Characteristics of naturally acquired avian malaria infections in naive juvenile African black-footed penguins (Spheniscus demersus)

Antibody responses to naturally acquiredPlasmodium relictum andP. elongatum infections, blood parasitemia, and disease signs were investigated in 23 naive juvenile African black-footed penguins (Spheniscus demersus). Anti-Plasmodium spp. immunoglobulins were detected by enzyme-linked immunosorbent assay (ELISA) usingP. falciparum antigens. All birds rapidly developed antibody toP. relictum andP. elongatum. Five penguins showed detectable parasitemia and signs of the disease. Parasitemia was not related to the timing of the maximal antibody response or to the antibody titer. Two of the five parasitemic birds died and gross examination revealed splenomegaly, hepatomegaly, and congested, edematous lungs. Although the other 17 birds were clearly exposed to the disease, none showed signs of infection. No subsequent episode of parasitemia was observed in individual penguins. A comparison of the fate of 1993 penguins with those from other years showed a great variability in the proportion of birds exhibiting signs of malaria.     

Novel relapsing fever <Emphasis Type="Italic">Borrelia</Emphasis> detected in African penguins (<Emphasis Type="Italic">Spheniscus demersus</Emphasis>) admitted to two rehabilitation centers in South Africa

The African penguin, Spheniscus demersus, the only penguin species that breeds in Africa, is endangered, and several diseases including avian malaria, babesiosis, and aspergillosis are common in some populations. From 2002 to 2010, spirochetes morphologically consistent with Borrelia were observed on thin blood smears from 115 of 8,343 (1.4%) African penguins admitted to rehabilitation centers in the Western Cape and Eastern Cape provinces of South Africa. Prevalence rates were significantly higher among chicks and juveniles compared with adults and for birds sampled during the summer months of October to February compared with winter months. The majority of infected birds were ultimately released, despite lack of antibiotic treatment; however, at least one bird is believed to have died of borreliosis based on characteristic gross and microscopic lesions. Analysis of partial flaB gene sequences indicated this was a relapsing fever Borrelia most similar to a Borrelia sp. detected in soft ticks from a seabird colony in Japan. This represents the fourth report of a relapsing fever Borrelia sp. in an avian species and highlights the need for additional studies of potentially pathogenic organisms infecting the African penguin in South Africa.     

Application of in-situ hybridization for the detection and identification of avian malaria parasites in paraffin wax-embedded tissues from captive penguins

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples.     

Evaluation of a commercial multiplex PCR test (SeptiFast) in the etiological diagnosis of community-onset bloodstream infections

The commercial polymerase chain reaction (PCR) test, SeptiFast, is designed to identify the DNA of individual bacterial and fungal pathogens in whole blood. We aimed to evaluate the usefulness of the test for the detection of community-onset bloodstream infections. We prospectively included adult patients who were subjected to blood culture (BC) at an infectious diseases department. For the evaluation, one BC/PCR set (two BC bottles and one PCR tube) per patient was used. When several sets were obtained and analyzed, the first set with any positive result was evaluated. Among 1,093 consecutively included patients, BC was positive in 138 and PCR was positive in 107. Fifty positive PCR results were supported by BC in the same BC/PCR set, ten were supported by other cultures, and, additionally, ten were supported by the clinical presentation. Compared with BC, PCR showed specificities and negative predictive values of >97% for all detectable pathogens. The following sensitivities and positive predictive values (PPVs) were noted: Staphylococcus aureus, 67% and 43%; Streptococcus pneumoniae, 12% and 67%; other Streptococcus species, 43% and 77%; Escherichia coli, 53% and 56%; and Klebsiella species, 43% and 23%. If support from other cultures and the clinical presentation were included in the reference standard, the PPVs for the detection of these bacteria were 57%, 100%, 92%, 75%, and 69%, respectively. Although the specificities were high, the low sensitivities and suboptimal PPVs noted in the present study discourage routine use of the test in its present form for the detection of community-onset bloodstream infections.     

Inhibition of the in vitro growth of plasmodium falciparum by human polymorphonuclear neutrophil leucocytes

Polymorphonuclear neutrophil leucocytes (PMN) from children with acute Plasmodium falciparum malaria significantly inhibited parasite growth in homologous and in non-immune sera. Phagocytosis of schizonts in vitro was observed. PMN from uninfected children and uninfected adults had no effect on parasite growth.     

