Cathepsin S Is More Abundant in Serum of Mycobacterium avium subsp. paratuberculosis-Infected Dairy Cows.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis, a chronic granulomatous enteritis leading to economic losses and posing a risk to human health due to its zoonotic potential. The pathogen cannot reliably be detected by standard methods, and immunological procedures during the infection are not well understood. Therefore, the aim of our study was to explore host-pathogen interactions in MAP-infected dairy cows and to improve diagnostic tests. Serum proteomics analysis using quantitative label-free LC-MS/MS revealed 60 differentially abundant proteins in MAP-infected dairy cows compared to healthy controls from the same infected herd and 90 differentially abundant proteins in comparison to another control group from an uninfected herd. Pathway enrichment analysis provided new insights into the immune response to MAP and susceptibility to the infection. Furthermore, we found a higher abundance of Cathepsin S (CTSS) in the serum of MAP-infected dairy cows, which is involved in multiple enriched pathways associated with the immune system. Confirmed with Western blotting, we identified CTSS as a potential biomarker for bovine paratuberculosis. This study enabled a better understanding of procedures in the host-pathogen response to MAP and improved detection of paratuberculosis-diseased cattle.

Evaluation of Foot and Claw Lesions and Claw Horn Growth in Piglets from Birth to End of Nursery.

The objective of this study is to evaluate foot lesions and claw horn growth of piglets from the day of birth until the end of the suckling period and describe the prevalence and extent in three different genetic lines. Therefore, bruising, dorsal horn lesions, claw horn growth, and weight gain were evaluated five times during growth, starting with the day of birth (day 0 of life) and ending with the end of nursery (day 68 ± 2 of life). Totally, 74 piglets of three genetic lines (German Landrace × Large White; Piétrain × Piétrain-Duroc; pure German Landrace) were examined. Bruising and dorsal horn lesions reached maximum levels at day 7 (±1) of life (with up to 91% of piglets having bruising marks and up to 94.1% of piglets having dorsal horn lesions). Differences among genetic lines were detected, with German Landrace × Large White crossbreds showing the highest percentage of bruising, but Piétrain × Piétrain-Duroc crossbreds showed the highest score for dorsal horn lesions at day 0. Until weaning (day 28 ± 1), front feet were more affected by bruising than hind feet (70.3% of the front feet and 64% of the hind feet showed bruising), but at the end of nursery (day 68 ± 2), hind feet showed a higher percentage of affected feet than front feet (65.5% vs. 41.3%). Several factors affect bruising scores in piglets, including body weight, age at examination, litter size, sex, parity, breed, and claw horn length. Additionally, significant differences for claw horn length were detected among the genetic lines from birth to end of nursery.

Feed Efficiency and Physiological Parameters of Holstein and Crossbred Holstein × Simmental Cows.

This study aimed to compare the feed efficiency (FE) and physiological parameters of Holstein and crossbred Holstein × Simmental cows in a confinement system during winter and summer. The study was conducted in a dairy farm in southern Brazil by including a total of 48 multiparous cows. The cows were studied for 21 days in two periods, summer and winter, and their daily dry matter intake (DMI), milk yield (MY), rectal temperature (RT), respiratory rate (RR), body weight, and body condition score were recorded. An analysis of variance was conducted using the SAS statistical package. The results showed that crossbred Holstein × Simmental cows have a similar FE as Holstein cows in a high-production system (1.83 × 1.81 kg DMI/kg MY, respectively), and they can achieve the same production levels as purebred Holstein cows (43.8 vs. 44.5 milk/cow/day). Our findings indicated a difference for the period as both genetic groups achieved higher FE in winter than in summer (1.98 vs. 1.67 DMI/kg MY, respectively). In addition, we found evidence that crossbred cows are better at dissipating body heat during heat-stress situations, as they have higher RR in summer compared to purebred cows, while Holstein cows have higher RT in summer afternoons than crossbred cows. Therefore, using crossbred Holstein × Simmental cows is an alternative for high-production systems.

