Protozoal DNA Research Articles

Effects of Garlic Oil and Cinnamaldehyde on Sheep Rumen Fermentation and Microbial Populations in Rusitec Fermenters in Two Different Sampling Periods.

Garlic oil (GO) and cinnamaldehyde (CIN) have shown potential to modify rumen fermentation. The aim of this study was to assess the effects of GO and CIN on rumen fermentation, microbial protein synthesis (MPS), and microbial populations in Rusitec fermenters fed a mixed diet (50:50 forage/concentrate), as well as whether these effects were maintained over time. Six fermenters were used in two 15-day incubation runs. Within each run, two fermenters received no additive, 180 mg/L of GO, or 180 mg/L of CIN. Rumen fermentation parameters were assessed in two periods (P1 and P2), and microbial populations were studied after each of these periods. Garlic oil reduced the acetate/propionate ratio and methane production (p < 0.001) in P1 and P2 and decreased protozoal DNA concentration and the relative abundance of fungi and archaea after P1 (p < 0.05). Cinnamaldehyde increased bacterial diversity (p < 0.01) and modified the structure of bacterial communities after P1, decreased bacterial DNA concentration after P2 (p < 0.05), and increased MPS (p < 0.001). The results of this study indicate that 180 mg/L of GO and CIN promoted a more efficient rumen fermentation and increased the protein supply to the animal, respectively, although an apparent adaptive response of microbial populations to GO was observed.

Ruminal Protozoal Populations of Angus Steers Differing in Feed Efficiency.

Simple SummaryThe rumen protozoa have been demonstrated to enhance methanogenesis and impact intraruminal recycling of microbial protein. However, they are also known to contribute to fiber degradation and the stabilization of ruminal pH changes. The apparent metabolic impact of ciliated protozoa in the rumen may contribute to the variation in feed efficiency. This study examined the relationship between the rumen protozoa and feed efficiency in beef steers. We monitored feed intake and body weight for a 70-day feed efficiency trial. Following the trial, rumen content was collected, protozoal DNA was extracted from the content, and the relationship between protozoa community diversity and species abundance with feed efficiency was examined. The ciliated protozoal community diversity differed between low- and high-feed efficient steers. Greater abundances of unidentified protozoa genera were detected in the low-feed efficient steers. These data suggest that unidentified protozoa and ciliated protozoal community diversity influence feed efficiency in beef steers. Feed accounts for as much as 70% of beef production costs, and improvement of the efficiency with which animals convert feed to product has the potential to have substantial financial impact on the beef industry. The rumen microbiome plays a key role in determining feed efficiency; however, previous studies of rumen microbiota have not focused on protozoal communities despite the estimation that these organisms represent approximately 50% of rumen content biomass. Protozoal communities participate in the regulation of bacterial populations and nitrogen cycling—key aspects of microbiome dynamics. The present study focused on identifying potential associations of protozoal community profiles with feed efficiency. Weaned steers (n = 50) 7 months of age weighing approximately 260 kg were adapted to a growing ration and GrowSafe for 2 weeks prior to a 70-day feed efficiency trial. The GrowSafe system is a feeding system that monitors feed intake in real time. Body weights were collected on the first day and then every 7 days of the feed efficiency trial, and on the final day, approximately 50 mL of rumen content were collected via orogastric tubing and frozen at −80 °C. Body weight and feed intake were used to calculate residual feed intake (RFI) as a measure of feed efficiency, and steers were categorized as high (n = 14) or low (n = 10) RFI based on ±0.5 standard deviations about the mean RFI. Microbial DNA was extracted, and the eukaryotic component profiled by amplification and sequencing of 18S genes using degenerate primers that can amplify this locus across a range of protists. The taxonomy of protozoal sequences was assigned using QIIME 1.9 and analyzed using QIIME and SAS 9.4 with significance determined at α ≤ 0.05. Greater abundances of unassigned taxa were associated with high-RFI steers (p = 0.03), indicating a need for further study to identify component protozoal species. Differences were observed between low- and high-RFI steers in protozoal community phylogenetic diversity, including weighted beta-diversity (p = 0.04), Faith’s phylogenetic diversity (p = 0.03), and observed Operational taxonomic unit (OTU) (p = 0.03). The unassigned taxa and differences in phylogenetic diversity of protozoal communities may contribute to divergences observed in feed efficiency phenotypes in beef steers.

Rangelia vitalii infection in a dog from São Paulo city, Brazil: case report

Canine rangeliosis is an extravascular hemolytic disease caused by the protozoan Rangelia vitalii, which is transmitted by ticks of the species Amblyomma aureolatum. Te most common clinical signs are apathy, hyperthermia and spontaneous bleeding. Anemia and thrombocytopenia are the most common hematological fndings. Tis work reports a clinical case of canine Rangeliosis treated at a private veterinary hospital, in São Paulo city in 2017. A dog was treated at a veterinary hospital in the north of São Paulo, with progressive weight loss, apathy and tail injury. Anemia and thrombocytopenia were observed on the hemogram. Rangelia vitalii DNA was detected in animal blood by real-time PCR (qPCR). In addition to the supportive treatment, doxycycline and subcutaneous imidocarb applications were used. Te sample collected afer treatment with the antibiotic continued to present protozoal DNA. Te disease should be considered as a diferential diagnosis and there is a great need for further studies about the therapy used.