Genetic characterization,distribution and prevalence of avian pox and avian malaria in the Berthelot’s pipit (<Emphasis Type="Italic">Anthus berthelotii</Emphasis>) in Macaronesia

Exotic pathogens have been implicated in the decline and extinction of various native-island-bird species. Despite the fact that there is increasing concern about the introduction of diseases in island ecosystems, little is known about parasites in the islands of Macaronesia. We focus on Berthelot’s pipit (Anthus berthelotii), an endemic and widespread Macaronesian bird species, using a combination of field studies and molecular techniques to determine: (1) the range and prevalence of avian pox and malaria in Berthelot’s pipits throughout the species’ distribution, (2) the genetic characterization of both parasites in order to ascertain the level of host specificity. We sampled 447 pipits across the 12 islands inhabited by this species. Overall, 8% of all individuals showed evidence of pox lesions and 16% were infected with avian malaria, respectively. We observed marked differences in the prevalence of parasites among islands both within and between archipelagos. Avian pox prevalence varied between 0–54% within and between archipelagos and avian malaria prevalence varied between 0–64% within and between archipelagos. The diversity of pathogens detected was low: only two genetic lineages of avian malaria and one lineage of avian pox were found to infect the pipit throughout its range. Interestingly, both avian malaria parasites found were Plasmodium spp. that had not been previously reported in the Macaronesian avifauna (but that had been observed in the lesser kestrel Falco naumannii), while the avian pox was a host specific lineage that had previously been reported on two of the Canary Islands.     

Malaria in penguins – current perceptions

Avian malaria is a mosquito-borne disease caused by protozoans of the genus Plasmodium, and it is considered one of the most important causes of morbidity and mortality in captive penguins, both in zoological gardens and rehabilitation centres. Penguins are known to be highly susceptible to this disease, and outbreaks have been associated with mortality as high as 50–80% of affected captive populations within a few weeks. The disease has also been reported in wild penguin populations, however, its impacts on the health and fitness of penguins in the wild is not clear. This review provides an overview of the aetiology, life cycle and epidemiology of avian malaria, and provides details on the strategies that can be employed for the diagnostic, treatment and prevention of this disease in captive penguins, discussing possible directions for future research.     

Pathological and molecular characterization of avian malaria in captive Magellanic penguins (Spheniscus magellanicus) in South America

Avian malaria is a mosquito-borne disease that affects multiple avian species and is caused by protozoans of the genus Plasmodium. An avian malaria infection caused by Plasmodium sp. in Magellanic penguins (Spheniscus magellanicus) with high mortality is described in a zoo in Southern Brazil. Clinically, three birds presented signs of inappetence, anorexia, pale mucosa, dyspnea, and opisthotonus, with death in a clinical course of 5–8 h. At the necropsy, all birds exhibited pale mucosa, marked splenomegaly and hepatomegaly, in addition to moderate leptomeningeal blood vessels ingurgitation in the brain. Microscopically, multiple exoerythrocytic meronts were observed in the cytoplasm of endothelial cells in the spleen, liver, heart, lungs, brain, kidneys, and pancreas. The spleen had a multifocal perivascular inflammatory infiltrate of lymphocytes, plasma cells, and macrophages, which also exhibited hemosiderosis and erythrophagocytosis. The liver had a multifocal periportal inflammatory infiltrate of lymphocytes, macrophages, and plasma cells, in addition to marked hemosiderosis in the hepatic sinusoids. Fragments of spleen, liver, brain, skeletal muscle, and lung were tested by the polymerase chain reaction technique for the detection of a fragment of the cytochrome B gene from haemosporidians, which resulted positive for Plasmodium spp. After sequencing, the samples were phylogenetically associated to Plasmodium sp. detected in Turdus albicollis (KU562808) in Brazil and matched to the lineage TURALB01 previously detected in T. albicollis. Avian malaria infections caused by Plasmodium sp. of lineage TURALB01 may occur in S. magellanicus with high mortality, and, thus, it is essential to detect and characterize the agent involved to obtain the differential diagnosis of the condition.

