W. Basso

Diagnosis of Sarcocystis cruzi , Neospora caninum , and Toxoplasma gondii infections in cattle

The aim of the study was to diagnose Sarcocystis sp. infections in cattle and to detect coinfections by Toxoplasma gondii and/or Neospora caninum. Blood, diaphragm, esophagus, and myocardium from 90 beef cattle from Argentina were collected. Histopathological, immunohistochemical, polymerase chain reaction assays, and direct microscopical examination were carried out. Sarcocysts from myocardium were measured and counted. Indirect fluorescent antibody test (IFAT) for the three protozoans was performed. Sarcocystis cruzi sarcocysts were found in 100% of myocardium samples. Sarcocysts per gram ranged from 8 to 380 with higher values found in adult cattle (p < 0.001). T. gondii and N. caninum were not detected by immunohistochemistry. T. gondii DNA was found in myocardium of 2/20 seropositive animals, while N. caninum DNA was not found. Antibodies against S. cruzi were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). It is concluded that serology by IFAT is a suitable method to diagnose these protozoan infections due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals.

Seroepidemiology of beef and dairy herds and fetal study of Neospora caninum in Argentina.

The purpose of the present work was to study the epidemiology of Neospora caninum in beef and dairy herds in the Humid Pampas of Argentina. The seroprevalence of N. caninum was evaluated in 2414 serum samples of cows from beef and dairy farms. An indirect fluorescent antibody test (IFAT) was used to determine specific antibodies. The sera was screened at a dilution >or=1:200 and >or=1:600 in cows with reproductive disease antecedents and without them, respectively. Cows without history of reproductive diseases from nine beef and fifteen dairy farms were grouped according to the percentage (> or <or= to 50%) of seropositive dogs. Additionally, the seroprevalence in beef and dairy cattle cohabiting in the same farm with these dogs was compared. Microscopic studies were performed in 188 aborted fetuses and/or their placentas. Formalin-fixed fetal tissues with microscopic lesions compatible with N. caninum were processed by immunohistochemistry (IHC). The seroprevalence in cows without reproductive diseases was 4.7% (19/400) for beef cattle and 16.6% (174/1048) for dairy cattle. The seroprevalence of N. caninum in dairy cattle was higher (P<0.05) in farms grouped according to the percentage (> or <or= to 50%) of seropositive dogs. The analysis of 966 serum samples from aborted cows, demonstrated positive 18.9% (41/216) and 43.1% (323/750) from beef and dairy herds, respectively. Microscopic lesions compatible with N. caninum were observed in 43 of 188 (22.8%) fetuses and/or placentas evaluated. The protozoan was identified in 29 of 43 (67.4%) aborted specimens, being the largest number of positive results in dairy fetuses. The results obtained demonstrate a high association between neosporosis and dairy herds, however, our data also reveals that N. caninum is an important risk factor for reproductive losses in the extensively farmed beef cattle in the Humid Pampas of Argentina.

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany

Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for gamma-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK(21), KH-R). The best growth of tachyzoites was observed in BHK(21) cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK(21) and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell lines may be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany.

Comparative evaluation of immunofluorescent antibody and new immunoblot tests for the specific detection of antibodies against Besnoitia besnoiti tachyzoites and bradyzoites in bovine sera

Besnoitia besnoiti, an apicomplexan parasite causes economically important disease in cattle in many countries of Africa and Asia is re-emerging in Europe. Serological identification of infected cattle is important because introduction of these animals into naive herds seems to play a major role in the transmission of the parasite. We report new, simplified immunoblot-based serological tests for the detection of B. besnoiti-specific antibodies. Antigens were used under non-reducing conditions in the immunoblots, because reduction of the antigen with beta-mercaptoethanol diminished the antigenicity in both, tachyzoites and bradyzoites. Ten B. besnoiti tachyzoite and ten bradyzoite antigens of 15-45 kDa molecular weight were recognized by B. besnoiti infected cattle, but not or only weakly detected by cattle infected with related protozoan parasites, Neospora caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, or Sarcocystis hirsuta. The sensitivity and specificity of B. besnoiti immunoblots were determined with sera from 62 German cattle with clinically confirmed besnoitiosis and 404 sera from unexposed German cattle including 214 sera from animals with a N. caninum-specific antibody response. Using a new scoring system, the highest specificity (100%) and sensitivity (90%) of the immunoblots were observed when reactivity to at least four of the ten selected tachyzoite or bradyzoite antigens was considered as positive. When a cut-off based on this scoring system was applied to both the tachyzoite- and the bradyzoite-based immunoblots, there was an almost perfect agreement with the indirect fluorescent antibody test with a titre of 200 as the positive cut-off. We identified and partially characterized 10 tachyzoite and 10 bradyzoite B. besnoiti antigens which may help to develop new specific and sensitive serological tests based on individual antigens and in the identification of possible vaccine candidates.

Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers

Neospora caninum infection is an important cause of bovine abortion. The infection can be transmitted transplacentally or by ingestion of oocysts shed by definitive hosts. There are few reports of dogs naturally shedding N. caninum oocysts and only some oocyst isolates were transferred into cell culture. The aim of the present study was to analyse N. caninum oocysts from the faeces of naturally infected dogs using a microsatellite-based typing technique and to compare them with cell culture-derived tachyzoites of the same isolates. To this end, N. caninum oocysts from six naturally infected dogs were inoculated into gamma-interferon knockout mice. After these mice had developed disease, tissue samples or peritoneal washings from necropsied mice were transferred into cell culture. Nested-PCR techniques were developed for the sensitive and specific amplification of N. caninum microsatellite-containing regions (MS1B, MS2, MS3, MS4, MS5 and MS10). DNA was extracted from oocysts and cell culture tachyzoites of each isolate, followed by amplification and sequence analysis of microsatellite-containing regions. Each parasite isolate examined yielded a unique microsatellite genotype, while no differences were revealed when data for N. caninum oocysts were compared with cultured tachyzoites of the same isolate. Our technique may allow the typing of clinical samples and different strains of N. caninum at the molecular level. This method may prove useful for the identification of infection sources in molecular epidemiological studies.

Microsatellite typing and avidity analysis suggest a common source of infection in herds with epidemic Neospora caninum-associated bovine abortion

Neosporosis is an important cause of reproductive failure in cattle worldwide. Two different abortion patterns associated with Neospora caninum infection have been observed in cattle herds: endemic and epidemic abortion outbreaks. The endemic pattern is characterized by an abortion problem in a herd persisting for several months or years, and is assumed to be caused by reactivation of a chronic infection. In epidemic outbreaks, abortions concentrate within a short period of time, most likely due to a recent point source exposure of naïve animals to N. caninum. The aim of the study was to characterize five N. caninum-associated epidemic abortion outbreaks in Germany by serological and molecular techniques, including a p38-avidity-ELISA and typing of N. caninum in clinical samples by multilocus-microsatellite analysis. DNA extracts from the brain of 18 N. caninum infected fetuses from epidemic abortion outbreaks were characterized using 10 N. caninum-microsatellite markers. Nested-PCR protocols were developed to amplify the marker regions MS1B, MS3, MS5, MS6A, MS6B, MS7, MS12 and MS21 from clinical samples for subsequent analysis by capillary electrophoresis. Microsatellites MS2 and MS10 were analyzed by previously reported sequencing techniques. Most dams which had aborted showed a low-avidity IgG response to the N. caninum p38-antigen, and in three of the five studied herds, the majority of the dams at risk, which had not aborted, had also low-avidity responses suggesting that infection with N. caninum had recently occurred in most animals. A common microsatellite pattern prevailed in all fetuses from each individual epidemic outbreak. This pattern was unique for each herd. Although the number of epidemic abortion outbreaks analyzed was limited, the observation of a common microsatellite pattern, accompanied by a low-avidity IgG response against N. caninum in the dams, supports the hypothesis of a recent infection from a common point source. The genetic diversity of N. caninum observed among these outbreaks may indicate that not a particular N. caninum genotype but the horizontal infection route determines the occurrence of epidemic abortions.

Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle

Sarcocystis cruzi and Neospora caninum infections in cattle are common throughout the world, and cause important economical losses. N. caninum can be transmitted horizontally by ingestion of oocysts or vertically from the infected dam to the fetus via the placenta. Vertical transmission for S. cruzi is infrequent and horizontal transmission is considered the most important route of infection. The objectives of this study were to evaluate the frequency of horizontal and vertical transmission for S. cruzi and N. caninum in a dairy cattle herd and to analyze IFAT titers as predictors of vertical transmission. Serum samples (n = 173) were collected from dairy calves at birth prior to colostrum ingestion and from their dams. In addition, 12 calves were also sampled after ingestion of colostrum, 25 female calves were sampled at 7 months, and 81 of the dams were also sampled at breeding. Sera were evaluated for S. cruzi and N. caninum antibodies by IFAT starting at 1:25 dilution. For S. cruzi, vertical transmission frequency was 1.7%, and all female calves evaluated at 7 months and cows were seropositive. Seroprevalence for N. caninum was 80.9% in cows and 30% in precolostrum calves. Vertical transmission frequency was 37.1%. Cows with high antibody titers (> or = 400) showed higher vertical transmission frequency (94.8%) than cows with low antibody titers (between 25 and 200) (14.8%). Negative precolostrum calves (7/12) had postcolostrum N. caninum titers 2-8 times higher than their dams. Estimated horizontal transmission frequency was 51 and 47%, based on differences of seroprevalences in calves and dams, and on the seroconversion of 9/19 negative precolostrum female calves when retested at 7 months, respectively. Average N. caninum titers of cows at breeding and calving were 120.6 and 320.9, respectively. Cows with a high titer at breeding had a high titer at calving. Therefore, N. caninum IFAT titers at breeding and calving could potentially be used as predictors of vertical transmission.

