Does avian malaria reduce fledging success: an experimental test of the selection hypothesis

Like many parasites, avian haematozoa are often found at lower infection intensities in older birds than young birds. One explanation, known as the “selection” hypothesis, is that infected young birds die before reaching adulthood, thus removing the highest infection intensities from the host population. We tested this hypothesis in the field by experimentally infecting nestling rock pigeons (Columba livia) with the malaria parasite Haemoproteus columbae. We compared the condition and fledging success of infected nestlings to that of uninfected controls. There was no significant difference in the body mass, fledging success, age at fledging, or post-fledging survival of experimental versus control birds. These results were unexpected, given that long-term studies of older pigeons have demonstrated chronic effects of H. columbae. We conclude that H. columbae has little impact on nestling pigeons, even when they are directly infected with the parasite. Our study provides no support for the selection hypothesis that older birds have lower parasite loads because parasites are removed from the population by infected nestlings dying. To our knowledge, this is the first study to test the impact of avian malaria using experimental inoculations under natural conditions.     

Geographical variation in blood parasites in feral pigeons: the role of vectors

Prevalence and intensity of blood parasites are known to vary in space within a same species, yet the causes underlying such variation are poorly known. Theoretically, blood parasites variation can be attributed to differences to exposure to parasite vectors and/or to differences in host susceptibility. Here, we show that prevalence of Haemoproteus columbae in feral pigeons Columba livia varied among five near-by populations (range 15%-100%), paralleled by variation in the abundance of its main vector, the louse flies Pseudolynchia canariensis. Geographic variation in intensity of blood parasites did not covary with abundance of vectors. Within populations, older individuals had a higher probability of being parasitized than younger ones, whereas younger birds, when infected, suffered higher intensities. Furthermore, we found no evidence of sex-related differences neither in prevalence nor in intensity of blood parasite infections. To demonstrate that geographical variation in prevalence was actually due to differences in vector exposure, we conducted two experiments based on translocation of unparasitized pigeons from a vector-free area to an area where both the parasite and vector were abundant. With the first experiment, we demonstrated that unparasitized pigeons were not resistant to the parasite because when transmission was possible pigeons became parasitized in a few months. With the second experiment, in which half of the pigeons were prevented from contacts with the vector, we ruled out the posibility that pigeons we considered as unparasitized would have suffered from latent infections. Therefore, both observational and experimental evidence supports the view that vector abundance is the major factor influencing the spatial variation in prevalence of H. columbae in pigeons.     

Serological responses and immunity to superinfection with avian malaria in experimentally-infected Hawaii amakihi

Six of seven Hawaii Amakihi (Hemignathus virens) with chronic malarial infections had no increases in peripheral parasitemia, declines in food consumption, or loss of body weight when rechallenged with the homologous isolate of Plasmodium relictum 61 to 62 days after initial infection. Five uninfected control amakihi exposed at the same time to infective mosquito bites developed acute infections with high parasitemias. Reductions in food consumption and loss of body weight occurred in all control birds and three of these individuals eventually died. When surviving birds were rechallenged >2 yr later with either the same parasite isolate or an isolate of P. relictum collected on the island of Kauai, all individuals were immune to superinfection. Chronically infected birds developed antibodies to a common suite of malarial antigens ranging in size from 22 to 170 kDa that were detectable as early as 8 days post infection on immunoblots of SDS-polyacrylamide gels. Antibodies to this suite of malarial antigens persisted as long as 1,248 days after initial infection and were consistently detectable at times when parasites were not easily found by microscopy on Giemsa-stained blood smears. The immunoblotting method that is described here appears to be an effective technique for identifying birds with chronic, low-intensity malarial infections when circulating parasites are not easily detectable by microscopy. Hawaiian honeycreepers that are capable of recovering from acute infections develop concomitant immunity to superinfection, making them functionally immune in areas where malaria transmission has become endemic.     

