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Abstract: Klebsiella spp. are gram-negative facultative anaerobic heavily encapsulated bacteria associated with opportunistic and primary infections in a wide range of species. We assessed a series of cases (n=8) of necrosuppurative peritonitis, lymphadenitis, and/or pyelonephritis in wild juvenile raccoons (Procyon lotor) that died under rehabilitator care in New York, New Jersey, and Wisconsin, US, between July 2020 and December 2023, plus a retrospective case of a juvenile raccoon necropsied from New York in August 2011. Gross necropsy (n=9) and histopathology (n=9) were performed to characterize the lesions, whereas bacterial culture (n=8) was used to identify and characterize the bacteria and associated phenotype. We observed gram-negative short rods and coccobacilli (7/9; 78%), fibrinosuppurative peritonitis of variable severity (7/9; 78%) correlated to gross pyoabdomen (5/9; 56%) or abscessation (2/9; 22%), lymphadenomegaly and associated necrosuppurative lymphadenitis (5/9; 56%), and urinary tract disease (3/9; 33%). Aerobic culture of affected tissues isolated Klebsiella pneumoniae (n=4), K. pneumoniae subsp. ozaenae (n=2), Klebsiella variicola (n=1), and Klebsiella sp. (n=1). Our study strongly suggests an association of bacteria within the K. pneumoniae complex with peritonitis, lymphadenitis, and pyelonephritis in raccoons. Disease might be associated with underlying nosocomial infection given that all animals were under rehabilitator care at the time of death., (© Wildlife Disease Association 2024.)

Antibiotic resistance poses a significant public health threat worldwide. The rise in antibiotic resistance and the sharp decline in effective antibiotics necessitate the development of innovative antibacterial agents. Based on the central symmetric structure of glycine-serine-glycine, combined with tryptophan and arginine, we designed a range of antimicrobial peptides (AMPs) that exhibited broad-spectrum antibacterial activity. Notably, AMP W 5 demonstrated a rapid and effective sterilization against methicillin-resistant Staphylococcus aureus (MRSA), displaying both a minimum inhibitory concentration and a minimum bactericidal concentration of 8 µM. Mechanistic studies revealed that AMP W 5 killed bacterial cells by disrupting the cytoplasmic membrane integrity, triggering leakage of cell contents. AMP W 5 also exhibited excellent biocompatibility in both in vitro and in vivo safety evaluations. AMP W 5 treatment significantly reduced skin bacterial load in our murine skin infection model. In conclusion, we designed a novel centrosymmetric AMP representing a promising medical alternative to conventional antibiotics for treating MRSA infections., Importance: Increasing antibiotic resistance and the paucity of effective antibiotics necessitate innovative antibacterial agents. Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing bacterial infections with high incidence and mortality rates, showing increasing resistance to clinical drugs. Antimicrobial peptides (AMPs) exhibit significant potential as alternatives to traditional antibiotics. This study designed a novel series of AMPs, characterized by a glycine-serine-glycine-centered symmetrical structure, and our results indicated that AMP W5 exhibited a rapid and effective bactericidal effect against MRSA. AMP W5 also demonstrated excellent biocompatibility and a bactericidal mechanism that disrupted membrane integrity, leading to leakage of cellular contents. The notable reduction in skin bacterial load observed in mouse models reinforced the clinical applicability of AMP W5. This study provides a promising solution for addressing the increasing threat of antibiotic-resistant bacteria and heralds new prospects for clinical applications., Competing Interests: The authors declare no conflict of interest.

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis., (© 2024. The Author(s).)

