Looks can deceive: Molecular identity of an intraerythrocytic apicomplexan parasite in Australian gliders

Two yellow-bellied gliders (Petaurus australis) had an intraerythrocytic parasite closely related to the cyst-forming coccidia (Apicomplexa: Sarcocystidae). The parasitaemia persisted for 3 months or more but was observed to clear within 3 years in captivity. The parasite appears not to significantly debilitate its infected host. Traditionally, using morphological identification, the intraerythrocytic parasite would have been classified within the Hepatozoon species typically found in red blood cells. However, molecular diagnostic techniques targeting the parasite's SSU rDNA and LSU rDNA demonstrated the unusual identity of this blood parasite and disputed its identity as a haemogregarine parasite of the genus Hepatozoon. The sequence was compared with available sequences from diverse mammalian and non-mammalian blood parasites (malaria, piroplasms, hemosporidia and sarcosporidia). The intraerythrocytic blood parasite was found to be most closely related to the cyst-forming coccidia including Besnoitia spp., Cystoisospora spp., Hammondia spp., Hyaloklossia lieberkuehni, Neospora caninum, Sarcocystis spp. and Toxoplasma gondii. The life cycle of this intraerythrocytic parasite remains unknown. The presented DNA identification demonstrates its suitability for an improved identification of blood parasites.     

Pigeons (Columba livia) are a suitable experimental model for Neospora caninum infection in birds

Neospora caninum infections in chickens have been recently described by epidemiological and experimental approaches, and these birds may be considered natural intermediate hosts of the parasite. It has been postulated that other bird species might perform this role in wildlife as well. To better understand the sylvatic life cycle of N. caninum, further studies are required. In that sense, this work aimed to observe infection kinetics in pigeons experimentally infected with N. caninum. Experimental infections were conducted in parallel with a related protozoan, Toxoplasma gondii, which has been already described as able to infect pigeons in nature. Our results demonstrated that N. caninum disseminated through various tissues of this host and induced parasite-specific IgG seroconversion. Infection parameters were similar to that observed in the T. gondii infected group, although N. caninum-infected pigeons presented lower IgG titers during acute phase. The results herein described demonstrate that pigeons are a suitable model for N. caninum infection, considering that these data are in agreement with those observed in chickens experimentally infected with this parasite. As pigeons may be revealed as important reservoirs for N. caninum infection in nature, future studies are necessary to determine the real prevalence of this parasite in this and other birds in wildlife.     

Prevalence and molecular analysis of Sarcocystis infections in cattle in Northwest Iran and the first global report of S. gigantea in cattle

Cattle are intermediate host for several species of Sarcocystis, including S. cruzi, S. hirsuta, and S. hominis with high prevalence worldwide. The present study aimed to determine the prevalence of Sarcocystis infection, species identification, and phylogenetic analysis of the parasite in cattle in Northwest Iran. The samples of diaphragm and esophagus from 290 cattle were collected from slaughterhouses in Northwest Iran and subjected to macroscopic, microscopic, and histopathology examinations, PCR-RFLP, sequencing and phylogenetic analyses. Tissue cysts of Sarcocystis spp. were detected in 92% of cattle by digestion and microscopic tests. Based on the PCR–RFLP and specific PCR, 87.9% and 1.03% of isolates were identified as S. cruzi, and S. hominis, respectively. Macrocyst was seen in a single sample that was identified as S. gigantea. The haplotype network exhibited the extension of the various haplotypes of S. cruzi between neighboring provinces in Northwest Iran. Heterogeneity analysis of S. cruzi 18S-rRNA sequences indicated genetic diversity among S. cruzi isolates (Haplotype diversity: 0.733-0.854) consisting 16 haplotypes; however, the nucleotide differences showed low diversity (0.01481 to 0.03351). Pair wise sequence distance matrix amongst S. cruzi sequences indicated an intra-species divergence of 0%-7.8% and identity of 92.6%-100%. Sarcocystis infection is highly prevalent in cattle in Northwest Iran, with the predominance of S. cruzi, and genetic variants of this species are unequivocally distributing in Northwest provinces. First global detection of S. gigantea in cattle reflects new insights of transmission dynamic and biology of this parasite in Iran.     

