New threats in the recovery of large carnivores inhabiting human-modified landscapes: the case of the Cantabrian brown bear (Ursus arctos).

Understanding mortality causes is important for the conservation of endangered species, especially in small and isolated populations inhabiting anthropized landscapes where both natural and human-caused mortality may hinder the conservation of these species. We investigated the mortality causes of 53 free-ranging brown bears (Ursus arctos) found dead between 1998 and 2023 in the Cantabrian Mountains (northwestern Spain), a highly human-modified region where bears are currently recovering after being critically threatened in the last century. We detected natural traumatic injuries in 52.63% and infectious diseases in 39.47% of the 38 bears for which the mortality causes were registered, with 21.05% of these cases presenting signs of both infectious diseases and traumas. More specifically, almost 30% of the bears died during or after intraspecific fights, including sexually selected infanticide (10.53%). In addition, primary infectious diseases such as infectious canine hepatitis, distemper, clostridiosis and colibacillosis caused the death of 15.79% of the bears. The number of direct human-caused deaths (i.e., shooting, poisoning, snare) decreased over the study period. This study also reveals three new mortality causes triggered by pathogens, two of which-Clostridium novyi and verotoxigenic Escherichia coli-not previously described in ursids, and the other one, canine distemper virus, never reported in brown bears as cause of death. New management strategies for the conservation of Cantabrian bears, which are urgently needed due to the rapid expansion of the population, should consider the mortality causes described in this study and must promote further research to elucidate how the high prevalence of infectious diseases may threaten the current recovery of the population.

Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Comparative pangenomic analysis of Campylobacter fetus isolated from Spanish bulls and other mammalian species.

Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). The latter causes bovine genital campylobacteriosis, a sexually-transmitted disease endemic in Spain that results in significant economic losses in the cattle industry. Here, 33 C. fetus Spanish isolates were whole-genome sequenced and compared with 62 publicly available C. fetus genomes from other countries. Genome-based taxonomic identification revealed high concordance with in silico PCR, confirming Spanish isolates as Cff (n = 4), Cfv (n = 9) and Cfv biovar intermedius (Cfvi, n = 20). MLST analysis assigned the Spanish isolates to 6 STs, including three novel: ST-76 and ST-77 for Cfv and ST-78 for Cff. Core genome SNP phylogenetic analysis of the 95 genomes identified multiple clusters, revealing associations at subspecies and biovar level between genomes with the same ST and separating the Cfvi genomes from Spain and other countries. A genome-wide association study identified pqqL as a Cfv-specific gene and a potential candidate for more accurate identification methods. Functionality analysis revealed variations in the accessory genome of C. fetus subspecies and biovars that deserve further studies. These results provide valuable information about the regional variants of C. fetus present in Spain and the genetic diversity and predicted functionality of the different subspecies.

High Exposure to Livestock Pathogens in Southern Pudu (Pudu puda) from Chile.

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

Outbreaks of Acute Necrotic Enteritis and Haemolytic Crisis and Renal Disease Associated with Clostridium perfringens in Captive Toucans (Ramphastos spp).

Four toco toucans (Ramphastos toco), one channel-billed toucan (Ramphastos vitellinus) and one white-throated toucan (Ramphastos tucanus) died in two disease outbreaks in the same aviary in 2011 and 2016. Post-mortem examination revealed diffuse necrotic enteritis (NE) as the cause of death of five of these six birds. Clostridium perfringens was identified by culture and real-time multiplex PCR for C. perfringens α-, ß-, ε- and ι-toxin genes in ligated intestine of one toucan from each outbreak. At another aviary, two keel-billed toucans (Ramphastos sulfuratus) died peracutely from severe haemolytic crisis with haemoglobinaemic nephrosis and cholestasis and acute tubulointerstitial nephritis. Mild NE was present in these birds and C. perfringens was demonstrated in liver by bacterial culture and real-time multiplex PCR for C. perfringens α-, ß-, ε- and ι-toxin genes. To the authors' knowledge, this is the first description of outbreaks of NE associated with C. perfringens in captive toucans. Although haemolytic crisis has been reported in humans with C. perfringens type A septicaemia and hepatic abscesses, this presentation appears not to have been described in C. perfringens infections in toucans or other avian species. The factors causing C. perfringens proliferation and disease in the toucans were not identified. PCR for C. perfringens NetB toxin and enterotoxin genes performed retrospectively on one of the C. perfringens isolates from the second outbreak and on paraffin-embedded tissues from one dead toucan from the first outbreak was negative. With the current C. perfringens toxin typing scheme, C. perfringens type A was identified in the first two outbreaks.

