Phenology and attraction of potential Culicoides vectors of bluetongue virus in Basque Country (northern Spain).

Bluetongue virus is transmitted by Culicoides biting midges (Diptera: Ceratopogonidae). Culicoides associated with livestock were captured using CDC blacklight traps at three BTV-infected farms in Basque Country between November 2007 and December 2008. Twenty-seven and nineteen Culicoides species were collected in outdoor and indoor habitats respectively. Indoor insect community represented 86.1% of the whole captured individual biting midges. Culicoides obsoletus/Culicoides scoticus (two sibling species of the Obsoletus complex) were dominant throughout all months and sexes with maximum phenological peaks in November 2007 and June-July 2008. Culicoides lupicaris was the second most dominant species followed by Culicoides pulicaris (both species of the Pulicaris complex). Few specimens of Culicoides imicola, the principal Afro-Mediterranean vector of BTV, as well as four new species recorded for the Iberian Peninsula, were also collected. BTV was detected by RT-PCR from pools of C. obsoletus/C. scoticus, C. lupicaris and C. pulicaris parous females. DL-Lactic acid significantly attracted more C. obsoletus/C. scoticus females and males, C. lupicaris females, C. pulicaris females and Culicoides punctatus females and males; whereas acetone increased only the captures of Culicoides achrayi.

Border disease virus seroprevalence correlates to antibodies in bulk-tank milk and reproductive performance of dairy sheep flocks.

There is a great need to establish effective tools to control border disease virus (BDV) in European dairy sheep flocks. Hence, our main aim was to investigate the accuracy of analyzing anti-BDV antibodies in bulk-tank milk (BTM) in detecting the real BDV seroprevalence in dairy sheep flocks. Furthermore, the relevance of BDV to reproductive performance of dairy sheep flocks prompted us to search for the association between BDV seroprevalence and reproductive parameters. For these purposes, 34 flocks were selected based on different percentages of antibody inhibition (AIP) values in BTM as estimated by ELISA. Serum samples from 10 replacement lambs older than 6 mo, 10 ewes 1 to 2 yr old, and 10 ewes > 2 yr old were collected and analyzed for the presence of anti-BDV antibodies by ELISA. A negative relationship between BDV AIP in BTM and within-flock seroprevalence was observed. Flocks with a high AIP (> 80%) had an average of 2.5% seropositive animals; flocks with a moderate AIP (46-79%) had 11.4% seropositive animals; and finally, flocks with an AIP < or = 45% showed a high flock seroprevalence (57.2%). Ten out of 34 flocks showed a high BDV seroprevalence in lambs, suggesting the presence of persistently infected animals in the flock. The observed AIP values in BTM from these likely BDV-infected flocks were indicative of a high seroprevalence. The analysis of reproductive-parameters data collected from these flocks showed no differences in fertility or prolificacy in relation to BDV circulation rates. Nonetheless, lamb mortality was significantly greater in flocks with low-moderate seroprevalence (10-30%), probably as a result of a first-time contact with BDV of previously naïve ewes. These findings suggest that testing of BTM samples may be useful in inferring the BDV seroprevalence in a flock.

Pathogenic characterization in mice of Neospora caninum isolates obtained from asymptomatic calves.

In this study, we characterized 8 new isolates obtained from healthy but congenitally infected calves using a BALB/c mouse model. Neospora caninum-infected mice survived without exhibiting any clinical signs of disease. Nevertheless, differences among isolates in parasite organ distribution, parasite burden and the severity of histopathological lesions were determined. Mice infected with the Nc-Spain 5H, Nc-Spain 7 and Nc-Spain 9 isolates showed higher parasite burdens and more severe brain lesions during the late phase of infection compared to mice infected with the Nc-Spain 2H, Nc-Spain 3H or Nc-Spain 6 isolates. Furthermore, differences in the immunoglobulin IgG1 and IgG2a isotype kinetics induced by these isolates were observed, with a more rapid IgG2a response seen in mice infected with the Nc-Spain 2H and Nc-Spain 3H isolates. These results confirm the intra-species variability of N. caninum pathogenicity.

