The growing concern surrounding antimicrobial resistance (AMR) in human bacterial infections has prompted increased investigation into the role of the agri-food sector in AMR transmission and control. While contaminated food is a recognized route of transmission for AMR bacteria, the potential for AMR mechanisms to confer cross-protection and enhance bacterial tolerance to food-relevant preservation stressors such as heat, acidification, or antimicrobial compounds remains poorly understood. In this study, nine resistant variants (RVs) of Salmonella enterica subsp. enterica serovar Typhimurium LT2 were isolated via adaptive laboratory evolution with increasing concentrations of antibiotics from distinct classes, including colistin (COL), amoxicillin (AMX), and erythromycin (ERY). These RVs displayed up to a 16-fold increase in minimum inhibitory concentrations relative to the parental strain. RVs frequently exhibited diverse cross-protection profiles against unrelated antibiotic families. Importantly, certain RVs also showed altered tolerance to food preservation stresses. One COL- and one ERY-RV showed a 10-fold increase in survival under heat treatment (54 °C, 30 min). All ERY-RVs exhibited marked cross-protection to carvacrol (200 μL/L, 30 min), with up to a 1000-fold increase in survival. One COL-RV showed a 100-fold increased tolerance to lactic acid (1% w/v, 40 min). Whole genome sequencing revealed mutations primarily in genes associated with several functional categories, including metabolism and signal processing. Among them, mutations in the efflux regulator ramR and in rfbH, involved in O-antigen synthesis, were identified as candidate determinants potentially linked to cross-protection. The findings suggest that AMR-related mutations in foodborne pathogens such as S. Typhimurium can modify responses to food preservation stresses, highlighting the need to better understand how antibiotic adaptation may influence bacterial persistence under food-related control conditions.
Toxoplasma gondii and Neospora caninum are apicomplexan parasites infecting cattle, with implications for public health and livestock productivity, respectively. Since effective vaccines against these parasites are not currently available, identifying epidemiological factors associated with infection is important for improving control strategies. This study aimed to estimate the seroprevalence of both parasites and to identify factors associated with seropositivity in intensive dairy cattle in central Chile. A cross-sectional study was conducted using serum samples from 567 cattle, analyzed by ELISA. Epidemiological data were collected through semi-structured surveys, and associations with seropositivity were evaluated using multivariable logistic regression models, including mixed-effects models to account for farm-level clustering. Seroprevalence was 7.6% for T. gondii and 22.4% for N. caninum. For T. gondii, factors associated with seropositivity included older age categories (OR = 7.09; 11.25) and the presence of dogs in pens (OR = 6.07). For N. caninum, straw bedding use (OR = 5.13) and cat presence (OR = 6.32) were associated with higher odds of seropositivity. An additional association with lower N. caninum seropositivity was observed for BCG vaccination (OR = 0.24). These findings provide updated epidemiological data for dairy cattle in Chile. The association observed with BCG vaccination should be interpreted cautiously, as the study design does not permit causal inference.
The objective of this study was to assess the infection rate of Anaplasma marginale in cattle by Rhipicephalus microplus. Two experiments were conducted for this purpose. Experiment 1 evaluated the infection rate by the intrastadial and transstadial routes. Calves naturally infected with A. marginale were infested with R. microplus and were called "positive with ticks" (PWT). These PWT animals were housed together with A. marginale-negative, tick-free calves, called "negative and free-tick" (NFT) for 31 consecutive days. On day 31, the PWT animals were removed from the pens and returned to the farm/herd of origin. The NFT animals remained in the pens until day 84, and the possible infection rate of these calves with A. marginale by cattle ticks via the transstadial or intrastadial route was investigated (using blood smears, PCR, and ELISA). During this period, we evaluated the transfer rate of R. microplus from PWT to NFT animals. As a result of Experiment 1, only adult male and female ticks were found on the NFT animals. The transfer rate of ticks from PWTs to NFT calves was 0.10% (31/28464), and 70% (7 of 10), and all the NFT animals housed along with PWT animals became infected by A. marginale. In Experiment 2, the infection rate of A. marginale in calves by R. microplus larvae was carried out in two steps. The first step involved the use of the first tick generation larvae, which were used to infest (at 40,000 larvae per animal) five NFT calves. If any of these five NFT animals became infected with this rickettsia, fully engorged females detached from these animals were collected to perform the second step of the Experiment 2, conducted with the second-generation larvae. The second-generation larvae came from the first generation. Another five NFT calves were infested with 40,000 larvae/animal from the second tick generation. As a result of Experiment 2, 100% of NFT calves were infected by A. marginale due to infestation with R. microplus larvae of the first and second generations. This tick species has epidemiological importance in the transmission of A. marginale to calves by the intra-transstadial or unfed larval route.