Objective: We aimed to study the antimicrobial and pro-healing potential of equine mesenchymal stromal cell secreted products (i.e. secretome), collected as conditioned media (mesenchymal stromal cell-conditioned media, MSC CM), in a novel in vivo model of methicillin-resistant Staphylococcus aureus (MRSA)-inoculated equine thorax wounds., Study Design: Prospective in vivo study., Animals: Two Thoroughbred geldings., Methods: Six full-thickness cutaneous wounds were created bilaterally on the dorsal thorax of two horses (n = 12 wounds/horse). Wounds on the left thoraces were inoculated with MRSA on day 0. All wounds were then treated with either mupirocin ointment, MSC CM, or vehicle control (n = 4 wounds per group) once daily for 3 days. Photographs were taken to quantify wound scores and sizes, as well as samples to determine bacterial colony forming units (CFUs), at days 0, 1, 2, 3, 7, 14, 21, and 28. The wound edge was biopsied on days 0, 7, and 28, and scored histologically., Results: Inoculated wounds had more bacterial CFUs at day 1 (p < .0001) and were larger in size at day 28 (p = .0009) than noninoculated wounds. Mupirocin-treated wounds were smaller than MSC CM and vehicle control-treated wounds at day 28 (p = .003). Mesenchymal stromal cell-conditioned media did not affect CFU numbers in inoculated and noninoculated wounds. Moreover, MSC CM did not affect the parameters of wound size or gross or microscopic wound scores over time., Conclusion: Mesenchymal stromal cell-conditioned media did not exhibit antimicrobial or pro-healing properties in the current study; however, the in vivo model of inoculated equine thorax wounds requires further optimization., Clinical Significance: This pilot study contributes to a growing understanding of the equine MSC secretome as an antimicrobial and pro-healing therapeutic for equine wounds., (© 2024 The Author(s). Veterinary Surgery published by Wiley Periodicals LLC on behalf of American College of Veterinary Surgeons.)

Ketosis, evidenced by hyperketonemia with elevated blood β-hydroxybutyrate (BHB) levels, is a significant metabolic disorder of dairy cattle, typically diagnosed within the first 6 weeks post-calving when high energy levels are essential to milk production. Our study aimed to identify genetic markers linked to hyperketonemia (HYK) patterns in Holstein cows during early lactation and compare these to HYK-negative cows. We screened 964 cows for HYK using a threshold of BHB ≥1.2 mmol/L during the first 2 weeks postpartum (screening period, SP). Cows that tested negative initially were retested the following week. Cows were deemed HYK-negative (CON group) if BHB levels were below 1.2 mmol/L in both tests, while those with BHB levels exceeding this threshold at any test were treated and classified as HYK-positive (HYK+). Post-treatment, HYK+ cows were monitored for two-week follow-up period (FP) and classified based on their recovery: cured (CUR; consistently low BHB), recurrent (REC; fluctuating BHB levels), severe (SEV; high initial BHB that decreased), or chronic (CHR; persistently high BHB). Using 489 cows that were genotyped, a GWAS was conducted using GCTA software, revealing significant associations of several SNPs across different HYK patterns when compared to the CON group. These SNPs were primarily linked to genes affecting milk traits and were enriched in biological pathways relevant to protein glycosylation, inflammatory response, glucose homeostasis, and fatty acid synthesis. Our findings highlight genomic regions, potential candidate genes, and biological pathways related to ketosis, underscoring potential targets for improving health management in dairy cattle. These insights could lead to better strategies for managing ketosis through genetic selection, ultimately enhancing dairy cattle welfare and productivity. Further research with a larger number of cows is recommended to validate these findings and help confirm the implicated SNPs and genes., (© 2024 The Author(s). Journal of Animal Breeding and Genetics published by John Wiley & Sons Ltd.)

The rostral cranial fossa (RCF) consists of the sphenoid and ethmoid bones, which accommodate the olfactory bulbs and nerves along the recesses of the cribriform plate. Neoplasms located in the vicinities of the RCF can compress and/or invade the cribriform plate. Here we describe the clinical and pathologic findings of neoplasms involving the cribriform plate in 32 dogs and 17 cats autopsied over a 13-y period. The average ages of affected dogs and cats were 9.2 y and 9.7 y, respectively. No sex or breed predisposition was evident in dogs, but 13 of 18 cats were spayed females and 14 of 18 were domestic shorthair cats. The main clinical signs were seizures (10 cases) and epistaxis (5 cases) in dogs, and red-to-brown nasal discharge (5 cases) and seizures (4 cases) in cats. In dogs, the 22 sinonasal neoplasms included adenocarcinoma (14 cases), transitional carcinoma (4), squamous cell carcinoma (2), lymphoma (1), and histiocytic sarcoma (1); the 10 intracranial neoplasms consisted of high-grade gliomas (3 cases), psammomatous meningiomas (2), histiocytic sarcomas (2), olfactory neuroblastomas (2), and a meningeal granular cell tumor (1). In cats, the 14 sinonasal neoplasms included lymphoma (8 cases), adenocarcinoma (4), adenosquamous carcinoma (1), and squamous cell carcinoma (1); the 3 intracranial neoplasms consisted of oligodendroglioma (1), transitional meningioma (1), and olfactory neuroblastoma (1)., Competing Interests: Declaration of conflicting interestsThe author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The eukaryotic Glucose Induced Degradation/C-Terminal to LisH (GID/CTLH) complex is a highly conserved E3 ubiquitin ligase involved in a broad range of biological processes. However, a role of this complex in host anti-microbial defenses has not been described. We exploited Mycobacterium tuberculosis (Mtb) induced cytotoxicity in macrophages in a FACS based CRISPR genetic screen to identify host determinants of intracellular Mtb growth restriction. Our screen identified 5 (GID8, YPEL5, WDR26, UBE2H, MAEA) of the 12 predicted members of the GID/CTLH complex as determinants of intracellular growth of both Mtb and Salmonella serovar Typhimurium. We show that the anti-microbial properties of the GID/CTLH complex knockout macrophages are mediated by enhanced GABAergic signaling, activated AMPK, increased autophagic flux and resistance to Mtb induced necrotic cell death. Meanwhile, Mtb isolated from GID/CTLH knockout macrophages are nutritionally starved and oxidatively stressed. Our study identifies the GID/CTLH complex activity as broadly suppressive of host anti-microbial responses against intracellular bacterial infections., (© 2024. The Author(s).)