Toxoplasma gondii: Congenital transmission in a hamster model

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non-invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n = 168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated. Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

Analysis of Toxoplasma gondii surface antigen 2 gene (SAG2). Relevance of genotype I in clinical toxoplasmosis

Toxoplasma gondii is one of the most successful protozoan parasites given its ability to manipulate the immune system and establish a chronic infection. It is a parasite with a significant impact on human health, mainly in immunocompromised patients. In Europe and North America, only a few clonal genotypes (I, II and III) seem to be responsible for the vast majority of Toxoplasma infections. Surface antigen 2 gene (SAG2) has been extensively used for genotyping T. gondii isolates. The analysis of this locus reveals that in Northern hemisphere, human disease causing isolates are mainly type II, whereas T. gondii isolated from different animals are both type II and III. Since the immune response depends on parasite genotype, it seems relevant to characterize parasites producing human toxoplasmosis in different geographical areas. The growing information about the prevalent T. gondii genotypes in South America mostly refers to domestic animals. This is the first report of genetic characterization of T. gondii isolates from clinical samples in Chile, South America. All the samples analyzed corresponded to SAG2 type I isolates, and they differ from classic SAG2 type I by genetic polymorphisms. This study contributes to the scarce available information on T. gondii at South America, and reinforces an emerging concept suggesting that SAG2 type I, rather than II, parasites are a frequent cause of clinical toxoplasmosis in this continent.     

Effects of Birds Ingesting Heterodera rostochiensis Cysts on Viability of Eggs and Larvae

Rate of passage through the digestive systems and effects of ingestion on viability of contents of cysts of Heterodera rostochiensis were determined in feeding trials with pigeons, thrushes, starlings, cowbirds, sparrows, and quails. Depending upon species of birds, 12-82% of the cysts ingested passed through the digestive system within 0.5 h. Pigeons required 6 h for complete evacuation. All other birds cmnpletely evacuated ingested cysts from their digestive systems within 3 h. Contents of cysts were nonviable if they were retained in the digestive system of starlings for more than 1.5 h, pigeons more than 1 h, or other birds more than 0.5 h. Cyst contents were nonviable if they remained in contact with excreta from cowbirds or quails for 4 h, thrushes for 96 h, or other species for 72 h after passage. Viability of contents of cysts was inversely related to exposure to excreta-filtrate concentration. Larvae failed to emerge from cysts that were exposed to a 25% concentration of excreta filtrate from starlings, 50% concentration from pigeons or thrushes, or 100% concentration of excreta filtrates from each of the other species. Cysts that were subjected to 44 C (avg. body temperature of cowbirds) for more than 3.5 h were nonviable. Cysts that passed through birds and collected with excrement on polyethylene or soil produced no infective larvae on potato.     

Experimental toxoplasmosis in budgerigars (Melopsittacus undulatus)

The susceptibility of budgerigars (Melopsittacus undulatus) to graded doses of Toxoplasma gondii oocysts was studied. Sixteen budgerigars were divided into 4 groups (A-D) of 4 each. Birds in groups A-C were fed 100,000, 1,000, or 100 infective oocysts of the VEG strain of T. gondii, respectively. Budgerigars in group D were not fed oocysts and served as controls. All 4 birds in group A died (or were killed) because of acute severe enteritis 5 or 6 days after feeding oocysts (DAFO). Three of the 4 birds in group B were killed (or died) because of toxoplasmosis 9 or 14 DAFO. One budgerigar in group C and the 4 budgerigars in group D remained healthy and were killed 35 or 39 DAFO. Toxoplasma gondii was demonstrated in tissues of all budgerigars fed oocysts. The control budgerigars remained clinically normal and showed no evidence of T. gondii exposure. These results indicate that, compared to other passerines, budgerigars are relatively resistant to clinical toxoplasmosis.     

Experimental Toxoplasmosis in Chimpanzees

Two chimpanzees were given by mouth large numbers of viable oocysts of Toxoplasma gondii obtained from the faeces of experimentally infected cats. Before the experiment the first chimpanzee had a positive dye test reaction (1:250), an indication that it had undergone an earlier infection of toxoplasmosis; the serum antibody titres remained unchanged, no evidence of illness was found, and oocysts did not appear in its faeces during the subsequent six weeks. The second chimpanzee showed a negative dye test reaction before infection, and this converted to positive on the 7th day, rose to a peak on the 35th day, and remained high for six months. This animal appeared unwell during the first week, and on the 7th day its blood proved infective to mice; on the 40th day the lymph nodes became enlarged and biopsy specimens of a node and muscle in the 11th week were also infective to mice. No oocysts were passed in the faeces. The presumed cycle in the chimpanzee and in man and the relationships between Toxoplasma and Isospora are discussed.     