Nutrient restriction (NR) increases small-for-gestational-age (SGA) offspring; however, some NR ewes deliver Non-SGA lambs. We evaluated whether fetal biometry and Doppler indices could distinguish divergent fetal growth trajectories. Ninety-five single-pregnant Corriedale ewes were assigned to NR grazing (n = 72) or supplemented Controls (n = 23) from gestational day (GD) 70 to 140. Fetal biparietal diameter (BPD), femur length (FL), thoracic height (TH), umbilical cord diameter (UCD), and resistance (RI) and pulsatility (PI) indices in umbilical (UA), cotyledonary (CA), and uterine (UtA) arteries were assessed at several GDs. Offspring within NR group was stratified by birth weight (BW) quartiles as SGA (n = 18) or Non-SGA (n = 18). At birth, BW differed (p < 0.05) among Control (4.95 ± 0.10 kg), Non-SGA (5.33 ± 0.06 kg), and SGA (3.79 ± 0.11 kg), with reduced BPD and FL in SGA lambs. Prenatal biometry did not differ. UA-RI at GD125 was higher in SGA (p < 0.005) and associated with BW (R2 = 0.15; p < 0.001). UtA indices were lower in SGA at GD110 and GD125 (p < 0.05) but weakly associated with BW (R2 ≤ 0.08). Doppler differences were detected before measurable growth divergence but have modest predictive value.
Canine leishmaniosis is an important vector-borne disease and a threat to dogs worldwide. Disease data are continuously being collected, with a consequent increase in the number of publications. This article provides practical guidelines for veterinary practitioners on the diagnosis, treatment, and prevention of leishmaniosis in dogs. These Canine Leishmaniosis Working Group (CLWG) recommendations aim to provide practical answers to the most common questions on the clinical management of this disease.
The liver coordinates metabolic processes that determine growth, health, and production efficiency in livestock species. In ruminants, hepatic metabolism is adapted to support glucose homeostasis, lipid partitioning, and nitrogen utilization during ruminal fermentation. Therefore, predictive experimental models are essential for studying nutrient-mediated regulation of liver function. However, conventional two-dimensional hepatocyte cultures fail to reproduce the structural organization and functional complexity of native liver tissue, limiting their translational value. Hepatic organoids were developed from the progenitor cells of the liver of a male Assaf lamb, and were characterized by histological and immunofluorescence analyses, followed by transcriptomic profiling using RNA-seq and functional enrichment analysis. The metabolic response was assessed by adding DL-methionine and/or betaine and analyzing, by RT-qPCR, the expression of genes encoding key metabolic enzymes (e.g., CPT1A and PDH1) and ribosomal proteins representative of cellular proliferation (e.g., RPL22L1). Hepatic organoids formed spherical epithelial structures with defined apical-basal polarity, intact tight junctions, albumin expression, and intracellular glycogen accumulation. Comparative transcriptomic analysis between organoids and native liver tissue supported the conservation of core cellular programs, although differential expression and functional enrichment analyses demonstrated that hepatic organoids exhibit a transcriptional profile biased toward cell proliferation, protein synthesis, and structural remodeling, while genes associated with mature liver metabolic functions, immune responses, and systemic homeostasis were relatively downregulated. Combined treatment with DL-methionine and betaine significantly increased the expression of CPT1A (associated with mitochondrial beta-oxidation) and RPL22L1 genes, indicating a synergistic metabolic response of both compounds. Ovine hepatic organoids reproduced structural and cellular features of liver tissue, supporting their relevance as an in vitro metabolic model for ruminants. Transcriptomic comparisons revealed incomplete functional maturation relative to adult liver, which is consistent with a progenitor-like hepatic state commonly observed in organoid systems. Nevertheless, the preserved metabolic response of organoids highlights their value for studying nutrient-gene interactions and evaluating dietary interventions prior to in vivo experimentation. This work represents the first report of sheep hepatic organoids and establishes a foundation for their use as predictive in vitro platforms to study hepatic metabolism, nutrient-gene interactions, and feed additive strategies in ruminant livestock.