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells.

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the "don't eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)-specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

Magnetic Resonance Imaging Used to Define the Optimum Needle Length in Pigs of Different Ages.

Intramuscular injections result in tissue destruction and alteration. Therefore, it is necessary to evaluate the optimum injection point for intramuscular injections. As animals­especially pigs­vary in size and explicit information about injection depth is not available. To determine the predicted optimum injection depth, magnetic resonance imaging was used in pigs of different ages and weight groups. In total, 730 magnetic resonance images of 136 pigs were used to calculate the optimum injection depth for intramuscular injections. Four age groups were evaluated: <29 days of age, 29−70 days of age, 71−117 days of age and >170 days of age. For fattening pigs (71−117 days of age), the present study recommends a needle length of 20 mm (range: 40−58 mm). For younger pigs (<70 days of age), a needle length of 12 to 14 mm (range: 10−18 mm), and for older pigs (>170 days of age), a needle length of 30 mm (range: 25−37 mm) is recommended. However, more data are needed. Therefore, further studies are necessary, especially in the youngest (suckling pigs) and oldest (sows) age groups, as these are the groups mainly injected/vaccinated. Additionally, age and weight should be examined in more detail compared to fat distribution in the neck, genetics and the sex of the animal.

A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1.

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

Bovine Peripheral Blood Derived Lymphocyte Proteome and Secretome Show Divergent Reaction of Bovine Immune Phenotypes after Stimulation with Pokeweed Mitogen.

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease "bovine neonatal pancytopenia" in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation.

Single-Domain Antibodies for Targeting, Detection, and In Vivo Imaging of Human CD4+ Cells.

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb1 in CD4+ T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

Identification and Functional Annotation of Genes Related to Bone Stability in Laying Hens Using Random Forests.

Skeletal disorders, including fractures and osteoporosis, in laying hens cause major welfare and economic problems. Although genetics have been shown to play a key role in bone integrity, little is yet known about the underlying genetic architecture of the traits. This study aimed to identify genes associated with bone breaking strength and bone mineral density of the tibiotarsus and the humerus in laying hens. Potentially informative single nucleotide polymorphisms (SNP) were identified using Random Forests classification. We then searched for genes known to be related to bone stability in close proximity to the SNPs and identified 16 potential candidates. Some of them had human orthologues. Based on our findings, we can support the assumption that multiple genes determine bone strength, with each of them having a rather small effect, as illustrated by our SNP effect estimates. Furthermore, the enrichment analysis showed that some of these candidates are involved in metabolic pathways critical for bone integrity. In conclusion, the identified candidates represent genes that may play a role in the bone integrity of chickens. Although further studies are needed to determine causality, the genes reported here are promising in terms of alleviating bone disorders in laying hens.

Keel Bone Damage in Laying Hens-Its Relation to Bone Mineral Density, Body Growth Rate and Laying Performance.

Keel bone damage is an important animal welfare problem in laying hens. Two generations of four layer lines, differing in phylogenetic background and performance level and kept in single cages or floor pens were weighed and scored for keel bone deformities (KBD) during the laying period. KBD, keel bone fractures (KBF) and the bone mineral density (BMD) of the keels were assessed post mortem. For BMD, relationships to laying performance and body growth were estimated. Caged hens showed more deformities, but fewer fractures and a lower BMD of the keel bone than floor-housed hens. White-egg layers had a lower BMD (0.140-0.165 g/cm2) and more KBD than brown-egg layers (0.179-0.184 g/cm2). KBF occurred more often in the high-performing lines than the moderate-performing ones. However, in the high-performing lines, BMD was positively related to total egg number from 18 to 29 weeks of age. The adult body weight derived from fitted growth curves (Gompertz function) had a significant effect (p < 0.001) on keels' BMD. The study contributes to the understanding of predisposing factors for keel bone damage in laying hens. It showed that the growth rate has a rather subordinate effect on keels' BMD, while the BMD itself greatly affects KBD.

