Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria.

Introduction: Influenza A viruses (IAVs) infect the respiratory tract of mainly humans, poultry, and pigs. Co-infections with pathogenic lung bacteria are a common event and contribute to the severity of disease progression. Neutrophils are a major cell type of the innate immune system and are rapidly recruited to the site of infection. They have several effector functions to fight invading pathogens such as the secretion of reactive oxygen species (ROS) or the release of neutrophil extracellular traps (NETs). NETs are known to promote the growth of Pasteurellaceae bacteria, especially if degraded by nucleases. Methods: In this study, bronchoalveolar lavage fluid (BALF) from 45 field-infected pigs was analyzed for 1) NET markers, 2) influence on growth of lung bacteria, and 3) impact on neutrophil functions. BALF samples from 21 IAV-positive pigs and 24 lung diseased but IAV-negative pigs were compared. Results: Here, we show that neutrophils in the lungs of IAV-positive pigs release vesicular NETs. Several NET markers were increased in the BALF of IAV-positive pigs compared with the BALF from IAV-negative pigs. The amount of NET markers positively correlated with the viral load of the IAV infection. Interestingly, the BALF of IAV-positive pigs enhanced the growth of bacteria belonging to the family of Pasteurellaceae as potential coinfecting bacteria. These effects were weaker with the BALF derived from IAV-negative pigs with other lung infections. The intensity of oxidative burst in neutrophils was significantly decreased by BALF from IAVpositive pigs, indicating impaired antimicrobial activity of neutrophils. Finally, the lung milieu reflected by IAV-positive BALF does not enable neutrophils to kill Actinobacillus pleuropneumoniae but rather enhances its growth. Discussion: In summary, our data show that an IAV infection is affecting neutrophil functions, in particular the release of NETs and ROS. Furthermore, IAV infection seems to provide growth-enhancing factors for especially coinfecting Pasteurellaceae and reduces the killing efficiency of neutrophils.

Comparison of NET quantification methods based on immunofluorescence microscopy: Hand-counting, semi-automated and automated evaluations.

Formation of neutrophil extracellular traps was first described in 2004, showing that NETs are composed of decondensed chromatin fibers and nuclear and granule components. Free DNA is often used to quantify NETs, but to differentiate NETosis from necrotic DNA-release, immunofluorescence microscopy with NET-specific markers is required. Although evaluation by hand is time-consuming and difficult to standardize, it is still widespread. Unfortunately, no standardized method and only limited software tools are available for NET evaluation. This study provides an overview of recent techniques in use and aims to compare two published computer-based methods with hand counting. We found that the selected semi-automated quantification method and fully automated quantification via NETQUANT differed significantly from results obtained by hand and exhibited problems in detection of complex NET structures with partially illogical results. In contrast to that, trained persons were able to adapt to varying settings. Future approaches aimed at developing deep-learning algorithms for fast and reproducible quantification of NETs are needed.

Studying the Interaction of Neutrophils and Glaesserella Parasuis Indicates a Serotype Independent Benefit from Degradation of NETs.

Glaesserella (G.) parasuis is one of the most important porcine pathogens causing Glaesser's disease. Neutrophil granulocytes are the major counteracting cell type of the innate immune system, which contribute to the host defense by phagocytosis or the formation of neutrophil extracellular traps (NETs). Recently, NET-formation has been shown to facilitate the survival of bacteria from the Pasteurellaceae family. However, the interaction of NETs and G. parasuis is unclear so far. In this study, we investigated the interplay of three G. parasuis serotypes with porcine neutrophils. The production of reactive oxygen species by neutrophils after G. parasuis infection varied slightly among the serotypes but was generally low and not significantly influenced by the serotypes. Interestingly, we detected that independent of the serotype of G. parasuis, NET formation in neutrophils was induced to a small but significant extent. This phenomenon occurred despite the ability of G. parasuis to release nucleases, which can degrade NETs. Furthermore, the growth of Glaesserella was enhanced by external DNases and degraded NETs. This indicates that Glaesserella takes up degraded NET components, supplying them with nicotinamide adenine dinucleotide (NAD), as this benefit was diminished by inhibiting the 5'-nucleotidase, which metabolizes NAD. Our results indicate a serotype-independent interaction of Glaesserella with neutrophils by inducing NET-formation and benefiting from DNA degradation.

New Insights into Neutrophil Extracellular Trap (NETs) Formation from Porcine Neutrophils in Response to Bacterial Infections.