The most common complication of arteriovenous fistula (AVF) is stenosis, which can result in poorer hemodialysis (HD) outcomes and thrombosis. It is important to identify those stenoses at high risk of thrombosis in order to perform elective treatment to restore their function. 1) To assess the degree of agreement between Doppler ultrasound (DU) and the temperature gradient technique (TGT) for blood flow measurement (QA). 2) To evaluate the relative importance of different monitoring and/or surveillance methods for detecting significant stenosis using a predictive model. A cross-sectional, comparative, observational study was conducted, in which the AVFs of 30 consecutive patients were assessed over a 3-month period, with monthly sampling. First-generation methods were applied, and second-generation methods using TGT and DU were used to measure QA. 80% were men and 20% women, with a mean age of 67.03 ± 10.25 years. 100% were hypertensive and 63.33% diabetic. 76.67% had a radiocephalic AVF and 23.33% a humeral AVF. The mean QA by TGT was 980.79 ± 63.86 ml/min and by DU 1098.43 ml/min. In the first sampling, 4 significant stenoses were detected, none in the second, and 3 in the third. In samplings 1 and 3, statistically significant differences were detected in the measurement of QA by TGT and ultrasound. No significant differences were found in any parameter measured by first-generation methods, except for PV in the first sampling. In the Bland-Altman analysis, a 96.7% agreement was observed between QA measurements by TGT and DU (p < 0.0001). The dot plot for the difference of each measurement from the mean for AVFs with QA > 1000 ml/min also showed agreement between both methods, with a deviation of only 4.34%. When first- and second-generation variables were introduced into the predictive model, four combinations with a significant Wilks' lambda were identified. The combination including QA determined by ultrasound was the most significant, with an overall correct classification of 80.5% when first-generation methods were included together with ultrasound-measured QA. In our study, the agreement between QA measurement by TGT and Doppler ultrasound is high at all flow levels. Furthermore, the predictive model demonstrates that second-generation methods are superior to first-generation methods for the early detection of clinically significant stenosis.
Arcobacter butzleri is an emerging zoonotic pathogen described in 1991. Despite its increasing incidence in clinical cases, there is still a need to better understand the underlying mechanisms of pathogenicity. Therefore, the aim of this study was to evaluate if different virulence profiles of A. butzleri are associated with different extent of virulence in the Galleria mellonella experimental model. Twenty-one A. butzleri strains isolated from various sources were characterized by searching for putative virulence determinants (PVDs) through genomic analysis and PCR (i.e., cadF, cj1349, ciaB, mviN, tlyA, pldA, irgA, hecA, hecB, and iroE genes). Seven virulence profiles were obtained (designated as Profile A to G). A representative strain from each profile was selected to conduct assays in the G. mellonella model. Even though the profile C was the most virulent, there was no correlation between the virulence profiles and G. mellonella mortality/melanization. Our study show that G. mellonella is a reliable and cost-effective model for studying the pathogenicity of A. butzleri. However, our results underscore the significance of evaluate additional virulence factors beyond the analyzed PVDs.