Clinical and laboratorial findings in pregnant ewes and their progeny infected with Border disease virus (BDV-4 genotype).

To evaluate the pathogenicity of local isolates of ovine pestiviruses (BDV-4 genotype), 13 virus- and antibody-negative, artificially inseminated pregnant ewes were challenged on days 108 (5 ewes), 76 (5 ewes) and 55 of pregnancy (3 ewes) with 2 ml of ovine pestivirus containing 10(6) TCID(50). Viraemia was detected by RT-PCR from 2 to 15 days pi in most ewes. No abortion due to the infection was observed but the number of stillbirths was high (32%), and bodyweight at lambing was significantly reduced compared to the experimental flock of origin used as control. Clinical symptoms in live lambs consisted on tremors, gait anomalies and inability to stand unaided. Skeletal abnormalities (brachygnathia, prognathia, arthrogryposis) were present in 44% of the lambs. Only 20% of the lambs were clinically normal. RT-PCR was a very sensitive technique compared to antigen ELISA in detecting viral presence in experimentally infected ewes and their progeny.

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.

Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses.

Three bulls with experimentally induced primary infection with Neospora caninum were re-infected intravenously with 10(8) live N. caninum tachyzoites of the NC-1 isolate at 300 days post-infection to investigate the presence of N. caninum in semen and blood, and the associated immune responses. In parallel, three bulls with experimentally induced primary infection with N. caninum and three non-infected bulls were also monitored. Re-infected and infected bulls showed an intermittent presence of N. caninum DNA in semen with a parasite load ranging from 0.1 to 15.6 (mean 4.4) and 0.1 to 11.1 (mean 4.1) parasites/ml, respectively. Re-infected bulls showed significant and persistent serum-specific IgM and IgG antibody responses. Specific IgG levels were detected in seminal plasma of all infected bulls, but the magnitude of the response was significantly higher in re-infected rather than in chronically infected animals. The mean specific IFN-gamma levels in re-infected bulls were significantly increased as early as 3 and 7 days after experimental infection when compared to bulls in other groups. This study showed that the intermittent presence and parasite load of N. caninum in the semen of re-infected bulls is very similar to that reported in chronically infected animals. The protozoa could not be isolated from BALB/c nu/nu mice inoculated with PCR-positive semen samples and inseminated heifers with pooled semen samples did not show seroconversion. Plasma IFN-gamma level seems to be a good indicator of a recent N. caninum infection.

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain.

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms.

AIMS: To determine prevalence and strain diversity of thermophilic campylobacters in healthy ruminants and swine. METHODS AND RESULTS: Faecal samples collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country were screened in pools for thermophilic campylobacters. Two hundred and three herds were positive (67.1% dairy cattle, 58.9% beef cattle, 55.0% sheep and 52.9% pig), and species-specific PCR identified Campylobacter jejuni in 20.7% of the herds and Campylobacter coli in 6.4%. Campylobacter coli was isolated from the four production systems and was the most prevalent species in swine, where C. jejuni was not found. Other thermophilic campylobacters were found in all production systems. Four hundred and ninety-three animals from 11 positive herds were individually analysed, detecting significantly higher within-herd prevalences in dairy cattle (66.7%) and swine (57.8%) than in sheep (8.8%) or beef cattle (5.4%). flaA PCR-RFLP and pulsed-field gel electrophoresis analysis of a selection of isolates showed high genetic diversity. CONCLUSIONS: Healthy swine, cattle and sheep are important reservoirs of thermophilic campylobacters of different species and high genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient farm-based intervention measures are needed to reduce risk of infection. Non-C. jejuni/C. coli species should be monitored to investigate their significance for infection.