Actinobacillus pleuropneumoniae (A.pp, Gram negative) and Streptococcus (S.) suis (Gram positive) can cause severe diseases in pigs. During infection, neutrophils infiltrate to counteract these pathogens with phagocytosis and/or neutrophil extracellular traps (NETs). NETs consist of a DNA-backbone spiked with antimicrobial components. The NET formation mechanisms in porcine neutrophils as a response to both of the pathogens are not entirely clear. The aim of this study was to investigate whether A.pp (serotype 2, C3656/0271/11) and S. suis (serotype 2, strain 10) induce NETs by NADPH oxidase- or CD18-dependent mechanisms and to characterize phenotypes of NETs in porcine neutrophils. Therefore, we investigated NET induction in porcine neutrophils in the presence and absence of NET inhibitors and quantified NETs after 3 h. Furthermore, NETosis and phagocytosis were investigated by transmission electron microscopy after 30 min to characterize different phenotypes. A.pp and S. suis induce NETs that are mainly ROS-dependent. A.pp induces NETs that are partially CD18-dependent. Thirty minutes after infection, both of the pathogens induced a vesicular NET formation with only slight differences. Interestingly, some neutrophils showed only NET-marker positive phagolysosomes, but no NET-marker positive vesicles. Other neutrophils showed vesicular NETs and only NET-marker negative phagolysosomes. In conclusion, both of the pathogens induce ROS-dependent NETs. Vesicular NETosis and phagocytosis occur in parallel in porcine neutrophils in response to S. suis serotype 2 and A.pp serotype 2.

In vivo oxygen measurement in cerebrospinal fluid of pigs to determine physiologic and pathophysiologic oxygen values during CNS infections.

During infection and inflammation, a reduced oxygen level clearly affects cellular functions. Oxygen levels during CNS infections are unknown. Here we established and evaluated an in vivo measurement system to characterize the oxygen level in parallel with bacterial numbers (CFU/mL), the cell number and pH level inside the CSF of healthy compared to Streptococcus suis-infected pigs. The animals were anesthetized over a seven-hour period with isoflurane in air/oxygen at physiologic arterial partial pressure of oxygen. Oxygen levels in CSF of anesthetized pigs were compared to euthanized pigs. The detected partial pressure of oxygen in the CSF remained constant in a range of 47-63 mmHg, independent of the infection status (bacterial or cell number). In contrast, the pH value showed a slight drop during infection, which correlated with cell and bacterial number in CSF. We present physiologic oxygen and pH values in CSF during the onset of bacterial meningitis.

How Long Does a Neutrophil Live?-The Effect of 24 h Whole Blood Storage on Neutrophil Functions in Pigs.

Neutrophils are important effector cells of the innate immune system, traditionally regarded to have a short life span. The goal of this study was to evaluate the effect of the whole blood storage on neutrophil functions, e.g., viability, antimicrobial effect, neutrophil extracellular trap (NET) formation and phagocytosis. Therefore, fresh porcine whole blood was compared to whole blood stored for 24 h in the dark at room temperature. Different cell parameters in whole blood and in isolated neutrophils were analyzed. The following parameters were analyzed: cell count, band and segmented neutrophil count, viability, cholesterol content, release of free DNA as a marker for cell death, phagocytic activity in whole blood and in isolated neutrophils, the transmigration rate of neutrophils to IL8 stimulus, the production of reactive oxygen species (ROS), and the formation of NETs. It was observed that the number of isolated neutrophils decreased over time, indicating cell death occurs during 24 h of blood storage. However, the surviving neutrophils isolated from stored blood reacted comparably or even showed enhanced antimicrobial activity in the case of phagocytosis of Streptococcus (S.) suis, ROS production, and transmigration. The slightly altered cholesterol level of the harvested neutrophils in stored blood when compared to fresh blood partially explains some of the detected differences.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

Degraded neutrophil extracellular traps promote the growth of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (A.pp) causes severe pneumonia associated with enormous economic loss in pigs. Peracute diseased pigs die in <24 h with pneumonia. Neutrophils are the prominent innate immune cell in this infection that massively infiltrate the infected lung. Here we show that neutrophils release neutrophil extracellular traps (NETs) as response to A.pp infection. Numerous NET-markers were identified in bronchoalveolar lavage fluid (BALF) of A.pp-infected piglets in vivo, however, most NET fibers are degraded. Importantly, A.pp is able to enhance its growth rate in the presence of NETs that have been degraded by nucleases efficiently. A.pp itself releases no nuclease, but we identified host nucleases as sources that degrade NETs after A.pp infection. Furthermore, the nucleases of co-infecting pathogens like Streptococcus suis increase growth of A.pp in presence of porcine NETs. Thus, A.pp is not only evading the antimicrobial activity of NETs, A.pp is rather additionally using parts of NETs as growth factor thereby taking advantage of host nucleases as DNase1 or nucleases of co-infecting bacteria, which degrade NETs. This effect can be diminished by inhibiting the bacterial adenosine synthase indicating that degraded NETs serve as a source for NAD, which is required by A.pp for its growth. A similar phenotype was found for the human pathogen Haemophilus (H.) influenzae and its growth in the presence of human neutrophils. H. influenzae benefits from host nucleases in the presence of neutrophils. These data shed light on the detrimental effects of NETs during host immune response against certain bacterial species that require and/or efficiently take advantage of degraded DNA material, which has been provided by host nuclease or nucleases of other co-infecting bacteria, as growth source.