NeutrobodyPlex-monitoring SARS-CoV-2 neutralizing immune responses using nanobodies.

In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

Detection of Local Tissue Reactions after Anti-GnRF Injection in Male Pigs Assessed Using Magnetic Resonance Imaging.

This study aimed at evaluating the local tissue reaction of an anti-GnRF product, which is used for the so called "immunocastration" in male pigs. A total of 34 pigs were injected two times (including a booster injection) with an anti-GnRF product. Injection was performed using the prescribed safety vaccinator. Injection sites were evaluated three times post injection using magnetic resonance imaging. Two examinations were performed after the first injection and one after the booster. The local tissue reaction was evaluated three-dimensionally by semi-automatic analyses, by linear measurements of injection depth and length, and by scoring the kind of tissue affected. Results showed a long-lasting reaction in affected muscle tissue. Therefore, needle length should be discussed, and an evaluation of the injection site after slaughter should be performed include behavioural scorings post injection to evaluate the impact on animal well-being and carcass quality.

Energy Balance Indicators during the Transition Period and Early Lactation of Purebred Holstein and Simmental Cows and Their Crosses.

Crossbreeding in dairy cattle has been used to improve functional traits, milk composition, and efficiency of Holstein herds. The objective of the study was to compare indicators of the metabolic energy balance, nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), glucose, body condition score (BCS) back fat thickness (BFT), as well as milk yield and milk composition of Holstein and Simmental cows, and their crosses from the prepartum period until the 100th day of lactation at the Livestock Center of the Ludwig Maximilians University (Munich, Germany). In total, 164 cows formed five genetic groups according to their theoretic proportion of Holstein and Simmental genes as follows: Holstein (100% Holstein; n = 9), R1-Hol (51-99% Holstein; n = 30), first generation (F1) crossbreds (50% Holstein, 50% Simmental; n = 17), R1-Sim (1-49% Holstein; n = 81) and Simmental (100% Simmental; n = 27). The study took place between April 2018 and August 2019. BCS, BFT blood parameters, such as BHBA, glucose, and NEFA were recorded weekly. A mixed model analysis with fixed effects breed, week (relative to calving), the interaction of breed and week, parity, calving year, calving season, milking season, and the repeated measure effect of cow was used. BCS increased with the Simmental proportion. All genetic groups lost BCS and BFT after calving. Simmental cows showed lower NEFA values. BHBA and glucose did not differ among genetic groups, but they differed depending on the week relative to calving. Simmental and R1-Sim cows showed a smaller effect than the other genetic groups regarding changes in body weight, BCS, or back fat thickness after a period of a negative energy balance after calving. There was no significant difference for milk yield among genetic groups, although Simmental cows showed a lower milk yield after the third week after calving. Generally, Simmental and R1-Simmental cows seemed to deal better with a negative energy balance after calving than purebred Holstein and the other crossbred lines. Based on a positive heterosis effect of 10.06% for energy corrected milk (ECM), the F1, however, was the most efficient crossbred line.

Correction: Jansen, S., et al. Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines. Animals 2020, 10, 850.

The authors wish to make the following corrections to this paper [...].

Egg Production and Bone Stability of Local Chicken Breeds and Their Crosses Fed with Faba Beans.

Poultry production is raising concerns within the public regarding the practice of culling day-old chicks and the importation of soy from overseas for feedstuff. Therefore, an alternative approach to poultry production was tested. In two consecutive experiments, two traditional chicken breeds, Vorwerkhuhn and Bresse Gauloise, and White Rock as a commercial layer genotype as well as crossbreds thereof were fed diets containing either 20% vicin-rich or vicin-poor faba beans, though addressing both subjects of debate. Hen performance traits and bone stability were recorded. All parameters were considerably influenced by the genotype with White Rock showing the significantly highest (p < 0.05) laying performance (99.4% peak production) and mean egg weights (56.6 g) of the purebreds, but the lowest bone breaking strength (tibiotarsus 197.2 N, humerus 230.2 N). Regarding crossbreds, the Bresse Gauloise × White Rock cross performed best (peak production 98.1%, mean egg weight 58.0 g). However, only limited dietary effects were found as only the feeding of 20% vicin-rich faba beans led to a significant reduction of egg weights of at most 1.1 g (p < 0.05) and to a significant reduction of the shell stability in the crossbred genotypes. In terms of dual-purpose usage, crossing of Bresse Gauloise with White Rock seems to be the most promising variant studied here.