Experimental neosporosis in bulls: parasite detection in semen and blood and specific antibody and interferon-gamma responses.

AIM: To investigate the presence of Neospora caninum in semen and blood, and the development of specific antibody and interferon-gamma (IFN-gamma) responses in experimentally infected bulls. METHODS: Eight bulls were intravenously infected with 10(8) live N. caninum tachyzoites of NC-1 isolate. The presence of N. caninum in semen and blood was assessed using a nested-PCR procedure. PCR-positive semen samples were bioassayed using a BALB/c nu/nu mouse model. Specific anti-N. caninum antibody and IFN-gamma responses were also examined. In parallel, eight seronegative bulls were studied as non-infected controls. All bulls were monitored for 26 weeks. RESULTS: All eight experimentally infected bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. Parasite load in semen ranged from 0.1 to 14.5 parasites/ml (mean 6.0). N. caninum could not be detected in BALB/c nu/nu mice inoculated with PCR-positive semen samples. A significant increase in mean serum specific IgM antibody response to N. caninum was detected between 10 and 28 days post-infection (p.i.). Serum specific IgG, IgG1, and IgG2 antibody levels in experimentally infected bulls were significantly different after 21, 10, and 14 days p.i. as compared to controls, respectively. Specific anti-N. caninum IgG were detected in seminal plasma from infected bulls and values obtained were different from controls after 25 days p.i. Mean specific IFN-gamma responses in experimentally infected bulls were significantly higher than controls 3 days p.i. CONCLUSIONS: This is the first study to report the presence of N. caninum DNA in the semen and blood of experimentally infected bulls. Our observations indicate an intermittent presence of N. caninum in low numbers in semen and associated with chronic stage of the infection. This study is also the first to report the detection of anti-N. caninum IgG in seminal plasma of experimentally infected bulls.

Disseminated Mycobacterium avium subsp. avium infection in a pet Korean squirrel (Sciuris vulgaris coreae).

A disseminated Mycobacterium avium subsp. avium infection was diagnosed in a pet Korean squirrel. Grossly, multiple small nodules in the lung, liver, spleen, and skin were observed. Adrenal glands were very enlarged. The only tissue exhibiting necrosis and calcification was a very enlarged bronchial lymph node. The remaining lymph nodes were slightly enlarged. Moderate ascites was also observed. Microscopically, a disseminated granulomatous inflammation with numerous lymphocytes was seen. Acid-fast bacilli were detected in macrophages, in giant cells, free in the interstitium, and in some lymphatic vessels, both within cells and free in the lumen. M. avium subsp. avium was isolated and identified by polymerase chain reaction-restriction endonuclease analysis.

Intrauterine Neospora caninum inoculation of heifers and cows using contaminated semen with different numbers of tachyzoites.