In vitro invasion efficiency and intracellular proliferation rate comprise virulence-related phenotypic traits of Neospora caninum.

In this study, we examined the in vitro invasion and proliferation capacities of the Nc-Liv and ten Spanish Neospora caninum isolates (Nc-Spain 1 H - Nc-Spain 10). The invasion rate was determined as the number of tachyzoites that completed their internalisation into MARC-145 cells at 2, 4, and 6 h post-inoculation (pi). The proliferation rate was evaluated by determining the doubling time during the exponential proliferation period. Significant differences in the invasion rates of these isolates were detected at 2 and 4 h pi (P < 0.0001, Kruskal-Wallis test). At 4 h pi, the Nc-Spain 4 H and Nc-Liv isolates displayed the highest, while the Nc-Spain 3 H and Nc-Spain 1 H isolates had the lowest invasion rates (by Dunn's test). Variations in the proliferation kinetics of these isolates were also observed. Between different isolates, the lag phase, which occurs before the exponential growth phase, ranged from 8 to 44 h, and the doubling time ranged from 9.8 to 14.1 h (P = 0.0016, ANOVA test). Tachyzoite yield, which combines invasion and proliferation data, was also assessed and confirmed marked differences between the highly and less prolific isolates. Interestingly, a direct correlation between the invasion rates and tachyzoite yields, and the severity of the disease that was exhibited by infected pregnant mice in previous works could be established for the isolates in this study (Spearman's coefficient > 0.62, P < 0.05). The results of this study may help us to explain the differences in the pathogenicity that are displayed by different isolates.

Faecal shedding and strain diversity of Listeria monocytogenes in healthy ruminants and swine in Northern Spain.

BACKGROUND: Listeria monocytogenes is among the most important foodborne bacterial pathogens due to the high mortality rate and severity of the infection. L. monocytogenes is a ubiquitous organism occasionally present in the intestinal tract of various animal species and faecal shedding by asymptomatically infected livestock poses a risk for contamination of farm environments and raw food at the pre-harvest stages. The aim of this study was to determine the prevalence and strain diversity of L. monocytogenes in healthy ruminants and swine herds. RESULTS: Faecal samples from 30 animals per herd were collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country and screened in pools by an automated enzyme-linked fluorescent immunoassay (VIDAS) to estimate the prevalence of positive herds. Positive samples were subcultured onto the selective and differential agar ALOA and biochemically confirmed. L. monocytogenes was isolated from 46.3% of dairy cattle, 30.6% beef cattle and 14.2% sheep herds, but not from swine. Within-herd prevalence investigated by individually analysing 197 sheep and 221 cattle detected 1.5% of faecal shedders in sheep and 21.3% in cattle. Serotyping of 114 isolates identified complex 4b as the most prevalent (84.2%), followed by 1/2a (13.2%), and PFGE analysis of 68 isolates showed a highly diverse L. monocytogenes population in ruminant herds. CONCLUSION: These results suggested that cattle represent a potentially important reservoir for L. monocytogenes in the Basque Country, and highlighted the complexity of pathogen control at the farm level.

Detection of Border disease virus in fetuses, stillbirths, and newborn lambs from natural and experimental infections.

The purpose of the present study was to evaluate the use of enzyme-linked immunosorbent assay (ELISA) antigen detection in blood or fetal fluids and reverse transcription polymerase chain reaction (RT-PCR) amplification in tissues for routine laboratory diagnosis of Border disease virus (BDV) infection. Samples from 67 fetuses, 6 stillbirths, and 11 lambs from 25 commercial flocks with suspicion of BDV abortion and 3 fetuses, 7 stillbirths, and 15 lambs obtained from an experimental infection with a local isolate (BDV genotype 4) were investigated. Presence of BDV was detected by RT-PCR in 7.9% of fetuses, 50% of stillbirths, and 50% of lambs from the commercial flocks analyzed, corresponding to 8 of the 25 farms (32%). A similar percentage of the lambs and stillbirths from the experimental infection were positive by RT-PCR of tissue samples (54.5%), and the highest positivity was detected in lymph node, thyroid gland, and kidney. The current study revealed that RT-PCR analysis of stillbirths and lambs with clinical symptoms is more suitable than the analysis of fetuses to confirm the presence of BDV in a flock. Pestiviral antigen was detected by antigen ELISA in a high proportion of fetuses (24/58) and stillbirths (3/4) from commercial flocks, but in lambs, the presence of colostral antibodies masked the detection of the antigen by ELISA. Nevertheless, in lambs from the experimental infection that were not fed colostrum, antigen ELISA was less efficient than RT-PCR in detecting viral presence in stillbirths and lambs. Antigen ELISA is therefore recommended for fetuses with advanced autolysis that can adversely affect RNA integrity.

