Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle.

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 documented5-12. 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.

Pathogenicity and transmissibility of bovine H5N1 influenza virus.

Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the USA, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1-4. Here we characterize an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species, however, this tropism was also observed for an older HPAI H5N1 virus isolate. Bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.

Lining the small intestine with mycobacteriophages protects from Mycobacterium avium subsp. paratuberculosis and eliminates fecal shedding.

Johne's disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry.

Variations in antibody repertoires correlate with vaccine responses.

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold the key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated data set, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study reveals that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

A locus conferring tolerance to Theileria infection in African cattle.

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa's most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

Multiomics reveals blood differential metabolites and differential genes in the early onset of ketosis in dairy cows.

Ketosis-a metabolic state characterized by elevated levels of ketone bodies in the blood or urine-reduces the performance and health of dairy cows and causes substantial economic losses for the dairy industry. Currently, beta-hydroxybutyric acid is the gold standard for determining ketosis in cows; however, as this method is only applicable postpartum, it is not conducive to the early intervention of ketosis in dairy cows. In this study, the sera of dry, periparturient, postpartum ketotic, and healthy cows were analyzed by both transcriptomics and metabolomics techniques. Moreover, changes of gene expression and metabolites were observed, and serum physiological and biochemical indexes were detected by ELISA. The purpose was to screen biomarkers that can be used to detect the incidence of dry or periparturient ketosis in cows. The results showed that ketotic cows had increased levels of glycolipid metabolism indexes, oxidizing factors, and inflammatory factors during dry periods and liver damage, which could be used as early biomarkers to predict the onset of ketosis. Transcriptomic results yielded 20 differentially expressed genes (DEGs) between ketotic and healthy cows during dry, peripartum, and postpartum periods. GO and KEGG enrichment analyses indicated that these DEGs were involved in amino acid metabolism, energy metabolism, and disease-related signaling pathways. The metabolomics sequencing results showed that ketotic cows mainly showed enrichment in tricarboxylic acid cycle, butyric acid metabolism, carbon metabolism, lysine degradation, fatty acid degradation, and other signaling pathways. Metabolites differed between ketotic and healthy cows in dry, pre-parturition, and post-parturition periods. Combined transcriptomics and metabolomics analyses identified significant enrichment in the glucagon signaling pathway and the lysine degradation signaling pathway in dry, periparturient, and postpartum ketotic cows. PRKAB2 and SETMAR-key DEGs of the glucagon signaling pathway and lysine degradation signaling pathway, respectively-can be used as key marker genes for determining the early onset of ketosis in dairy cows.

Proinflammatory CD14highCD16low monocytes/macrophages prevail in Treponema phagedenis-associated bovine digital dermatitis.

Digital dermatitis (DD) is a skin disease in cattle characterized by painful inflammatory ulcerative lesions in the feet, mostly associated with local colonization by Treponema spp., including Treponema phagedenis. The reason why most DD lesions remain actively inflamed and progress to chronic conditions despite antibiotic treatment remains unknown. Herein, we show an abundant infiltration of proinflammatory (CD14highCD16low) monocytes/macrophages in active DD lesions, a skin response that was not mitigated by topical treatment with oxytetracycline. The associated bacterium, T. phagedenis, isolated from DD lesions in cattle, when injected subcutaneously into mice, induced abscesses with a local recruitment of Ly6G+ neutrophils and proinflammatory (Ly6ChighCCR2+) monocytes/macrophages, which appeared at infection onset (4 days post challenge) and persisted for at least 7 days post challenge. When exploring the ability of macrophages to regulate inflammation, we showed that bovine blood-derived macrophages challenged with live T. phagedenis or its structural components secreted IL-1ß via a mechanism dependent on the NLRP3 inflammasome. This study shows that proinflammatory characteristics of monocytes/macrophages and neutrophils dominate active non-healing ulcerative lesions in active DD, thus likely impeding wound healing after antibiotic treatment.

Adherence and metal-ion acquisition gene expression increases during infection with Treponema phagedenis strains from bovine digital dermatitis.

