Isolation and genetic characterization of Neospora caninum from asymptomatic calves in Spain.

Neospora caninum is a cyst-forming parasite that causes abortion in cattle. Despite this parasite's ubiquitous distribution and wide host range, the number of N. caninum isolates obtained to date is limited. In vitro isolation of the parasite is arduous and often unsuccessful. In addition, most isolates have been obtained from clinically affected hosts and therefore could be biased towards more virulent isolates. In this report, an improved isolation approach from transplacentally infected newborn calves was undertaken and 9 new isolates were obtained. Moreover, a microsatellite technique was applied to investigate the genetic diversity of these isolates. Most isolates showed specific genetic profiles. However, the Nc-Spain10 isolate was identical to the previously described Nc-Spain1H isolate and Nc-Spain3H was identical to Nc-Spain4H. These isolates were likely to have identical genotypes because they were isolated from distinct calves of the same herd. Future pathogenic characterization of these isolates will contribute to the investigation of the relationship between isolate virulence and the outcome of infection, as well as other epidemiological features, such as transmission.

Seroprevalence and risk factors associated with Neospora caninum infection in different dog populations in Spain.

In this study, Neospora caninum seroprevalence and some associated risk factors were investigated in four different dog populations in Spain. N. caninum seropositivity was significantly higher in farm dogs (51%, 51/100) (P<0.001) and lower in household dogs (2.9%, 3/102) (P<0.0001). The seroprevalence in hunting (23%, 23/100) and stray (24.5%, 23/94) dogs was moderate, and no significant differences were observed between these two populations (P>0.05). A significantly higher number of dogs showed titres of 1:50-1:100 (68%, 68/100) than >or=1:200 (33%, 33/100) titres (P<0.0001). N. caninum antibodies were more often detected in mixed breed than pure breed dogs (P<0.01), but when data were stratified by dog type a significant association was not found (P>0.05). A significantly higher prevalence of N. caninum was observed in dogs over 1 year old (P<0.01), indicating that horizontal transmission may be the most important route of infection. The presence of N. caninum antibodies was significantly more frequent in Leishmania infantum-seropositive hunting (P<0.05) and stray dogs (P<0.00001). This study confirms that farm, stray and hunting dogs can be considered at-risk dog populations for N. caninum infection in Spain.

Role of adult sheep in transmission of infection by Cryptosporidium parvum to lambs: confirmation of periparturient rise.

In sheep farms, oocyst shedding by asymptomatic adult carriers is one of the mechanisms which may explain maintenance of infections by Cryptosporidium parvum between lambing periods. The objective of this work was to investigate this hypothesis and the existence of a periparturient rise in oocyst shedding. Fourteen pregnant sheep were randomly selected from two farms with a history of neonatal diarrhoea caused by C. parvum and samples were collected from the 6th week before birth until 2 weeks after birth. Faecal samples were filtered, concentrated and examined for oocysts using an indirect immunofluorescence assay. The kinetics of anti-C. parvum antibodies (IgG and IgA) were studied using an indirect enzyme-linked immunosorbent assay. All except one animal excreted C. parrum oocysts at some time during the experimental period. The percentage of animals passing oocysts increased in the first week post-partum (farm 1) and in the first week before birth (farm 2). The numbers of oocysts excreted ranged from 20-440 oocysts g(-1) of faeces. In contrast, no significant changes in the anti-C. parvum immunoglobulin levels were observed over the sampling period. Finally, a high percentage of lambs (71%) born to these ewes acquired infection in the first 2 weeks of life.

Seroprevalence of Neospora caninum infection in dairy and beef cattle in Spain.

In recent years, neosporosis has been identified as a major cause of abortion in dairy and beef cattle. Although the disease has been described worldwide, there is a Jack of information concerning the prevalence of this infection in different cattle production systems. The aim of this study was to investigate the seroprevalence of Neospora caninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study was undertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were considered. Using a 95% level of confidence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143 dairy herds were randomly selected and sampled. Nine animals (> 1 year old) were randomly sampled in each herd to detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for specific anti-N. caninum IgG using an ELISA. Specific antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds. Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattle production system (dairy or beef) was statistically significant [(chi2)Y= 29.21, P < 0.001, OR = 4.04 (2.35-6.99)]. Herd size of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection was associated with herd size in beef cattle (chi2 = 12.79, P < 0.01). Finally, no association was found between replacement or pasture management and infection in beef herds.

Pattern of recognition of Neospora caninum tachyzoite antigens by naturally infected pregnant cattle and aborted foetuses.

