Inducible nitric oxide synthase expression in Leishmania-infected dog macrophages

Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-γ and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes, zymodeme MON1. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab′)₂ goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS–PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-γ and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-γ and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-γ and LPS in NOS2 induction also in this animal model.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

Induction of inflammatory host immune responses by organisms belonging to the genera Chlamydia/Chlamydophila

Chlamydia/Chlamydophila are a family of intracellular gram-negative bacteria that infect their hosts primarily via mucosal epithelia. Chronic disease associated with bacterial persistence, inflammation and tissue damage are common sequelae of infection with these organisms. Human epithelial cell lines respond to infection by releasing pro-inflammatory cytokines and chemokines such as interleukin (IL)-6 and IL-8, and upregulating the expression of mRNA encoding Iκ-Bα, the endogenous inhibitor of NF-κB. However, Iκ-Bα is not upregulated in response to bacterial lipopolysaccharide (LPS). The failure of epithelial cells to respond to LPS is associated with the absence of surface expression of CD14. Identification of the components of Chlamydia/Chlamydophila that can induce pro-inflammatory mediators coupled with the mechanisms by which epithelial cells detect infection and respond accordingly will advance the development of preventative strategies.     

布鲁氏菌LPS对小鼠巨噬细胞中NLRP3炎症小体转录水平的影响

为研究布鲁氏菌LPS对巨噬细胞中NLRP3炎症小体的影响,本试验提取布鲁氏菌2308、RB51和△WbkA的LPS,以不同浓度与小鼠巨噬细胞相互作用,荧光定量PCR检测其对NLRP3、ASC、Caspase1、IL1-β、IL18转录水平的影响。结果显示RB51LPS和△WbkA LPS上调NLRP3炎症小体相关基因的转录水平,且呈浓度依赖性,而浓度对2308LPS调节NLRP3炎症小体相关基因转录的作用不大;且同一浓度下,RB51LPS和2308LPS比△WbkA LPS更好的调节Caspase1、IL1-β、IL18的转录水平。     

Infection of the equine population by Leishmania parasites

Infection of equids by Leishmania (L.) parasites was previously described in both the Old and New World, particularly in Central and South America. Equine cutaneous leishmaniasis (CL) is caused by the Leishmania species, L. Viannia (V.) braziliensis and L. infantum, previously identified in humans and other parasite hosts living in the same geographic endemic areas. Sporadic autochthonous clinical cases, with no travel history, were documented in several countries including Germany, Portugal, Spain, Texas and Brazil; L. infantum and L. (Mundinia) martiniquensis were the infectious species. Prevalence of subclinical infections is extremely low and CL is observed in only a small proportion of infected animals with the appearance of single or multiple cutaneous lesions located on the head, external ear, scrotum, legs and the neck. To date, there has been no report of visceral abnormalities. However, the mild clinical profile of the disease and its spontaneous regression may indicate that skin lesions related to Leishmania infection is underdiagnosed. Importantly, although the prevalence of Leishmania infections in the equine population is low, a risk may rise from its potential involvement in the parasite transmission cycles as a source of infection for phlebotomine vectors and susceptible mammalian hosts. This review article summarises our current knowledge of the epidemiology, clinical presentation and diagnosis of Leishmania-infected equids.     

Resistance to Rhipicephalus evertsi evertsi in immunosuppressed rabbits

Rabbits treated with goat anti-rabbit thymocyte serum acquired less resistance to Rhipicephalus evertsi evertsi infestation than untreated controls. This inferior resistance was manifested as higher engorged weights of ticks and higher biotic potential, undue persistence of the ticks on the hosts and poor anti-tick antibody and delayed-type hypersensitivity responses. These observations highlight the significance of host T-cells in mediating resistance of ticks.     

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.     

