.05). The proportion of seropositive pregnant cows increased with trimester (P=0.02).     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

Peripheral white blood cell counts throughout pregnancy in non-aborting Neospora caninum-seronegative and seropositive high-producing dairy cows in a Holstein Friesian herd

Pregnancy is characterized by transient changes in the maternal immune system, also evident at peripheral level. The present study analyzes the kinetics and possible factors affecting peripheral white blood cell populations throughout pregnancy in a herd of high-producing dairy cows chronically infected or not with Neospora caninum. We examined 54 pregnant parous cows: 29 Neospora-seronegative and 25 Neospora-seropositive cows. Blood samples were collected on Days 90, 120, 150, 180 and 210 of gestation. General Linear Model (GLM) repeated measures analysis of variance showed that the interaction Neospora-seropositivity × parity significantly affected total leukocyte, neutrophil and monocyte counts with lower levels of total leukocytes, lower neutrophil and higher monocyte counts recorded in primiparous Neospora-seropositive cows. In addition, N. caninum-seropositive cows had significantly increased monocyte counts on Day 180 of gestation compared to seronegative ones. Other factors significantly associated with changes in total and/or differential leukocyte profiles were period of pregnancy, season, twin pregnancy and milk production. In conclusion, a parity-associated effect of chronic N. caninum infection was observed on peripheral blood cell profiles in dairy cattle during gestation.     

Prevalence of antibodies to Neospora caninum in local and imported cattle breeds in the Kars province of Turkey

This study investigates the seroprevalence of Neospora caninum in cattle in the province of Kars in north-eastern Turkey. A total of 301 serum samples, 228 from local breeds of cows with a history of recent abortion and the remaining 73 collected at random from Simmental cows imported from Germany or their offspring, were tested for anti-Neospora antibodies by ELISA. All the serum samples from local breeds were negative for N. caninum antibodies, apart from one which tested inconclusive. In contrast six of the samples (8.2% with 95%CI: 2-14.5%) from Simmental cows tested positive. There was a significant difference (P<0.001) in the seroprevalence of N. caninum between local and Simmental breeds, even if the inconclusive test was considered positive, and the odds ratio of its occurrence in the Simmental breed was 20.3. In conclusion, it is unlikely that N. caninum contributes to abortion in local cattle breeds and our results also suggest that N. caninum was introduced to the region by the importation of Simmental cattle and that the vertical transmission of the parasite in cattle is important in the region.     

Host/parasite relationship in the in vitro infection of rat gliocytes by Neospora caninum: evaluation of cell respiration

Neospora caninum is a protozoon that causes abortion in cattle and neuromuscular lesions in dogs, with the formation of cysts mainly in the central nervous system. Since N. caninum is an intracellular parasite with tropism for the cells of nervous system, this study evaluated the respiratory metabolism of glial cells infected by this* parasite. Glial cultures obtained from the cerebral cortex of newborn rats were kept in DMEM enriched with 10% fetal bovine serum, 1 mM pyruvic acid and 2 mM of L-glutamine. They were infected at a ratio of approximately 1:1 (cell/parasite). Oxygen consumption was evaluated by polarography in the non infected and N. caninum infected groups, 24 and 72 h following infection. Glial cell respiration after 24 and 72 h was 307.2 +/- 34.7 and 308.9 +/- 64.1 microL of oxygen per mug of total protein per minute, and 566.2 +/- 54.6 and 579 +/- 117.5 microL O2/microg of total protein/minute in the control and infected groups, respectively. These results show that N. caninum does not interfere with glial respiration in vitro.     

Effects of crossbreed pregnancies on the abortion risk of Neospora caninum-infected dairy cows

