Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV " and FeLV + asymptomatic cats compared with FeLV + symptomatic cats.Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV + symptomatic cats were higher than those in unstimulated neutrophils from FeLV " and FeLV + asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV + cats and that this feature could be considered a marker of disease progression in FeLV infection.

Visceral leishmaniasis is a chronic disease that affects humans and dogs as well. Dogs, the domestic reservoir of Leishmania , play a central role in the transmission of visceral leishmaniasis, the most severe form of this disease. Neutrophils are the most abundant leukocytes in blood and interact with the parasite after infection. Here, we evaluate the effector properties of neutrophils from healthy and naturally Leishmania infantum- infected dogs. Our results showed that the parasite induced neutrophil extracellular trap (NET) release from neutrophils in both groups. Additionally, phagocytosis and NETs contributed differently to parasite killing by neutrophils from healthy and infected animals, and IFN-γ, IL-8, IL-4 and TNF-α production by neutrophils from both groups were differentially modulated by the parasite. Our results contribute to a better understanding of the complex role played by neutrophils in canine visceral leishmaniasis, which may favor the development of more effective therapies.

Up to 50% of patients with the multibacillary form of leprosy are expected to develop acute systemic inflammatory episodes known as type 2 reactions (T2R), thus aggravating their clinical status. Thalidomide rapidly improves T2R symptoms. But, due to its restricted use worldwide, novel alternative therapies are urgently needed. The T2R triggering mechanisms and immune-inflammatory pathways involved in its pathology remain ill defined. In a recent report, we defined the recognition of nucleic acids by TLR9 as a major innate immunity pathway that is activated during T2R. DNA recognition has been described as a major inflammatory pathway in several autoimmune diseases, and neutrophil DNA extracellular traps (NETs) have been shown to be a prime source of endogenous DNA. Considering that neutrophil abundance is a marked characteristic of T2R lesions, the objective of this study was to investigate NETs production in T2R patients based on the hypothesis that the excessive NETs formation would play a major role in T2R pathogenesis. Abundant NETs were found in T2R skin lesions, and increased spontaneous NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression was shown to be higher in T2R neutrophils, suggesting that DNA recognition via TLR9 may be one of the pathways triggering this process during T2R. Finally, treatment of T2R patients with thalidomide for 7 consecutive days resulted in a decrease in all of the evaluated in vivo and ex vivo NETosis parameters. Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, will contribute to the emergence of novel alternative therapies and the identification of prognostic reactional markers in the near future.

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25Up to 50% of patients with the multibacillary form of leprosy is expected to 26 develop acute systemic inflammatory episodes known as type 2 reactions (T2R), 27 thus aggravating their clinical status. Thalidomide rapidly improves T2R 28 symptoms. But, due to its restricted use worldwide, novel alternative therapies 29 are urgently needed. A hallmark of T2R lesions is the presence of a neutrophil-30 rich inflammatory infiltrate. In this study, the potential involvement of neutrophil 31 extracellular traps (NETs) production in T2R pathogenesis was investigated. 32Abundant NETs were found in T2R skin lesions, and increased spontaneous 33NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae 34 whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 35 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression 36 was shown to be higher in T2R neutrophils, suggesting that DNA recognition via 37 TLR9 may be one of the pathways triggering this process during T2R. Finally, 38 treatment of T2R patients with thalidomide for 7 consecutive days resulted in 39 decreasing all of the evaluated in vivo and ex vivo NETosis parameters. 40Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, 41 will contribute to the emergence of novel alternative therapies in the near future. 42 43 44 45 46 3 Author summary 47 48Leprosy is caused by a mycobacterium that has a predilection for skin and nerve 49 cells. The chronic course of the disease may be interrupted by acute 50 inflammatory episodes known as reactions, despite effective bacterial killing with 51 antibiotics. Reactions aggravate patient's clinical status and may become a 52 medical emergency. Type 2 reactions (T2R) only occur in patients with high 53 bacterial burden and is treated with thalidomide and/or corticosteroids. We are 54 interested in understanding how inflammation is triggered and amplified during 55 T2R. In this study we investigated the potential role of extracellular DNA released 56 by neutrophils (known as NETs) in T2R, since they have been shown to cause 57 inflammation. Abundant NETs were found in T2R skin lesions, and increased 58 spontaneous NETs formation was observed in neutrophils present in the blood of 59 T2R patients. Moreover, bacterial constituents were able to induce NETs 60 production. Finally, treatment of T2R patients with thalidomide resulted in 61 decreased NET formation. Altogether, our findings shed light on the 62 pathogenesis of T2R, which, it is hoped, will contribute to the identification of 63 biomarkers for early diagnosis and emergence of novel alternative therapies in 64 the near future. 65 66 67 68 69

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Plague is a flea-borne zoonosis that affects a wide range of mammals and still causes outbreaks in human populations yearly across several countries. While crucial for proper treatment, early diagnosis is still a major challenge in low- and middle-income countries due to poor access to laboratory infrastructure in rural areas. To tackle this issue, we developed and evaluated a new F1-based rapid diagnostic test (RDT) as an alternative method for plague diagnosis in humans and other mammals in the field. In this study, 187 serum samples from humans, dogs, rodents and rabbits were retrospectively assessed using the Plague RDT method. To calculate its performance rates, results were confronted to those obtained by hemagglutination (HA) and ELISA, considered as the reference standards. Remarkably, the results from RDT were in full agreement with those from the ELISA and HA assays, resulting in 100% (CI 95% = 95.5-100%) of sensitivity and 100% (CI 95% = 96.6-100%) of specificity. Accordingly, the Cohens Kappa test coefficient was 1.00 (almost perfect agreement). Moreover, the RDT showed no cross-reaction when tested with sera from individuals positive to other pathogens, such as Yersinia pseudotuberculosis, Yersinia enterocolitica, Anaplasma platys, Erliquia canis and Leishmania infantum. Although preliminary, this study brings consistent proof-of-concept results with high performance rates of the Plague RDT when compared to other methods well-established in the plague routine serodiagnosis. Although further human and animal population-based studies will be necessary to validate these findings, the data presented here show that the Plague RDT is highly sensitive and specific, polyvalent to several mammal species and simple to use in field surveillance or point-of-care situations with instant results.