Understanding the dynamics of multihost pathogens, such as Mycobacterium tuberculosis complex (MTC), requires considering not only host interaction patterns but also variation in infectiousness across species. Network analysis is a useful tool to assess contact structure and disease risk, but it often depends on invasive methods. Camera trapping offers a noninvasive alternative to build co-occurrence networks in complex communities. In this study, we applied a novel approach that integrates shedding into ecological networks, weighing links between species pairs according to their co-occurrence frequency and shedding capacity, to evaluate tuberculosis (TB) risk across 18 study sites in the Iberian Peninsula. At the community level, TB risk was positively associated with mean strength-out, a local centrality measure of the frequency of interspecific contacts and their infectious potential. Species-specific models revealed that community TB risk increased with the strength-out of wild boar, red deer, and cattle, and with the closeness of red fox and badger. Furthermore, the community TB risk was jointly explained by shedding-weighted connectivity and spatial aggregation of wild boar, whereas red deer mainly contributed through their local abundance. In contrast, shedding-weighted centrality of badgers, foxes, and cattle explained TB risk, suggesting that ecological and management factors may influence TB spread. We defined epidemiological scenarios according to latitude, population factors, and infection pressure. These findings highlight the importance of including host infectiousness in ecological network analyses, as well as combining centrality measures and population data to understand and manage TB risk in complex host communities, with potential applications to other wildlife diseases and multihost systems.
The type VI secretion system (T6SS) is a contact-dependent multiprotein apparatus widely distributed in Gram-negative bacteria that contributes to interbacterial competition and pathogenesis via a contractile mechanism. In Salmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22, which are differentially distributed among serotypes. One of the most studied and widely distributed T6SS corresponds to that encoded in SPI-6 (T6SSSPI-6), which contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, there is still limited information available regarding the total number of effector proteins encoded within SPI-6 of different Salmonella enterica serotypes. In the present study, we characterized the SPI-6 T6SS gene cluster encoded in Salmonella enterica subspecies enterica serotype Tennessee (S. Tennessee), a pathogen frequently associated with foodborne gastrointestinal outbreaks. Interbacterial competition assays demonstrated that T6SSSPI-6 of S. Tennessee displays antibacterial activity. Additionally, we performed comparative genomic and bioinformatic analyses and identified an antibacterial Effector/Immunity protein (E/I) module encoding a putative effector with DNase activity (RhsA-HNHc) and its cognate immunity protein within the variable region 3 (VR3) of the SPI-6 T6SS gene cluster. Interbacterial competition assays confirmed the antibacterial activity of this novel E/I pair. In addition, heterologous expression assays showed that induction of the RhsA-HNHc effector led to significant E. coli growth inhibition, while co-expression with its putative immunity protein fully restored bacterial growth, thus demonstrating protection against toxicity. Finally, a nuclease activity assay demonstrated that RhsA-HNHc possesses DNase activity. Altogether, this study expands the experimentally validated SPI-6 T6SS effector repertoire beyond well-studied Salmonella serotypes, providing the first functional characterization of a DNase-type Rhs effector in S. Tennessee.
The aim of this study was to determine whether the socio-productive characteristics of Sheep Production Units (SPU) in the State of Mexico are related to the level of animal welfare (AW) of their animals. A survey was conducted with 37 sheep producers to identify their profiles, sheep management practices, and understanding of aspects of AW. The second level of the AWIN Protocol was used to measure the health status of 710 sheep by assessing body condition, mucosal color, fleece and/or hair cleanliness and loss, fecal soiling, and tail length, using a semi-quantitative intensity scale. A hierarchical agglomerative clustering analysis was performed to integrate similarities in clusters (CLs). Four CLs were defined based on differences in socio-productive characteristics, management, and facilities, none of which affected the sheep's health status. The relationship between the CL and the data collected in the survey and from the animals was determined. In conclusion, there was no evidence of a relationship between the socio-productive characteristics of the SPUs in the State of Mexico and the indicators used to assess the AW of their sheep, except for fleece and/or hair cleanliness. The SPUs with the worst punctuation in this indicator were those where animals were confined, where facilities differed, and where sheep producers were less experienced.
