Treatment with chloroquine is retinotoxic in captive African penguins (Speniscus demersus). Attenuation and recovery of electroretinographic responses.

PURPOSE: To evaluate the effect of prophylactic anti-malarial chloroquine treatment, and its cessation, on electroretinographic (ERG) responses of captive African penguins. METHODS: A brief ERG protocol ("QuickRetCheck") was recorded under mesopic conditions with manual restraint and no sedation or pupil dilation. Birds were recorded on two separate occasions, first while being treated with a daily chloroquine dose of 10 mg/kg for 12 days (n = 15, treatment group) and second after 4 months without chloroquine treatment (n = 6, off-treatment group). Three birds were recorded on both occasions. Three other birds from the flock that died were studied histopathologically. RESULTS: Scotopic responses were unmeasurable in either recording and therefore were not analyzed. Mean a- and b-wave amplitudes of the mixed rod-cone responses to standard (3 cd·s/m2 ) and high (10 cd·s/m2 ) intensity flashes were higher in the off-treatment group. No difference in implicit times was observed. Sex, age, and number of previous chloroquine treatments did not affect ERG responses. Histopathology revealed Plasmodium spp.in the lungs, liver, and brain, but not in the eyes, of the necropsied birds, and there were no signs of retinitis or retinopathy. CONCLUSIONS: Daily chloroquine treatment was associated with attenuated ERG responses in penguins, which improve following cessation of treatment. Further work is warranted to establish a chloroquine dose that is efficacious yet has minimal adverse effects. Our results suggest that ERG responses of captive penguins undergoing ERG for any indication (such as prior to cataract surgery), must be evaluated in light of the birds' anti-malaria treatment status.

Primaquine-thiazolidinones block malaria transmission and development of the liver exoerythrocytic forms.

BACKGROUND: Primaquine is an anti-malarial used to prevent Plasmodium vivax relapses and malaria transmission. However, PQ metabolites cause haemolysis in patients deficient in the enzyme glucose-6-phosphate dehydrogenase (G6PD). Fifteen PQ-thiazolidinone derivatives, synthesized through one-post reactions from primaquine, arenealdehydes and mercaptoacetic acid, were evaluated in parallel in several biological assays, including ability to block malaria transmission to mosquitoes. RESULTS: All primaquine derivatives (PQ-TZs) exhibited lower cell toxicity than primaquine; none caused haemolysis to normal or G6PD-deficient human erythrocytes in vitro. Sera from mice pretreated with the test compounds thus assumed to have drug metabolites, caused no in vitro haemolysis of human erythrocytes, whereas sera from mice pretreated with primaquine did cause haemolysis. The ability of the PQ-TZs to block malaria transmission was evaluated based on the oocyst production and percentage of mosquitoes infected after a blood meal in drug pre-treated animals with experimental malaria caused by either Plasmodium gallinaceum or Plasmodium berghei; four and five PQ-TZs significantly inhibited sporogony in avian and in rodent malaria, respectively. Selected PQ-TZs were tested for their inhibitory activity on P. berghei liver stage development, in mice and in vitro, one compound (4m) caused a 3-day delay in the malaria pre-patent period. CONCLUSIONS: The compound 4m was the most promising, blocking malaria transmissions and reducing the number of exoerythrocytic forms of P. berghei (EEFs) in hepatoma cells in vitro and in mice in vivo. The same compound also caused a 3-day delay in the malaria pre-patent period.

Artesunate-tafenoquine combination therapy promotes clearance and abrogates transmission of the avian malaria parasite Plasmodium gallinaceum.

