Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Provider reported implementation of nutrition-related practices in childcare centers and family childcare homes in rural and urban Nebraska.

Approximately 15 million children under age 6 are in childcare settings, offering childcare providers an opportunity to influence children's dietary intake. Childcare settings vary in organizational structure - childcare centers (CCCs) vs. family childcare homes (FCCHs) - and in geographical location - urban vs. rural. Research on the nutrition-related best practices across these childcare settings is scarce. The objective of this study is to compare nutrition-related best practices of CCCs and FCCHs that participate in the Child and Adult Care Food Program (CACFP) in rural and urban Nebraska. Nebraska providers (urban n = 591; rural n = 579) reported implementation level, implementation difficulty and barriers to implementing evidence-informed food served and mealtime practices. Chi-square tests comparing CCCs and FCCHs in urban Nebraska and CCCs and FCCHs in rural Nebraska showed sub-optimal implementation for some practices across all groups, including limiting fried meats and high sugar/ high fat foods, using healthier foods or non-food treats for celebrations and serving meals family style. Significant differences (p < .05) between CCCs and FCCHs also emerged, especially with regard to perceived barriers to implementing best practices. For example, CCCs reported not having enough money to cover the cost of meals for providers, lack of control over foods served and storage problems, whereas FCCHs reported lack of time to prepare healthier foods and sit with children during mealtimes. Findings suggest that policy and public health interventions may need to be targeted to address the unique challenges of implementing evidence-informed practices within different organizational structures and geographic locations.

Early preterm infants with abnormal psychomotor neurodevelopmental outcome at age two show alterations in amplitude-integrated electroencephalography signals.

INTRODUCTION: Recent studies showed that neurodevelopment in preterm infants can be predicted by using amplitude-integrated electroencephalography (aEEG)-derived parameters. In our previous study we demonstrated that aEEG could be useful in predicting neurodevelopmental outcome in very preterm infants at the corrected age of 2 years. AIM: The aim of this study was to further evaluate aEEG for predicting neurodevelopmental outcome at the at the corrected age of 2 years in preterm infants. METHODS: Between July 2010 and June 2016 440 very preterm infants were eligible for the study at Innsbruck Medical University Hospital. The aEEG was evaluated for the Burdjalov score in 306 preterm infants (mean gestational age 29.5 weeks; range: 24.1-31.9 weeks). At the corrected age of 2 years outcome was assessed by the Bayley Scales of Infant and Toddler Development. RESULTS: The cohort was divided into three subgroups: 248 infants with normal outcome, 40 infants with delayed outcome and 18 infants with abnormal outcome. Burdjalov scores were lower in infants with delayed outcome than in infants with normal outcome and even lower in infants with abnormal outcome. Post-hoc analysis showed significant differences between normal and delayed psychomotor outcome at 18-24 h (5 (3;6) versus 3 (3;5), p = .024), 30-36 h (6 (4;8) versus 4 (4;6), p = .033), 42-48 h (7 (5;8.5) versus 4 (4;7), p = .003), 54-60 h (7 (6;9) versus 5 (4;7), p = .003), 66-72 h (8 (6;9) versus 6.5 (4.25;7.75), p = .027) and week one (8 (7;10) versus 6.5 (5;8), p = .021). Additionally, when comparing normal to abnormal outcome, a significant difference was found at week four (12 (9;12) versus 8 (7;10), p = .024). The Burdjalov score was only predictive for a delayed psychomotor outcome, presenting the highest area under the curve (0.690) at week two of life. CONCLUSION: We observed differences in aEEG signals and neurodevelopmental outcome at the corrected age of 2 years, especially for psychomotor outcome. The predictive value of the Burdjalov score regarding neurodevelopmental outcome at the corrected age of 2 years in preterm infants was low.

Amplitude-integrated electroencephalography shows alterations in children born preterm displaying poor literacy precursor skills.