The molecular epidemiology of Cryptosporidium and Giardia infections in coyotes from Alberta,Canada, and observations on some cohabiting parasites

Coyotes from southern Alberta and Saskatchewan, Canada, were examined for the presence of Giardia and Cryptosporidium and cohabiting helminths. Toxascaris was present in over 90% of the 70 animals examined, and Taenia sp. in 6.5–25% of the two groups of animals studied. Giardia (12.5–21.7%) and Cryptosporidium (0–17.4%) were also common and molecular characterisation revealed both zoonotic and host-adapted genotypes of Giardia, whereas the Cryptosporidium proved to be a variant of the canine species C. canis. The seasonal variation observed in the occurrence of Cryptosporidium may be related to stress-induced shedding of the parasite.     

Survey of <Emphasis Type="Italic">Sarcocystis</Emphasis> infection in slaughtered cattle in Kerman,Iran

The parasite of genus Sarcocystis is one of the most commonly found parasite in domestic animals worldwide. Some species of Sarcocystis cause important economic loss when causing clinical and sub clinical disease. The aim of this study was to determine the prevalence of Sarcocystis in slaughtered Cattle in Kerman, Iran. The prevalence of Sarcocystis spp. infection was investigated in 480 cattle, slaughtered from May 2005 to February 2006 in the Kerman, Iran using naked eye examination for macroscopic Sarcocysts, and peptic digestion, muscle squash, squeezing methods for microscopic types. Muscles from heart, tongue, and esophagus, cervical and abdominal muscles of 480 slaughtered cattle were examined for Sarcocystis cysts. The prevalence of microscopic Sarcocystis cysts in cattle was detected in 100% and there was no macroscopic cyst in examined cattle.     

Gastrointestinal parasites of cavy (Cavia aperea aperea) in southern Brazil

The aim of this study was to evaluate the gastrointestinal parasitism in Cavia aperea aperea (cavy), captured in the central region of Rio Grande do Sul State. Fecal samples from five free-living cavies were collected for research of parasites. Samples were analyzed by the centrifugal-flotation method with zinc sulfate and parasites were identified microscopically based on (oo)cyst and egg size and morphology. Cysts of Giardia sp. and (oo)cysts of Cryptosporidium sp. and Cystoisospora sp. were observed in one or more cavies. Eggs of Paraspidodera uncinata were observed in three of the five rodents. All infected animals showed mild infection by parasite. This is the first report of Giardia sp., Cryptosporidium sp. and Cystoisospora sp. in Cavia a. aperea.     

Cryopreservation of Trypanosoma evansi after DEAE-cellulose purification: Evaluation of infective parameters

Cryopreservation is a method of keeping parasites alive in a laboratory. However, this technique may also damage the parasite. Alternatively, parasites may be maintained by in vitro culture. Unfortunately, for Trypanosoma evansi no effective medium that is able to maintain the parasite for more than 4 months has been described. In this study, we examined the effect of purifying trypomastigote through DEAE-cellulose chromatography before and after cryopreservation, by analyzing the pre-patent period, longevity, parasitemia, and count of viable parasites. Our results showed a three-times increase in the concentration of viable trypomastigote in DEAE-purified cryopreserved parasites as compared to non-DEAE-purified cryopreserved parasites. This indicates that DEAE-cellulose chromatography followed by cryopreservation is an effective method for the storage and preservation of T. evansi, with the advantage that the stocked parasites will be ready to use in molecular biology procedures.     