Clinical manifestations of malaria infection in vertebrate hosts arise from the multiplication of the asexual stage parasites in the blood, while the gametocytes are responsible for the transmission of the disease. Antimalarial drugs that target the blood stage parasites and transmissible gametocytes are rare, but are essentially needed for the effective control of malaria and for limiting the spread of resistance. Artemisinin and its derivatives are the current first-line antimalarials that are effective against the blood stage parasites and gametocytes, but resistance to artemisinin has now emerged and spread in various malaria endemic areas. Therefore, a novel antimalarial drug, or a new drug combination, is critically needed to overcome this problem. The objectives of this study were to evaluate the efficacy of a relatively new antimalarial compound, tafenoquine (TQ), and a combination of TQ and a low dose of artesunate (ATN) on the in vivo blood stage multiplication, gametocyte development and transmission of the avian malaria parasite Plasmodium gallinaceum to the vector Aedes aegypti. The results showed that a 5-d treatment with TQ alone was unable to clear the blood stage parasites, but was capable of reducing the mortality rate, while TQ monotherapy at a high dose of 30mg/kg was highly effective against the gametocytes and completely blocked the transmission of P. gallinaceum. In addition, the combination therapy of TQ+ATN completely cleared P. gallinaceum blood stages and sped up the gametocyte clearance from chickens, suggesting the synergistic effect of the two drugs. In conclusion, TQ is demonstrated to be effective for limiting avian malaria transmission and may be used in combination with a low dose of ATN for safe and effective treatment.

Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes.

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a ß-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a ß-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.

Plasmodium spp. In a captive raptor collection of a Safaripark in northwest Italy.

Blood parasites infect all vertebrates (Clayton and Moore 1997). Avian malaria parasites (Plasmodium spp., Plasmodiidae) are cosmopolitan in their distribution and are responsible for severe diseases in domestic and wild birds.In September 2009, nine raptorial birds that either arrived recently or were maintained as permanent residents at the Safaripark Pombia (northwest Italy) showed loss of stamina, developing listlessness, anorexia and regurgitation. Within one month three animals died and were necropsied.Following the diagnosis of Plasmodium infection all other raptorial birds were treated: clinical improvement was observed in all birds, and blood smears made after one month resulted negative for parasites.

Effects of artesunate treatment on Plasmodium gallinaceum transmission in the vectors Aedes aegypti and Culex quinquefasciatus.

In the absence of vaccines, chemotherapy is an effective and economical way for controlling malaria. Development of anti-malarial drugs that target pathogenic blood stage parasites and gametocytes is preferable for the treatment as it can alleviate the host's morbidity and mortality and block transmission of the Plasmodium parasite. Recently, our laboratory has developed an in vivo transmission blocking assay that involves administration of 7 consecutive daily doses of a test compound into domestic chickens (Gallus gallus domesticus) infected with the avian malaria parasite Plasmodium gallinaceum with 10% parasitaemia and 1% gametocytaemia. To compromise the cost and time for artesunate (ATN) treatment, this study aimed to investigate effects of a 5-day consecutive administration of 10 milligrams per kilogram (mg/kg) ATN on P. gallinaceum infection in chickens and transmission to two natural vectors, Aedes aegypti and Culex quinquefasciatus. Our study showed that the treatment with 10 mg/kg ATN for 7 days, but not 5 days, completely eliminated blood stage infections, prevented recrudescence and blocked gametocyte production and transmission of P. gallinaceum to its vectors, thereby confirming the potent schizontocidal and gametocytocidal activities of ATN. This regimen should be further evaluated in field trials.

Highly potential antiplasmodial restricted peptides.

Malaria is an infectious disease responsible for approximately one million deaths annually. The antimalarial effects of angiotensin II and its analogs against Plasmodium gallinaceum and P. falciparum have recently been reported. To evaluate antiplasmodial activity, we synthesized five angiotensin II-restricted analogs containing disulfide bridges. To accomplish this, peptides containing two inserted amino acid residues (cysteine) were synthesized by the Fmoc solid-phase method, purified by liquid chromatography, and characterized by mass spectrometry. Conformational studies were performed by circular dichroism. The results indicated that two of the analogs had higher antiplasmodium activity (92% and 98% activity) than angiotensin II (88% activity), measured by fluorescence microscopy. Results showed that the insertion position must be selected, to preserve the hydrophobic interactions between the non-polar residues, as this affects antiplasmodial activity. The circular dichroism studies suggested that the active analogs as well as the native angiotensin II adopt a ß-turn conformation in different solutions. This approach provided insight for understanding the effects of restricting the ring size and position on the bioactivity of angiotensin II and provides a new direction for the design of potential chemotherapeutic agents.

