A new inactivated Tritrichomonas foetus vaccine that improves genital clearance of the infection and calving intervals in cattle.

Bovine trichomonosis is a sexually transmitted disease that is a primary cause of early reproductive failure in cattle. The aim of the present study was to develop a vaccine formulation based on Tritrichomonas foetus trophozoites inactivated by lyophilization and Quil-A-adjuvanted. The safety, immunogenicity and efficacy of this new vaccine formulation (Trichobovis®) administered by two routes (subcutaneous: SC, and intravulvar: IVU) were compared with a commercial vaccine (TrichGuard®) in a well-established experimental bovine model of genital T. foetus infection. The new vaccine was considered safe in cattle because only mild local reactions were found in the vaccination area, which disappeared 3 weeks after administration. Cows immunized with Trichobovis cleared the infection faster than the non-immunized/challenged group (27-28 vs. 60 days; P < 0.05). Not significant differences were observed with the commercial vaccine respect to the positive control group, or between SC and IVU routes. The new vaccine stimulated high serum anti-T. foetus IgG and genital IgA levels and generated an IgG booster effect similar to TrichGuard. IgA levels were associated with significantly earlier genital clearance of T. foetus in cows immunized with Trichobovis by SC route (G1A) or TrichGuard (G2). The strongest association was found in the group G1A on day 14 post-infection (p.i.) (r = -0.74) and in G2 on day 35 p.i. (r = -0.71). The efficacy of vaccination using Trichobovis on the reproductive performance was also investigated under field conditions in a herd where T. foetus was present. The calving intervals were significantly reduced by 45.2 days (P < 0.05), calves were born 28 days earlier (P < 0.05) and an increase of 8.7% in the calving rate (P > 0.05) was observed in the vaccinated group. These results demonstrate that Trichobovis improved the reproductive performance under field conditions in herds where T. foetus infection is present.

Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation.

Bovine neosporosis is currently considered one of the main causes of abortion in cattle worldwide and the outcome of the infection is, in part, determined by Neospora caninum isolate virulence. However, the dam and foetal immune responses associated with this factor are largely unknown. We used a model of bovine infection at day 110 of gestation to study the early infection dynamics (10- and 20-days post-infection, dpi) after experimental challenge with high- and low-virulence isolates of N. caninum (Nc-Spain7 and Nc-Spain1H, respectively). In the present work, dam peripheral cellular immune responses were monitored twice a week from -1 to 20 dpi. At different time points, IFN-γ and IL-4 production was investigated in stimulated dam blood and the percentage of monocytes, NK cells, B cells and T cells (CD4+, CD8+ and γδ) in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry. In addition, maternal iliofemoral lymph nodes and foetal spleen and thymus were collected at 10 and 20 dpi for the study of the same cell subpopulations. Peripheral immune response dynamics were similar after the infection with both isolates, with a significant increase in the percentage of CD4+ T cells at 6 and 9 dpi in PBMC, coincident with the higher levels of IFN-γ and IL-4 release. However, the levels of IFN-γ were significantly higher and an increase in CD8+ T cells at 9, 13 and 20 dpi was observed in the dams infected with Nc-Spain7. Nc-Spain1H infection induced higher IL4 levels in stimulated blood and a higher CD4+/CD8+ ratio in PBMC. The analysis of the maternal iliofemoral lymph node showed a significant enhancement in the percentage of NK, CD4+ and CD8+ T cells for the animals infected with the highly virulent isolate and euthanized at 20 dpi. Regarding the foetal responses, the most remarkable result was an increase in the percentage of monocytes at 20 dpi in the spleen of foetuses from both infected groups, which suggests that foetuses were able to respond to N. caninum infection at mid gestation. This work provides insights into how isolate virulence affects the maternal and foetal immune responses generated against N. caninum, which may influence the course of infection.

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection.

Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring.

Correction to: Effects of challenge dose and inoculation route of the virulent Neospora caninum Nc-Spain7 isolate in pregnant cattle at mid-gestation.

In the original publication of this article [1], there are error in the Fig. 5, the "ml" should be replaced by "mL" (Fig. 5A) and "IFNγ" should be "IFN-γ" in Fig. 5A, B. The correct figure is below.

Early Neospora caninum infection dynamics in cattle after inoculation at mid-gestation with high (Nc-Spain7)- or low (Nc-Spain1H)-virulence isolates.

Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 107 tachyzoites of high (G-NcSpain7)- or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.

Effects of challenge dose and inoculation route of the virulent Neospora caninum Nc-Spain7 isolate in pregnant cattle at mid-gestation.

Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 107 parasites, n = 6; IV2, 105, n = 6; IV3, 103, n = 6; and IV4, 102, n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 105 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation.