Phenotyping of the Visceral Adipose Tissue Using Dual Energy X-Ray Absorptiometry (DXA) and Magnetic Resonance Imaging (MRI) in Pigs.

The objective of this study was to phenotype visceral adipose tissue (VAT) in pigs. In this context, the ability to detect VAT by using the DXA CoreScan mode within the enCORE software, version 17 (GE Healthcare) was evaluated in comparison with MRI measurements (Siemens Magnetom C!) of the same body region. A number of 120 crossbred pigs of the F1 and F2 generation, with the parental breeds Large White, Landrace, Piétrain, and Duroc, were examined at an age of 150 days. A whole-body scan in two different modes ("thick", "standard") was carried out by a GE Lunar iDXA scanner. Very strong relationships (R2 = 0.95, RMSE = 175cm3) were found for VAT between the two DXA modes. The comparison of VAT measured by MRI and DXA shows high linear relationships ("thick": R2 = 0.76, RMSE = 399.25cm3/"standard": R2 = 0.71, RMSE = 443.42cm3), but is biased, according to the Bland-Altman analysis. A variance analysis of VAT shows significant differences for both DXA modes and for MRI between male and female pigs, as well as between F1 and F2. In conclusion, DXA "CoreScan" has the ability to estimate VAT in pigs with a close relationship to MRI but needs bias correction.

Relationship between Bone Stability and Egg Production in Genetically Divergent Chicken Layer Lines.

Impaired animal welfare due to skeletal disorders is likely one of the greatest issues currently facing the egg production industry. Reduced bone stability in laying hens is frequently attributed to long-term selection for increased egg production. The present study sought to analyse the relationship between bone stability traits and egg production. The study comprised four purebred layer lines, differing in their phylogenetic origin and performance level, providing extended insight into the phenotypic variability in bone characteristics in laying hens. Data collection included basic production parameters, bone morphometry, bone mineral density (BMD) and bone breaking strength (BBS) of the tibiotarsus and humerus. Using a multifactorial model and regression analyses, BMD proved to be of outstanding importance for bone stability. Only for the tibiotarsus were morphometric parameters and the bone weight associated with BBS. Within the chicken lines, no effect of total eggshell production on BBS or BMD could be detected, suggesting that a high egg yield itself is not necessarily a risk for poor bone health. Considering the complexity of osteoporosis, the estimated genetic parameters confirmed the importance of genetics in addressing the challenge of improving bone strength in layers.

"Sex" and body region effects on bone mineralization in male pigs.

Lameness in pigs is one of the major reasons for culling and early losses in pigs. This can be linked to osteoporosis due to pathologic alterations in bone mineral density (BMD) or bone mineral content (BMC) and may also be linked to the sex. Dealing with the ban on piglet castration without anaesthesia in Germany 2021, we have three male "sex" types: entire boars (EB), immunocastrated boars (IB), and surgically castrated boars (SB). The hypothesis of the present study is that BMC or BMD varies between different male sex types. If sex has an effect on bone mineralization (BMC or BMD) and if this affects leg health, it could result in more lameness and problems during fattening in the negatively affected sex type. The present study evaluated bone mineralization (in terms of BMD and BMC) and body composition traits using dual-energy X-ray absorptiometry (DXA) three times during growth at 30, 50, and 90 kg live body weight. Nine body regions were analysed for bone mineral traits and compared for different male sex types and the fattening season. Significant differences were found regarding BMD (and BMC) among EB, IB, and SB for whole-body BMD (BMC). Additionally significant differences were found in the front and lower hind limbs, where SB showed a significantly higher BMD compared to EB, with IB in between. Additionally regional differences were detected among the groups. Further studies are needed to evaluate the effect of these differences in bone mineralization on leg health.