AIM: To assess whether amplitude-integrated electroencephalography (aEEG) alterations in the newborn period are associated with poor precursor skills of literacy at five years of age in children born preterm. METHODS: Between October 2007 and September 2011 248 preterm infants were eligible for the study at Innsbruck Medical University Hospital. aEEG was analysed for dominating background activity, calculation of the percentage of continuous activity, the Burdjalov scoring system, the minimum, mean and maximum amplitude. At the age of five years, we evaluated preterm born children by the Bielefelder screening (BISC) to assess for early diagnosis of reading problems and weak spelling and classified them as normal performers (n = 64) or poor performers (n = 20). Completion of testing was not possible for one infant. RESULTS: The minimum amplitude was significantly lower in the poor BISC performance group as compared to the normal BISC performance group at postnatal week two. The percentage of continuous background activity was significantly higher in infants with normal BISC performance than in infants with poor BISC performance at postnatal week three. CONCLUSION: Children with poor developed precursor skills of literacy showed alterations in aEEG signals. The aEEG could be useful in further diagnosing preterm infants at risk for developmental complications.

Mif-deficiency favors an atheroprotective autoantibody phenotype in atherosclerosis.

The inflammatory cytokine macrophage migration-inhibitory factor (MIF) promotes atherosclerosis via lesional monocyte and T-cell recruitment. B cells have emerged as important components in atherogenesis, but the interaction between MIF and B cells in atherogenesis is unknown. Here, we investigated the atherosclerotic phenotype of Mif-gene deletion in Apoe-/- mice. Apoe-/- Mif-/- mice fed a Western diet exhibited strongly reduced atherosclerotic lesions in brachiocephalic artery (BC) and abdominal aorta compared with controls. This phenotype was accompanied by reduced circulating B cells. Flow cytometry revealed a B-cell developmental defect with increased premature and immature B-cell counts in bone marrow (BM) of Apoe-/- Mif-/- mice and diminished B-cell numbers in spleen. This finding was linked with a decreased expression of Baff-R and differentiation-driving transcription factors at the immature B-cell stage, whereas peritoneal B cells exhibited unchanged CD80 and CD86 expression but vastly decreased CD9 and elevated CD23 levels, indicating that the developmental block favors the generation of immature, egressing, and reactive B cells. Mif deficiency did not affect absolute B-cell numbers in the vessel wall but favored a relative increase of B cells in the atheroprone BC region and the appearance of periadventitial B-cell-rich clusters. Of note, Mif-/- mice exhibited a significant increase in oxidized low-density lipoprotein (oxLDL)-specific antibodies after the injection of oxLDL, indicating that Mif deficiency is associated with higher sensitivity of B cells against natural-occurring antigens such as oxLDL. Importantly, Apoe-/- mice adoptively transplanted with Apoe-/-Mif-/- BM showed reduced peripheral B cells compared with Apoe-/- BM transplantation but no atheroprotection in the BC; also, whereas there was a selective increase in atheroprotective IgM-anti-oxLDL-antibodies in global Mif deficiency, BM-specific Mif deficiency also led to elevated proatherogenic anti-oxLDL-IgG. Together, these findings reveal a novel link between MIF and B cells in atherogenesis. Protection from atherosclerosis by Mif deficiency is associated with enhanced B-cell hypersensitivity, which in global but not BM-restricted Mif deficiency favors an atheroprotective autoantibody profile in atherosclerotic mice. Targeting MIF may induce protective B-cell responses in atherosclerosis.-Schmitz, C., Noels, H., El Bounkari, O., Straussfeld, E., Megens, R. T. A., Sternkopf, M., Alampour-Rajabi, S., Krammer, C., Tilstam, P. V., Gerdes, N., Bürger, C., Kapurniotu, A., Bucala, R., Jankowski, J., Weber, C., Bernhagen, J. Mif-deficiency favors an atheroprotective autoantibody phenotype in atherosclerosis.

