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Mastitis remains the most prevalent and costly disease to dairy producers. Granulocytes are the primary host innate immune cell responders during infectious mastitis. Here we examine three mid-lactation Holsteins challenged with ~ 150 CFU of Staphylococcus aureus (Newbould) that developed chronic mastitis as assessed by bacteria and somatic cell counts in a single quarter. Single-cell RNA-sequencing (scRNA-seq) of blood and milk cells identified immune cell populations of interest from both tissues, and the proportion of cell types recovered via scRNA-seq were highly similar to those recovered via flow cytometry. Granulocytes were the predominating cell type in both blood and milk samples; however granulocytes identified via scRNA-seq revealed several clusters comprised primarily of milk-derived cells. Milk-enriched granulocyte clusters were further investigated to identify gene signatures indicative of the granulocyte-specific localized immune responses in the mammary gland during chronic mastitis infection. Biological process enrichment analysis of gene signatures further revealed relevant networks such as granulocyte migration, myeloid cell differentiation, and inflammatory responses. In total, the work describes the immune landscape occurring at both peripheral and local sites of cattle with mastitis and identified important granulocyte-specific features of the localized immune response occurring during chronic infection.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a health risk for humans and other domestic and wildlife species. Recently, North American elk have been identified as seropositive for SARS-CoV-2, thus posing a potential threat to humans and other mammals. In this work, we characterized the peripheral transcriptomic response to experimental SARS-CoV-2 infection in calves and adult elk at different time points. Results Significantly differentially expressed genes were identified at 2-, 5-, and 14-days post inoculation (pi) for both age groups. Adult elk presented the greatest number of differentially expressed (DE) genes at all time points, including many genes associated with viral response, immune activation, antibody production, as well as genes associated with coronavirus disease (COVID-19), and coronavirus GO terms and KEGG pathways. Calves presented DE genes associated with viral responses at 5 days pi as well as neurodegenerative-associated genes at 14 days pi. Both adults and calves showed predicted activation of the ISGF3 and IFN type I pathways at day 2 pi and, globally, increased activity related to the coronavirus pathway disease at 5 and 14 days pi. Conclusions Collectively, this work provides valuable data characterizing the cervid immune response of elk to viral diseases as well as the response of wildlife to SARS-CoV-2 infection.

The COVID-19 pandemic represents one of the most significant public health events of the last century. As with other coronaviruses (SARS, MERS) the role of animals is of intense interest. Believed to have originated in bats, the role of other animals in the epidemiology of the SARS-CoV-2 pandemic is still unclear, as is the range of susceptible hosts. American bison were intranasally infected with SARS-CoV-2 and monitored for seroconversion and the presence of viral RNA in oronasal secretions and feces. Although clinical signs were not seen, permissiveness of bison to infection with SARS-CoV-2 was manifest by seroconversion, the presence of viral RNA in oronasal secretions, persistence of viral RNA in lymphoid tissue, and viral associated interstitial pneumonia. Retrospective sequencing of the inoculum revealed a common in vitro adaptation in the furin cleavage site of the spike protein that may have reduced in vivo viral fitness. As such, we cannot exclude the possibility that use of an isolate with an intact furin cleavage motif would more efficiently infect bison.

Leptospirosis is a global zoonotic disease affecting humans, wildlife, companion, and domestic animals. Incidental hosts can contract the disease directly or indirectly from asymptomatic reservoir hosts, most commonly small rodents. The Golden Syrian hamster is recognized as the dominant rodent model for acute leptospirosis because the animals are susceptible to many serovars and are used to maintain laboratory strains and test bacterin vaccine efficacy. However, hamsters are primarily used in survival-based studies, and investigations into host immune response and disease pathogenesis are limited. We found that Peromyscus leucopus white-footed deer mice are susceptible to acute leptospirosis, and thus might be an alternative rodent model. Furthermore, similar to hamsters, deer mice produce circulating foamy macrophages in response to Leptospira challenge. Deer mice exhibit differences in response to different serovars, clinical disease severity, kidney and liver lesions, and an overall sex effect, with male mice demonstrating more severe clinical signs and higher bacterial burden.

Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira , allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

IntroductionBrucella abortus and Mycobacterium bovis , the causative agents of bovine brucellosis and tuberculosis respectively, are zoonotic bacterial pathogens that both contribute to major economic losses in the cattle industry and pose a human health risk worldwide. Co-infections of cattle with B. abortus and M. bovis have been identified in various developing countries, necessitating the development of an efficacious strategy for controlling both important zoonotic diseases even in the event of co-infection. Brucella abortus strain RB51, a live attenuated vaccine for bovine brucellosis that is currently used in the US, is highly effective at preventing reproductive failure due to brucellosis in cattle. Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine strain of M. bovis that provides protection against bovine tuberculosis in cattle but is not currently licensed for use in the US.MethodsThe study presented here compares functional Th1 responses of RB51 + BCG vaccinated cattle to responses of RB51-only and BCG-only vaccinated cattle to evaluate the feasibility of a combined vaccination strategy for controlling both bovine brucellosis and tuberculosis.ResultsThis work identified that peripheral blood mononuclear cells (PBMC) from RB51 vaccinates proliferate not only in response to stimulation with killed RB51 but also in response to mycobacterial antigen PPDb. Combination vaccinates show significantly more CD4+ T cell proliferation than single BCG vaccinates when stimulated with PPDb, while no differences were observed between RB51 and combination vaccinates stimulated with RB51.Discussion/conclusionSignificantly enhanced BCG-specific Th1 responses in combination vaccinates compared to BCG-only vaccinates suggest that combining vaccinations for B. abortus and M. bovis may alter the host CD4+ T cell response.