Latitudinal variability in the seroprevalence of antibodies against Toxoplasma gondii in non-migrant and Arctic migratory geese

Toxoplasma gondii is an intracellular coccidian parasite found worldwide and is known to infect virtually all warm-blooded animals. It requires a cat (family Felidae) to complete its full life cycle. Despite the absence of wild felids on the Arctic archipelago of Svalbard, T. gondii has been found in resident predators such as the arctic fox and polar bear. It has therefore been suggested that T. gondii may enter this ecosystem via migratory birds. The objective of this study was to identify locations where goose populations may become infected with T. gondii, and to investigate the dynamics of T. gondii specific antibodies. Single blood samples of both adults and juveniles were collected from selected goose species (Anser anser, A. brachyrhynchus, Branta canadensis, B. leucopsis) at Arctic brood-rearing areas in Russia and on Svalbard, and temperate wintering grounds in the Netherlands and Denmark (migratory populations) as well as temperate brood-rearing grounds (the Netherlands, non-migratory populations). A modified agglutination test was used on serum, for detection of antibodies against T. gondii. Occasional repeated annual sampling of individual adults was performed to determine the antibody dynamics. Adults were found seropositive at all locations (Arctic and temperate, brood-rearing and wintering grounds) with low seroprevalence in brood-rearing birds on temperate grounds. As no juvenile geese were found seropositive at any brood-rearing location, but nine month old geese were found seropositive during spring migration we conclude that geese, irrespective of species and migration, encounter T. gondii infection in wintering areas. In re-sampled birds on Svalbard significant seroreversion was observed, with 42% of seropositive adults showing no detectable antibodies after 12 months, while the proportion of seroconversion was only 3%. Modelled variation of seroprevalence with field data on antibody longevity and parasite transmission suggests seroprevalence of a population within a range of 5.2–19.9%, in line with measured values. The high occurrence of seroreversion compared to the low occurrence of seroconversion hampers analysis of species- or site-specific patterns, but explains the absence of an increase in seroprevalence with age and the observed variation in antibody titre. These findings imply that even though infection rate is low, adults introduce T. gondii to the high Arctic ecosystem following infection in temperate regions.     

Growth hormones therapy in immune response against Trypanosoma cruzi

Growth hormone (GH) is an important hypophyseal hormone that is primarily involved in body growth and metabolism. In mammals, control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. To explore the possibility that GH might be effective in the treatment of Chagas’ disease, we investigated its effects on the course of T. cruzi infection in rats, focusing our analyses on its influences on parasitemia, NO, TNF-α and IFN-γ concentration and on histopathological alterations and parasite burden in heart tissue. T. cruzi-infected male Wistar rats were intraperitoneally treated with 5 ng/10 g body weight/day of GH. Animals treated with GH showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals (P < 0.05). For all experimental days (7, 14 and 21 post infection) of the acute phase, infected and GH treated animals reached higher concentrations of TNF-α, IFN-γ and nitric oxide as compared to untreated and infected counterparts (P < 0.05) Histopathological observations of heart tissue revealed that GH administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that GH can be considered as an immunomodulator substance for controlling parasite replication and combined with the current drug used may represent in the future a new therapeutic tool to reduce the harmful effects of Chagas’ disease.     

An outbreak of massive mortality among farm rabbits associated with Cryptosporidium infection

Cryptosporidium in farm rabbits is not often recognised due to a low prevalence and asymptomatic course of infection. Nonetheless, incidences of fatal diarrhoeic diseases are frequently noticed in the rabbitries. In this article, we report an outbreak where there was massive mortality among farm rabbits associated with Cryptosporidium infection. The disease was characterised by profuse diarrhoea resulting in the death of rabbits. A pooled faecal sample was screened for a presence of parasites using microscopy methods. In the tested sample no other parasites other than Cryptosporidium oocysts were found. Further identification of the parasite species was performed at a molecular level, using the 18 SSU rRNA, COWP and LIB13 PCR followed by a subtyping at the GP60 gene locus. Sequence analysis of GP60 gene fragment revealed the presence of a novel subtype VbA24 of Cryptosporidium cuniculus. In this outbreak a Cryptosporidium protozoan parasite played a major role in the etiology of the gastrointestinal disorders in rabbits resulting in massive mortality of the infected animals.     