CD27 co-stimulation increases the abundance of regulatory T cells and reduces atherosclerosis in hyperlipidaemic mice.

Aims: The co-stimulatory receptor CD27 modulates responses of T cells, B cells, and NK cells. Various T cell subsets participate in atherogenesis. However, the role of CD27 in atherosclerosis remains unexplored. Methods and results: Here we investigated the effect of bone marrow-derived and systemic CD27 deficiency in Apolipoprotein E-deficient (Apoe-/-) mice in early and advanced stages of atherosclerosis. Lethally-irradiated Apoe-/- mice reconstituted with Cd27-/-Apoe-/- bone marrow and consuming an atherogenic diet displayed a markedly increased plaque size and lesional inflammation compared to mice receiving Cd27+/+Apoe-/- bone marrow. Accordingly, chow diet-fed Cd27-/-Apoe-/- mice showed exacerbated lesion development and increased inflammation at the age of 18 weeks. At a more advanced stage of atherosclerosis (28 weeks), lesion size and phenotype did not differ between the two groups. Systemic and bone marrow-derived CD27 deficiency reduced the abundance of regulatory T cells (Treg) in blood, lymphoid organs, and the aorta. Numbers of other immune cells were not affected while expression of inflammatory cytokine genes (e.g. IL-1ß and IL-6) was increased in the aorta when haematopoietic CD27 was lacking. In vitro, Tregs of CD27-deficient mice showed similar suppressive capacity compared with their wild-type controls and migrated equally towards CCL19 and CCL21. However, thymic Cd27-/- Tregs underwent increased apoptosis and expressed fewer markers of proliferation in vivo. Reconstitution of Cd27-/-Apoe-/- mice with Cd27+/+Apoe-/- Tregs reversed the increase in atherosclerosis. Conclusion: We demonstrate that CD27 co-stimulation increases the number of Tregs and limits lesion development and inflammation in experimental atherosclerosis, particularly during early stages of disease. Thus, our study suggests that promotion of CD27 function may mitigate atherosclerosis.

Constitutive CD40 Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption.

The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr-/-) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr-/- chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr-/- (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

Use of 23.4% Saline in Symptomatic Vasospasm and Cushing's Triad to Prevent Herniation and Death: A Case Report.

A 53-year-old woman with migraines presented with Hunt-Hess grade 5 and Fisher grade 4 subarachnoid hemorrhage with intraventricular hemorrhage. She experienced severe vasospasm requiring intra-arterial medications. Continued vasospasm and edema resulted in Cushing's triad with profound tachypnea. Three percentage saline was administered twice without improvement. Despite the general practice to wait until complete neurologic deterioration before administering 23.4% saline, it was administered on 2 separate occasions, once after the failure of the 2 boluses of 3% saline and once on the reappearance of Cushing's triad 24 hours later, and on each occasion produced overall clinical improvement. The patient was subsequently discharged to a rehabilitation facility and then home. A paradigm shift to earlier intervention with 23.4% saline may improve overall outcomes in patients with severe intracranial hypertension refractory to 3% saline and impending herniation.

Angioedema: Perioperative management.

OBJECTIVE: To describe the perioperative management of a patient with acquired angioedema (AAE). METHODS: A 66-year-old Caucasian male presented from an outside hospital with a history of acquired angioedema and gastrointestinal stromal tumor-related intractable urticaria and mastocytosis. He was admitted for urgent laparoscopic partial gastrectomy, secondary to gastric outlet obstruction symptomatology. Previous combined attacks were characterized by a widespread rash, abdominal pain and respiratory distress resulting in hospitalization. Following preoperative consultation with the patient's allergist and a hospital pharmacist, he was treated preoperatively with fresh frozen plasma and his home prednisone dose. C1-inhibitor (Berinert®) was on standby along with epinephrine, given that the underlying etiology (C1- inhibitor deficiency vs histaminergic) was not known. RESULTS: There were no intraoperative complications, and the patient was discharged home 3 days after the procedure. CONCLUSIONS: Optimization of perioperative outcomes in patients, especially during urgent or emergent surgery, with a history of angioedema requires the development of a patient-specific perioperative plan, including prophylaxis, rescue therapies and opioid-sparing strategies.