Background: Comparative pharmacokinetics and pharmacodynamics (PK/PD) of apixaban and rivaroxaban have not been studied in dogs and the propensity of these drugs to cause hypercoagulability after discontinuation is unknown., Hypothesis: Compare the PK/PD of clinical dosing regimens of PO apixaban and rivaroxaban administered repeatedly to healthy dogs and assess the effect of abrupt drug discontinuation on coagulation., Animals: Six University-owned, purpose-bred, middle-aged, mixed-breed dogs (4 male, 2 female)., Methods: Dogs were given apixaban or rivaroxaban PO at 0.5 mg/kg q12h for 7 days with a 14-day washout period between drugs. Plasma drug concentrations were quantitated, and anticoagulant effects were measured using clotting times, calibrated anti-Xa bioactivity assays, and measurements of thrombin generation. The potential for rebound hypercoagulability was assessed by measuring D-dimers, thrombin-antithrombin (TAT) complexes, and antithrombin activity after drug discontinuation., Results: Plasma drug concentrations and anti-Xa bioactivities were closely correlated for both drugs, but drug concentrations varied considerably among dogs, despite consistent dose regimens. Thrombin generation variables were significantly correlated with the anti-Xa bioactivity of both drugs and no significant differences in the effects of apixaban and rivaroxaban on thrombin generation were observed. Drug discontinuation had no effect on D-dimer concentrations. The concentration of TAT complexes decreased after apixaban discontinuation and did not change after rivaroxaban discontinuation., Conclusions and Clinical Importance: Repeated PO administration of apixaban or rivaroxaban to healthy dogs produced comparable anticoagulant effects measured by inhibition of thrombin formation. Rebound hypercoagulability after drug discontinuation was not observed and weaning of these drugs in clinical patients might not be necessary., (© 2024 The Author(s). Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018-2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020-2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

Schwannosis is a rare, non-neoplastic, perivascular proliferation of aberrant Schwann cells within the CNS with simultaneous partial myelination of axons. A single report exists in veterinary medicine of schwannosis in the spinal cord of 3 foals and 1 calf. Here we describe a case of schwannosis in the brain of a 1-d-old Holstein-Friesian calf, submitted for autopsy due to arthrogryposis and premature death, with no other gross abnormalities observed. Histologically, the brain had multifocal, mainly perivascular, spindle-cell proliferations within the white matter of the medulla oblongata and focally within the gray matter of the midbrain. These cells immunolabeled with periaxin, myelin protein zero, SOX10, S100, and equivocally for vimentin, indicating Schwann cell origin. No changes were identified within other organs. Ancillary tests did not support an infectious etiology. Schwannosis should be considered as a differential diagnosis when investigating cases of arthrogryposis in calves with negative ancillary tests for infectious conditions., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Equine herpesvirus type 1 (EHV-1) is one of the most prevalent respiratory pathogens in horses with a high impact on animal health worldwide. Entry of the virus into epithelial cells of the upper respiratory tract and rapid local viral replication is followed by infection of local lymphoid tissues leading to cell-associated viremia and disease progression. Pre-existing mucosal immunity has previously been shown to reduce viral shedding and prevent viremia, consequently limiting severe disease manifestations. Here, nasopharyngeal transcriptomic profiling was used to identify differentially expressed genes following EHV-1 challenge in horses with different