Bartonella melophagi in Melophagus ovinus (sheep ked) collected from sheep in northern Oromia,Ethiopia

Melophagus ovinus (sheep ked) is one of the most common ectoparasites that contributes to enormous economic losses in the productivity of sheep in many countries. The present study was conducted from January 2012 to July 2013 on M. ovinus collected from sheep at three sites in Ethiopia. Of the sheep studied, 65.7% (88/134) were infested with M. ovinus. The prevalence of M. ovinus was 76% (76/100), 47% (8/17) and 23.5% (4/17) at the Kimbibit, Chacha and Shano sites, respectively. An overall number of 229 M. ovinus specimens (138 females, 86 males and five pupae) and 554 M. ovinus specimens (272 females, 282 males) were collected from young and adult sheep, respectively. Bartonella DNA was detected in 89% (694/783) of M. ovinus using a quantitative Bartonella genus-specific PCR assay targeting the 16S/23S rRNA intergenic spacer region. The sequencing of the PCR products of fragments of the gltA and rpoB genes showed 99.6–100% and 100% homology, respectively, with B. melophagi. Statistically significant variation was not noted in the overall prevalence of Bartonella DNA between female and male M. ovinus. All of the sheep infested with M. ovinus 100% (88/88) harbored at least one M. ovinus specimen that contained Bartonella DNA. This study highlights that B. melophagi in M. ovinus from sheep in highlands in Ethiopia possibly has certain zoonotic importance.     

Effect of zinc supplementation in pregnant mice during experimental Trypanosoma cruzi infection

Zinc is an essential micronutrient and has significant effects on human growth, development, and immune function. Zinc supplementation or deficiency may affect the course of infection. Zinc enhances immune response against a wide range of viral, bacterial, and parasitic pathogens. In the present study, we investigated the effects of zinc sulphate (ZnSO₄) supplementation (20 mg/kg/day) during pregnancy in mice, Swiss Webster strain infected by the Y strain of Trypanosoma cruzi. Oral supplementation of zinc sulphate in pregnant and non-pregnant infected animals did not affect the count of blood parasites as well as tissue parasitism in the heart, liver, and spleen. Zinc supplementation did not alter female body weight, the length of fetuses and neonates, placental size/weight and mortality rate. Among zinc supplied animals, no significant plasmatic zinc concentrations were observed. Concerning to tissue zinc concentrations, only the liver displayed enhanced values as compared to other organs. For placental parasitism, zinc supplied group displayed a significant decrease in amastigote burdens (P < 0.05). However due to the reduced number of parasite burdens in placenta of animals supplied with zinc, these data suggest that zinc was partially effective in up-regulating the host’s immune response against parasite, probably attenuating the infection in fetuses.     

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca²⁺ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca²⁺ concentration in the cytosol of the parasite cells. The increased intracellular Ca²⁺ concentration following these treatments was confirmed using live cell Ca²⁺ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca²⁺ signalling pathway in the egress of B. bovis merozoites.     

First isolation of Neospora caninum from the faeces of a dog from Portugal

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of γ-interferon knockout mice with 10² sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.     

Prevalence and antibiotic resistance profiles of Enterococcus species in chicken at slaughter level; absence of vanA and vanB genes in E. faecalis and E. faecium

The prevalence of enterococci in neck skin samples of poultry from Ankara region in Turkey was investigated and their antibiotic resistance patterns were determined. In the study, 83 of 106 analyzed neck skin samples were positive for Enterococcus, with E. faecium as the most prevalent species (48%) followed by E. durans (23%) and E. faecalis (19%). Lower numbers were detected for E. gallinarum, E. hirae, E. mundtii and E. casseliflavus. Using the disc diffusion method, it was established that over 90% of E. faecium and E. faecalis isolates were high-level resistant against erythromycin and tetracycline. Four E. faecium isolates were additionally resistant to chloramphenicol, gentamicin and streptomycin, though they were susceptible to penicillin G. The most frequently observed multiple resistance in E. faecium (25%) was against erythromycin, tetracycline, chloramphenicol and streptomycin. Of the E. faecalis isolates, 44% were multiple resistant to erythromycin, tetracycline and streptomycin. Vancomycin resistance could not be demonstrated phenotypically and vanA or vanB genes were not detected by multiplex PCR in any of the isolates. Nevertheless, the observed resistance patterns are of concern for public health.     