     

Clinico-pathological studies of Plasmodium falciparum and Plasmodium vivax — malaria in India and Saudi Arabia

Malaria is one of the most devastating diseases of tropical countries with clinical manifestations such as anaemia, splenomegaly, thrombocytopenia, hepatomegaly and acute renal failures. In this study, cases of thrombocytopenia and haemoglobinemia were more prominent in subjects infected with Plasmodium falciparum (Welch, 1897) than those with Plasmodium vivax (Grassi et Feletti, 1890). However, anaemia, jaundice, convulsions and acute renal failure were significantly high (3–4 times) in subjects infected with P. falciparum than those infected with P. vivax. The incidence of splenomegaly and neurological sequelae were 2 and 6 times higher in P. falciparum infections compared to the infections of P. vivax. Both in P. vivax and P. falciparum malaria, the cases of splenomegaly, jaundice and neurological sequelae were almost double in children (<10 years) compared to older patients. The liver enzymes were generally in normal range in cases of low and mild infections. However, the AST, ALT, ALP activities and serum bilirubin, creatinine, and the urea content were increased in P. falciparum and P. vivax malaria patients having high parasitaemia, confirming liver dysfunction and renal failures in few cases of severe malaria both in India and Saudi Arabia.     

Prevalence of avian malaria parasite in mosquitoes collected at a zoological garden in Japan

Several species of captive birds at zoological gardens of Japan were found to be infected with avian Plasmodium. However, incriminated vector mosquito species have not been identified yet. To indicate the competent vectors of avian malaria parasite, we collected mosquitoes at a zoological garden in Japan and examined for the avian malaria parasite DNA. Totally, 1,361 mosquitoes of 11 species were collected in the zoological garden of Kanagawa, the south of Tokyo in Japan in 2005. Captured mosquitoes were pooled by each species, date collected, and location and used for DNA extraction. Eight out of 169 DNA samples were positive for the nested PCR of avian Plasmodium cyt b gene. Estimated minimum infection rates of mosquitoes were 5.9 per 1,000. The PCR positive mosquito species were Culex pipiens group and Lutzia vorax. Some DNA sequences amplified from collected mosquitoes were identical to avian Plasmodium lineages detected from captive birds in the same zoological garden studied. Our results suggest that C. pipiens group and L. vorax could be incriminated vectors of avian malaria parasite transmitting in captive birds kept in the zoological garden in Japan.     

Comparative diagnostic performance of two commercial rapid tests for malaria in a non-endemic area

In the study reported here, the diagnostic performance of two new rapid tests for the diagnosis of malaria was evaluated in symptomatic patients in a non-endemic area. Of 557 consecutive patients, 109 (19.6%) had documented malaria. For the NOW ICT MALARIA P.f./P.v. (Binax, Portland, ME, USA) and OptiMAL IT (Diamed, Cressier, Switzerland) tests, respectively, sensitivity values were 96.3% and 79.8% (P-value, 0.0001), and specificity values were 98.8% and 98.4%. The NOW ICT test did not detect two of 80 Plasmodium falciparum infections, and it generated false-positive results for five patients. The OptiMAL IT test failed to detect ten of the P. falciparum infections, and it generated seven false-positive results. The results suggest that these rapid diagnostic tests for malaria may be useful, but they cannot replace microscopic examination of blood films.     

Free-flow electrophoresis for the separation of malaria infected and uninfected mouse erythrocytes and for the isolation of free parasites (Plasmodium vinckei): A new rapid technique for the liberation of malaria parasites from their host cells

Summary After aggregation of erythrocytes from malaria infected mice, the parasites (Plasmodium vinckei) could be set free using gentle mechanical forces. The mixture of freed parasites, infected and non-infected erythrocytes, and membraneous material was separated by free-flow electrophoresis. The free parasites produced were very pure and infectious. Morphological and enzymatic data on the separated fractions are presented. Free-flow electrophoresis also allowed the separation of infected and uninfected erythrocytes.     

Development of a DIVA (Differentiating Infected from Vaccinated Animals) strategy using a vaccine containing a heterologous neuraminidase for the control of avian influenza

The present paper reports of the development and validation of a control strategy for avian influenza infections in poultry. The "DIVA" (Differentiating Infected from Vaccinated Animals) strategy is based on the use of an inactivated oil emulsion vaccine containing the same haemagglutinin (H) subtype as the challenge virus, but a different neuraminidase (N). The possibility of using the heterologous N subtype, to differentiate between vaccinated and naturally infected birds, was investigated through the development of an "ad hoc" serological test based on the detection of specific anti-N1 antibodies. This was achieved using a baculovirus expressing a recombinant N1 protein. The A/ck/Pakistan/H7N3 virus was used as a vaccine and birds were challenged with the HPAI A/ty/Italy/4580/V99/H7N1 strain. The homologous H group ensured a clinical protection of 93% regardless of the vaccination scheme used, and was able to prevent viraemia and muscle colonization in the clinically healthy challenged birds. However, it was not able to prevent viral shedding. The "ad hoc" serological assay was developed as an indirect immunofluorescence test, and was validated using 608 field sera, and showed an "almost perfect agreement" (Kappa value) with the HI test, with relative sensitivity and specificity values of 98.1 and 95.7, respectively. The results of the present investigation suggest that the "DIVA" control strategy may represent a tool for the control of avian influenza infections in poultry.     