This study describes embryo developmental progression and morphokinetic patterns in embryos derived from oocytes of young and aged domestic cats, a model for endangered felids, using a time-lapse monitoring system. Because maternal aging is associated with reduced oocyte competence, possibly linked to mitochondrial dysfunction that may impair fertilization and embryo development, the potential influence of the SIRT1 activator SRT1720 during in vitro maturation on embryo developmental progression was also evaluated under these conditions. Oocytes from young (1.13 ± 0.79 years) and old (7.0 ± 1.10) cats were matured in vitro with SRT1720 (1-μM, 0.1-μM, and 0.05-μM) and without, followed by in vitro fertilization. 8-Day embryo in vitro culture was noninvasively monitored using the MIRI® time-lapse system. Embryos from aged cats exhibited accelerated progression up to the morula stage; in contrast, embryos from young cats showed faster progression at later preimplantation stages and reached the hatched blastocyst stage. In young queens, embryos exposed to 0.1-μM showed numerically higher first cleavage and morula formation rates than controls, while 1-μM was associated with altered first cleavage timing and short cell cycles. In older queens, 0.1-μM maintained cleavage timing patterns comparable to untreated embryos but fail to produce hatched blastocysts, whereas 1-μM yielded a single hatched blastocyst despite signs of altered cell cycle dynamics. Overall, SRT1720 supplementation did not significantly influence embryo development under the tested conditions. Morphokinetic monitoring provided descriptive observations suggesting distinct age-related developmental profiles. These findings provide baseline information on feline developmental kinetics and may support further investigation of metabolic modulators in feline assisted reproduction.
Neurocysticercosis (NCC), a parasitic brain infection caused by Taenia solium larvae, remains a leading cause of preventable epilepsy globally. Although calcified brain lesions were formerly considered as the quiescent end stage of NCC, they may act as epileptogenic foci. It has been suggested that parasitic antigens within calcified lesions may act as potential triggers of inflammation and subsequent seizure activity. In this study, we developed and optimized immunohistochemistry (IHC) assays employing anti-Taenia solium monoclonal antibodies (mAbs) to detect residual cyst antigens in calcified lesions in a porcine NCC model and assessed antigen persistence for up to 12 months after successful antiparasitic treatment. Six mAbs raised against T. solium whole cyst (TsW5, TsW8, and TsW12), vesicular fluid (TsV3 and TsV4), and excretory/secretory products (TsE1) were used for IHC assay development and tested in brain sections containing viable brain cysts from NCC pigs and uninfected tissue from controls to optimize assay conditions, blocking, primary and secondary antibody dilutions. Optimized assays were subsequently performed in selected calcified granulomas (n = 20) obtained from NCC-infected pigs sacrificed at 4, 8, and 12 months after antiparasitic treatment to identify residual cyst antigens as well as their localization and area of reactivity. We observed residual cyst antigens in 65-80% of calcified granulomas, with TsW8 and TsV3 showing the highest percentages of immunoreactivity. Antigen localization followed two patterns, one with antigens entirely located within the calcified lesions (TsW5, TsW8, TsW12, and Tsv4) and another with antigens located outside the cyst in the perilesional brain tissue (TsV3 and TsE1). Antigen detection and the extent of reactivity declined progressively after antiparasitic treatment but persisted at detectable immunoreactive areas in calcified granulomas up to month 12 months after treatment. T. solium antigens remain detectable in calcified granulomas and in the perilesional tissue for up to 12 months after antiparasitic treatment in the pig model.
