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Understanding the pulmonary adaptive immune system of pigs is important as respiratory pathogens present a major challenge for swine producers and pigs are increasingly used to model human pulmonary diseases. Single-cell RNA sequencing (scRNAseq) has accelerated the characterization of cellular phenotypes in the pig respiratory tract under both healthy and diseased conditions. However, combining scRNAseq with recovery of paired T cell receptor (TCR) α and β chains as well as B cell receptor (BCR) heavy and light chains to interrogate their repertoires has not to our knowledge been demonstrated for pigs. Here, we developed primers to enrich porcine TCR α and β chains along with BCR κ and λ light chains and IgM, IgA, and IgG heavy chains that are compatible with the 10x Genomics VDJ sequencing protocol. Using these pig-specific assays, we sequenced the T and B cell receptors of cryopreserved lung cells from CD1D -expressing and -deficient pigs after one or two infections with influenza A virus (IAV) to examine whether natural killer T (NKT) cells alter pulmonary TCR and BCR repertoire selection. We also performed paired single-cell RNA and receptor sequencing of FACS-sorted T cells longitudinally sampled from the lungs of IAV-vaccinated and -infected pigs to track clonal expansion in response to IAV exposure. All pigs presented highly diverse repertoires. Pigs re-exposed to influenza antigens from either vaccination or infection exhibited higher numbers of expanded CD4 and CD8 T cell clonotypes with activated phenotypes, suggesting potential IAV reactive T cell populations. Our results demonstrate the utility of high throughput single-cell TCR and BCR sequencing in pigs., Competing Interests: Competing Interest Statement: Authors declare that they have no competing interests.

Influenza virus infections are a major cause of respiratory disease in humans. Neuraminidase inhibitors (NAIs) are the primary antiviral medication used to treat ongoing influenza infections. However, NAIs are not always effective for controlling virus shedding and lung inflammation. Other concerns are the emergence of NAI-resistant virus strains and the risk of side effects, which are occasionally severe. Consequently, additional anti-influenza therapies to replace or combine with NAIs are desirable. Here, we compared the efficacy of the NAI oseltamivir with the invariant natural killer T (iNKT) cell superagonist, α-galactosylceramide (α-GalCer), which induces innate immune responses that inhibit influenza virus replication in mouse models. We show that oseltamivir reduced lung lesions and lowered virus titers in the upper respiratory tract of pigs infected with A/California/04/2009 (CA04) pandemic H1N1pdm09. It also reduced virus transmission to influenza-naïve contact pigs. In contrast, α-GalCer had no impact on virus replication, lung disease, or virus transmission, even when used in combination with oseltamivir. This is significant as iNKT-cell therapy has been studied as an approach for treating humans with influenza., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Madrid, Gu, Artiaga, Yang, Loeb, Hawkins, Castleman, Lednicky, Richt and Driver.)