Antibody response to plasmodium falciparum malaria. Comparisons of immunoglobulin concentrations, antibody titres and the antigenicity of different asexual forms of the parasite

Antibody titres of children and adults living in a malaria endemic region were measured by the indirect fluorescent antibody technique, using fluorescein-conjugated monospecific immunoglobulin antisera. The antigenicity of Plasmodium falciparum trophozoites was compared with that of schizonts obtained by in vitro culture of infected blood. Malarial antibody was detected with antisera to IgG, IgM and, at low levels, to IgA but not with antisera to IgD or IgE. Higher titres were obtained with schizonts as the source of antigen than with trophozoites. Adults in whom IgM antibodies were detected had a mean IgM concentration significantly higher than that for individuals who were IgM antibody negative, and there was a positive correlation between IgG fluorescent antibody titres and total IgG in the children examined. Serial plasma samples from children with acute P. falciparum infections were examined to determine changes in immunoglobulin concentrations, antibody titres and the levels of serum antigens as a result of the infection.     

Amniotic Fluid Granulocyte Colony Stimulating Factor Levels: A Rapid Marker for Diagnosing Chorioamnionitis

PROBLEM: To assess the usefulness of amniotic fluid (AF) granulocyte colony stimulating factor (G-CSF) levels as a rapid marker for diagnosing chorioamnionitis. METHOD OF STUDY: AF levels were obtained from term and preterm patients with and without chorioamnionitis (CAM). Patients with urinary tract, respiratory tract, and other infections were excluded. Results obtained from the AF G-CSF assays were compared with those from other parameters used for diagnosing CAM: maternal fever, leukocytosis, tachycardia, fetal tachycardia, AF glucose levels, white blood cell count, Gram stain, and aerobic and anaerobic cultures. The sensitivity, specificity, and predictive values were calculated. RESULTS: In the uninfected AF samples, G-CSF levels were present but low, ranging from 400 to 1600 pg/ml. Levels in the infected samples, however, were markedly increased, ranging from 1600 to 14,000 pg/ml; P < 0.05. When a cutoff of 2000 pg/ml was used as a clear marker for CAM, the sensitivity was 67%, the specificity was 100%, and the positive and negative predictive values were 100% and 86%, respectively. The comparison of the other AF G-CSF laboratory parameters also revealed high sensitivity, specificity, and predictive values for detecting CAM. CONCLUSION: (i) AF G-CSF levels are elevated in CAM. (ii) An AF G-CSF level >2000 pg/ml is a strong positive predictor of CAM. (iii) Elevated AF G-CSF levels appear to be more reliable in predicting CAM than any other single test currently used in clinical practice.     

Protein changes in bovine lymphoblastoid cells induced by infection with the intracellular parasite Theileria parva

Protein and glycoprotein changes induced in bovine lymphoblasts by infection with Theileria parva were analyzed by high-resolution two-dimensional gel electrophoresis. Uninfected and infected cloned bovine T and B lymphoblasts were biosynthetically labeled with [35S]methionine and their two-dimensional autoradiographic patterns were compared with each other and with the pattern obtained using purified labeled schizonts. Ten proteins were found in infected cells which were not present in uninfected cells, and seven of these were detected in preparations of purified schizonts. Four glycoproteins were detected on the surface of infected cells labeled with [3H]borohydride while a major glycoprotein present on uninfected cells disappeared or was reduced in infected cells. Other minor changes in protein and glycoprotein patterns were also observed.     

Malaria infection alters the expression of B‐cell activating factor resulting in diminished memory antibody responses and survival

Malaria is a major cause of morbidity worldwide with reports of over 200–500 million infected individuals and nearly 1 million deaths each year. Antibodies have been shown to play a critical role in controlling the blood stage of this disease; however, in malaria‐endemic areas antibody immunity is slow to develop despite years of exposure to Plasmodium spp. the causative parasite. Using rodent Plasmodium yoelii YM, we provide evidence that malarial infections result in a decrease in the proportion of DCs that express the B‐cell survival factor, BAFF, resulting in a decreased ability of these DCs to support memory B‐cell differentiation into antibody secreting cells (ASCs) and/or the survival of ASCs. Further, compared with infected WT mice, ASC numbers were significantly increased in malaria‐infected transgenic mice that either overexpressed BAFF or mice with BAFF‐independent B‐cell survival (B‐cell‐restricted TRAF3 deletion). Remarkably, BAFF‐overexpressing mice were protected from lethal malaria infections, indicating the significance of the role BAFF plays in determining the outcome of malaria infections. These findings describe a previously unappreciated mechanism by which Plasmodium spp. can depress the generation of protective antibody responses.     