The Toxoplasma MAG1 peptides induce sex-based humoral immune response in mice and distinguish active from chronic human infection

To distinguish active from inactive/chronic infection in Toxoplasma gondii-seropositive individuals, we have developed an enzyme-linked immunosorbent assay (ELISA) using specific peptides derived from Toxoplasma matrix antigen MAG1. We used this assay to measure matrix specific antibodies and pilot studies with infected mice established the validity of two peptides. The immune response against MAG1 occurs in about 12 days postinfection and displays a sex difference later on in mouse model, with males producing higher antibody titers than females. Serum samples from 22 patients with clinical toxoplasmosis and from 26 patients with serological evidence of past exposure to Toxoplasma (more than one year infection history) were analyzed. Both MAG1 peptides detected antibodies significant frequently and robustly from active stage than from the chronic stage of toxoplasmosis. The results indicate that both MAG1 peptides may be used as a tool to differentiate active from inactive infection. It also may be considered in the design of potential vaccines in humans.     

A novel luciferase-linked antibody capture assay (LACA) for the diagnosis of Toxoplasma gondii infection in chickens

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the most common parasitic zoonosis worldwide in multiples species of mammals and birds. Although free-range chickens may play a role as an important reservoir for T. gondii, there is no reliable and commercially available diagnostic test for this disease in chickens. In this study, we aimed to develop a novel Luciferase-linked Antibody Capture Assay (LACA) for the serodiagnosis of Toxoplasma infection in chickens. Recombinant nanoluciferase fused-T. gondii dense granule antigen 8 (rNluc-GRA8) was produced and applied to LACA assay as a diagnostic antigen. GRA8-LACA was tested with the sera from uninfected and experimentally infected chickens with T. gondii and other parasitic pathogens and showed unexpectedly high sensitivity (90.5%) and specificity (95.4%). Interestingly, E. coli lysate expressing rNluc-GRA8 could be applied in GRA8-LACA with 85.7% sensitivity and an increased specificity (96.9%) that gave better diagnostic performance compared to conventional ELISA. We applied our diagnostic system to examine 267 free-range chicken sera collected from 12 farms and 100 closed-house broiler chicken sera from local poultry abattoirs. The overall seroprevalence of toxoplasmosis in free-range chickens was 10.9% (95% CI: 10.6%–11.1%), while no positive case was found in broiler chickens. GRA8-LACA could be a useful diagnostic technique for T. gondii infection in chickens. The detection of T. gondii seropositive chickens in this study warns a potential risk of Toxoplasma transmission by the consumption of raw or undercooked chicken meat.     

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2?μM to as low as 110?nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5?brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.     

Contrasting Effect of Prepulse Signals on Performance of Toxoplasma-Infected and Toxoplasma-Free Subjects in an Acoustic Reaction Times Test

Background

About 30% of people on Earth have latent toxoplasmosis. Infected subjects do not express any clinical symptoms, however, they carry dormant stages of parasite Toxoplasma for the rest of their life. This form of toxoplasmosis is mostly considered harmless, however, recent studies showed its specific effects on physiology, behaviour and its associations with various diseases, including psychiatric disorders such as schizophrenia. Individuals who suffer from schizophrenia have about 2.7 times higher prevalence of Toxoplasma-seropositivity than controls, which suggests that some traits characteristic of schizophrenic patients, including the sex difference in schizophrenia onset, decrease of grey matter density in specific brain areas and modification of prepulse inhibition of startle reaction could in fact be caused by toxoplasmosis for those patients who are Toxoplasma-seropositive.

Methodology/Principal Findings

We measured the effect of prepulse inhibition/facilitation of the startle reaction on reaction times. The students, 170 women and 66 men, were asked to react as quickly as possible to a startling acoustic signal by pressing a computer mouse button. Some of the startling signals were without the prepulse, some were 20 msec. preceded by a short (20 msec.) prepulse signal of lower intensity. Toxoplasma-seropositive subjects had longer reaction times than the controls. Acoustic prepulse shorted the reaction times in all subjects. This effect of prepulse on reaction times was stronger in male subjects and increased with the duration of infection, suggesting that it represented a cumulative effect of latent toxoplasmosis, rather than a fading out after effect of past acute toxoplasmosis.