The efficacy of artesunate, chloroquine, doxycycline, primaquine and a combination of artesunate and primaquine against avian malaria in broilers.

The efficacy of 5 antimalarial drugs was evaluated on P. gallinaceum infected broilers. One hundred and forty-seven 19-day-old broilers were divided into 7 groups of 21 chicks each. Group 1 was the unmedicated, uninfected control. Groups 2-6 were infected and medicated with artesunate, chloroquine, doxycycline, primaquine and an artesunate-primaquine combination, respectively. Group 7 was the unmedicated, infected control. Infectivity, mortality, parasitemia, schizonts in tissues and body weight gain were monitored. The results revealed that the two most effective drugs for treating P. gallinaceum at the asexual erythrocyte stage were chloroquine and doxycycline. Tissue schizonts of P. gallinaceum in all the medicated groups were significantly fewer than the unmedicated, infected control (P<0.05). The mortality rate of all the medicated groups was significantly lower than the unmedicated, infected control (P<0.05).

Chickens treated with a nitric oxide inhibitor became more resistant to Plasmodium gallinaceum infection due to reduced anemia, thrombocytopenia and inflammation.

Malaria is a serious infectious disease caused by parasites of the Plasmodium genus that affect different vertebrate hosts. Severe malaria leads to host death and involves different pathophysiological phenomena such as anemia, thrombocytopenia and inflammation. Nitric oxide (NO) is an important effector molecule in this disease, but little is known about its role in avian malaria models. Plasmodium gallinaceum-infected chickens were treated with aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase, to observe the role of NO in the pathogenesis of this avian model. AG increased the survival of chickens, but also induced higher parasitemia. Treated chickens demonstrated reduced anemia and thrombocytopenia. Moreover, erythrocytes at different stages of maturation, heterophils, monocytes and thrombocytes were infected by Plasmodium gallinaceum and animals presented a generalized leucopenia. Activated leukocytes and thrombocytes with elongated double nuclei were observed in chickens with higher parasitemia; however, eosinophils were not involved in the infection. AG reduced levels of hemozoin in the spleen and liver, indicating lower inflammation. Taken together, the results suggest that AG reduced anemia, thrombocytopenia and inflammation, explaining the greater survival rate of the treated chickens.

Identification of Plasmodium relictum causing mortality in penguins (Spheniscus magellanicus) from São Paulo Zoo, Brazil.

This study reports avian malaria caused by Plasmodium relictum in Magellanic Penguins (Spheniscus magellanicus) from São Paulo Zoo. The disease was highly infective among the birds and was clinically characterized by its acute course and high mortality. The penguins of São Paulo Zoo were housed for at least 2 years without malaria; however, they had always been maintained in an enclosure protected from mosquito exposure during the night period. When they presented pododermatitis, they were freed at night for a short period. São Paulo Zoo is located in one of the last forest remnants of the city, an area of original Atlantic forest. In the winter, the space destined for Zoo birds is shared with migratory species. Hence the possibility exists that the disease was transmitted to the penguins by mosquitoes that had previously bitten infected wild birds. Avian malaria parasites are transmitted mainly by mosquitoes of the genera Aedes and Culex, common vectors in the Atlantic forest. In this study, one Culex (Cux.) sp. was found, infected with P. relictum. There are diverse problems in housing distinct species of animals in captivity, principally when occupying the same enclosure, since it facilitates the transmission of diseases with indirect cycles, as is the case of Plasmodium spp., because certain species that cause discrete infections in some bird species can become a serious danger for others, especially penguins, which do not possess natural resistance. Thus, serious implications exist for periodically testing and administrating malaria therapy in captive penguins potentially exposed to mosquitoes during the night period, as well as other captive birds from São Paulo Zoo.

Chronic malaria infections increase family inequalities and reduce parental fitness: experimental evidence from a wild bird population.