A survey of food-borne pathogens in free-range poultry farms.

A survey of the occurrence of Campylobacter, Salmonella, Listeria and Shiga toxin-producing E. coli was performed on 60 flocks of free-range chicken from 34 farms in the Basque Country (Northern Spain). Campylobacter was the most prevalent of the four pathogens, isolated in 70.6% of the farms, followed by L. monocytogenes (26.5%), and Salmonella (2.9%). No E. coli O157 or other STEC were isolated. In total 48 flocks from 26 farms were positive for at least one pathogen: 31 of them for a single pathogen (64.6%), and 17 for more than one species (35.4%). C. coli was more prevalent than C. jejuni (15 vs. 13 farms), and both species of Campylobacter were found in 3 farms. L. monocytogenes isolates were identified as serotype 4b complex, and the only Salmonella isolated was serovar Enteritidis. flaA PCR-RFLP performed on 91 Campylobacter isolates (36 C. jejuni and 55 C. coli) yielded 26 patterns, with higher diversity among the C. jejuni isolates. More than one pattern was found in 11 farms, and in 8 of them several patterns were found within the same flock. The findings of the present study suggest that the free-range rearing conditions described herein might have an advantageous effect on diminishing Salmonella but not on Campylobacter or L. monocytogenes flock contamination.

Unilateral orchitis and epididymitis caused by Salmonella enterica subspecies diarizonae infection in a ram.

A case of unilateral suppurative epididymo-orchitis associated with Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) infection is described in a 2-year-old ram. Gross lesions were characterized by severe enlargement of the scrotal contents, fibrous adhesions between testicular layers, coexistence of epididymal abscesses and foci of fibrinous exudate, and testicular atrophy. Microscopically, testicular and epididymal microabscesses and diffuse inflammatory infiltrates with abundant macrophages containing short Gram-negative rods were observed. Superimposed on the chronic lesions were fibrin deposits with clusters of neutrophils, as well as walled-off granulation tissue. Bacterial colonies were also identified in thrombosed spermatic cord vessels, scrotal lymph nodes, lung, and liver. S. enterica subsp. diarizonae serovar 61:k:1,5,(7) was isolated from the affected testis. To the authors' knowledge, this is the first report of infection of the testis and epididymis by Salmonella in rams. This organism must be taken into account in the differential diagnosis of ovine genital infections.

Carcass condemnation causes of growth retarded pigs at slaughter.

Condemnation causes of growth retarded pigs were studied in a Spanish abattoir. A total of 513 carcasses out of 6017 (8.5%) were rejected during inspection. The main reasons for condemnation were abscesses, cachexia, catarrhal bronchopneumonia, vertebral osteomyelitis, arthritis, pleuritis, peritonitis and pleuropneumonia. Positive relationships were found between tail lesions and arthritis (OR=5.23) or vertebral osteomyelitis (OR=24.81), while no relationships were found between tail lesions and abscesses. Lower risks were observed among carcasses condemned for cachexia, and were as follows: abscesses (OR=0.18), arthritis (OR=0.32), vertebral osteomyelitis (OR=0.06). Arcanobacterium pyogenes, either alone or in combination with other agents, was the main bacterial species isolated from abscesses, osteomyelitis and arthritis (73.5% of lesions). Direct economical losses associated with condemnation were calculated to be 30,000 Euro.

Influence of the stage of pregnancy on Neospora caninum distribution, parasite loads and lesions in aborted bovine foetuses.

In the present work we have studied in Neospra caninum aborted bovine foetuses the influence of foetal age (first, second and third gestational periods) on parasite distribution by nested PCR, parasite loads by real-time PCR and N. caninum associated lesions. For this purpose, a total of 220 aborted foetuses were analysed and detection of N. caninum infection was accomplished by nested-PCR in brain, heart and liver, detecting the presence of the parasite in 72 (32.7%) bovine foetuses. When the different age classes were compared, parasite DNA-detectability in heart and liver was reduced over time of gestation (P < 0.05, Fisher F-test). N. caninum distribution, parasite loads and lesions were studied on 34 out of 72 N. caninum-infected foetuses selected according to the stage of pregnancy and organs recovered. A higher number of positive-PCR tissue samples were observed in the foetuses corresponding to the first and second pregnancy periods. In the last trimester, the parasite could only be detected in the brain and, sporadically, in the diaphragm, heart and lymph nodes. The parasite loads decreased during pregnancy and the foetuses from the first period had higher parasite burdens in brain, heart, kidney and lung (P < 0.05, Kruskal-Wallis H-test) than in those corresponding to the other two trimesters of pregnancy. In addition, the observed lesions were more severe in foetuses from the first and second pregnancy periods than those from the third period (P > 0.05, Kruskal-Wallis H-test). Our results confirm the influence of N. caninum foetal age on pathogenesis in natural N. caninum infections.