Conclusions

Different sensitivity of Toxoplasma-seropositive and Toxoplasma-seronegative subjects on effect of prepulses on reaction times (the toxoplasmosis-prepulse interaction) suggested, but of course did not prove, that the alternations of prepulse inhibition of startle reaction observed in schizophrenia patients probably joined the list of schizophrenia symptoms that are in fact caused by latent toxoplasmosis.     

The prevalence and intensity of external parasites in domestic pigeons (Columba livia domestica) in Egypt with special reference to the role of deltamethrin as insecticidal agent

This study aimed to investigate the prevalence and intensity of external parasites in domestic pigeons in Giza, Egypt, from January 2020 to December 2020. A total of 300 domestic pigeons (25 pigeons per month) were examined. The birds were divided into groups based on their age. The oxidative stress parameters; serum zinc concentration, serum malondialdehyde (MDA), and serum Nitric oxide were evaluated in single and mixed external parasitic infestations. The prevalence of external parasites in examined pigeons was 80.3%. The detected parasites were Pseudolynchia canariensis (P. canariensis), Hippobosca equina (H. equina), Columbicola columbae (C. columbae), Menopon gallinae (M. gallinae), Knemidocoptes species (spp.) and Dermanyssus gallinae (D. gallinae); their incidences were 41.6, 26, 7, 5,0.33 and 0.33%, respectively. The highest infestation was recorded in both spring and summer. . The incidence of disease was higher in squabs and young birds than in adults. The mixed external parasitic infestation was recorded in this study. The infected birds showed decreased serum zinc concentration and elevated MDA and serum Nitric oxide levels. In conclusion, regular monthly treatment with deltamethrin is recommended as an effective drug in the treatment of the infested birds and succeeded in reducing the incidence of externalparasites in the treated birds; in addition, pigeon management measures must be implemented to reduce the risk of external parasites.     

Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus)

Brugia pahangi has been found to be primarily a lymphatic-dwelling parasite in jirds when infections are induced by the subcutaneous injection of infective larvae or by allowing infected Aedes aegypti to feed.Migration to the regional lymphatics occurred as early as 1–4 days. Although some injected larvae remained in the skin for as long as 30 days and some became localized in the heart, lungs, pleural cavity, or peritoneal cavity, about three-fourths of the recovered filariae were found in the regional lymphatics. In contrast, when larvae were injected peritoneally they remained largely in the peritoneal cavity for at least 30 days.The relevant lymphatics and their drainage patterns in jirds have been described.The major pathological changes noted in jirds involved the regional lymphatic vessels and nodes, which were severely affected when they contained dead worms. Pulmonary granulomas due to dead microfilariae and occasionally to dead larvae or adult worms were noted.Observations are included on the susceptibility and course of B. pahangi infections in jirds.     

Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.     

Characterization of ROP18 alleles in human toxoplasmosis

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis.     

Toxoplasmosis: stages of the protozoan life cycle and risk assessment in humans and animals for an enhanced awareness and an improved socio-economic status

Toxoplasma gondii is a protozoan parasite distributed globally. It causes toxoplasmosis, which is prevalent in animals, birds, and soil. T. gondii infection leads to severe pathological impacts in immunodeficient patients and congenital cases. This review indicated that high prevalence groups had close contact with cats, dogs, consumed uncooked raw fruits, meat, or vegetables and the socio-economic level noted to be one of the crucial factors that influence toxoplasmosis. Toxoplasmosis infection is high in low-income countries and low in developed European countries. Immunosuppressed groups and pregnant women were the highly vulnerable groups. The epidemiology of the parasite enumerated various routes of infections; but consumption of T. gondii contaminated food was the major route of disease transmission. However, the role of meat and meat-producing animals on disease transmission remained unclear. Unfiltered water acts as the primary reservoir of toxoplasmosis transmission. The diagnostic methods for determining T. gondii infection are not the gold standard, and different approaches have been prescribed to analyze the infected populations based on the organs affected. Although toxoplasmosis was reported before 70 years, no appropriate solution noted to be recommended to treat this disease. Based on the present analyses, it concluded that the eradication of toxoplasmosis would be challenging from the world until people''s socio-economic level is improved. The main aim of the present study was to analyze and update the disease transmission, epidemiology, and possible clinical interventions of toxoplasmosis.