Alterations in systemic levels of Th1, Th2, and Th17 cytokines in overweight adolescents and obese mice.

BACKGROUND: Obesity represents a major problem for patients and health care systems in most industrialized countries. A chronic inflammatory state in obese individuals leads to disease conditions associated with activation of cellular immune mechanisms. Here, we sought to investigate the role of Th1-, Th2-, and Th17-related cytokines in overweight adolescents and mice on a high-fat diet. METHODS: Plasma samples were obtained from 79 male adolescents aged 13-17 years. Thirty-seven of them had a body mass index (BMI) above the 90th age-specific percentile. Th1, Th2, and Th17 cytokines were measured using Bio-Plex multiplex technology (Bio-Rad, Hercules, USA). In an experimental approach, mice were fed with high-fat (HFD) or normal chow for 15 weeks. RESULTS: Interleukin (IL)-17 concentrations were significantly decreased in overweight adolescents compared to lean controls [99.8 ± 7.3 pg/mL standard error of the mean (SEM) vs 146.6 ± 11.5 pg/mL SEM P = .001]. Levels of IL-17 correlated significantly with anthropometrical parameters of obesity. A concordant response was found in mice consuming a HFD for 15 weeks compared to controls (861 ± 165 pg/mL SEM vs 1575 ± 187 pg/ml SEM, P = .0183). However, a biphasic response was evident for most Th1, Th2, and Th17 cytokines as levels initially increased within the first 5 weeks on HFD and showed a decline afterwards. CONCLUSIONS: In contrast to previous studies showing elevated levels of IL-17 in obese adults, we found a decreasing trend in overweight adolescents. This difference could possibly be related to the fact that disease conditions associated with obesity such as hypertension, vascular pathologies, diabetes, and a triggering of the Th1/Th17 axis were not yet present in overweight teenagers.

CD70 limits atherosclerosis and promotes macrophage function.

The co-stimulatory molecule CD70 is expressed on activated immune cells and is known to modulate responses of T, B, and NK cells via its receptor CD27. Until now, there is only limited data describing the role of CD70 in atherosclerosis. We observed that ruptured human carotid atherosclerotic plaques displayed higher CD70 expression than stable carotid atherosclerotic plaques, and that CD70 expression in murine atheroma localized to macrophages. Lack of CD70 impaired the inflammatory capacity (e. g. reactive oxygen species and nitric oxide production) of bone marrow-derived macrophages, increased both M1-like and M2-like macrophage markers, and rendered macrophages metabolically inactive and prone to apoptosis. Moreover, CD70-deficient macrophages expressed diminished levels of scavenger receptors and ABC-transporters, impairing uptake of oxidised low-density lipoprotein (oxLDL) and cholesterol efflux, respectively. Hyperlipidaemic Apoe-/- mice reconstituted with CD70-deficient bone marrow displayed a profound increase in necrotic core size, plaque area, and number of lesional macrophages as compared to mice receiving control bone marrow. Accordingly, 18 week-old, chow diet-fed CD70-deficient Apoe-/- mice displayed larger atheroma characterised by lower cellularity and more advanced plaque phenotype than Apoe-/- mice. In conclusion, CD70 promotes macrophage function and viability and is crucial for effective phagocytosis and efflux of oxLDL. Deficiency in CD70 results in more advanced atheroma. Our data suggest that CD70 mitigates atherosclerosis at least in part by modulating macrophage function.

Methylene Blue for Acute Septic Cardiomyopathy in a Burned Patient.