Septicaemia secondary to infection by Corynebacterium macginleyi in an Indian python (Python molurus).

A seven-year-old female Indian python (Python molurus) weighing about 35kg was euthanased after several clinical episodes of stomatitis, pneumonia, ophthalmitis and dystocia over a period of four years. The animal had been maintained in a terrarium in a circus truck at an adequate temperature. During shows, however, the snake was considered to be exposed to stressful conditions for several hours at a time at low temperatures and with noise and bright lights. A post-mortem examination indicated ulcerative stomatitis, osteomyelitis, severe pneumonia and numerous granulomata and multifocal necrosis in stomach and spleen. Corynebacterium macginleyi was isolated in pure culture from the ulcerative stomatitis, and mixed with Stenotrophomonas maltophilia from the lungs and spleen. The findings indicated that the snake had died from a septicaemic process caused by C. macginleyi, probably originating from the stomatitis. The role of S. maltophilia as a secondary agent is discussed. The stress of the circus show and poor husbandry may have predisposed the animal to infection and septicaemia. This is the first report of C. macginleyi causing disease in a snake.

A Pasteurella multocida strain affecting nulliparous heifers and calves in different ways.

Pasteurella multocida isolates from dairy cattle on a farm in Spain were associated with pneumonia of calves (six isolates) and mastitis of heifers (five isolates). The objective was to determine if the P. multocida isolates retrieved from both disease scenarios were the same strain or whether more than one strain was present. The isolates were identified by a species-specific polymerase chain (PCR) assay, serotyped by the Heddleston scheme and then typed by a number of molecular genotyping assays including multi-locus sequence typing (MLST). The 11 isolates were confirmed as P. multocida but failed to react with any of the 16 Heddleston antisera. The PCR targeting the genes associated with the lipopolysaccharide outer core biosynthesis locus assigned all the isolates to L3-the type that contains Heddleston serovars 3 and 4. The MLST analysis showed all isolates belonging to ST 79 within the clonal complex of ST13. Only one of the isolates showed a slight different profile by the repetitive extragenic palindromic PCR. The conclusion was that the same strain was associated with pneumonia in calves and mastitis in heifers.

Comparison of blood polymerase chain reaction and enzyme-linked immunosorbent assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep.

A study was carried out to compare the performance of enzyme-linked immunosorbent assay (ELISA) and blood polymerase chain reaction (PCR) for diagnosis of paratuberculosis in cattle and sheep. For cattle, a set of 278 samples from 1 paratuberculosis-affected Friesian farm was used; it included 80 ELISA-positive samples and 198 ELISA-negative samples from an age-matched group. Ninety-four samples were from heifers and 184 were from 2-5-year-old cows. The overall analysis showed a clear association (Fisher exact test [FET] P = 0.0049) but a weak negative agreement (45.3%, kappa = -0.1665 +/- 0.0994) between the 2 tests. It reflected a moderate agreement among heifers (87.7%, kappa = 0.4471 +/- 0.2435) and a moderate disagreement among cows (62.7%, kappa = -0.3670 +/- 0.1057). For sheep, 496 blood samples from 53 Latxa dairy flocks were used; 180 of the blood samples were from dam/offspring pairs. The overall association between the 2 tests on ovine samples was strong (FET, P = 0.0005), whereas the agreement was low (kappa = 0.1622 +/- 0.1188). There was slightly better agreement for ewes (kappa = 0.2135 +/- 0.1992) than for lambs (kappa = 0.1193 +/- 0.1301). There was also a highly unlikely proportion of dam/offspring positive results (FET, P < 0.0001, kappa = 0.6269 +/- 0.1854). Four of 6 lambs that were necropsied 1 year after testing had paratuberculosis microscopic lesions in the ileocecal valve (3 lambs) or a PCR-positive result (4 lambs). These results suggest that blood PCR testing might be a potentially useful new approach in paratuberculosis diagnosis, especially in young animals.

Detection of Neospora caninum in the semen and blood of naturally infected bulls.