Avian malaria parasites (Plasmodium) occur commonly in wild birds and are an increasingly popular model system for understanding host-parasite co-evolution. However, whether these parasites have fitness consequences for hosts in endemic areas is much debated, particularly since wild-caught individuals almost always harbour chronic infections of very low parasite density. We used the anti-malarial drug Malarone to test experimentally for fitness effects of chronic malaria infection in a wild population of breeding blue tits (Cyanistes caeruleus). Medication caused a pronounced reduction in Plasmodium infection intensity, usually resulting in complete clearance of these parasites from the blood, as revealed by quantitative PCR. Positive effects of medication on malaria-infected birds were found at multiple stages during breeding, with medicated females showing higher hatching success, provisioning rates and fledging success compared to controls. Most strikingly, we found that treatment of maternal malaria infections strongly altered within-family differences, with reduced inequality in hatching probability and fledging mass within broods reared by medicated females. These within-brood effects appear to explain higher fledging success among medicated females and are consistent with a model of parental optimism in which smaller (marginal) offspring can be successfully raised to independence if additional resources become available during the breeding attempt. Overall, these results demonstrate that chronic avian malaria infections, far from being benign, can have significant effects on host fitness and may thus constitute an important selection pressure in wild bird populations.

Antimalarial activity of the novel quinoline/6-thiopurine conjugate in Gallus gallus Linnaeus, infected experimentally by Plasmodium (Novyella) juxtanucleare.

This study aimed to evaluate the 4-(6'-thiopurine)-7-chloroquinoline, a novel quinoline/6-thiopurine conjugate, for the treatment of Gallus gallus experimentally infected with Plasmodium juxtanucleare, an avian malaria agent. The avian group treated with 4-(6'-thiopurine)-7-chloroquinoline showed a significative parasite clearance and maintained a low level of parasitaemia, when compared with the untreated control group and to the chloroquine treated avian group.

Plasmodium relictum (lineage P-SGS1): Further observation of effects on experimentally infected passeriform birds, with remarks on treatment with Malarone.

Plasmodium relictum (lineage P-SGS1) is a widespread malaria parasite that causes disease of different severity in different species of birds. However, experimental studies on the effects of this parasite on avian hosts are uncommon. We investigated development of this lineage in experimentally infected greenfinches Carduelis chloris and compared the obtained data with the literature information about the virulence of the same parasite lineage for phylogenetically closely related bird species. We also used an opportunity to test the efficacy of the antimalarial drug Malarone in treatment of the experimental infection. The cryopreserved strain of the lineage P-SGS1 was multiplied in 4 experimentally infected chaffinches. Light parasitemia developed in these birds; the parasites were then inoculated to 6 uninfected recipient greenfinches. Six uninfected greenfinches were used as negative controls. Light parasitemia developed in all experimental greenfinches. There were no significant effects of malaria on the body mass of greenfinches, but haematocrit value was slightly lower in experimental birds than in control ones; the infection did not cause mortality or morbidity in these birds. According to available data, all investigated fringillid birds are susceptible to P. relictum (P-SGS1), but the same malaria parasite develops markedly differently in different bird species, even closely related hosts. Thus, the observed effects of the same malaria lineage on one species of bird cannot be generalized to others, even closely related ones. The cure with Malarone was highly efficient for blood stages of P. relictum, but exoerythrocytic stages were unaffected.

An outbreak of avian malaria in captive yellowheads/mohua (Mohoua ochrocephala).