OBJECTIVE: To investigate the potential of different Neospora caninum tachyzoite doses to infect heifers (experiment 1) and cows (experiment 2) when administered in utero by artificial insemination via contaminated semen. METHODS: In experiment 1, five groups of 5, 7, 8, 9, and 5 cyclic heifers were hormonally synchronized and artificially inseminated with semen containing 0 (A, controls), 10(2) (B), 5 x 10(3) (C), 5 x 10(4) (D), and 5 x 10(5) (E) live N. caninum NC-1 isolate-tachyzoites, respectively. Experimental infection was followed for 100 days. Parasitaemia and specific serum IgG, and interferon-gamma (IFN-gamma) responses were studied. In experiment 2, four groups of 9, 10, 9, and 9 adult multiparous cows with confirmed infertility problems of diverse aethiology were hormonally synchronized and artificially inseminated with semen containing 0 (a, controls), 10(2) (b), 5 x 10(3) (c), and 5 x 10(5) (d) live N. caninum NC-1 isolate-tachyzoites, respectively. Experimental infection was followed for 63 days. Parasitaemia and specific serum IgG responses were studied. RESULTS: In experiment 1, parasitaemia was detected in 1, 2, and 3 heifers from groups B, C, and D, respectively, between 9 and 23 days after insemination. Persistent specific serum antibody responses were detected in 2 and 3 heifers from groups D and E, respectively. Transient specific serum antibody responses were detected in 2, 1 and 1 heifers from groups C, D, and E, respectively. In addition, 1 heifer from group B showed a serum-specific antibody level higher than cut off value at 21 days post-insemination. Heifers seroconverted between 23 and 47 days after insemination. Specific IFN-gamma levels were detected in 1, 4, 6, and 3 heifers from groups B, C, D, and E, respectively, between 9 and 55 days after insemination. Pregnancy rate in the control group (60%) was higher than those observed in inoculated heifers (0-42.9%). Pregnancy rates in inoculated heifers were lower when the tachyzoite dose was increased (B 42.9%, C 12.5%, D 11.1%, and E 0%). In experiment 2, no Neospora DNA in blood nor specific serum IgG to N. caninum were detected in any of the cows studied, except in one cow inoculated with 5 x 10(5) tachyzoites (group d) which showed a relative index x100 (RIPC) values of 9.4, 18.9, and 18.1 at 42, 56, and 63 days after insemination, respectively. CONCLUSIONS: This study provides evidence that the intrauterine infection via contaminated semen using 5 x 10(4) and 5 x 10(5) tachyzoites caused persistent serum-specific antibody responses in some heifers. On the basis of serological data, a dose-response effect was also observed. In addition, N. caninum would be a probable cause of early foetal death in inoculated heifers. In contrast, results obtained in a similar experiment with cows showing confirmed infertility indicate that higher doses, such as of 5 x 10(5) tachyzoites, were necessary to induce seroconversion in at least one animal.

Incidence of ovine abortion by Coxiella burnetii in northern Spain.

The infectious causes of ovine abortion occurring in 148 farms in northern Spain between 1999 and 2003 were investigated. Laboratory analysis included microbiological, serological, pathological and molecular techniques. Border disease was diagnosed in 16% of the flocks, toxoplasmosis in 15%, chlamydiosis in 12%, salmonellosis in 10%, Q fever in 3%, miscellaneous infections in 7% (Yersinia spp., Listeria spp., Brucella spp.), and inflammatory lesions compatible with an infectious cause were seen in 7% of the flock. In an additional 1% of the flocks non-infectious causes were identified, and a diagnosis was not reached in 38% of the flocks. When a PCR retrospective study was carried out to investigate the possible implication of Coxiella burnetii in the cases without diagnosis, including those with inflammatory lesions, the prevalence of this pathogen increased from 3% up to 9% of the flocks, revealing the importance of this zoonotic pathogen as a small-ruminant abortifacient agent. Placenta was the most commonly positive sample, but other fetal tissues were also of value for C. burnetii DNA detection. The present results update information about the situation of abortion in sheep farms in northern Spain, and highlight the relevance of molecular diagnostic tools in routine laboratory analysis of abortions by C. burnetii.

Flock-prevalence of border disease virus infection in Basque dairy-sheep estimated by bulk-tank milk analysis.

Bulk-tank milk (BTM) samples from 154 sheep flocks were used to estimate BDV prevalence in the Basque Country in Spain using an ELISA and a RT-PCR test. The proportion of antibody-positive flocks was 68% but varied significantly between provinces and was 93% in Araba and 54-55% in Bizkaia and Gipuzkoa. Most ELISA-positive flocks had very low antibody inhibition percentage (AIP) indicating high seroprevalence and recent BDV exposure. However, only 9% flocks were PCR-positive suggesting few infected ewes were being milked at the time of sampling. Phylogenetic analysis of the 5' NCR sequences of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study in the region, whereas the remaining isolate was closest to BDV type A. These results suggest that BDV strains in most Basque flocks have a common origin and differences in prevalence between provinces are associated to recent events affecting BDV spread such as use of communal pastures and sheep trading. The widespread distribution of BDV in the region, advocates for the implementation of BDV control strategies and highlights the potential risk of sheep as a pestivirus reservoir for other species.