EHV-1 immune statuses. Immune horses ( n = 4) did neither develop clinical disease nor viremia and did not shed virus after experimental infection, while non-immune horses ( n = 4) did all the above. RNA sequencing was performed on nasopharyngeal samples pre- and 24 hours post-infection (24hpi). At 24hpi, 109 and 44 genes were upregulated in immune horses and non-immune horses, respectively, and three genes were explored in further detail. Antileukoproteinase ( SLPI ) gene expression increased 2.1-fold within 24 hours in immune horses in concert with protein secretion. Interferon (IFN)-induced proteins with tetratricopeptide repeats 2 ( IFIT2 ) and 3 ( IFIT3 ) were upregulated in non-immune horses, corresponding with nasal IFN-α secretion and viral replication. By contrast, neither IFIT expression nor IFN-α secretion was induced by EHV-1 infection of immune horses. Transcriptomic profiling offered a tool to identify, for the first time, the role of SLPI in innate immunity against EHV-1, and further emphasized the central role of the type I IFN response in the anti-viral defense of non-immune horses., Importance: Equine herpesvirus type 1 (EHV-1) remains a considerable concern in the equine industry, with yearly outbreaks resulting in morbidity, mortality, and economic losses. In addition to its importance in equine health, EHV-1 is a respiratory pathogen and an alphaherpesvirus, and it may serve as a model for other viruses with similar pathogenicity or phylogeny. Large animal models allow the collection of high-volume samples longitudinally, permitting in-depth investigation of immunological processes. This study was performed on bio-banked nasopharyngeal samples from an EHV-1 infection experiment, where clinical outcomes had previously been determined. Matched nucleic acid and protein samples throughout infection permitted longitudinal quantification of the protein or related proteins of selected differentially expressed genes detected during the transcriptomic screen. The results of this manuscript identified novel innate immune pathways of the upper respiratory tract during the first 24 hours of EHV-1 infection, offering a first look at the components of early mucosal immunity that are indicative of protection., Competing Interests: The authors declare no conflict of interest.

Great Crested Flycatchers (Myiarchus crinitus), migratory passerines with a breeding range throughout the northeastern, midwestern, and southern US, are banded annually at the Braddock Bay Bird Observatory located on the southern shore of Lake Ontario, New York, USA. In 2016, a Great Crested Flycatcher was observed with distinct lesions in the gular and ventral neck region, which prompted evaluation for similar lesions in subsequently trapped flycatchers and other passerine species. From 2016 to 2023, 62/102 banded Great Crested Flycatchers had their gular region examined, and seven were found to have lesions (11.3% incidence). Similar lesions were not found in any other species. Lesions were localized to the gular region and included extensive feather loss with thickened, corrugated, pale-yellow skin. Grossly visible 1- to 2-mm-diameter, raised, white-to-yellow foci throughout the affected region corresponded microscopically to feather follicles that were massively dilated with mites. Morphologic analysis of mites obtained from skin scrapes revealed that this mite species belongs to the family Harpirhynchidae. Mites in this family have restricted avian host ranges and cause varying clinical presentations in passerines, though many species remain unidentified. PCR efforts were unsuccessful in yielding a species-level identification. Further monitoring of Great Crested Flycatchers and other avian species is warranted, as the fitness implications of this ectoparasitism at the individual and population levels are not known., (© Wildlife Disease Association 2024.)