The objective of this case summary was to describe the use of methylene blue (MB) in a burned patient with acute septic cardiomyopathy. A 60-year-old Caucasian man was admitted to the Burn Intensive Care Unit with 45% TBSA burns after a house explosion. During the course of his care, he experienced hypotension that was refractory to fluid therapy and vasoactive medications. Echocardiography and right heart catheterization showed new acute systolic dysfunction with concurrent elevated systemic vascular resistance (SVR). High-dose inotropic agents did not improve cardiac function, and septic shock rendered him a poor candidate for mechanical intra-aortic balloon pump support. MB was administered to sensitize the myocardium to catecholamines and improve contractility with the goal of weaning the other vasoactive medications and diuresing for afterload reduction when hemodynamic stability was achieved. MB has been described in critical care medicine predominately for vasoplegia after cardiopulmonary bypass and vasodilatory septic shock., Our patient had acute septic cardiomyopathy that was refractory to standard pharmacologic approaches to inotropy with concurrent elevated SVR. Hypothesizing the differential temporal effect of inducible nitric oxide synthase on the vasculature and myocardium, we administered MB to improve contractility and support the impending vasodilatory effects of distributive shock. Although MB is not a new drug, the application for septic cardiomyopathy with a supranormal SVR is a unique application. Because of the risk profile associated with MB, we recommend drug monitoring utilizing serial echocardiography and/or right heart catheterization.

Regulatory T cells in atherosclerosis: critical immune regulatory function and therapeutic potential.

Atherosclerosis is a chronic inflammatory disease that is mediated by innate and adaptive immune responses. The disease is characterized by sub-endothelial accumulation and modification of lipids in the artery wall triggering an inflammatory reaction which promotes lesion progression and eventual plaque rupture, thrombus formation, and the respective clinical sequelae such as myocardial infarction or stroke. During the past decade, T-cell-mediated immune responses, especially control of pro-inflammatory signals by regulatory T cells (Tregs), have increasingly attracted the interest of experimental and clinical researchers. By suppression of T cell proliferation and secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor-ß, Tregs exert their atheroprotective properties. Atherosclerosis-prone, hyperlipidemic mice harbor systemically less Tregs compared to wild-type mice, suggesting an imbalance of immune cells which affects local and systemic inflammatory and potentially metabolic processes leading to atherogenesis. Restoring or increasing Treg frequency and enhancing their suppressive capacity by various modulations may pose a promising approach for treating inflammatory conditions such as cardiovascular diseases. In this review, we briefly summarize the immunological basics of atherosclerosis and introduce the role and contribution of different subsets of T cells. We then discuss experimental data and current knowledge pertaining to Tregs in atherosclerosis and perspectives on manipulating the adaptive immune system to alleviate atherosclerosis and cardiovascular disease.

Intravenous warfarin and heparin-induced thrombocytopenia: making the diagnosis, management, modern monitoring, and multidisciplinary care.

OBJECTIVE: To describe the diagnosis, management, and monitoring of a patient with heparin-induced thrombocytopenia (HIT) with thrombosis and simultaneous bleeding risk treated with argatroban and transitioned to intravenous (IV) warfarin secondary to the inability to administer enteral medications. CASE SUMMARY: A 71-year-old man was admitted to the surgical intensive care unit (SICU) following aortic valve repair, coronary artery bypass, and ascending aortic aneurysm repair. On postoperative day 9, he was found to have a pulmonary embolism, and therapeutic heparin was started. The following day, his platelet count was found to have dropped precipitously. HIT was diagnosed, heparin was discontinued, and argatroban was initiated. On postoperative day 22, anticoagulation was discontinued because of massive gastrointestinal bleeding. On postoperative day 35, multiple venous thromboses were found, and argatroban was restarted. The patient developed a high-output enterocutaneous fistula, eliminating the option of enteral route of medication administration. The multidisciplinary SICU team transitioned the patient from argatroban to IV warfarin for long-term anticoagulation. The international normalized ratio was monitored and remained therapeutic throughout his admission without further thrombotic complications. DISCUSSION: HIT occurs when antibodies develop to heparin-platelet factor 4 complexes, causing simultaneous hypercoagulability and thrombocytopenia. It is diagnosed based on both clinical factors and laboratory testing. Treatment includes discontinuation of all forms of heparin; initiation of a nonheparin anticoagulant, such as argatroban; and transition to warfarin. CONCLUSIONS: IV warfarin is a therapeutic option for patients with malabsorption issues. A multidisciplinary team in an intensive care setting optimizes cost-effective, patient-centered, and safe care.