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 gamma-interferon knockout mice with 10(2) 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.

Evaluation of a commercial ELISA for the specific detection of antibodies against Besnoitia besnoiti

Bovine besnoitiosis is an economically important disease in cattle caused by the protozoan parasite Besnoitia besnoiti, which occurs endemically in many countries of Africa and Asia and is spreading in Europe. Serological identification of subclinically infected cattle is important to avoid the introduction of infected animals into naive herds. Here we determine the sensitivity and specificity of the PrioCHECK(®) Besnoitia Ab, a serological test recently introduced into the European market. Analytical specificity was examined using sera from animals experimentally infected with parasites related to B. besnoiti (n=27). Three animals experimentally infected with Neospora caninum or Toxoplasma gondii showed inconclusive reactions in the ELISA (percent positivity relative to the positive control [PP] 10% ≤ 20%) while all other sera reacted negative (PP<10%). An estimate of the diagnostic specificity was obtained by analysing field sera from bovine herds without besnoitiosis but with abortion problems associated to N. caninum (n=403). The analysis revealed a specificity of 94.3% or 96.8% depending on the applied cut-off (PP 10% or 20%, respectively). Sensitivity was assessed with sera from 110 animals of a herd in Germany where clinical bovine besnoitiosis was first diagnosed in September 2008. A positive serological reference standard was defined regarding sera from animals as reference positive, if these animals had tested positive in at least two of a panel of three other serological tests (two different B. besnoiti immunoblots and one immunofluorescence antibody test) on both of two sampling dates, November 2008 and April 2009. A diagnostic sensitivity of 91.8% or 75.5% was determined for sera collected in November 2008 and a sensitivity of 82.7% or 50% for sera collected in April 2009 (cut-off PP 10% or PP 20%, respectively). The marked drop in sensitivity from November 2008 to April 2009 was predominantly observed in reference-positive cattle without clinical signs. We conclude that PrioCHECK(®) Besnoitia Ab is a valuable diagnostic tool to detect clinically infected animals. Thus it may be used to support control measures, e.g., for the separation of infected animals from the remaining herd to avoid a further transmission of the infection within the herd.

Seroprevalence of Neospora caninum, Toxoplasma gondii and Sarcocystis sp. in llamas (Lama glama) from Jujuy, Argentina.

Llamas (Lama glama) are South American camelids described as intermediate hosts of Neospora caninum, Toxoplasma gondii and Sarcocystis aucheniae. Due to the potential role of these protozoan infections as a cause of economic losses, the aim of this study was to determine the seroprevalence for T. gondii, N. caninum and Sarcocystis sp. in llamas from Argentina. Serum samples from 308 llamas (>2 years old) were collected between 2005 and 2007. A total of 55 farms located in six departments of Jujuy province, Argentina were sampled. Presence of antibodies to N. caninum, T. gondii and Sarcocystis sp. was determined by the indirect fluorescent antibody test (IFAT). For Sarcocystis, 2 different bradyzoites-based antigens were prepared using S. aucheniae and S. cruzi. Sera were tested at dilutions 1:25 and 1:50. Antibodies to N. caninum were found in 4.6% serum samples. Fifty percent of departments and 14.5% of farms had positive animals. Antibodies to T. gondii were found in 30% of samples, distributed in 66% of departments and 43.6% of farms. Antibodies to Sarcocystis sp. were detected in 96% of samples and all departments and farms had positive animals, suggesting frequent contact between llamas and canids. Co-infection with N. caninum, T. gondii and Sarcocystis sp. was also recorded. Low seroprevalence of N. caninum in llamas detected in this study could be related to climatic and geographical conditions that limit cattle breeding activity, reducing the source of infection for definitive hosts. Seroprevalence of T. gondii and the positive animal distribution suggest frequent contamination of grass with felid faeces. In conclusion, this is the first report of combined seroprevalence for N. caninum, T. gondii and Sarcocystis sp. in llamas. Further studies are needed to determine the potential role of these protozoan infections as cause of abortion in Argentina as well as presence of these protozoans in llama meat used for human consumption.