Comparison of Neospora caninum distribution, parasite loads and lesions between epidemic and endemic bovine abortion cases.

It has been suggested that the abortion herd pattern could influence bovine foetal neosporosis. Here, a comparison of (i) Neospora caninum DNA-detectability by PCR, (ii) N. caninum-associated lesions and (iii) parasite loads in target organs was made between epidemic and endemic abortion cases. We observed that N. caninum DNA was predominantly detected in more than one organ in the foetuses from herds with epizootic rather than endemic abortion cases (P<0.05, Fisher F-test). The highest parasite burdens were found in the heart in foetuses from outbreaks of epidemic abortion and in the brain in endemic cases (P<0.05, Kruskal-Wallis H-test). Moreover, foetuses from epidemic outbreaks had significantly higher parasite burdens in heart (P<0.05, Mann-Whitney U-test) than endemic abortion cases. Epidemic abortion cases showed higher lesion frequencies in liver (P<0.05, Fisher F-test). This report confirms that the abortion herd pattern is an important factor that influences pathogenesis in natural N. caninum infections.

Pathological and aetiological studies of multifocal interstitial nephritis in wasted pigs at slaughter.

Multifocal interstitial nephritis in pigs has been associated with several infectious agents. The objective of the present study was to investigate several different potential infectious agents associated with "white-spotted" kidneys in pigs suffering from wasting at slaughter (aged 6-8 months). Twenty-nine case kidneys (with a "white-spotted" gross appearance) classified into 3 macroscopic lesional grades, and 15 control kidneys (lacking gross lesions), were obtained from a pig abattoir. Laboratory analyses to detect potential associations with the aforementioned pathological condition with Leptospira spp., porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bacteria, were carried out. Microscopically, interstitial nephritis with a lymphofollicular inflammatory pattern (follicular nephritis) was observed in both case and control kidneys, with a higher frequency seen in the former ones. No leptospires were identified, although antibodies to the Pomona and Bratislava serovars were detected. Some pyogenic bacteria were also isolated from both case and control kidneys. PCV2 nucleic acid was only detected in 1 case kidney. PRRSV antigen was not found in any tested sample. Some pigs were tested positive for PPV by serology. Apparently, none of the studied agents were specifically associated as being the potential cause of the renal lesions in the studied wasted pigs. The fact that these chronic lesions may have been the consequence of a previous infection with one of these studied microorganisms, or more, and eventually with other non-tested infectious agents during the growing-finishing period, cannot be ruled out.

Intrauterine Neospora caninum inoculation of heifers.

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.

Serological evidence of Leptospira interrogans serovar Bratislava infection and its association with abortions in cattle in northern Spain.

A cross-sectional study was carried out in the Basque Country of Spain to determine the seroprevalence of 10 Leptospira serovars in a population of dairy cattle with poor fertility, and a case-control study was carried out in another northern area to investigate the role of Leptospira interrogans serovar Bratislava in abortions. L. Bratislava was the most prevalent serovar in the cross-sectional study, with 25.4 per cent of the cows testing positive in the microagglutination test when a cut-off of 1:10 or higher was applied, followed by Leptospira Hardjo (8.2 per cent), Leptospira Pomona (7.7 per cent), Leptospira Autumnalis (0.7 per cent) and Leptospira Copenhageni (0.1 per cent). In the case-control study the seroprevalence of L. Bratislava was significantly higher among the cows which had aborted when a titre of 1:300 or more was used as a cut-off (9.7 per cent v 3.4 per cent, P=0.008); 69 per cent of the L. Bratislava-infected cows that had aborted apparently aborted as a result of the infection.