Effect of parasite dose and host age on the infection with Besnoitia besnoiti tachyzoites in cattle.

Bovine besnoitiosis is continuing to spread in Europe. Therefore, the development of ruminant animal models of infection is urgently needed to evaluate therapeutic and prophylactic tools. Herein, we studied the effect of parasite dose and host age on the infection dynamics with Besnoitia besnoiti tachyzoites in cattle in two independent experimental infections. In experiment A, twelve 3-month-old male calves were inoculated intravenously with either three different doses of tachyzoites (G1: 108 ; G2: 107 ; G3: 106 ) or with PBS (G4). In experiment B, six 14-month-old bulls were inoculated with 106 tachyzoites based on results obtained in experiment A. In both trials, clinical signs compatible with acute and chronic besnoitiosis were monitored daily; blood and skin samples were collected regularly for 70-115 days post-infection (pi). Finally, animals were killed, and tissues were collected for lesion and parasite detections. Infected animals developed mild-moderate signs compatible with acute besnoitiosis. Lymphadenopathy and fever were observed in both calves (from 12 hr until 7 days pi) and bulls (from 6 days until 9 days pi). Seroconversion was detected at 16-19 days pi, and antibody levels remained high. Infected animals did not developed characteristic clinical signs and macroscopic lesions of chronic besnoitiosis. However, successfully, parasite-DNA was detected in a reduced number of target tissues: conjunctiva, ocular sclera, epididymis, skin of the scrotum and carpus in calves (n = 10, 6 of which belonged to G3), and pampiniform plexus and testicular parenchyma in bulls. Remarkably, one tissue cyst and mild microscopic lesions were also detected. In summary, inoculated animals developed the acute besnoitiosis and chronic infection was evidenced by microscopic findings. However, our results suggest that tachyzoite dose and host age are not key variables for inducing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Thus, a further refinement of this model should evaluate other parasite- and host-dependent variables.

Efficacy of a control program for bovine trichomonosis based on testing and culling infected bulls in beef cattle managed under mountain pastoral systems of Northern Spain.

Bovine trichomonosis (BT) is a sexually transmitted disease that is considered a cause of early reproductive failure in cattle under extensive management conditions. Recently, Tritrichomonas foetus was detected in 41.5% of herds from one representative beef cattle breed (Asturiana de la Montaña; AM) reared in traditional mountain systems in Spain. The objective of the present study was to evaluate the effect of BT on reproductive performance and the economic consequences in AM herds. The benefits of a control program based on testing and culling infected bulls were also studied by comparing T. foetus prevalence and reproductive data before and after the implementation of the control measures. In infected herds, T. foetus infection increased calving intervals by 79 days (P<0.0001) and resulted in a higher percentage of cows-not-in calf (36% vs. 19%; P<0.001). An economic analysis showed that BT could reduce income by 68.7% in AM herds. The implementation of the control program decreased calving intervals (P<0.0001) and increased calving percentage (P<0.05). T. foetus prevalence showed a significant decline compared with the prevalence before implementing the control program (P<0.05). Nevertheless, after 2 years, the herd prevalence did not decrease (12.7-13.6%; P>0.05) and the herd incidence was 22.72%. The testing and culling policy was effective in improving reproductive efficiency but the complete elimination of BT without substantial changes in management appears unlikely because putative risk factors associated with the disease are present in the management of this breed.

Differences in the prevalence of Tritrichomonas foetus infection in beef cattle farmed under extensive conditions in northern Spain.

Bovine trichomonosis (BT) is a sexually transmitted disease of cattle caused by infection with Tritrichomonas foetus. In a recent study, T. foetus infection was detected in 41.5% of herds of an endangered beef breed, the Asturiana de la Montaña (AM), which is farmed under extensive, mountain pastoral systems in northern Spain. The objective of this study was to determine the prevalence of this pathogen in the more production-centred Asturiana de los Valles (AV) beef breed farmed in the same region, and to identify potential associated management risk factors. Infection was detected in a significantly smaller number (5.2%) of AV herds, despite the fact that both populations share the same ecological niche. Communal grazing was not identified as significant risk factor and study results suggest the prevalence of BT is likely to vary considerably depending on how the cattle are managed.

Identification of biomarkers of meat tenderisation and its use for early classification of Asturian beef into fast and late tenderising meat.