Many aspects of the porcine immune system remain poorly characterized, which poses a barrier to improving swine health and utilizing pigs as preclinical models. Here, we employ single-cell RNA sequencing (scRNA-seq) to create a cell atlas of the early-adolescent pig thymus. Our data show conserved features as well as species-specific differences in cell states and cell types compared with human thymocytes. We also describe several unconventional T cell types with gene expression profiles associated with innate effector functions. This includes a cell census of more than 11,000 differentiating invariant natural killer T (iNKT) cells, which reveals that the functional diversity of pig iNKT cells differs substantially from the iNKT0/1/2/17 subset differentiation paradigm established in mice. Our data characterize key differentiation events in porcine thymopoiesis and iNKT cell maturation and provide important insights into pig T cell development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Many aspects of the bovine immune system remain poorly characterized, which poses an obstacle to improving dairy cow health. Herein, we describe two flow cytometry panels that included antibodies against CD8α, CD4, TCR-δ, CD172α, CD14, MHCII, CD21, CD62L, and CD11b. These panels were used to characterize the phenotype of leukocyte subpopulations from the peripheral blood of 30-day old Holstein calves and Holstein cows at 260 d of gestation and calving. No leukocyte subset differences were found between the pre- and post-partum cows. However, calf leukocytes presented a higher proportion of CD3 + lymphocytes, γδ T-cells, CD8 + γδ T-cells, and monocytes when compared with mature cows. Conversely, cow lymphocytes had a higher proportion of CD4 + and CD8 + T-cells, and B-cells than calf lymphocytes. The proportion of CD4 + T-cells and B-cells expressing CD62L was greater in calves than in cows, while cow B-cells expressed greater levels of CD11b than calf B-cells. In contrast, calf polymorphonuclear cells (PMN) and monocytes expressed greater levels of CD11b compared to cows. Moreover, calf monocytes expressed higher levels of MHCII compared with those of cows. Collectively, our data provides a resource to better understand the bovine immune system as well as immune-related diseases that affect dairy cattle., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bovine trypanosomosis has been spreading in Brazil. In the present study, we evaluated the spatial distribution, prevalence and risk factors of this disease in the state of Goiás, Brazil, and performed both molecular and phylogenetical analyses of Trypanosoma vivax. A total of 4049 blood samples were collected from cattle for a period of 2 years. The parasitological diagnosis was performed using the Woo method and a questionnaire was administered to the farmers to document risk factors associated with the disease in the herd. Positive samples were DNA sequenced and compared to GenBank codes. The prevalence of T. vivax was 8.84%, occurring on 24 ranches only in dairy cattle and mainly in the central and southern portions of the state. The acquisition of new animals infected with T. vivax and the administration of exogenous oxytocin to cows using the same syringe and needle were the main associated factors (P ≤ 0.05). After an outbreak, milk production decreased by 39.62%. The presence of biting flies (tabanids, Haematobia irritans and Stomoxys calcitrans) was not a risk factor (P > 0.05) for the occurrence of T. vivax. The epidemiological data demonstrate the importance of restricting the practice of auctions as well as eliminating the use of exogenous oxytocin in animals during milking. The samples tested by polymerase chain reaction were positive for T. vivax and were genetically homologous with T. vivax found in different states of Brazil and west Africa based on the 18S rRNA gene.

The aim of the present study was to compare the infection capacity of Trypanosoma vivax experimentally inoculated through different routes in calves naturally infected with latent Anaplasma marginale. On Day 0 of the study, 25 calves (breed: Girolando) were divided into five groups. The first four groups of five calves each received approximately 1 × 10 6 trypomastigotes of T. vivax through the intradermal, subcutaneous, intramuscular and intravenous routes. Another five animals remained unaffected to serve as A. marginale naturally infected controls. The study of T. vivax was performed on all calves from D+1 to D+30 using the Woo, Brener and blood smear techniques. PCR was performed on Days +1, +3, +4, +5, +28, +29 and + 30. The results indicated that T. vivax was capable of infecting and developing the disease in the calves independent of the inoculation route. A positive correlation was found between T. vivax and rectal temperature (P ≤ 0.05) and a negative correlation was seen between the protozoan and globular volume (P ≤ 0.05). Latent A. marginale in the calves acted as co-infection for T. vivax. Jaundice was seen only in calves with a high parasitemia by A. marginale. Therefore, in areas with the confirmed presence of T. vivax in bovines, this protozoan should be included in the complex denominated "Bovine Parasitic Sadness", which currently encompasses only Anaplasma and Babesia., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Trypanosomiasis caused by Trypanosoma vivax has increased the reports in Brazil in the last decade. An outbreak is herein first reported in the state of Goiás, from May 2016 to January 2017. The outbreak start occurred in the city of Ipameri (Goiás) after the introduction of 18 auctioned cows from the state of Minas Gerais. Direct parasitological test (blood smears) and polymerase chain reactions targeting the catL genes diagnosed T. vivax infection. Fifty six cows from a herd of 161 were infected; 12 died during the outbreak and 44 animals persistently positive (by blood smears) even after chemical treatment were discarded. After this first case, five other cases were detected in state of Goiás. The spread of this disease can be linked to the commercialization of animals carrying T. vivax, allied to the iatrogenic transmission practice, using a single needle and syringe for all cows, during oxytocin administration before each milking.