Different aspects of Neospora tachyzoite antigen recognition by Neospora-infected heifers and cows and aborted foetuses were studied. The pattern of antigen recognition and the relationship between IFAT titres and number of Neospora antigens detected, were evaluated. In addition, the tachyzoite antigens involved in the humoral immune response developed against infection in normal cows and cows that aborted were also characterised throughout pregnancy. Comparison of tachyzoite antigen recognition was carried out in 13 thoracic and/or abdominal fluids from Neospora aborted foetuses and 33 sera from Neospora infected cows that had aborted. The kinetics of Neospora-antigen recognition was studied in Neospora-infected heifers and cows that had aborted foetuses (7) or not (14) during pregnancy. Based on the frequency and intensity of recognition, four IDAs-17-18, 34-35, 37 and 60-62kDa antigens-have been described. Moreover, a correlation was found between Western blot results and IFAT titres in both age groups. In relation to antigen recognition throughout pregnancy by seropositive cows that had aborted or not, the antibody fluctuations throughout pregnancy described in the literature could be due to differences in the intensity and frequency of recognition of particular antigens, especially the 17-18kDa antigen. We emphasize the important role that the 17-18kDa antigen could play in the serological diagnosis of Neospora infection in cattle as this was intensely detected in 100% of the animals.

Prevalence of Tritrichomonas foetus infection in beef bulls in northwestern Spain.

The purpose of this study was to investigate the prevalence of Tritrichomonas foetus infection in beef bulls in north-western Spain. The study area comprised of 350 herds (5909 cows and 102 bulls) managed under extensive or semi-extensive systems where natural mating alone or alternated with artificial insemination are used. The targets of this survey were bulls of 1 year of age or older. Preputial smegma samples were taken from 70 bulls (68.6% of bull population) servicing a total of 184 herds (52.6%) and 4136 cows (69.9%). Data collected at sampling included farm location, herd size, age, breed, private or communal bull and previous infertility problems in the herd. The percentage of infected bulls was 2.9% (2 of 70). Age of infected bulls was 5 and 7 years and both were of the local breed, Asturiana de los Valles. These results confirm the presence of T. foetus infection in Spain and the necessity to include this disease in the differential diagnosis of reproductive failure in rangeland grazing cattle.

Evaluation of ovine abortion associated with Toxoplasma gondii in Spain by different diagnostic techniques.

A total of 173 aborted ovine foetuses and seven aborted caprine foetuses, submitted from different points of north and central Spain, were analysed to determine the role of T. gondii in abortion and to compare the utility of the most widely used techniques in diagnosis of the congenital infection (histopathology, serology--IFAT and ELISA--and a nested-PCR). Parasite infection was diagnosed in 40 (23.1%; n = 173) ovine foetuses by at least one of the diagnostic techniques used. A higher percentage of foetuses were diagnosed using serological techniques (IFAT and ELISA) (28.3%; n = 106) than by histologic examination (8.7%; n = 173) or PCR (6.9%; n = 173). No significant association between infection and the foetal age categories was found (P > 0.05). In this study, 106 aborted foetuses were analysed by all of the three diagnostic techniques. When we compared serological results, perfect agreement between ELISA and IFAT was obtained. On the contrary, slight to fair agreements were observed when histology results were compared with those obtained by serology and PCR techniques. All the positive foetuses were aborted in the mid (60%) or last (40%) term of pregnancy, but no significant differences were found between ages of the infected and non-infected foetuses (P > 0.05). This report indicates that toxoplasmosis may be a common cause of small ruminant abortion and neonatal death in Spain and points out the necessity of using different and complementary techniques to increase the probability of detecting Toxoplasma infection in an aborted foetus.

Teaching veterinary parasitology throughout the world.

In order to understand how the teaching of parasitology in veterinary schools and faculties in the world is carried out, a questionnaire was sent to all centres listed in the W.H.O. World Directory. A total of 91 replies were received. Additional information was obtained from the report of a symposium held in Hannover, Germany in 1978 and from the Education Committee of the American Association of Veterinary Parasitologists in 1981. The academic level, the place of parasitology in the veterinary curriculum, textbooks and practical instruction, evaluation techniques, teaching staff, institute organization and publications are discussed.

Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain.