Effects of GnRH in combination with PGF2α on the dynamics of follicular and luteal cells in post-pubertal Holstein heifers

Holstein heifers were randomly allotted by weight, age and body condition score to one of three treatments to test the hypothesis that GnRH administration concurrent with PGF_2α injection would advance follicle or corpus luteum (CL) development parallel to an induced luteolysis of the pre-existing CL. Heifers in the control group (n = 14) received two treatments of PGF_2α(25 mg, im) given 10 days apart. Groups 2 (n = 14) and 3 (n = 14) received an additional treatment of GnRH (100 μg, im) after the first and second PGF_2α respectively. Estrus detection began immediately after PGF_2α and continued for 80 h. Blood sampling was initiated 7 days prior to the first PGF_2α (day − 7) and continued on days 0, 7, 10 (prior to the second PGF_2α), 17 and 24. Heifers were artificially inseminated after the second PGF_2α and pregnancy diagnosed at 60 days. There was a trend (P < .10) toward a lower estrus response in group 3 when compared to the other groups. Pregnant heifers in group 2 had lower progesterone (0.44 ± 0.09 vs. 1.72 ± 0.56 ng/ml) a week after the second PGF_2α than the non-pregnant animals in that group (P < .05). Similar results were observed in the control group but only within the responding heifers (0.61 ± 0.08 vs. 0.93 ± 0.03 ng/ml; P < .05). Progesterone in heifers in group 2 remained high on day 0, 7, and 10 (1.48 ± 0.37, 1.23 ± 0.39, 1.96 ± 0.36 ng/ml) in spite of the treatment with PGF_2α. This data suggest that administration of GnRH following PGF_2α alters bovine luteal and/or follicular cell function.     

Caseous Lymphadenitis in Goats: The Pathogenesis,Incubation Period and Serological Response after Experimental Infection

Twenty goats, in two groups of 10, were injected intradermally with Corynebacterium pseudotuberculosis. The doses of infection were 1×10⁵ and 5×10⁴ colony-forming units (cfu) for groups 1 and 2, respectively. Thereafter, a goat from each group was killed every 2–3 days and examined for gross and microscopic caseous lesions in the draining lymph nodes. Bands or zones of macrophages and polymorphonuclear granulocytes were observed microscopically on the second day of infection in both groups. Gross caseous lesions were observed from days 8 and 9 of infection, respectively. Positive bacterial agglutination test and haemolysis inhibition test titres were detected after 15–17 days and 20–25 days of infection, respectively. These results indicated that caseous lymphadenitis is a subacute disease with an incubation period of 8–9 days, but that it is not detectable serologically until after 15 days of infection.     

Some properties of beta toxin produced by Clostridium haemolyticum strain IRP-135

Six culture media were evaluated for the optimization of β-toxin production by Clostridium haemolyticum (strain IRP-135) using both batch and dialysis culture techniques. The lethal component of β-toxin remained active for 13 days when maintained at 37°C but was inactivated by heating at 60° for 20 min. A 1 : 10,000 dilution of trypsin inactivated the toxin in 15 min. Preliminary data from electrophoresis in SDS acrylamide gel indicate the molecular weight of the β-toxin to be approximately 32,000.     

The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood

Recent research has demonstrated the high sensitivity of real time PCR (qPCR) in the diagnosis of Leishmania infantum infection. The goal of this study was to develop and evaluate a qPCR detection system for the diagnosis of visceral leishmaniosis (VL) in dogs. Specific primer sets were developed for the Leishmania donovani complex, in which a fragment of 132 bp of kDNA from L. infantum was amplified. The reaction was performed using the ABI PRISM 7000 system with ABI PRISM software used to carry out the analysis. When canine blood samples were assessed using this system the detection limit of the method was found to be 0.07 parasites per reaction, the efficiency was 94.17% (R² = 0.93, slope = −3.47) and the sensitivity and specificity were 100% and 83.33% respectively. The use of such a sensitive, reproducible and rapid qPCR-based assay will be useful in the diagnosis and control of L. infantum infection in endemic areas, where serological surveys often underestimate true disease prevalence.     