Digital dermatitis (DD) is an ulcerative foot lesion on the heel bulbs of dairy cattle. DD is a polymicrobial disease with no precise etiology, although Treponema spirochetes are found disproportionally abundant in diseased tissue. Within Treponema, several different species are found in DD; however, the species Treponema phagedenis is uniformly found in copious quantities and deep within the skin layers of the active, ulcerative stages of disease. The pathogenic mechanisms these bacteria use to persist in the skin and the precise role they play in the pathology of DD are widely unknown. To explore the pathogenesis and virulence of Treponema phagedenis, newly isolated strains of this species were investigated in a subcutaneous murine abscess model. In the first trial, a dosage study was conducted to compare the pathogenicity of different strains across three different treponemes per inoculum (TPI) doses based on abscess volumes. In the second trial, the expression levels of 11 putative virulence genes were obtained to gain insight into their involvement in pathogenesis. During the RT-qPCR analysis, it was determined that genes encoding for two metal-ion import lipoproteins and two adherence genes were found highly upregulated during infection. Conversely, two genes involved in motility and chemotaxis were found to not be significantly upregulated or utilized during infection. These results were supported by gene expression data from natural M2 lesions of dairy cattle. This gene expression analysis could highlight the preference in strategy for T. phagedenis to persist and adhere in the host rather than engage in motility and disseminate.

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

An autosomal recessive variant in PYGM causes myophosphorylase deficiency in Red Angus composite cattle.

BACKGROUND: Between 2020 and 2022, eight calves in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) displayed exercise intolerance during forced activity. In some cases, the calves collapsed and did not recover. Available sire pedigrees contained a paternal ancestor within 2-4 generations in all affected calves. Pedigrees of the calves' dams were unavailable, however, the cows were ranch-raised and retained from prior breeding seasons, where bulls used for breeding occasionally had a common ancestor. Therefore, it was hypothesized that a de novo autosomal recessive variant was causative of exercise intolerance in these calves. RESULTS: A genome-wide association analysis utilizing SNP data from 6 affected calves and 715 herd mates, followed by whole-genome sequencing of 2 affected calves led to the identification of a variant in the gene PYGM (BTA29:g.42989581G > A). The variant, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon (p.Arg650*). The protein product of PYGM, myophosphorylase, breaks down glycogen in skeletal muscle. Glycogen concentrations were fluorometrically assayed as glucose residues demonstrating significantly elevated glycogen concentrations in affected calves compared to cattle carrying the variant and to wild-type controls. The absence of the PYGM protein product in skeletal muscle was confirmed by immunohistochemistry and label-free quantitative proteomics analysis; muscle degeneration was confirmed in biopsy and necropsy samples. Elevated skeletal muscle glycogen persisted after harvest, resulting in a high pH and dark-cutting beef, which is negatively perceived by consumers and results in an economic loss to the industry. Carriers of the variant did not exhibit differences in meat quality or any measures of animal well-being. CONCLUSIONS: Myophosphorylase deficiency poses welfare concerns for affected animals and negatively impacts the final product. The association of the recessive genotype with dark-cutting beef further demonstrates the importance of genetics to not only animal health but to the quality of their product. Although cattle heterozygous for the variant may not immediately affect the beef industry, identifying carriers will enable selection and breeding strategies to prevent the production of affected calves.

Metabolomics and proteomics insights into subacute ruminal acidosis etiology and inhibition of proliferation of yak rumen epithelial cells in vitro.

BACKGROUND: Untargeted metabolomics and proteomics were employed to investigate the intracellular response of yak rumen epithelial cells (YRECs) to conditions mimicking subacute rumen acidosis (SARA) etiology, including exposure to short-chain fatty acids (SCFA), low pH5.5 (Acid), and lipopolysaccharide (LPS) exposure for 24 h. RESULTS: These treatments significantly altered the cellular morphology of YRECs. Metabolomic analysis identified significant perturbations with SCFA, Acid and LPS treatment affecting 259, 245 and 196 metabolites (VIP > 1, P < 0.05, and fold change (FC) ≥ 1.5 or FC ≤ 0.667). Proteomic analysis revealed that treatment with SCFA, Acid, and LPS resulted in differential expression of 1251, 1396, and 242 proteins, respectively (FC ≥ 1.2 or ≤ 0.83, P < 0.05, FDR < 1%). Treatment with SCFA induced elevated levels of metabolites involved in purine metabolism, glutathione metabolism, and arginine biosynthesis, and dysregulated proteins associated with actin cytoskeleton organization and ribosome pathways. Furthermore, SCFA reduced the number, morphology, and functionality of mitochondria, leading to oxidative damage and inhibition of cell survival. Gene expression analysis revealed a decrease the genes expression of the cytoskeleton and cell cycle, while the genes expression associated with inflammation and autophagy increased (P < 0.05). Acid exposure altered metabolites related to purine metabolism, and affected proteins associated with complement and coagulation cascades and RNA degradation. Acid also leads to mitochondrial dysfunction, alterations in mitochondrial integrity, and reduced ATP generation. It also causes actin filaments to change from filamentous to punctate, affecting cellular cytoskeletal function, and increases inflammation-related molecules, indicating the promotion of inflammatory responses and cellular damage (P < 0.05). LPS treatment induced differential expression of proteins involved in the TNF signaling pathway and cytokine-cytokine receptor interaction, accompanied by alterations in metabolites associated with arachidonic acid metabolism and MAPK signaling (P < 0.05). The inflammatory response and activation of signaling pathways induced by LPS treatment were also confirmed through protein interaction network analysis. The integrated analysis reveals co-enrichment of proteins and metabolites in cellular signaling and metabolic pathways. CONCLUSIONS: In summary, this study contributes to a comprehensive understanding of the detrimental effects of SARA-associated factors on YRECs, elucidating their molecular mechanisms and providing potential therapeutic targets for mitigating SARA.