CASE HISTORY: Eight mohua, or yellowheads (Mohoua ochrocephala), were held in a large open aviary over the summer months of 2003-2004, following their capture for captive breeding purposes. Two birds died of transportation trauma shortly after arrival, one became ill and died a month later, and another four died within a 2-week period in February 2004. The eighth bird also became ill at this time but survived for a year following treatment with chloroquine and doxycycline. CLINICAL AND PATHOLOGICAL FINDINGS: The affected birds were depressed, lethargic and dyspnoeic. Necropsy of three birds showed a slightly pale and swollen liver and spleen. Impression smears of the liver of one bird revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes, which was confirmed histopathologically. Similar protozoal organisms were seen within splenic histiocytes and pulmonary endothelial cells of 5/6 birds. Electron microscopy identified these as protozoal schizonts containing merozoites of similar size and structure to those of Plasmodium spp. DIAGNOSIS: The birds were infected with a protozoal haemoparasite resembling Plasmodium spp.; asexual stages within hepatocytes and endothelial cells of the lung and spleen were typical of this organism. CONCLUSIONS AND CLINICAL RELEVANCE: The mohua captured from west Otago were highly susceptible to avian malaria as they came from an isolated population that was likely to be naïve and have had no previous contact with this organism. The birds were probably infected by bites from mosquitoes feeding off local populations of blackbirds subsequently found to be infected with Plasmodium spp.

[Malaricide activity of the quercetin in Gallus gallus L., 1758 immunocompromised infected by Plasmodium (Bennettinia) juxtanucleare Versiani and Gomes, 1941]. / Atividade malaricida da quercetina em Gallus gallus L., 1758 imunossuprimidos infectados por Plasmodium (Bennettinia) juxtanuclear Versiani e Gomes, 1941.

The aim of this study was comparatively to evaluate the malaricide activity of the quercetin to the action of the chloroquine in Gallus gallus experimentally infected for Plasmodium juxtanucleare and immunocompromised. Thirty- four hens had been used, previously infected for P. juxtanucleare and immunocompromised by the administration of 26 mg/kg of metilprednisolon 40mg/ml in the pectoral muscle. These had been divided in three groups: 1- control, 2- treated with chloroquine and 3- with quercetin. The administration of substances occurred way gavagem to four consecutive days. The malaricide action of the drugs was evaluated in agreement the parasitemia for P. juxtanucleare in blood smears, during 30 days after the infection. The groups that had received treatment with chloroquine and quercetin had presented significant reduction (p < 0,01 in both) of the parasitaemia comparative to the group control, it suggesting that the drugs had acted as malaricide in the infection for P. juxtanucleare.

The efficacy of a mixture of trimethoprim and sulphaquinoxaline against Plasmodium gallinaceum malaria in the domesticated fowl Gallus gallus.

The apicomplexan parasite Plasmodium gallinaceum has not been much studied from the veterinary standpoint. Although it causes malaria in domesticated chickens, no effective drugs appear to be commercially available. A mixture of trimethoprim and sulphaquinoxaline (TMP/SQX, ratio 1:3), with a wide spectrum of activity against bacteria and coccidia, is here shown to be also efficacious against blood-induced P. gallinaceum malaria when administered therapeutically in the feed of chickens for 5-day periods, beginning on the day before infection, or on the day of infection, or up to four days after infection. Chickens were protected against mortality and reduction of weight gain. Three other criteria of efficacy, which showed good correlation with each other and also with the two commercial performance criteria, were the production of green diarrhoea (due to biliverdin), parasitaemia and reduced haematocrit values. When TMP/SQX treatments were initiated sooner than five days after infection, parasites were almost entirely eliminated from the blood, whereas treatments initiated later than four days after infection failed to protect birds against clinical disease. Birds protected by TMP/SQX against primary infection with P. gallinaceum were immune to clinical malaria when exposed to a severe blood-induced challenge of P. gallinaceum 28 days later.

Subclinical avian malaria infections in African black-footed penguins (Spheniscus demersus) and induction of parasite recrudescence.