BACKGROUND: The objective of this work was to study the post-mortem evolution of potential biomarkers (µ-calpain activity and proteolytic profile) of meat tenderisation in bovine longissimus dorsi (LD) muscle from several biotypes coming from two beef breeds ('Asturiana de los Valles' and 'Asturiana de la Montaña') and showing different levels of muscular hypertrophy (mh/mh, mh/+, + /+). RESULTS: LD samples were taken at 2, 12, 24 and 48 h and 3, 7, 14 and 21 days post-mortem. The presence of muscular hypertrophy produced a faster rate of pH decline, faster exhaustion of µ-calpain activity and earlier occurrence of proteolytic changes. Changes in the electrophoretic pattern of some peptides from sarcoplasmic (glyceraldehyde-3-phosphate dehydrogenase) and myofibrillar (troponin T and troponin I) muscle extracts within the first 24 h significantly correlated with meat toughness and allowed accurate discrimination of meat products into two groups: (1) fast tenderising meat, coming from mh-biotypes, and (2) late tenderising meat, from normal (+/+) biotypes. CONCLUSION: Early monitoring (within 24 h after slaughter) of selected biomarkers in LD muscle allowed accurate prediction of ultimate meat toughness and could be used in the meat industry as a tool for early classification of beef into fast and late tenderising meat.

High prevalence of Tritrichomonas foetus infection in Asturiana de la Montaña beef cattle kept in extensive conditions in Northern Spain.

Bovine trichomonosis (BT) and bovine genital campylobacteriosis (BGC) are sexually transmitted diseases that can be important infectious causes of reproductive failure in extensively managed beef cattle where natural mating is a common practice. However, their prevalence in Europe was thought to be insignificant or very low. The purpose of this study was to investigate the prevalence and risk factors associated with BT and BCG in a representative beef cattle breed, Asturiana de la Montaña (AM), which is usually managed extensively in the mountain areas of Northern Spain and putative risk factors associated with the two diseases are present on most farms holding AM cattle. Preputial smegma samples were collected from 103 bulls belonging to 65 herds. Pathogen detection was undertaken using culture and PCR. Two scraping methods for sample collection (AI pipette and plastic scraper), as well as different culture media and DNA extraction methods were evaluated on field samples. Campylobacter fetus veneralis infection was not detected in any animal in any herd. However, Tritrichomonas foetus infection was demonstrated in 32% (33/103) and 41.5% (27/65) of bulls and herds tested, respectively. AM bulls older than 3 years (39.7%) were more likely to be infected than young bulls (16%) (OR=3.45, CI=1.07-11.19). An increase in repeat breeder cows was reported in herds from which T. foetus was detected (OR=5.2, CI=1.5-17.18). These findings highlight the re-emergence of this disease in extensively managed beef cattle in Spain. For routine diagnosis, the use of a culture technique and PCR in combination is advisable for testing smegma samples under field conditions.

Double-muscling character influences the trans-18:1 and conjugated linoleic acid profiles in concentrate-fed yearling bulls.

Seventy-five yearling bulls of the "Asturiana de los Valles" northern-Spanish beef breed (double-muscled, mh/mh, n=24; heterozygous, mh/+, n=26; normal, +/+, n=25) were produced under intensive conditions and the effects of double-muscling genotype on trans-18:1 and CLA isomer profiles were examined. Total trans-18:1 contents, measured as percentages of total fatty acid methyl esters, were 10.98-15.07% in backfat and 8.64-9.46% in muscle, and the major isomer was 10t-18:1 in all animals. In mh/+ animals, 11t-18:1 was the second most abundant trans isomer in muscle whereas in mh/mh and +/+ animals 11t-18:1 and 13t/14t-18:1 were relatively similar. Total CLA percentages were 0.71-0.85% in backfat and 0.32-0.40% in muscle. The highest total CLA percentage found was in mh/+, intermediate in +/+ and lowest in mh/mh animals (P<0.05). Heterozygous animals had significantly higher percentages of 9c,11t- (P<0.01), 11t,13c- (P<0.01) and 9c,11c-18:2 (P<0.05) and also significantly higher percentages of the sum of 9c,11t-/11t,13c-/11t,13t-18:2. Overall, mh/+ animals produced meat with a slightly better trans and CLA profile (i.e., less 10t-18:1 and more 11t-18:1 and 9c,11t-18:2) than other genotypes, however, further improvements would still be necessary to achieve a profile with a positive health image.

Arthropod fauna on grassland-heathland associations under different grazing managements with domestic ruminants.