Eighty foetuses from some of the main cattle-producing regions in Spain were analysed to investigate the participation of Neospora caninum in cases of bovine abortion. Diagnosis of the infection was determined by histopathological analysis complemented with immunohistochemistry, serology (IFAT and ELISA) and PCR tests. A total of 38.8% of the bovine foetuses analysed were considered to be infected by at least one of the diagnostic techniques used. Microscopic lesions consistent with Neospora infection in brain were identified in 31.3% of the samples, whereas only 10.7 and 15.3% were positive using serological and PCR analysis, respectively. Perfect agreement was shown between IFAT and ELISA, although there was little agreement among results of the other diagnostic techniques. Gestational age of aborted foetuses checked ranged from <3 to 9 months, with a mean of 5.9 months, and no difference in age was found between infected and non-infected foetuses (P>0.05). This study confirms the importance of N. caninum as a cause of abortion in Spain and underlines the need to use different diagnostic techniques to increase the chance to detect the infection in aborted foetuses.

Herd-level risk factors associated with Leptospira spp. seroprevalence in dairy and beef cattle in Spain.

Our aim in this cross-sectional study was to investigate the seroprevalence of Leptospira spp. infection in herds and cattle and the relationships between seroprevalence and beef versus dairy, size, replacement policy and grazing management in a representative area of beef- and dairy-cattle production in Spain. Herds were the initial sampling unit. Blood samples were collected from 762 dairy cattle belonging to 81 herds and 1238 beef cattle from 134 herds; sera were tested for antibodies against 11 serovars of Leptospira (autumnalis, ballum, bratislava, canicola, castellonis, copenhagheni, grippotyphosa, hardjo, louisiana, pomona and tarassovi) using the microagglutination test. Forty-three percent (36.2-49.5%) of the herds and 8% (6.4-8.8%) of the individuals were seropositive against one or more of the serovars studied. Bratislava was the most-prevalent serovar (24% of the herds and 4% of the individuals) followed by hardjo (11 and 1%, respectively). Grippotyphosa, copenhagheni and tarassovi were more prevalent in dairy than in beef herds (P<0.001, P<0.05, P<0.05, respectively) -- but no significant association was found between herd-size and Leptospira seroprevalence for any of the serovars considered.

Effects of Neospora caninum infection at mid-gestation on placenta in a pregnant mouse model.

Neospora caninum is one of the more-efficient transplacentally-transmitted organisms. The goal of the present study was to investigate the pathologic and immunologic changes that occur at the materno-fetal interphase in pregnant BALB/c mice infected with N. caninum at mid-gestation. Parasite DNA was detected in feto-placentary units 3 days post-infection (PI). On day 7 PI, the DNA detection level and parasite burden were significantly higher in the placentas than in the fetuses, which may indicate that the parasite is mainly multiplying in the placenta during the initial infection. In the spleens of infected dams, we observed an increase in IFN-γ, IL-10, and IL-4. However, only IL-4 was upregulated in placentas from the infected dams; this may enhance susceptibility to N. caninum at the materno-fetal interphase and favor transmission to the progeny. Finally, an increase in TNF-α expression in nested-PCR-positive placentas combined with necrosis may compromise the viability of the fetuses.

Celozoic myxosporidians (Myxidium spp. and Chloromyxum spp.) of cyprinids from the river Esla (León, NW Spain). I. Description of the species.

In the examination of 417 Cyprinids, belonging to 7 species, caught in the Esla river from April 1977 to December 1979, the following celozoic myxosporidians were found: Myxidium macrocapsulare Auerbach, 1910 in Leuciscus cephalus cabeda and Chondrostoma polylepis polylepis; Myxidium carinae n. sp. in Barbus barbus bocagei; Chloromyxum complicatum n. sp. in B. barbus bocagei and L. cephalus cabeda; Chloromyxum chondrostomi n. sp. in Ch. polylepis polylepis; and Chloromyxum cyprini Fujita, 1927 in B. barbus bocagei. All of them were found in the gall bladder, except Chl. cyprini that also parasitizes the liver. The myxosporidians are described and drawings and photomicrographs are presented. Data on the prevalence and intensity of their infections are also given.

Celozoic myxosporidians (Myxidium spp. and Chloromyxum spp.) of cyprinids from the river Esla (León, NW Spain). II. Population dynamics.

In the analysis of the population dynamics of various celozoic Myxosporidians found in Cyprinids of the river Esla (León, NW Spain) a bimodal seasonality was observed for Myxidium macrocapsulare Auerbach, 1910, Myxidium carinae Alvarez-Pellitero et al., 1983 and Chloromyxum complicatum Alvarez-Pellitero et al., 1983. We did not observe a clear influence of the age and sex of the host on the infections. An apparently positive association was observed for Myxidium carinae and Chloromyxum complicatum in Barbus barbus bocagei, and for Myxidium macrocapsulare and Chloromyxum complicatum in Leuciscus cephalus cabeda, which we consider due probably to ecological factors.