Luis Vicente Monteagudo

One ram of the Rasa Aragonesa breed (M) exhibited low reproductive performance throughout his reproductive life (ewes that lambed/ewes inseminated: median: 0.57, interquartile range: 0.35; lambs born/ewes inseminated: 0.96 ± 0.513). In this exploratory case comparison, our objective was to determine the causes of M’s reproductive problems. Another ram (O) was chosen as the reference animal due to its history of high fertility and fecundity (fertility: median: 0.73, interquartile range: 0.33; fecundity: 1.22 ± 0.523). Semen samples from both males were submitted to a complete quality study. M exhibited lower sperm concentration, reduced membrane integrity, altered ROS/live–dead sperm profiles, increased phosphatidylserine inversion, and reduced DNA integrity. RNA sequencing, mapping against the reference genome (Ovis aries, GCF_016772045.2), differential gene expression, and GO enrichment analysis were performed on testicular biopsies of M and O. Despite the limitations of this study, which is based solely on two rams with extreme reproductive performances, it was detected that the KRT2 gene was downregulated in M with respect to O, with possible deleterious effects on membranes. Since only two single individuals were considered, this transcriptomic finding should be interpreted as hypothesis-generating rather than confirmatory. Therefore, for KRT2 to be considered a likely candidate to explain the observed differences in sperm quality and fertility outcomes, a larger number of animals with either high or low fertility should be considered, and validation using qRT-PCR should be performed.

Tick-borne haemopathogens pose a persistent threat to the cattle industry, causing significant economic losses. A comprehensive molecular study was conducted on 291 blood samples from symptomatic cattle across Spain and Portugal (2018–2023). Samples were analysed using group-specific (Anaplasma spp, Piroplasmida) and species-specific qPCR assays for Anaplasma marginale, Babesia bovis, B. bigemina, Theileria annulata, and Mycoplasma wenyonii. Additionally, a representative subset (n=137) was screened for Theileria orientalis, followed by genetic characterisation of the Major Piroplasm Surface Protein (MPSP) gene in 16 positive specimens. The overall infection rate was 82.1% (239/291). M. wenyonii (38.5%) and A. marginale (32.3%) were the most prevalent species. Notably, T. orientalis exhibited a high prevalence of 40.9% in the tested subset, surpassing T. annulata (28.5%). Phylogenetic analysis confirmed the circulation of two distinct T. orientalis genotypes in the region: Type 1 (Chitose) and Type 3 (Buffeli). Co-infections were frequent (48.5%), with triple infections in 13.4% of cases. Adult cattle showed significantly higher prevalence rates compared to younger animals (P<0.05). This study provides the first molecular identification of M. wenyonii and T. orientalis in symptomatic cattle from the Iberian Peninsula, alongside the first large-scale characterisation of T. orientalis genotypes in the region. The dominance of T. orientalis over T. annulata and the high rate of multi-pathogen infections reveal a hidden diversity that traditional diagnostic approaches fail to capture. These findings highlight the urgent need to update regional surveillance programs and diagnostic panels to include these emerging pathogens, especially in the context of shifting vector dynamics and global trade.

Merino sheep exhibit a high follicular density, enhancing wool yield but predisposing them to inherited cutaneous disorders, such as breech wrinkle, which is characterized by the appearance of multiple skin folds in the breech area. This study provides the first clinicopathological description of the so-called body wrinkle, a congenital skin condition in Merino lambs where the folds affect the whole-body skin. Four affected lambs displayed generalized alopecia with excessive skin folds. Hematological analysis showed neutrophilia, lymphopenia, and eosinopenia. Histological examination revealed follicular dysplasia, follicular keratinization and keratosis, and degeneration of follicular epithelium, particularly in the outer root sheath. The information obtained from pedigree analysis suggested an autosomal recessive inheritance pattern. All affected lambs exhibited severe OrfV lesions in the oral cavity and muzzle, persisting from 2 weeks of age until death at 1 to 2 months. Our results suggest that body wrinkle condition might favor a more prolonged clinical course and more severe outcome of OrfV infection in lambs. Further genetic and immunopathological investigations are needed.