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Understanding temporal patterns of Leishmania infection in sand fly populations is essential for assessing transmission risk and improving surveillance and control in endemic regions. This study investigated infection dynamics in natural populations of Phlebotomus perniciosus and Phlebotomus ariasi, two established vectors of Leishmania infantum in the western Mediterranean and evaluated the utility of pooled sampling for vector surveillance. Sand flies were tested for Leishmania infection using a TaqMan real-time kinetoplast PCR either individually or in pools. Infection rates from pooled samples were estimated using maximum likelihood methods and analyzed alongside individual infection data using mixed-effects regression models to assess the influence of seasonality and female reproductive status on infection probability. Both individual and pooled analyses showed similar seasonal trends. Infection probability peaked in late summer, likely reflecting high parasite prevalence in reservoir hosts, and was highest in gravid females, suggesting cumulative parasite acquisition as sand flies age and complete successive gonotrophic cycles. Individual testing, although more resource-intensive, provided additional resolution by revealing species-specific patterns, with P. ariasi exhibiting higher infection rates than P. perniciosus, offering a deeper understanding of parasite circulation and species-specific infection patterns. We conclude that pooled sampling combined with appropriate statistical modelling can reliably capture seasonal infection patterns. Integrating reproductive status indicators with molecular surveillance represents a novel approach to refine transmission risk assessments and support more targeted and effective vector monitoring strategies in Leishmaniosis-endemic regions.
Limited understanding of African swine fever (ASF) immunity remains a major barrier to the rational development of safe and effective vaccines. While antibody-mediated protection is still poorly defined, growing evidence highlights a central role for cellular immunity, particularly cytotoxic cells, as components associated with ASF virus (ASFV) infection control. However, the contribution of individual cytotoxic subsets across different virological and immunological contexts is not well characterised. Here, we investigated cytotoxic responses during BA71ΔCD2 live attenuated vaccine-induced protection and during late-stage lethal ASFV infection. Protective immunity was characterised by antigen-specific cytotoxic T-cell responses, whereas acute ASF was associated with broad cytotoxic activation. Early increases in perforin-positive CD4-CD8αβ⁺ T cells coincided with the onset of protection, and elevated recall responses correlated with survival following lethal challenge, supporting their association with protective immunity. Additional correlates of protection included CD4⁺CD8αβ⁺ cytotoxic T cells, IFNγ-producing cells, and specific antibodies, illustrating the multifactorial nature of protective immunity. In contrast, pigs with acute ASF showed broad cytotoxic expansion, perforin-positive natural killer and γδ T cells showing the strongest association with viremia. Together, these findings advance our understanding of cytotoxic responses to ASFV and identify perforin-positive T cells, alongside complementary immune components, as potential correlates of protection for future vaccine development.
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This study evaluated the effects of equine chorionic gonadotropin (eCG) dose and application timing after progestogen-sponge removal on estrus characteristics and ovulation rate in ewe‑lambs of three hair‑sheep breeds under dry-tropical conditions. Two hundred sixteen ewe‑lambs (62 Dorper, 69 Katahdin, 85 Pelibuey; 8-10 months old) were studied during a high (n = 91) and a low (n = 125) breeding season. Animals received intravaginal progestogen sponges (Cronolone, 20 mg) for 12 days and were assigned to a 2×2 factorial treatment: eCG 200 IU or 300 IU administered either 24 h before (-24 h) or at sponge withdrawal (0 h). Estrus signs (mount acceptance and vulvar swelling) were recorded every 4 h for 48 h; ovulation was confirmed by laparoscopic corpora lutea (Cl) count eight days later. Overall, 82% of ewe‑lambs exhibited estrus within 48 h. There were no main effects of eCG dose or application time on overall estrus rate. A significant dose×time interaction affected interval to estrus (P = 0.012): mean interval was shorter after -24 h application (30.2 ± 1.0 h) than after 0 h application (34.6 ± 1.0 h). Breed influenced estrus expression and silent ovulation: Dorper ewe‑lambs were more likely to exhibit estrus than Pelibuey (odds ratio (OR) = 9.74, 95% confidence interval (CI) = 1.93-49.19; P = 0.0059) and had lower silent ovulation (3.4% vs. 24.7%; OR = 0.56, 95% CI = 0.02-0.59; P = 0.0094). Mean ovulation rate exceeded 95% across groups and was higher for Pelibuey (2.4 ± 0.1 Cl) than Dorper (2.0 ± 0.1 Cl) and Katahdin (1.9 ± 0.1 Cl; P < 0.05). Under the conditions tested, administering 300 IU eCG at -24 h sponge removal provided a practical balance for synchronized estrus and high ovulation rates across seasons.