Prostaglandins have many roles in the equine reproductive tract, including but not limited to luteolysis, luteal support, ovulation, transport through the uterine tube, uterine contraction, embryonic mobility, inflammation, and fibrosis. Altered secretion of inflammatory proteins are likely to disrupt the balance of endometrial function and could impair fertility. Our overall goal was to measure the expression of several prostaglandin- and inflammation-related genes in mares with different degrees of endometrial histological changes. Our hypothesis was that mares with neutrophilic and lymphocytic plasmocytic inflammation, fibrosis, or different biopsy grades would have altered concentrations of prostaglandin E2 (PGE2) and F2α (PGF2α), as well as altered expression of inflammation- and prostaglandin-related genes, compared to mares with minimal to no histological changes on biopsy evaluation. Forty-five endometrial biopsies from estrous mares were assessed by a reproductive pathologist for the degree of neutrophilic inflammation, lymphocytic and plasmocytic inflammation, and fibrosis, and a biopsy grade was assigned based on the Kenney-Doig system. A low-volume uterine lavage was collected from a subset of twenty-six mares prior to biopsy collection and was used to measure PGE2 and PGF2α concentrations via ELISA. Total RNA was extracted from biopsies and mRNA expression was evaluated for twenty-five genes of interest. A restricted maximum likelihood linear model was used to compare differences of mRNA expression, with a statistical significance set at P < 0.05. There was no difference in the abundance of PGE2 or PGF2α between any of the variables tested. Mares with endometrial biopsy grade I had lower expression of NF-kB, PTGS1 and HPGD compared to grade IIA or IIB (P < 0.05). Mares with neutrophilic inflammation had decreased expression of NF-kB, PTGS1, PTGER4, CBR1, mPGES2 and PTGIS compared to mares without inflammation. Mares with mild or minimal endometrial fibrosis had increased expression of mPGES2 and PTGIS, compared to mares with moderate endometrial fibrosis. In conclusion, several genes were identified to be differentially expressed in mares with histological changes compared to mares with no to minimal histological changes. The presence of inflammation and fibrosis may alter the concentration of prostaglandins in endometrial tissue, which could impair many of the uterine reproductive and immune functions during estrus, affecting early embryo survival., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The highly pathogenic avian influenza (HPAI) H5N1 virus clade 2.3.4.4b has caused the death of millions of domestic birds and thousands of wild birds in the USA since January 2022 (refs. 1-4 ). Throughout this outbreak, spillovers to mammals have been frequently documented 5-12 . Here we report spillover of the HPAI H5N1 virus to dairy cattle across several states in the USA. The affected cows displayed clinical signs encompassing decreased feed intake, altered faecal consistency, respiratory distress and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of the HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the ability of the virus to cross species barriers., (© 2024. The Author(s).)

The widespread circulation of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b in wild birds in North America since late 2021 has resulted in multiple outbreaks in commercial and backyard poultry leading to major economic losses. Since the emergence of the virus in North America, multiple reassortment events have led to the emergence of many new variant genotypes that have been isolated from wild birds, with several viruses spilling over into poultry and other terrestrial and aquatic mammalian hosts. Notably, the most recent emerging HPAI H5N1 reassortant genotype B3.13 spilled over into dairy cattle ( Bos taurus ), resulting in unprecedented efficient transmission of the virus in this species, the first case of sustained transmission in a mammalian species. The transmission pathways involved in the spread of the virus from its first detection in Texas to several other states are complex. However, movement of subclinically infected cattle likely played a major role in virus spread. Infection in dairy cattle is characterized by the virus's tropism for milk-secreting cells in the mammary gland, leading to high viral load and shedding in milk. Replication of the virus in milk-secreting cells results in destruction of infected cells leading to severe viral mastitis, which is characterized by marked changes in milk quality (altered consistency and color) and pronounced decline in milk production by clinically affected animals. Here, we provide an overview of the HPAI H5N1 panzootic virus and discuss its host range and the current knowledge of its pathogenesis in the new bovine host., (© 2024.)

Our study aimed to compare Bovine Respiratory Disease (BRD) morbidity, mortality, and growth in dairy and dairy beef cross-bred calves during the commingle period, 81-120 days of age, using two different BRD prevention strategies. The calves (n = 1799) were randomly assigned into groups: (1) Control (CON; received no vaccine or metaphylaxis); (2) Tulathromycin metaphylaxis (TUL; Increxxa TM , Elanco Animal Health Inc., Greenfield, IN, USA); and (3) Mannheimia haemolytica vaccine (VACC; Nuplura ® PH, Elanco Animal Health Inc., Greenfield, IN, USA). Calves were individually weighed three times during the study to estimate average daily gain (ADG). Deep nasopharyngeal swabs, thoracic ultrasonography, health scores, and treatment records were collected during the study. Ultrasound and health score results were not different across treatments. In this study, the TUL group had a lower cumulative BRD incidence than CON. The cumulative incidence and 95% CI of BRD during the commingle period, 81-120 days of age for CON, TUL, and VACC were 0.43 (0.38 to 0.47), 0.36 (0.38 to 0.40), and 0.39 (0.35 to 0.43), respectively. The ADG for CON, TUL, and VACC were 0.25 ± 0.15, 0.32 ± 0.15, and 0.17 ± 0.15 kg, respectively. There was no difference among the treatment groups for ADG. Management and environmental conditions were variable at this operation throughout the study period and likely impacted the calves. Earlier interventions may be needed when the BRD incidence is elevated in high-risk calves.