Antibody response and viral shedding profile of Egyptian geese (Alopochen aegyptiacus) infected with low pathogenicity H7N1 and H6N8 avian influenza viruses.

Egyptian geese (Alopochen aegypticus), a duck species endemic to sub-Saharan Africa and occasionally implicated in the transmission of avian influenza viruses (AIV) to farmed ostriches, were experimentally infected with low pathogenicity H7N1 and H6N8 viruses to assess viral shedding and immune profiles. Following the first infection with H7N1 virus, high titers of virus were shed from both the tracheae and cloacae for at least 7 days postinfection, and tracheal shedding lasting until day 14. All detectable shedding from both tracheae and cloacae had ceased within 28 days of infection. Antibody titers peaked at day 7 postinfection, but the initial immune response was short-lived. Birds that received a second challenge with the homologous H7N1 virus mounted a more robust response that lasted beyond 66 days postchallenge, and H7N1 virus was detected, albeit at much lower levels, until day 28 post secondary infection (psi) in the cloaca and beyond day 28 psi in the trachea. Birds that received an initial infection with H7N1 virus were also challenged with H6N8 virus, and because a comparable shedding pattern to the H7N1 challenge group was observed, we concluded that the effect of any nonspecific immunity was negligible.

The ecology of influenza A viruses in wild birds in southern Africa.

Avian influenza viruses (AIVs) are pathogens of global concern, but there has been little previous research on avian influenza in southern Africa and almost nothing is known about the dynamics of AIVs in the region. We counted, captured and sampled birds regularly at five sites, two in South Africa (Barberspan and Strandfontein) and one in each of Botswana (Lake Ngami), Mozambique (Lake Chuali) and Zimbabwe (Lakes Manyame and Chivero) between March 2007 and May 2009. The South African and Zimbabwean sites were visited every 2 months and the sites in Botswana and Mozambique every 4 months. During each visit we undertook 5-7 days of standardised bird counts followed by 5-10 days of capturing and sampling water-associated birds. We sampled 4,977 birds of 165 different species and completed 2,503 half-hour point counts. We found 125 positive rRT-PCR cases of avian influenza across all sites. Two viruses (H1N8 and H3N8) were isolated and additional H5, H6 and H7 strains were identified. We did not positively identify any highly pathogenic H5N1. Overall viral prevalence (2.51%) was similar to the lower range of European values, considerable spatial and temporal variation occurred in viral prevalence, and there was no detectable influence of the annual influx of Palearctic migrants. Although waterbirds appear to be the primary viral carriers, passerines may link wild birds and poultry. While influenza cycles are probably driven by the bird movements that result from rainfall patterns, the epidemiology of avian influenza in wild birds in the subregion is complex and there appears to be the possibility for viral transmission throughout the year.

Persistence of low pathogenic avian influenza virus in waterfowl in a Southern African ecosystem.

Waterfowl were counted and sampled in a Zimbabwean wetland over 24 months. LPAI strains were detected during 20 consecutive months, providing evidence of regional yearly persistence of LPAI. We discuss the role of Afro-tropical ducks in viral maintenance and transmission, and attempt to explain the observed patterns.

Phylogenetic analysis of influenza A viruses (H6N8, H1N8, H4N2, H9N2, H10N7) isolated from wild birds, ducks, and ostriches in South Africa from 2007 to 2009.

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.