Comparative Effects of Milk Containing A1 versus A2 ß-Casein on Health, Growth and ß-Casomorphin-7 Level in Plasma of Neonatal Dairy Calves.

Research has shown that digestion of A1 ß-casein (ß-CN) affects gastrointestinal motility and opioid activity through the release of the peptide ß-casomorphin-7 (ß-CM7). In the case of the A2 variant, the cleavage of ß-CM7 does not occur or occurs at a very low rate. Therefore, the aim of the study was to compare the effects of milk containing either homozygote A1 or A2 ß-CN on health and growth parameters of dairy calves. Forty-seven neonatal calves (24 females, 23 males) of the breeds German Holstein (GH, n = 9), German Simmental (GS, n = 33) and their crossing (GH × GS, n = 5) were used in a 21-day feeding study. Fecal score (FS), respiratory frequency (RF), and rectal body temperature (BT) were recorded daily, whereas body weight was measured at birth and at day 21 to estimate the average daily weight gain (ADG). Additionally, blood was collected from calves three times during the experimental period and, for the first time, the respective plasma samples were analyzed for intact ß-CM7. Consumption of A2-milk led to a lower daily milk intake (dMI) (p < 0.05). Furthermore, fecal consistency was softer for calves fed A2-milk (p < 0.05). Although 44% of A2-calves had diarrhea or revealed a tendency towards it (FS ≥ 3), A1-calves had a prevalence of 21%. Calves with a FS of 4 were offered an electrolyte solution and received a dietary food supplement for the stabilization of the fluid and electrolyte balance. Nevertheless, similar ADG and end weights (EW) of calves fed A1- or A2-milk (p > 0.05) indicate that A2-milk may compensate higher diarrhea rates and lower dMI due to the associated higher protein content. This is the first report of intact ß-CM7 in plasma of calves fed milk of either A1 or A2 ß-CN. Evidence from this study suggests that due to the change in the amino-acid sequence, A2-milk might be able to prevent or, at least, to minimize the cleavage of ß-CM7 in calves.

Reproductive and productive performance, udder health, and conformation traits of purebred Holstein, F1, and R1 crossbred Holstein × Simmental cows.

The objective of this study was to compare the reproductive performance, milk yield and composition, and udder health and conformation traits of Holstein (Ho), F1, and R1 crossbred Ho × Simmental (Sim) cows. Three commercial dairy farms in south Brazil were used as the research units. All farms held Ho, F1, and R1 crossbred Ho × Sim (¾ Ho × » Sim and ¾ Sim × » Ho) cows. The collection of milk samples and evaluation of udder conformation traits occurred during four visits to each farm. In addition to the actively collected data, retrospective reproduction records of the farms served as the basis for the statistical analysis using analysis of variance models using SAS. The F1 crossbred Ho × Sim cows and ¾ Sim (first rotational crossbreeding generation = R1 using Sim semen) cows had a shorter calving interval and calving to first service interval compared to the Ho cows (P < 0.0001). Milk yield did not differ among the genetic groups except for R1 (¾ Sim) that produced approximately 10% less milk than the other groups (P = 0.0245). Fat plus protein yield and somatic cell score did not differ among the genetic groups. Ho cows had shallower udders (P < 0.0001) and a higher udder clearance (P < 0.0001) than the other groups. F1 and R1 crossbred Ho × Sim cows had shorter reproduction intervals than purebred Ho cows. Although udder conformation traits lacked high-quality scores in crossbred cows, somatic cell scores reached the same level as in purebred Ho cows.