The effects of two grazer species (cattle or sheep) and two flock types (single or mixed with goats) on vegetation and arthropod fauna were studied in a factorial design on eight plots which comprised two thirds of mechanically cleared heathland and one third of improved ryegrass-clover grassland. After six grazing seasons, the shrubland areas were dominated by gorse (Ulex gallii) in all treatments. Herbaceous cover was higher under mixed than under single grazing, and under sheep than under cattle grazing. Higher captures of Opiliones, Julida, Lithobiomorpha, Microcoryphia and Carabidae were recorded in shrublands than in grasslands, while the reverse was observed for Linyphiidae, Lycosidae and Hemiptera. Within shrublands, fauna responded to the flock type but not to the grazer species. More arthropod groups favoured the patchier areas with higher herbaceous biomass generated by mixed herds with goats. Within grasslands, species-specific responses to the grazer species were observed. Mixed grazing schemes which include goats within partially improved heathlands could contribute to maintain higher biodiversity levels in these marginal areas.

Responses of arthropod fauna assemblages to goat grazing management in northern Spanish heathlands.

Changes in arthropod fauna assemblages after different goat grazing treatments (breeds and stocking rates) and responses to grazing cessation were studied in a heath-gorse shrubland located in northern Spain. Three treatments (low grazing pressure and high grazing pressure with Cashmere breed and high grazing pressure with local Celtiberic breed) with three replicates were randomly allocated to nine plots. Fauna data were collected three times per year during 3 grazing yr (2003, 2004, and 2005) and three times during 2007, i.e., 2 yr after grazing cessation. Arthropods were collected by 12 pitfall traps per plot, whereas vegetation cover and height were estimated by 100 random contacts per plot. Arthropod community composition was mostly affected by sampling year during the grazing period (between 2003 and 2005) but also between 2005 and 2007 (after cessation). Species composition differed between treatments, although the differences were not attributed to the stocking rates or to the goat breeds along those periods. Differences between treatments remained constant from 2003 to 2005 and between 2005 and 2007. Heather height explained most of the variance in arthropod species data during the last grazing year (2005), whereas heather cover was the most explanatory environmental variable 2 yr after grazing cessation (2007). Grazing effects still remained on both vegetation and fauna 2 yr after grazing cessation.

The influence of breed and mh-genotype on carcass conformation, meat physico-chemical characteristics, and the fatty acid profile of muscle from yearling bulls.

The influence of breed and mh-genotype on carcass conformation, meat physico-chemical characteristics and the fatty acid profile of muscle were studied. Samples from 16 yearling bulls from "Asturiana de los Valles" (AV, n=12) and "Asturiana de la Montaña" (AM, n=4) were collected. AV animals were classified into three groups according to the presence of the gene causing double-muscling (AV double-muscled (mh/mh), n=4; AV heterozygous (mh/+), n=4; AV normal (+/+), n=4). Double-muscled animals displayed better carcass traits, lower total fat (comprised of subcutaneous (SC), intermuscular (IT) and intramuscular (IM) deposits), higher lean, moisture and drip loss, and lighter meat than AV normal animals. Heterozygous animals showed intermediate characteristics. AM animals, being a more rustic and smaller breed, showed lower conformation, higher total fat (SC, IT and IM), lower moisture and darker meat. According to the intramuscular fatty acid profile, mh/mh animals showed a lower proportion of SFA and MUFA, and a higher proportion of PUFA with an equal proportion of CLA in total fatty acid content. The P/S ratio increased with increasing number of mh alleles (or double-muscling character), while no differences between animal groups were found for the n-6/n-3 ratio.

Effect of muscular hypertrophy on physico-chemical, biochemical and texture traits of meat from yearling bulls.

The effect of the presence of the double-muscling gene either homozygous (mh/mh) or heterozygous (mh/+) on the physico-chemical, biochemical and texture traits of Longissimus thoracis muscle of yearling bulls of the Asturiana de los Valles (AV) breed was studied. Meat of mh/mh bulls had lower amount of intramuscular fat (p<0.001) and also lower total (p<0.01) and insoluble collagen (p<0.05), although the double-muscling genotype did not affect collagen solubility. Homozygous animals had lower pigment content (p<0.05) and a lighter meat, showing lower water holding capacity, estimated as expressible juice under pressure (p<0.001). Genotype affected significantly the metabolic traits of muscle, with mh/mh animals having higher muscle glycolytic metabolism, assessed by a higher (p<0.001) lactate dehydrogenase (LDH) activity and lower (p<0.001) oxidative activity of the isocitrate dehydrogenase (ICDH). The percentage of myosin heavy chain I in muscle was lower, although not significantly, for mh/mh bulls compared to mh/+ bulls. Texture measurements performed on raw material showed that meat of mh/mh bulls had lower resistance to total and 80% compression, which means lower background or collagen toughness. However, there were no differences between genotypes on shear force of cooked meat. Therefore, the physico-chemical, biochemical and texture traits of meat from mh/mh and mh/+ bull are in general significantly different, which could affect the sensorial quality of meat and hence the consumer acceptance.