A prospective study was designed to investigate the presence of Neospora caninum in semen and blood of eight bulls seropositive to N. caninum using nested-PCR procedures. Positive semen and blood samples were bioassayed in a BALB/c nu/nu mouse model. Specific anti-N. caninum serological and interferon-gamma (IFN-gamma) responses were also studied. In parallel, five seronegative bulls acted as non-infected controls. All bulls were located in a collaborating AI centre and monitored for 22 weeks. Six of eight seropositive bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. In all positive semen samples, we consistently found Neospora-DNA in the cell fraction and not in seminal plasma. Parasite load, as determined by a real-time PCR in nested-PCR positive semen samples, ranged from 1 to 10 parasites/ml. We found no association between the presence of N. caninum DNA in semen and blood. N. caninum could not be detected in the BALB/c nu/nu mice inoculated with PCR-positive semen or blood samples. Specific IgG antibody levels in seropositive bulls fluctuated over time, at times falling below cut-off level. The response was predominantly IgG2, with significant differences compared to control bulls (P < 0.05). The overall mean specific IFN-gamma response in seropositive bulls was also higher than those observed in the control group (P < 0.05), although extensive variation in individual responses was observed among bulls and over time. No significant association was found between bulls showing Neospora DNA in semen, blood, or both, and specific IgG, IgG1, IgG2, IgM and IgA levels or IFN-gamma response. This study is the first to report the presence of Neospora DNA in semen and blood of naturally-infected bulls. Our observations indicate intermittent presence of N. caninum in blood and semen and shedding in semen in low numbers.

First detection of bovine viral diarrhoea virus type 2 in cattle in Spain.

BACKGROUND: Bovine viral diarrhoea virus (BVDV) is a member of the genus Pestivirus that belongs to the family Flaviviridae. BVDV is found worldwide in cattle population and causes significant economic losses to the dairy and beef industries. Two distinct genotypes of BVDV exist: BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). OBJECTIVE: The aim of the present study was to investigate retrospectively the presence of BVDV-2 in Spain. RESULTS: With this objective, 47 blood samples that had tested positive in an ELISA for BVDV antigen were selected. Samples had been submitted by practitioners to the Diagnostic Service of NEIKER. The 18 herds of origin were all located in the northern half of Spain. BVDV positive samples were genotyped by reverse transcription-PCR. BVDV-1 was detected with the highest frequency (46/47), in contrast to BVDV-2 (2/47). In one blood sample, both pestivirus genotypes, BVDV-1 and BVDV-2, were detected. Sequencing of a viral genomic region, 5' untranslated region, confirmed the identity of the BVDV-2 isolate. CONCLUSIONS: So far as the authors know, this is the first reported presence of BVDV-2 in cattle herds in Spain. This finding may have important implications for the epidemiology, diagnosis and control of BVDV infection in the country.

Detection of Neospora caninum in semen of bulls.

In cattle, transplacental infection is the main route of Neospora caninum transmission, but postnatal transmission by the oral uptake of sporozoite-containing oocysts shed by dogs may also be possible. Other routes of horizontal transmission, such as the venereal route, have not been investigated. In this study, we evaluated the presence of N. caninum DNA by a nested-PCR in fresh non-extended semen and frozen extended semen straws of five Holstein-Friesian bulls with naturally-acquired neosporosis. The infection status was assessed by an immunofluorescent antibody test (IFAT) and confirmed by immunoblotting (IB). Because of inhibitory components of semen, a protocol was developed to purify N. caninum DNA from bovine semen. Sporadically, N. caninum DNA was detected in non-extended fresh semen samples and frozen extended semen straws of the five seropositive bulls. In all positive samples, specific DNA was consistently found in the cell fraction of semen and not in seminal plasma. The parasite mean load in positive fresh semen samples determined by a real-time PCR was low oscillating between 1 and 2.8 parasites/ml of semen (maximum parasite load detected in one sample was 7.5 parasites/ml of semen). In parallel, another three similar but uninfected bulls acted as controls and no N. caninum DNA was amplified in any of their fresh and straw semen samples assayed. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined.

Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls.

Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI.

Molecular identification of a new pestivirus associated with increased mortality in the Pyrenean Chamois (Rupicapra pyrenaica pyrenaica) in Spain.

Pestivirus infection was identified in 16 of 17 chamois during an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in northeastern Spain in 2001-02. By analysis of the 5' noncoding regions of the virus, we assigned it to the border disease virus cluster with pairwise similarity values ranging from 82.1% to 88.1%. It will be important to investigate the association of this pestivirus with disease in Pyrenean chamois.