Influenza D virus (IDV) and co-circulating respiratory pathogens were investigated using RT-qPCR in 316 samples collected from 210 Spanish cattle farms with bovine respiratory disease (BRD) outbreaks between July 2023 and September 2024. Thirty-eight IDV-positive samples, from 30 farms across 15 provinces throughout Spain, were identified. IDV was significantly more frequent in bronchoalveolar lavage samples (22.1%) and nasal swabs (13.5%) compared to lung tissues (5%) and other sample mixtures (5%). All IDV-positive specimens exhibited co-infections, with most (94.7%) harboring three to seven viral and/or bacterial pathogens, highlighting the complexity of BRD outbreaks. Cramer’s V analysis revealed moderate IDV association with Mycoplasma bovis (V = 0.255) and Pasteurella multocida (V = 0.223), and a weaker IDV-bovine coronavirus association (V = 0.202), while IDV association with Histophilus somni (V = 0.025) and bovine herpesvirus 1 (V = 0.000) was negligible. Partial sequences of the hemagglutinin-esterase (HEF) gene from a subset of 13 IDV-positive samples (Ct < 32) were obtained. This confirmed the presence of the two major genetic lineages detected among cattle in Europe, D/OK and D/660, with D/660 exhibiting higher genetic diversity, as determined by DNAsp 6.12 software. This is the first report of IDV infection in Spanish cattle, confirming the circulation of the D/OK and D/660 lineages within the cattle population.

Limited molecular data exist on the prevalence and subtype distribution of Blastocystis spp., the most prevalent parasite in human and animal feces worldwide. A total of 44 different subtypes (STs) of Blastocystis are currently recognized based on the sequence of the small subunit ribosomal RNA (SSU-rRNA) gene. This is a molecular study of Blastocystis spp. in hospitalized patients with gastrointestinal symptoms in northern Spain. We analyzed 173 Blastocystis-positive patients with gastrointestinal symptoms by using nested PCR for molecular detection, subtype identification, phylogenetic analyses, and genetic diversity assessment. ST2 (34.1%) and ST3 (34.7%) predominated, followed by ST1 (15.6%) and ST4 (15.6%). Mixed infections with different subtypes were observed in some patients. Sequence analysis revealed for the first time in European humans the allele 88 (a variant of ST1). In other cases, alleles commonly found in animal samples were detected (allele 9 in ST2, allele 34 in ST3, and allele 42 in ST4). Phylogenetic analysis showed high variability in ST1 and ST2, suggesting a polyphyletic origin, while both ST3 and ST4 exhibited higher genetic homogeneity, indicating a possible monophyletic origin and recent transmission to humans. These data confirm Blastocystis spp. subtype diversity and may help in understanding the evolutionary processes and potential zoonotic transmission of this parasite.

Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, pose economic challenges to herds in Spain and worldwide. The aetiological agent for FR is Dichelobacter nodosus, while CODD is caused by pathogenic Treponema phylogroups. We detail the findings derived from the analysis by qPCR of 105 pooled samples from 100 ovine and five caprine herds in Spain and Portugal, alongside 15 samples from healthy flocks in order to identify Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). Treponema spp. were detected in all 120 pools, including samples from the 15 healthy flocks where only one positive result for F. necrophorum was recorded. Mixed infections by agents different from Treponema spp. were identified in 68.57% of samples. Positive results for F. necrophorum and/or D. nodosus, were obtained for 91.4% of the pools, whereas the presence of the three pathogenic Treponema phylogroups was rare: each of them appeared in isolation in a single pool, while they were found in 18 pools in combination with other agents. While F. necrophorum was the sole finding in 16.2% of samples from affected herds, D. nodosus (the footrot causative agent) was only detected in 61% of affected farms. An improved qPCR protocol was implemented to determine the serogroups of D. nodosus in the samples and found all of them (except the G serogroup), often in combined infections (35.1%). This report concludes with comprehensive proposals for diagnosing, preventing, and treating hoof ailments, remarking the interest of the information about D. nodosus serogroups in order to improve the efficiency of immunization by choosing appropriate vaccine protocols.