Previous studies have shown that the use of beef bull semen significantly reduces the rate of abortions due to Neospora caninum in artificially inseminated (AI) seropositive dairy cows. In addition, certain beef breeds could be more resistant to N. caninum infection and abortion than others. The aim of the present study was to determine whether different crossbreed pregnancies, those derived from Limousin, Charolais, Piedmontese or Belgian Blue semen, carry different risks of abortion in Neospora-infected dairy cows. The effects of possible interactions between maternal levels of N. caninum antibodies and the different breed crosses were also evaluated. The study was performed on five commercial Holstein–Friesian dairy herds in Northeast Spain with previously confirmed diagnoses of N. caninum infection in aborted foetuses. The study population was comprised of 1115 pregnancies: 633 pregnancies recorded after AI using Holstein–Friesian semen from 18 bulls and 482 after AI using beef semen from 27 bulls (304 inseminations using semen from Limousin bulls, 191 from Belgian Blue bulls, 89 from Piedmontese bulls and 49 from Charolais bulls). Abortion rates were 32.2% (155/482) and 15.2% (96/633) for seropositive cows inseminated with Holstein–Friesian and beef breed semen, respectively. Logistic regression analysis revealed the herd and the interaction between maternal N. caninum antibody titre and the different crossbreeds as significant factors affecting the abortion rate. Lowest abortion rates, similar to that shown by seronegative animals in the analysed herds (3.2%, 239/7432), were observed in dams AI using Limousin semen that had low (<30 relative index (RI) units) N. caninum antibody titres (2.1% abortion, 3/145) and these cows were used as reference. Compared to the cows used as reference, cows with low N. caninum antibody titres (<30 RI units) showed a similar risk of abortion when inseminated with Piedmontese or Charolais bull semen, but higher risk of abortion when inseminated with Holstein (17.9 times) or Belgian Blue (7.2 times) bull semen. All cows with high N. caninum antibody titres (≥30 RI units) had a higher risk of abortion, ranging from 8.9 times (cows inseminated with Limousine semen) to 37.8 times (cows inseminated with Piedmontese semen), compared to the cows used as reference. In conclusion, different crossbreed pregnancies carried different abortion risks in Neospora-infected dairy cows. The use of beef bull semen dramatically reduced the risk of abortion in dairy cows, especially if Limousin breed semen was used. Moreover, this reduction was found to be dependent on the N. caninum antibody titre such that the lowest incidence of abortions was recorded in Limousin semen inseminated cows with low antibody titres. Insemination of Neospora-seropositive cows with beef bull semen could both reduce the risk of abortion and avoid breeding replacements for infected cattle.     

Prevalence of Neospora caninum and bovine viral diarrhoea virus in dairy cows in Southern Vietnam

The main purpose of this study was to investigate the prevalence of Neospora caninum and bovine viral diarrhoea virus (BVDV) in some dairy herds in Southern Vietnam, and to ascertain whether there were differences in seroprevalences between herds with imported and locally bred cows. Serum samples collected on five state farms and 97 smallholder herds were analysed for the presence of antibodies to N. caninum and BVDV. All BVDV antibody-negative sera were further tested by antigen-ELISA in order to identify persistently infected individuals. The N. caninum prevalence varied between 16% and 53% in the state herds, and was higher in the four herds that had imported cows than in the herd that only had locally bred cows. Nineteen percent of the samples collected on smallholder farms, which all had only locally bred cows, had antibodies to N. caninum. The BVDV seroprevalence varied between 58% and 93% on the state farms. In smallholder herds, the prevalence of BVDV among the sampled cows was 18% and even lower on the state farms. Despite the high seroprevalence for BVDV in the state herds, no persistently BVDV infected cows were found. Given the high prevalence for Neospora and BVDV among herds with imported cows, it seems advisable to test for both infections before cattle are imported into the country.     

Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model

Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 10⁶ tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.     

Seroepidemiology of Neospora caninum infection in dairy cattle herds in Mashhad area, Iran

A seroepidemiology study of Neospora caninum was conducted in Mashhad area, Khorasan province, Iran on 337 cattle from 30 dairy using ELISA. All herds had seropositive cattle and antibodies were formed in 46% of the cattle. Abortion was significantly associated with seropositivity of cattle. The highest risk of abortion was observed in the 1-2-year-old cows. Our results indicate that Neospora infection is widespread in Iran.     

新疆和静县黑头羊犬新孢子虫的检测及分子鉴定

犬新孢子虫是一种细胞内原生动物寄生虫,可导致包括绵羊和山羊在内的多种哺乳动物流产和新生胎儿死亡。本研究为了预估巴音郭楞蒙古自治州和静县黑头羊中犬新孢子虫的流行情况,采集了8 个乡镇的165 份血样,运用聚合酶链式反应（PCR）扩增犬新孢子虫Nc5基因片段,并对部分阳性产物进行基因测序。结果显示,在165 只黑头羊中有13 只（7.9%）检测到犬新孢子虫阳性;年龄、流产史、饲养犬只与感染率有统计学差异（P< 0.05）;2 岁以上的母羊感染率最高,有流产史的母羊,新孢子虫感染率较高。饲养场地周边犬的存在对感染率也有所影响,有饲养犬的羊群感染率11.4%,高于未饲养犬的羊群。将Nc5 基因的部分序列与GenBank 上传的基因序列进行比对,结果显示同源性为92%~100%。本研究是首次对和静县黑头羊犬新孢子虫进行系统进化分析,为犬新孢子虫病防控方案的制订提供参考,对减少畜牧业的经济损失具有实际意义。