In dairy cows, the period from the end of lactation through the dry period and into the transition period, requires vast physiological and immunological changes critical to mammary health. The dry period is important to the success of the next lactation and intramammary infections during the dry period will adversely alter mammary function, health and milk production for the subsequent lactation. MicroRNAs (miRNAs) are small non-coding RNAs that can post transcriptionally regulate gene expression. We sought to characterize the miRNA profile in dry secretions from the last day of lactation to 3, 10, and 21 days post dry-off. We identified 816 known and 80 novel miRNAs. We found 46 miRNAs whose expression significantly changed (q-value < 0.05) over the first three weeks of dry-off. Additionally, we examined the slopes of random regression models of log transformed normalized counts and cross analyzed the 46 significantly upregulated and downregulated miRNAs. These miRNAs were found to be associated with important components of pregnancy, lactation, as well as inflammation and disease. Detailing the miRNA profile of dry secretions through the dry-off period provides insight into the biology at work, possible means of regulation, components of resistance and/or susceptibility, and outlets for targeted therapy development.

Background Mycobacterium bovis BCG is the human tuberculosis vaccine and is the oldest vaccine still in use today with over 4 billion people vaccinated since 1921. The BCG vaccine has also been investigated experimentally in cattle and wildlife by various routes including oral and parenteral. Thus far, oral vaccination studies of cattle have involved liquid BCG or liquid BCG incorporated into a lipid matrix. Lyophilization is an established technique used for stabilizing bioproducts such as vaccines. Methods In the current study, cattle were vaccinated in two phases. In each phase, cattle were divided into three groups. Group 1 received BCG injected SQ, Group 2 received liquid BCG delivered to the posterior oral cavity, Group 3 orally consumed lyophilized BCG contained within a gelatin capsule placed within a small amount of a commercial alfalfa product. Results No vaccinated cattle were positive by an interferon gamma release assay. All but 4 animals were negative by tuberculin skin testing prior to vaccination: the 4 non-negative animals being categorized as suspects. Sixteen weeks post-vaccination all but 1 animal was negative, it being categorized as a suspect. An in vitro antigen stimulation assay and flow cytometry were used to detect antigen-specific CD4, CD8 and γδ T cell responses following vaccination. Oral vaccination of animals with lyophilized BCG did not result in any increases in the frequency of CD4, CD8 or γδ T cell proliferative or IFN-γ responses at any of the time points analyzed in either phase 1 or 2. In contrast, vaccination with BCG SQ and liquid BCG delivered to the posterior pharynx, resulted in an increase in the frequency of proliferating and IFN-γ-producing CD4 T cells with peak responses at 9–12 weeks post-vaccination. Similar to oral lyophilized BCG vaccinated animals, we did not observe any significant increases in the frequency of CD8 and γδ T cell proliferative and IFN-γ responses following SQ or oral liquid vaccinated animals. Conclusions These data would suggest that vaccination with oral lyophilized BCG does not induce a measurable, antigen-specific cell mediated responses in the periphery, when compared to BCG administered SQ or liquid BCG administered via the oral route. However, vaccination with either SQ or liquid BCG delivered to the posterior pharynx does induce measurable CD4 T cell responses in the periphery.

Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.

Background Mastitis is the most common health concern plaguing the modern dairy cow and costs dairy producers estimates of two billion dollars annually. Staphylococcus aureus infections are prevalent, displaying varied disease presentation and markedly low cure rates. Neutrophils are considered the first line of defense against mastitis causing bacteria and are frequently targeted in the development of treatment and prevention technologies. We describe a case of naturally occurring, chronic mastitis in a Holstein cow (1428), caused by a novel strain of S. aureus that was not able to be cleared by antibiotic treatment. Case presentation The infection was identified in a single quarter, 2 months into the cow’s first lactation. The infection persisted for the following 20 months, including through dry off, and a second calving and lactation. This case of mastitis was associated with a consistently high somatic cell count, however presented with no other clinical signs. This cow was unsuccessfully treated with antibiotics commonly used to treat mastitis, consisting of two rounds of treatment during lactation and an additional round at the beginning of dry off. The chronic infection was also unchanged through an experimental mid-lactation treatment with pegylated granulocyte-colony stimulating factor (PEG-gCSF) and an additional periparturient treatment with PEG-gCSF. We isolated milk neutrophils from 1428 and compared them to two cows challenged with experimental S. aureus, strain Newbould 305. Neutrophils from 1428’s milk had higher surface expression of myeloperoxidase compared to experimental Newbould challenged animals, as well as increased presence of Neutrophil Extracellular Traps. This suggests a heightened activation state of neutrophils sourced from 1428’s naturally occurring infection. Upon postmortem examination, the affected quarter revealed multifocal abscesses separated by fibrous connective tissues. Abscesses were most common in the gland cistern and collecting duct region. Microscopically, the inflammatory reaction was pyogranulomatous to granulomatous and consistent with botryomycosis. Colonies of Gram-positive cocci were found within the eosinophilic matrix of the Splendore-Hoeppli reaction within granulomas and intracellularly within the acinar epithelium. Conclusions Collectively, we describe a unique case of chronic mastitis, the characterization of which provides valuable insight into the mechanics of S. aureus treatment resistance and immune escape.