Conformational teat traits such as teat barrel shape and teat-end shape have long been recognized as possible risk factors for elevated somatic cell count and clinical mastitis in dairy cows. However, the association between udder health and these teat traits is still under debate. Our objective of this ambidirectional cohort study was to investigate the relationship between teat shape and the occurrence of clinical mastitis in dairy cows. For this purpose, we analyzed quarter-level data from 14,948 quarters of 3,913 Holstein cows from 1 commercial dairy farm in New York State. Cows were milked 3 times daily, housed in free-stall pens, bedded with manure solids, and fed a total mixed ration. Teat shape was assessed visually and classified based on teat-barrel and teat-end shape into 1 of 4 categories as follows: (1) triangular barrel and pointed teat end (TP), (2) square barrel and round teat end (SR), (3) square barrel, round teat end, and flat in the area of the teat orifice (SRF), and (4) square barrel and flat teat end (SF). Data on the occurrence of clinical mastitis were obtained from the dairy management software. To test the hypothesis that teat shape was associated with the occurrence of the first clinical mastitis event during the first 305 d in milk, a multivariable semiparametric proportional hazards model was built. Our results showed that teat shape was associated with the occurrence of clinical mastitis. Compared with quarter with teats with SR, the clinical mastitis hazards (95% confidence intervals) were TP, 1.66 (1.25-2.19); SF, 1.58 (1.14-2.18); and SRF, 1.05 (0.88-1.26). We conclude that teat shape could be useful to identify cows at increased risk of clinical mastitis. This could allow farmers to employ targeted monitoring of these high-risk animals and develop management strategies that mitigate their risk., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The influence of diet composition on the degree of adipose and lean muscle mobilization and concentrations of circulating AA has been demonstrated during the transition period. Altering the MP supply might offer a strategy to control tissue mobilization and increase circulating AA availability, but the optimum supply of MP fed pre- and postpartum remains unknown. We investigated the effect of increasing the MP supply in the prepartum, postpartum, or both diets on plasma AA concentrations and ultrasound and circulating indicators of tissue mobilization. Multiparous Holstein cows (n = 96) were assigned to 1 of 4 treatment groups at 28 d before expected calving following a randomized block design. Prepartum diets were formulated to contain either a control (C; 85 g of MP/kg DM; 1,175 g of MP/d) or high (H; 113 g of MP/kg DM; 1,603 g of MP/d) level of estimated MP. From calving to 21 DIM, fresh diets were formulated to contain either a control (C; 104 g of MP/kg DM; 2,044 g of MP/d) or high (H; 131 g of MP/kg DM; 2,685 g of MP/d) level of estimated MP. To control the potential confounding effect of Met and Lys supply, diets were formulated to supply an equal amount at 1.24 and 3.84 g/Mcal of ME in both prepartum diets and 1.15 and 3.16 g/Mcal of ME in both postpartum diets, respectively. The combination of a pre- and postpartum diet resulted in 4 treatment groups: 1) CC (n = 23), 2) CH (n = 24), 3) HC (n = 22), and 4) HH (n = 23). A common lactation diet (113 g of MP/kg DM; 2,956 g of MP/d) was fed from 22 DIM to the end of the observation period at 42 DIM. Transcutaneous ultrasonography was used to determine the longissimus dorsi muscle diameter and backfat thickness. Concentrations of plasma AA, 3-methylhistidine (3MH), and creatinine were determined on a subset of cows (n = 60) using ultra-high-performance liquid chromatography and mass spectrometry. Treatment did not affect the longissimus dorsi muscle diameter from -14 to 21 d relative to calving, but the diameter was greater in CH compared with HH at 40 DIM. Backfat thickness and the ratio of 3MH to creatinine did not differ by treatment. Concentrations of EAA were greater at -13 d relative to calving in HH compared with CC and CH and at -6 d relative to calving EAA concentrations were higher in HC compared with CC. Cows fed the H diet postpartum had elevated EAA concentrations at 6 and 20 DIM compared with cows fed the C postpartum diet but EAA concentration did not differ at 40 DIM. Total NEAA concentrations were higher in CH compared with HC and HH at -6 d relative to calving, but NEAA