Canine trypanosomiasis in an endemic Costa Rican community: Demonstration of the active infection cycle.

A cross-sectional study was conducted to determine the prevalence of canine trypanosomiasis in an endemic community of Costa Rica. The indirect hemagglutination and indirect immunofluorescence assay yielded positive results in 6.4% (20/314) of canine samples analyzed; polymerase chain reaction (PCR) and light microscopy yielded positive results in one dog. Subsequently, a longitudinal study was carried out with 55 negative T. cruzi canines in the cross-sectional study. These dogs were divided into two groups: Group 1, which consisted of 25 individuals that lived in dwellings where triatomines were found in their homes; and Group 2, which consisted of 30 dogs that lived in dwellings where triatomines were not found during the previous study in their homes. Seroconversion occurred in six dogs (10.9%) in Group 1 in the first months of the year (dry season); these dogs remained seropositive until the end of the study. Only one of the six seropositive canines was also found positive once in T. cruzi PCR. The analysis of the amplified T. cruzi sequences of dogs and triatomines showed that all of them belonged to the TcI lineage. It is recommended that residents be made aware of the need to eliminate vectors in their homes and their surroundings.

Comparing Cathelicidin Susceptibility of the Meningitis Pathogens Streptococcus suis and Escherichia coli in Culture Medium in Contrast to Porcine or Human Cerebrospinal Fluid.

Host defense peptides or antimicrobial peptides (AMPs), e.g., cathelicidins, have recently been discussed as a potential new treatment option against bacterial infections. To test the efficacy of AMPs, standardized methods that closely mimic the physiological conditions at the site of infection are still needed. The aim of our study was to test the meningitis-causing bacteria Streptococcus suis and Escherichia coli for their susceptibility to cathelicidins in culture medium versus cerebrospinal fluid (CSF). Susceptibility testing was performed in analogy to the broth microdilution method described by the Clinical and Laboratory Standard Institute (CLSI) to determine minimum inhibitory concentrations (MICs) of antimicrobial agents. MICs were determined using cation-adjusted Mueller-Hinton broth (CA-MHB), lysogeny broth (LB), Roswell Park Memorial Institute medium (RPMI) or Dulbecco's Modified Eagle's Medium (DMEM) (the latter two supplemented with 5% CA-MHB or blood) and compared with MICs obtained in porcine or human CSF. Our data showed that MICs obtained in CA-MHB as recommended by CLSI do not reflect the MICs obtained in the physiological body fluid CSF. However, the MICs of clinical isolates of S. suis tested in RPMI medium supplemented with CA-MHB, were similar to those of the same strains tested in CSF. In contrast, the MICs in the human CSF for the tested E. coli K1 strain were higher compared to the RPMI medium and showed even higher values than in CA-MHB. This highlights the need for susceptibility testing of AMPs in a medium that closely mimics the clinically relevant conditions.

Extracellular Trap Formation in Response to Trypanosoma cruzi Infection in Granulocytes Isolated From Dogs and Common Opossums, Natural Reservoir Hosts.