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Background/Objectives: Combination of antiparasitic drugs with different mechanisms of action has been suggested as an effective strategy to delay the development of parasite resistance. Considering the need to understand the pharmacological basis of drug combinations, the current study evaluated the potential pharmacokinetic (PK) interactions and the clinical efficacy (pharmacodynamic response) occurring after the subcutaneous administration of ivermectin (IVM) and levamisole (LEV), administered either as single treatments or concurrently to different groups of parasitized calves on three commercial farms (A, B and C). Methods: Forty-five (45) male calves naturally infected with gastrointestinal nematodes were randomly allocated into three groups (n = 15): IVM, treated with IVM by subcutaneous injection (0.2 mg/kg); LEV, treated subcutaneously with LEV (8 mg/kg); IVM + LEV, simultaneously treated with IVM and LEV (two subcutaneous injections at the same dose rates). Seven animals from each treated group (farm C) were randomly selected to perform the PK study. Drug concentrations were measured by HPLC. The therapeutic response (efficacy) was determined at 14 days after treatment by the fecal egg reduction test. Results: The mean area under the concentration vs time curve (AUC) for IVM obtained after administration of IVM alone (274 ± 65.1 ng.d/mL) was similar to that obtained when IVM was co-administered with LEV (295 ± 111 ng.d/mL). Likewise, mean LEV AUC values were similar after LEV administration alone (8.90 ± 2.69 µg.h/mL) or combined with IVM (9.11 ± 1.82 µg.h/mL). No adverse PK interactions were observed after the combined treatment, with similar PK parameters (p > 0.05) obtained between the single-drug and the combination-based strategies. On farm A, the overall fecal egg reductions were 38% (IVM), 99% (LEV) and 100% (IVM + LEV). While Cooperia spp. and Haemonchus spp. showed reduced susceptibility to IVM treatment, LEV demonstrated high efficacy against both genera, with only a minimal proportion of Haemonchus spp. remaining after treatment. Similarly, total fecal egg reductions were 42% (IVM), 99% (LEV) and 100% (IVM + LEV) on farm B, and 54% (IVM), 99% (LEV) and 100% (IVM + LEV) on farm C. On those farms, IVM was ineffective against Cooperia spp. and/or Haemonchus spp., while LEV failed to control Ostertagia spp. Remarkably, the combination of both molecules was the only treatment that achieved 100% efficacy against all nematode genera (Cooperia, Ostertagia, Haemonchus and Oesophagostomum spp.). Conclusions: Based on the described PK and pharmacodynamic (PD) assessments, the IVM + LEV combination appears to be a promising pharmacological option for controlling resistant gastrointestinal nematodes in cattle, with the additional potential to delay the progression of nematode anthelmintic resistance. Overall, the study provides original and robust pharmacokinetic and efficacy data that contribute to the optimization of parasite control strategies in cattle. This drug combination strategy may enhance treatment efficacy and contribute to improved parasite control in cattle production systems.