Crab-eating fox (Cerdocyon thous), a South American canid, as a definitive host for Hammondia heydorni

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15 mu m, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70 kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. (C) 2009 Elsevier B.V. All rights reserved

Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice

<div><p>Malaria remains one of the greatest burdens to global health, causing nearly 500,000 deaths in 2014. When manifesting in the lungs, severe malaria causes acute lung injury/acute respiratory distress syndrome (ALI/ARDS). We have previously shown that a proportion of DBA/2 mice infected with <i>Plasmodium berghei</i> ANKA (PbA) develop ALI/ARDS and that these mice recapitulate various aspects of the human syndrome, such as pulmonary edema, hemorrhaging, pleural effusion and hypoxemia. Herein, we investigated the role of neutrophils in the pathogenesis of malaria-associated ALI/ARDS. Mice developing ALI/ARDS showed greater neutrophil accumulation in the lungs compared with mice that did not develop pulmonary complications. In addition, mice with ALI/ARDS produced more neutrophil-attracting chemokines, myeloperoxidase and reactive oxygen species. We also observed that the parasites <i>Plasmodium falciparum</i> and PbA induced the formation of neutrophil extracellular traps (NETs) <i>ex vivo</i>, which were associated with inflammation and tissue injury. The depletion of neutrophils, treatment with AMD3100 (a CXCR4 antagonist), Pulmozyme (human recombinant DNase) or Sivelestat (inhibitor of neutrophil elastase) decreased the development of malaria-associated ALI/ARDS and significantly increased mouse survival. This study implicates neutrophils and NETs in the genesis of experimentally induced malaria-associated ALI/ARDS and proposes a new therapeutic approach to improve the prognosis of severe malaria.</p></div

<i>Plasmodium berghei</i>-iRBCs promote more NETs than RBCs in mouse neutrophils.

<p>(A) Immunofluorescence of non-stimulated (NS) DBA/2 mouse neutrophils and of neutrophils stimulated with PMA [positive control (PC)], red blood cells (RBCs), and <i>P</i>. <i>berghei</i> ANKA (iRBCs) stained with Sytox Green (630x, scale bar 20 μm). (B) Quantification of extracellular DNA (NETs) using Sytox Green based on units of arbitrary fluorescence (UAF). Measurements were performed after 60, 120 and 180 minutes of stimulation. The data are representative of three independent experiments with mean ± SEM (Kruskal-Wallis test where *** p <0.001, between iRBC and RBC).</p

Neutrophil interactions in malaria-associated ALI/ARDS.

<p>Following <i>P</i>. <i>berghei</i> ANKA infection in DBA/2 mice, neutrophils promote the pathogenesis of ALI/ARDS. In particular, the release of myeloperoxidase and reactive species of oxygen and the formation of neutrophil extracellular traps determines the cause of death of these mice.</p

CXCR4 antagonist treatment protects DBA/2 mice from ALI/ARDS.

<p>(A) Survival and (B) parasitemia curves in <i>P</i>. <i>berghei</i> ANKA-infected mice treated with 5 mg/kg of AMD3100 or DMSO + saline solution (control group) on the 1^st, 3^rd, 5^th and 7^th days post-infection (dpi). (C) Lung tissues from untreated mice (CTR) (died on the 9^th dpi with ALI/ARDS) and AMD3100-treated mice (died on the 21^st dpi due to hyperparasitemia) (400x, scale bar 25 μm). (D) Number of neutrophils in the lungs, measured via flow cytometry, from infected AMD3100-treated mice and control mice (CTR). (E) Enhanced respiratory pause (Penh) and (F) respiratory frequency (RF) data for these mice. Data are representative of three independent experiments and are expressed as the mean ± SEM. The data from (D) to (F) were collected on the 7^th dpi. (A, log-rank test and Wilcoxon-Gehan-Breslow test, p<0.05; Mann Whitey-test, *p<0.05, ** p<0.01 and *** p<0.001; n = 10–15 mice/experiment). The dashed line represents the mean value for the non-infected mice (NI).</p

Early depletion of neutrophils protects DBA/2 mice from ALI/ARDS.

<p>(A) Survival and (B) parasitemia curves in <i>P</i>. <i>berghei</i> ANKA-infected mice treated with 0.2 mg/kg of anti-GR1 or IgG antibodies (control group) on the 1^st day post-infection (dpi). Respiratory parameters (C) enhanced pause (Penh) and (D) respiratory frequency on the 7^th dpi. (E) Photomicrographs of lung tissue on the day of death from non-infected mice, control mice (8^th dpi) and anti-GR1 treated mice (18^th dpi) (400x, scale bar 25 μm). Number/frequency of neutrophils in the blood from infected control (black) and anti-GR1-treated (gray) mice measured via (F) flow cytometry and (G) an analysis of the blood smears. Data are representative of three independent experiments and are expressed as the mean ± SEM (A, log-rank test and Wilcoxon-Gehan-Breslow test, p<0.05; Mann Whitey-test, * p <0.05, ** p <0.01 and *** p <0.001; n = 15–20 mice/experiment). The dashed line represents the mean value for the non-infected mice (NI).</p