Comparative study of clinical signs, haematology and prevalence of trypanosomiasis in Holstein Friesian and White Fulani Zebu cattle exposed to natural infection in a rain forest zone of Nigeria

A comparison of the symptomatology, haematology and prevalence of trypanosomiasis in Holstein Friesian and White Zebu cattle exposed to natural infections on a ranch in a rain forest zone of Nigeria is presented. 23 (44%) of the adult Friesians had trypanosome infections with a generally heavy parasitaemia. Infection was light in the Friesian calves and the Zebus with infection rates of 2 (6.9%) and 3 (15%) respectively. Trypanosoma vivax was the sole species encountered on the farm. There was a general unthrifty appearance in the adult Friesian herd as well as classical signs of trypanosomiasis. The Friesian calves and Zebus were generally in good conditions with an appearance of good health except one each of the infected animals which showed apparent symptoms of the disease. There was a marked reduction of the red cell values of the infected adult Friesian and Zebu cattle. The red cell values of the uninfected adult Friesians were equally depressed suggesting cryptic infections or that they were cured parasitologically by the recently administered trypanocide. The Friesian calves had normal red cell values for both the infected and uninfected. Leucocyte counts were generally high on the Farm and higher for the infected animals.     

Helminth‐induced CD19+CD23hi B cells modulate experimental allergic and autoimmune inflammation

Numerous population studies and experimental models suggest that helminth infections can ameliorate immuno‐inflammatory disorders such as asthma and autoimmunity. Immunosuppressive cell populations associated with helminth infections include Treg and alternatively‐activated macrophages. In previous studies, we showed that both CD4⁺CD25⁺ Treg, and CD4^– MLN cells from Heligmosomoides polygyus‐infected C57BL/6 mice were able to transfer protection against allergic airway inflammation to sensitized but uninfected animals. We now show that CD4^–CD19⁺ MLN B cells from infected, but not naïve, mice are able to transfer a down‐modulatory effect on allergy, significantly suppressing airway eosinophilia, IL‐5 secretion and pathology following allergen challenge. We further demonstrate that the same cell population can alleviate autoimmune‐mediated inflammatory events in the CNS, when transferred to uninfected mice undergoing myelin oligodendrocyte glycoprotein_(p35–55)‐induced EAE. In both allergic and autoimmune models, reduction of disease was achieved with B cells from helminth‐infected IL‐10^?/? donors, indicating that donor cell‐derived IL‐10 is not required. Phenotypically, MLN B cells from helminth‐infected mice expressed uniformly high levels of CD23, with follicular (B2) cell surface markers. These data expand previous observations and highlight the broad regulatory environment that develops during helminth infections that can abate diverse inflammatory disorders in vivo.     

Plasmodium falciparum: surface modifications of infected erythrocytes from clinical isolates

 Infections of human erythrocytes with the mature asexual blood stages of Plasmodium falciparum result in antigenic changes in the host cell membrane that, by virtue of their position, length of exposure, and close association with functional changes critical to pathogenesis, are a potential important target for host effector mechanisms. These parasite-induced antigens expressed on the surface of infected erythrocytes have been shown to exhibit considerable polymorphism. An antibody-mediated agglutination assay using malaria serum samples from different regions of Venezuela has been developed to examine the extent of antigenic diversity of infected red blood cells (IRBC) taken from subjects with naturally acquired P. falciparum infections. An important humoral immune recognition of surface molecules from red blood cells infected with a wide variety of clinical isolates of P. falciparum was observed even when sera from individuals experiencing a single episode of malaria were used. A process of in vivo antigenic variation of surface molecules is postulated, since agglutination of IRBC was observed with acute heterologous but not autologous sera. When sera obtained from Amerindians inhabiting the Venezuelan Amazon were assayed, a strong immune response to different parasite isolates, including those of another geographic region, was observed, suggesting the recognition of highly conserved immunogenic parasitic epitopes in people exposed to multiple malaria infections. Received: 20 June 1995 / Accepted: 3 November 1995