Crimean-Congo Hemorrhagic Fever Virus in Ticks Collected from Cattle, Corsica, France, 2023.

We report the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) in Corsica, France. We identified CCHFV African genotype I in ticks collected from cattle at 2 different sites in southeastern and central-western Corsica, indicating an established CCHFV circulation. Healthcare professionals and at-risk groups should be alerted to CCHFV circulation in Corsica.

Deforestation and Bovine Rabies Outbreaks in Costa Rica, 1985-2020.

In Latin America, rabies virus has persisted in a cycle between Desmodus rotundus vampire bats and cattle, potentially enhanced by deforestation. We modeled bovine rabies virus outbreaks in Costa Rica relative to land-use indicators and found spatial-temporal relationships among rabies virus outbreaks with deforestation as a predictor.

Avian Influenza A(H5N1) Virus among Dairy Cattle, Texas, USA.

During March and April 2024, we studied dairy cattle specimens from a single farm in Texas, USA, using multiple molecular, cell culture, and next-generation sequencing pathogen detection techniques. Here, we report evidence that highly pathogenic avian influenza A(H5N1) virus strains of clade 2.3.4.4b were the sole cause of this epizootic.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024.

We report highly pathogenic avian influenza A(H5N1) virus in dairy cattle and cats in Kansas and Texas, United States, which reflects the continued spread of clade 2.3.4.4b viruses that entered the country in late 2021. Infected cattle experienced nonspecific illness, reduced feed intake and rumination, and an abrupt drop in milk production, but fatal systemic influenza infection developed in domestic cats fed raw (unpasteurized) colostrum and milk from affected cows. Cow-to-cow transmission appears to have occurred because infections were observed in cattle on Michigan, Idaho, and Ohio farms where avian influenza virus-infected cows were transported. Although the US Food and Drug Administration has indicated the commercial milk supply remains safe, the detection of influenza virus in unpasteurized bovine milk is a concern because of potential cross-species transmission. Continued surveillance of highly pathogenic avian influenza viruses in domestic production animals is needed to prevent cross-species and mammal-to-mammal transmission.

Rift Valley Fever Epizootic, Rwanda, 2022.

A Rift Valley fever epizootic affected livestock in Rwanda during March-October 2022. We confirmed 3,112 infections with the virus, including 1,342 cases, 1,254 abortions, and 516 deaths among cattle, goats, and sheep. We recommend a One Health strategy for investigations and response to protect animal and human health.

Group B Streptococcus Sequence Type 103 as Human and Bovine Pathogen, Brazil.

Group B Streptococcus sequence type 103 is known primarily as a bovine mastitis pathogen. In Brazil, it has circulated in cattle and humans since the 1990s. It lacks scpB and, in humans, was found only among carriage isolates. Bovine-human interspecies transmission may have contributed to its evolution and spread.

Outbreak of Intermediate Species Leptospira venezuelensis Spread by Rodents to Cows and Humans in L. interrogans-Endemic Region, Venezuela.

Leptospirosis is a common but underdiagnosed zoonosis. We conducted a 1-year prospective study in La Guaira State, Venezuela, analyzing 71 hospitalized patients who had possible leptospirosis and sampling local rodents and dairy cows. Leptospira rrs gene PCR test results were positive in blood or urine samples from 37/71 patients. Leptospira spp. were isolated from cultured blood or urine samples of 36/71 patients; 29 had L. interrogans, 3 L. noguchii, and 4 L. venezuelensis. Conjunctival suffusion was the most distinguishing clinical sign, many patients had liver involvement, and 8/30 patients with L. interrogans infections died. The Leptospira spp. found in humans were also isolated from local rodents; L. interrogans and L. venezuelensis were isolated from cows on a nearby, rodent-infested farm. Phylogenetic clustering of L. venezuelensis isolates suggested a recently expanded outbreak strain spread by rodents. Increased awareness of leptospirosis prevalence and rapid diagnostic tests are needed to improve patient outcomes.