concentration did not differ by treatment at -13, 6, 20, or 40 d relative to calving. In conclusion, increasing the supply of MP fed prepartum, postpartum, or both had minimal effects on tissue mobilization but influenced concentrations of plasma AA., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this study was to investigate the effect of increasing MP supply in the prepartum, postpartum, or both diets on intake, performance, and metabolic indicators. Multiparous Holstein cows (n = 96) were assigned to 1 of 4 treatment groups at 28 d before expected calving following a randomized block design. Prepartum diets were formulated to contain either a control (C; 85 g of MP/kg DM) or high (H; 113 g of MP/kg DM) level of estimated MP. From calving to 21 DIM, diets were formulated to contain either a control (C; 104 g of MP/kg DM) or high (H; 131 g of MP/kg DM) level of estimated MP. To control the potential confounding effect of Met and Lys supply, diets were formulated to supply an equal amount at 1.24 and 3.84 g/Mcal of ME in both prepartum diets and 1.15 and 3.16 g/Mcal of ME in both postpartum diets, respectively. The combination of a pre- and postpartum diet resulted in 4 treatment groups: 1) CC (n = 23), 2) CH (n = 24), 3) HC (n = 22), and 4) HH (n = 23). A common lactation diet (113 g of MP/kg DM) was fed from 22 DIM to the end of the observation period at 42 DIM. Milk yield and DMI were collected daily, and plasma metabolic indicators (BHB, fatty acids [NEFA], urea nitrogen [PUN], and glucose) were determined twice weekly from -28 to 28 d relative to calving and once weekly from 29 to 42 DIM. Samples with BHB ≥1.2 mmol/L between 3 and 10 DIM were considered hyperketonemia events. Milk composition was determined weekly. Milk yield during 1 to 21 DIM was greater in HH (44.7 ± 1.0 kg/d) compared with CC (39.2 ± 1.0 kg/d) and HC (38.0 ± 1.0 kg/d) and milk yield in CH (42.4 ± 0.9 kg/d) was greater than HC, respectively. From 22 to 42 DIM, milk yield was greater in CH (53.3 ± 1.0 kg/d) and HH (54.1 ± 1.0 kg/d) compared with CC (49.6 ± 1.0 kg/d) and HC (49.3 ± 1.0 kg/d). Dry matter intake (% of BW) and concentrations of milk protein, fat, and total solids were not affected by treatment. Prepartum concentrations of PUN were greater in H compared with C. From 1 to 21 DIM, PUN concentrations were greater in CH and HH compared with CC and HC. From 1 to 21 DIM, glucose concentrations were lower in HH compared with HC and BHB were greater in CH and HH compared with HC. Concentrations of NEFA, as well as the number of hyperketonemia events did not differ by treatment during this time. From 22 to 42 DIM, concentrations of NEFA were greater in HH compared with HC and concentrations of BHB were greater in CH and HH compared with HC. Overall, feeding CH or HH increased lactation performance without altering intake or hyperketonemia events. Results from this study support formulating a fresh diet to reduce the negative MP balance during early lactation., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA 1 . Rapid dissemination to more than 190 farms in 13 states followed 2 . Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 isolates. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 10 8 TCID 50 /mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n = 15). All patients received remdesivir and some also received nirmatrelvir-ritonavir (n = 3) or therapeutic monoclonal antibodies (n = 4). Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient sequentially treated with nirmatrelvir-ritonavir and remdesivir. In vitro characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility in vivo., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The recent incursion of highly pathogenic influenza viruses into dairy cattle opens new insights for influenza virus ecology and its interspecies transmission and may have a significant impact on public health and agriculture. The aim of this study was to determine the stability of a bovine highly pathogenic avian influenza H5N1 virus isolate in the milk byproduct lactose and to evaluate two inactivation methods using industrial procedures. The bovine isolate of the highly pathogenic avian influenza H5N1 virus was stable for 14 days in a concentrated lactose solution under refrigerated conditions. Heat or citric acid treatments successfully inactivated the virus in lactose. This study highlights the persistence of HPAIV in lactose and its efficient inactivation under industrial standards.