Granulocytes mediate the first line of defense against infectious diseases in humans as well as animals and they are well known as multitasking cells. They can mediate antimicrobial activity by different strategies depending on the pathogen they encounter. Besides phagocytosis, a key strategy against extracellular pathogens is the formation of extracellular traps (ETs). Those ETs mainly consist of DNA decorated with antimicrobial components and mediate entrapment of various pathogens. In the last years, various studies described ET formation as response to bacteria, viruses and parasites e.g., Trypanosma (T.) cruzi. Nevertheless, it is not fully understood, if ET formation helps the immune system to eliminate intracellular parasites. The goal of this study was to analyze ET formation in response to the intracellular parasite Trypanosma (T.) cruzi by granulocytes derived from animals that serve as natural reservoir. Thus, we investigated the ET formation in two T. cruzi reservoirs, namely dogs as domestic animal and common opossums (Didelphis marsupialis) as wild animal. Granulocytes were harvested from fresh blood by density gradient centrifugation and afterwards incubated with T. cruzi. We conducted the analysis by determination of free DNA and immunofluorescence microscopy. Using both methods, we show that T. cruzi efficiently induces ET formation in granulocytes derived from common opossum as well as dog blood. Most ETs from both animal species as response to T. cruzi are decorated with the protease neutrophil elastase. Since T. cruzi is well known to circulate over years in both analyzed animals as reservoirs, it may be assumed that T. cruzi efficiently evades ET-mediated killing in those animals. Therefore, ETs may not play a major role in efficient elimination of the pathogen from the blood of dogs or common opossums as T. cruzi survives in niches of their body. The characterization of granulocytes in various animals and humans may be helpful to understand the anti-pathogenic capacity and overall role of ETs against zoonotic pathogens like T. cruzi.

Serological detection of antibodies to Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis and of Dirofilaria immitis antigen in dogs from Costa Rica.

In a study in Costa Rica 314 serum samples from dogs throughout all seven provinces were tested using a commercial kit for the detection of circulating antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato and Ehrlichia canis, and of circulating antigen of Dirofilaria immitis. A total of 6.4% (20/314) and 38.2% (120/314) were positive for Anaplasma spp. (An) and E. canis (Ec) antibodies. Overall, 8.0% (25/314) were positive for D. immitis (Di) antigen. One single dog reacted positive with B. burgdorferi s.l. (Bb) antigen (0.3%, 1/314). E. canis positive dogs were detected in all provinces (highest percentages in Guanacaste, Puntarenas [both significantly different compared to the overall] and Limón). Guanacaste and Puntarenas also showed the highest prevalences of Anaplasma spp. (both significantly different compared to the overall). The highest prevalence of D. immitis was detected in Puntarenas (significantly different compared to the overall). Double pathogen exposure (Ec plus An; Ec plus Di; Ec plus Bb) were recorded in 8.9% (28/314). Two dogs showed a triple pathogen exposure (0.6%, 2/314; An, Ec and Di). There was a significant difference between male (11.5%, 18/156) and female (4.4%, 7/158) animals for D. immitis positive results. There was also a significant difference between breed and no breed dogs regarding the characteristics of a general positive test, as well as seropositivity to the single pathogens of Anaplasma spp., E. canis and D. immitis. Finally there was a significant difference in the presence of clinical signs again regarding the characteristics of a general positive test, as well as seropositivity to Anaplasma spp., E. canis and D. immitis. Practitioners in Costa Rica should be aware of the canine vector-borne diseases mentioned as dogs are at risk of becoming infected. Concerning the positive B. burgdorferi s.l. dog, an autochthonous occurrence cannot be confirmed due to a history of adoption and an unusual tattoo number. Veterinary advice to protect dogs and limit transmission of vector-borne pathogens, also to humans, by using prophylactic measures is strongly recommended.

Characterization of Anaplasma spp. infection in dogs from Costa Rica.

A cross-sectional study combining serological and molecular techniques for detecting selected Anaplasma species was conducted between 2011 and 2012 in dogs and ticks sampled in all provinces of Costa Rica. Global Anaplasma spp. seroprevalence was 2.7% (11/408) by indirect immunofluorescence assay. The 16S rRNA PCR confirmed active A. phagocytophilum infection only in one dog (0.3%, 1/374); however, the same sample was negative to groEL PCR. Out of 122 Rhipicephalus sanguineus s.l. ticks analyzed, one (0.8%) was found positive to A. phagocytophilum 16S rRNA PCR but negative when tested by groEL PCR; this tick was collected from a seronegative and PCR negative dog. Both 16S rRNA sequences were 100% (510/510bp) identical to A. phagocytophilum strains isolated in different countries from different hosts. The presence of A. platys was established in four dogs (1%, 4/374) by both 16SrRNA and groEL PCR. Ticks collected from the same dogs tested negative by PCR. The 16S rRNA sequences were 100% identical to the corresponding sequences of A. platys strains isolated from dogs in Croatia and Brazil, however groEL sequences showed variable similarity levels (99-100%) with different strains of A. platys isolated in Chile, Japan and Thailand, pointing out the possible presence of different variants in Central America. Collectively data indicate low prevalence of A. phagocytophilum and A. platys in dogs from Costa Rica. Furthermore, infections seem to occur without clinical signs but with some hematological changes, and seem to resolve without treatment.