Identification of novel immunogenic proteins in pathogenic Haemophilus parasuis based on genome sequence analysis

Haemophilus parasuis causes contagious porcine Glässer's disease, which is occurring worldwide and leads to severe losses in the pig industry. To identify novel antigen candidates against this disease, 22 surface-exposed or secreted proteins were selected from the annotated H. parasuis genome by reverse vaccinology strategy. Expression of these proteins in Escherichia coli was attempted. Immunogenicity of the expressed candidates was assessed using Western blot analysis with mouse-derived antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5. Three ABC-type transporters (OppA, YfeA and PlpA) and 1 curli protein assembly (CsgG) were identified as potent immunogenic proteins. The proteins show cross-reactions when tested with sera raised against serovars 4 and 5 of H. parasuis.     

Evidence for a relationship between Leishmania load and clinical manifestations

Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmaniainfantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis.The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis.Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

Update on the pathogenesis of Haemophilus parasuis infection and virulence factors

Haemophilus parasuis is the causative agent of Glässer's disease in pigs, a severe systemic disease that has led to increasing economic losses in the pig industry worldwide. The H. parasuis genome sequence has been completed, but the function and essentiality of the annotated genes remain largely unknown, especially virulence factors. The recent developments in the efficient genetic manipulation of H. parasuis have greatly facilitated the study of gene function, pathogenesis mechanisms and virulence factors. In this review, we provided update information regarding that (i) how the pathogen overcome host immune responses and cell barriers which were tightly associated with the pathogenesis, and (ii) the several recent identification of virulence factors were involved in evading the immune responses and cell barriers in H. parasuis.     

Anti-Leishmania IgA in urine samples from dogs with clinical leishmaniasis

Recently, anti-Leishmania IgG has been detected in urine samples from Leishmania-infected dogs and its concentrations have been correlated with impairment of renal function. The presence and relationship with other anti-Leishmania Ig isotypes in urine have not yet been investigated. The current study analyzed the concentrations of anti-Leishmania IgA and IgG in sera (Ig-S) and urine (Ig-U) samples by ELISA in 64 untreated dogs with clinical leishmaniasis. All 64 serum samples tested were positive for anti-Leishmania IgG. Fifty of them (78.1%) were also positive for anti-Leishmania IgA. The results showed the presence of anti-Leishmania IgA-U in 38% of the 50 dogs that were positive for specific IgA-S. Thirty-eight of the 64 dogs positive for Leishmania-specific IgG-S (59.4%) were also positive for Leishmania-specific IgG in urine (IgG-U). The concentrations of anti-Leishmania IgA-U were significantly correlated with urine protein/creatinine (uP/C) ratio (ρ = 0.542; P < 0.001) and with serum biochemical parameters, such as γ-globulins, urea and creatinine. Goldmann–Witmer coefficient (C value) indicated that detection of specific IgA in urine samples from dogs with leishmaniasis might not only be due to impairment of filtration of the glomerular barrier but also be due to local production of this isotype, which might reflect a local immunological response to the presence of the parasite in the genitourinary tract. Anti-Leishmania IgG-U concentrations were highly correlated with uP/C ratio (ρ = 0.779; P < 0.001) and C value did not support in any case local production of this isotype. IgG isotype might be a more suitable and specific tool to evaluate renal damage due to the lower IgA-U sensitivity and correlation coefficients and evidence of IgA local production. However, dogs found positive for both Ig isotypes in urine presented significantly higher specific IgG-U concentrations and higher uP/C ratios than dogs found positive only for IgG-U, thus suggesting that the first group suffered more severe renal damage. This fact makes it necessary to evaluate the prognosis of dogs showing both anti-Leishmania IgA-U and IgG-U in future studies.