Leishmania infantum secreted iron superoxide dismutase purification and its application to the diagnosis of canine Leishmaniasis

Leishmania spp. are digenetic parasites whose infection occurs inside the mononuclear phagocitary system. The iron superoxide dismutase secreted (Fe-SODe) by promastigotes of Leishmania spp. seems to plays an important role in the defense to environmental detoxification and neutralization of oxidative stress damage caused by reactive oxygen species (ROS) produced by macrophages during the infection. Parasites Fe-SODe is involved in establishing the infection and manifestation of Leishmaniasis. Its high immunogenicity makes it a useful molecular marker in diagnosing trypanosomatids infections. The aim of this study is demonstrate that purified Fe-SODe from Leishmania infantum is much more sensitive than un-purified Fe-SODe for diagnosis canine Leishmaniasis. We have purified a Fe-SODe of L. infantum using an ion exchange and a molecular sieve chromatographies and its application in diagnosis of canine Leishmaniasis was tested. One hundred and forty-five dogs’ sera from Andalusia Autonomous Community, Spain were tested by ELISA and Western blot and the antigen Fe-SODe purified is compared with two different antigens: the total parasites soluble lysate and the unpurified Fe-SODe. To validate the results obtained using the Fe-SODe purified we tasted 10 L. infantum infected dogs’ sera from Lombardy, Italy as positive control.     

Application of a recombinant protein for the serological diagnosis of canine leishmaniasis

This work reports the results obtained by a new enzyme-linked immunosorbent assay (ELISA) test developed for the serological diagnosis of canine leishmaniasis.The new ELISA is based on a recombinant protein obtained by joining different antigens of Leishmania infantum.Test performances have been evaluated through the screening 227 sera of dogs, infected and uninfected by L. infantum. The new ELISA test has been compared to the indirect immunofluorescent-antibody test (IFAT) as a reference assay of canine leishmaniasis, and to a commercial ELISA.Excluding from the total number of IFAT positive sera the 27 sera with IFAT titre 1:40 (considered doubtful), the recombinant ELISA showed 97.0% specificity, 93.9% sensitivity and 95.5% agreement with IFAT. The commercial ELISA showed 78.2% specificity, 94.9% sensitivity and 86.5% agreement with IFAT.The results demonstrate a higher performance of the new recombinant ELISA test for the detection of negative samples, with a greater agreement with the reference test (IFAT).     

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen

The nature of the local immune response was assessed studying the distribution of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes, IgM⁺ B cells, IL-4⁺ and IFN-γ⁺ cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-γ⁺ lymphoid cells was absent or very occasional in HLN where the number of IL-4⁺ cells was higher than that of IFN-γ, suggesting a polarized Th2 response in immunised and in infected control group.     

Pancytopenia in a cat with visceral leishmaniasis

Abstract: A 4‐year‐old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune‐mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.     

Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells

Changes in the proportion of peripheral blood T cell subsets after subcutaneous inoculation of cattle with Mycobacterium bovis Bacille Calmette-Guerin (BCG) were studied. Calves were injected with approximately 8 × 10⁶ BCG bacillus and blood samples collected at weekly intervals for flow-cytometric analyses to determine the proportion of CD4⁺, CD8⁺ and γδ T cells. In addition, whole blood samples were stimulated in vitro with M. bovis purified protein derivative (PPD) and the secreted IFN-γ quantified by ELISA. Results showed cellular and cytokine changes which could be categorized into three phases. The first phase occurred within the first 2 weeks after vaccination involving an increase in proportion of WC1⁺ γδ T cells and a concomitant increase in the secretion of IFN-γ. These two responses peaked at 2 weeks and waned thereafter. The second phase involved an increase in the CD4/CD8 ratio as a result of an increase in the proportion of CD4⁺ T cells between 4 and 6 weeks. The third phase involved a decrease in the CD4/CD8 ratio due to an increase in the proportion of CD8⁺ T cells between 8 and 10 weeks. Surprisingly, the IFN-γ response was associated with changes in the γδ rather than the CD4⁺ or CD8⁺ T cells, suggesting that this cytokine was secreted by γδ-T cells. These results are consistent with the reported ability of γδ T cells to act rapidly and bridging the innate and classically adaptive immune responses.     

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n = 53), 29% of foxes (n = 48), 29% of stone martens (n = 21) and in 25–50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.