The objectives of this study were to assess the effect of flow-responsive vacuum and pulsation, in conjunction with the early attachment of the milking unit (TRT), on teat tissue conditions and milking characteristics in dairy cows. In a switch-back trial, 5,235 Holstein cows milked 3 times daily in a rotary parlor were assigned to the TRT or control (CON) group. The trial lasted 84 d and was comprised of 4 alternating 3-week periods of TRT and CON. For both groups, premilking udder preparation consisted of teat brushing, forestripping and predipping, and wiping of teats, resulting in a stimulation time of 4 s. In the TRT group, the preparation lag time was 58 s, and in the CON group, it was 91 s for early- and mid-lactation cows and 105 s for late-lactation animals. Upon milking unit attachment, the TRT cows were milked at a lower vacuum (37.6 kPa) and pulsation (50 cycles/min, pulsation ratio of 30:70). The vacuum and pulsation settings were changed to milking mode when the milk flow reached 0.5 kg/min (pulsation switch-point) and 1.6 kg/min (vacuum switch-point). For milking mode, the vacuum setting was 47.7 kPa, and the pulsation rate was 60 cycles/min at a ratio of 65:35. The CON cows were milked with a flow-responsive vacuum, using the same vacuum settings as the TRT group. We assessed machine milking-induced short-term teat tissue changes and teat-end hyperkeratosis by palpation and visual inspection postmilking. Electronic on-farm milk meters were used to assess milking characteristics. Generalized linear mixed models were used to analyze the effect of treatment on the outcome variables. Compared with cows in group CON, the odds ratios (95% confidence interval; 95% CI) of short-term teat-tissue changes in early-, mid-, and late lactation cows in group TRT were 0.62 (0.52-0.76), 0.61 (0.48-0.77), and 0.93 (0.76-1.14), respectively. The least squares means [LSM, (95% CI)] for milking unit-on time in early-, mid-, and late lactation animals, respectively, were 251 (248-253), 236 (234-238), and 220 (218-222) s for group TRT and 247 (245-249), 232 (230-234), and 214 (213-216) s for the CON group. The LSM (95% CI) of peak milk flow rate in early-, mid-, and late lactation animals, respectively, were 5.75 (5.68-5.82), 5.77 (5.70-5.84), and 5.54 (5.48-5.59) kg/min for the TRT cows and 5.65 (5.58-5.72), 5.74 (5.68-5.81), and 5.45 (5.40-5.51) kg/min for the CON cows. The odds ratios (95% CI) of forced take-off in group TRT for early-, mid-, and late lactation cows, respectively, were 0.39 (0.37-0.41), 0.32 (0.30-0.34), and 0.47 (0.44-0.52) compared with their respective CON groups. In this study, cows that were milked using flow-responsive vacuum and pulsation with early attachment of the milking unit had lower odds of short-term teat tissue changes and forced take-off, as well as a higher peak milk flow rate. Our data suggest that the application of flow-responsive vacuum and pulsation facilitates early attachment of the milking unit, improves teat tissue condition, and has the potential to improve parlor efficiency., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

We report the design, development, and characterization of a miniaturized version of the photonic resonator absorption microscope (PRAM Mini), whose cost, size, and functionality are compatible with point-of-care (POC) diagnostic assay applications. Compared to previously reported versions of the PRAM instrument, the PRAM Mini components are integrated within an optical framework comprised of an acrylic breadboard and plastic alignment fixtures. The instrument incorporates a Raspberry Pi microprocessor and Bluetooth communication circuit board for wireless control and data connection to a linked smartphone. PRAM takes advantage of enhanced optical absorption of ∼80 nm diameter gold nanoparticles (AuNP) whose localized surface plasmon resonance overlaps with the ∼625 nm resonant reflection wavelength of a photonic crystal (PC) surface. When illuminated with wide-field low-intensity collimated light from a ∼617 nm wavelength red LED, each AuNP linked to the PC surface results in locally reduced reflection intensity, which is visualized by observing dark spots in the PC-reflected image with an inexpensive CMOS image sensor. Each AuNP in the image field of view can be easily counted with digital resolution. We report upon the selection of optical/electronic components, image processing algorithm, and contrast achieved for single AuNP detection. The instrument is operated via a wireless connection to a linked mobile device using a custom-developed software application that runs on an Android smartphone. As a representative POC application, we used the PRAM Mini as the detection instrument for an assay that measures the presence of antibodies against SARS-CoV-2 infection in cat serum samples, where each dark spot in the image represents a complex between one immobilized viral antigen, one antibody molecule, and one AuNP tag. With dimensions of 23 × 21 × 10 cm 3 , the PRAM Mini offers a compact detection instrument for POC diagnostics., Competing Interests: B. Cunningham is a founder of Atzeyo Biosensors, which holds a license for the PRAM technology., (© 2024 Optica Publishing Group.)