The subclinical and clinical Plasmodium elongatum and Plasmodium relictum infections of captive-reared African black-footed penguins (Spheniscus demersus) were evaluated in nine adult and 29 juvenile penguins in the Baltimore Zoo (Maryland, USA) during summer 1988 and winter 1989. Two diagnostic methods were used: Giemsa-stained thin blood films, and subinoculation of penguin blood into 1-day-old Peking ducklings. Chloroquine and primaquine treatment was applied to all parasitemic juvenile penguins. Twenty-nine parasite-free, juvenile penguins were monitored for parasitemia by Giemsa-stained thin blood films every two weeks for 26 weeks of their first outdoor exposure. Eighteen of 29 penguins experienced naturally acquired malaria; 14 were infected with P. elongatum, three with P. relictum, and one bird had a mixed P. relictum and P. elongatum infection. Eleven of 18 juveniles became parasitemic again after chloroquine and primaquine treatments. Based on Giemsa-stained thin blood smears and subinoculation of penguin blood into 1-day-old ducklings, performed in a mosquito-free environment in winter, nine adult penguins had no evidence of Plasmodium spp. infection. After dexmethasone-induced immunosuppression, four of six of these nonparasitemic adult penguins were found to be infected with P. relictum by the blood inoculation method.

Plasmodium relictum as a cause of avian malaria in wild-caught magellanic penguins (Spheniscus magellanicus).

Avian malaria (Plasmodium relictum) caused significant mortality in wild-caught Magellanic penguins (Spheniscus magellanicus) in 1986 at the Blank Park Zoo in Des Moines, Iowa (USA). In early winter, wild birds were captured off the southern coast of Chile and flown to Detroit, Michigan for a 38 day quarantine. After quarantine, 18 birds were dispersed to Lansing, Michigan, six to a facility in Maine, and 46 to Des Moines, Iowa. Upon arrival in Des Moines, several penguins became weak and inactive, had to be force-fed, and died after 2 days. Gross lesions at postmortem included splenomegaly, hepatomegaly, and pulmonary edema. Histopathological examination revealed numerous intraendothelial schizonts in spleen, lung, liver, heart and kidney. Schizonts were generally 16 to 28 micron by 11 to 16 micron and contained merozoites of two distinct sized (macromerozoites, nuclei 1.0 micron; micromerozoites, nuclei 0.5 micron). Based on the morphology of the abundant exoerythrocytic forms, a tentative diagnosis of avian malaria (Plasmodium sp.) was made. Subsequent transmission electron microscopic examination of schizonts in formalized tissue revealed merozoites with tear-shaped rhoptries. Antimalarial therapy was initiated early but deaths continued for 5 mo. Mortality, which eventually totaled 83%, occurred in three distinct waves, each separated by a hiatus of approximately 1 mo. Despite examinations of repeated blood smears, intraerythrocytic Plasmodium relictum was not detected until late in the outbreak. Diagnosis was based on morphologic characteristics including schizonts with eight to 12 merozoites/segmenter and round gametocytes that displaced and turned the infected erythrocyte nucleus. In addition to malaria, penguins showed evidence of aspergillosis, bacterial enteritis (Escherichia coli; Proteus sp.; and Edwardsiella sp.), and helminthiasis (Contracaecum sp. and Tetrabothrius sp.). Based on gross and histological lesions, disease prevalence in this group of penguins was malaria 58%, aspergillosis 61%, enteritis 60%, helminthiasis 26%. Epidemiologic investigation including group transport history, disease prevalence in co-quarantined birds not sent to Des Moines and climatological data implicated Des Moines as the likely site for initial exposure, although information is not conclusive. Stress and concurrent disease certainly contributed to the severe mortality in this group of penguins infected with P. relictum.

Plasmodium gallinaceum: avian screen for drugs with radical curative properties.

Existing primary screens for radical curative antimalarial drugs fail to adequately detect many compounds which affect the latent, exoerythrocytic hypnozoite, the stage of the parasite responsible for relapse. At the same time, these screens falsely identify a wide range of compounds with no radical curative activity. The avian malaria, Plasmodium gallinaceum, and Aedes aegypti mosquitos were used in a screen which measures the effects of candidate compounds on gametocytes and their development within the mosquito. Sporontocidal and gametocytocidal effects could be differentiated by this screen. In a blind study, those compounds shown to be exclusively gametocytocidal were those same drugs which had previously been shown to have radical curative effects against true relapsing malarias. The chicken malaria gametocyte screen was more sensitive than the rodent screens in detecting useful compounds, with a minimum of false positive identifications.