Effects of modifications to retain protozoa in continuous-culture fermenters on ruminal fermentation, microbial populations, and microbial biomass assessed by two different methods

An important limitation of continuous-culture fermenters is their inability of maintaining microbial populations similar to those observed in the rumen, especially protozoa numbers, which usually decrease markedly or even disappear. Two approaches (a polyurethane-sponge (SP) and a filter system (FIL) for additionally retaining protozoa were tested in continuous culture system already designed to retain protozoa (Muetzel et al., 2009), and their effects on microbial populations, fermentation parameters and microbial biomass were assessed. Two 14-day incubation runs were carried out with 6 fermenters, and in each run two fermenters were randomly assigned to each of the experimental treatments (control, SP and FIL). Total protozoa numbers assessed by microscopic counting were 1.7 and 2.1 times greater in SP and FIL fermenters than in control ones on day 14, although differences did not reach the significance level (P = 0.855). Protozoal DNA concentration on day 14 were 1.6 and 1.4 times greater in SP and FIL fermenters, respectively, than in control ones, but differences were not significant (P = 0.524). Results from protozoal DNA concentrations determined in each fermenter on the different sampling days (n = 48) were positively correlated (P < 0.001) with the numbers of total (r = 0.826), entodiniomorphid (r = 0.824) and holotrich (r = 0.675) protozoa determined by microscopic counting, indicating that both methods are valid to assess protozoa populations. The proportion of holotrich in FIL-fermenters was relatively constant over the incubation period (ranging from 10.5 to 13.3% of total protozoa), but decreased with time in control (from 10.9% at day 2 to 6.7% at day 14) and SP (from 10.9 to 6.9%) fermenters. Neither the bacterial DNA concentration nor the relative abundance of fungal and archaeal DNA were influenced by any of the modifications tested, but values changed over the sampling period (days 10–14). Bacterial DNA concentration increased (P < 0.001) from day 10 to 14 in all fermenters, whereas the relative abundance of fungal and archaeal DNA decreased (P < 0.001). The tested modifications did not affect (P > 0.05) fermentation parameters, which reached a steady-state after 6 days of incubation. Values of microbial biomass determined using purine bases as a microbial marker were significantly correlated with the amount of bacterial plus protozoal DNA (r = 0.794; P = 0.002; n = 12) in each fermenter. In conclusion, the two tested modifications increased the protozoa numbers in continuous-culture fermenters, and the FIL allowed maintaining a proportion of holotrich protozoa similar to that in the ruminal fluid used as inoculum.

Vector-borne and zoonotic diseases of dogs in North-west New South Wales and the Northern Territory, Australia

BackgroundVector-borne diseases of dogs in Australian Aboriginal communities are relatively unexplored. These dogs represent a unique group with variable ecto- and endo-parasitic burdens, nutritional stresses and a general lack of veterinary intervention. We investigated haemoprotozoal and bacterial pathogen prevalences in relation to erythrocyte and platelet numbers in dogs from North-West New South Wales (N-W NSW) and the Northern Territory (NT; Central Australia).MethodsReal-time PCR (qPCR) amplification of Anaplasma platys, Babesia vogeli, Mycoplasma haemocanis, Candidatus Mycoplasma haematoparvum and Bartonella spp., serological screening for Coxiella burnetii, and Bartonella spp. and haematological analyses were performed on dogs from the two cohorts (96 dogs in total). Brucella suis serology was determined additionally for the N-W NSW cohort.ResultsAnaplasma platys (n = 26 dogs), Babesia vogeli (n = 7), Candidatus Mycoplasma haematoparvum (n = 10 dogs), and Mycoplasma haemocanis (n = 14) were detected in the sample population (n = 96) using qPCR. There were significant associations between (i) A. platys and anaemia (OR 8.7, CI 2.4–31.7; P < 0.001), thrombocytopenia (OR 12.1, CI 3.4–43.2; P < 0.001) and breed (OR 16.1, CI 2.1–121.5; P = 0.007), and (ii) between B. vogeli and anaemia (OR 11.8, CI 2.3–61.6; P = 0.003). Neither protozoal nor bacterial DNA loads, estimated using qPCR, were positively correlated with anaemia or thrombocytopenia. Haemotropic mycoplasmas were not associated with any haematologic abnormality. Four dogs from the NT were seropositive for Coxiella burnetii, while no dogs were seropositive for Brucella suis or to a panel of Bartonella spp. antigens. Despite directed efforts, Bartonella DNA was not detected in blood from any of the cohorts studied. A sample of dogs from the NT recruited specifically for Bartonella α-proteobacteria growth medium enrichment blood culture were also Bartonella PCR negative.ConclusionsVector-borne pathogens occur in dogs free ranging near Aboriginal communities, with higher detection rates in NT than N-W NSW. The preponderant haematologic abnormalities were anaemia and thrombocytopenia, likely attributable to A. platys and B. vogeli infections, but also probably affected by nutritional, parasitic, lactational and environmental stressors. The absence of Bartonella spp. is of importance to the Australian setting, and work needs to be extended to tropical coastal communities where fleas are present as well as ticks. Dogs living in and around Aboriginal communities may provide valuable sentinel information on disease infection status of human public health significance.

Shifts in microbial populations in Rusitec fermenters as affected by the type of diet and impact of the method for estimating microbial growth (15N v. microbial DNA)

Rusitec fermenters are in vitro systems widely used to study ruminal fermentation, but little is known about the microbial populations establishing in them. This study was designed to assess the time evolution of microbial populations in fermenters fed medium- (MC; 50% alfalfa hay : concentrate) and high-concentrate diets (HC; 15 : 85 barley straw : concentrate). Samples from solid (SOL) and liquid (LIQ) content of fermenters were taken immediately before feeding on days 3, 8 and 14 of incubation for quantitative polymerase chain reaction and automated ribosomal intergenic spacer analysis analyses. In SOL, total bacterial DNA concentration and relative abundance of Ruminococcus flavefaciens remained unchanged over the incubation period, but protozoal DNA concentration and abundance of Fibrobacter succinogenes, Ruminococcus albus and fungi decreased and abundance of methanogenic archaea increased. In LIQ, total bacterial DNA concentration increased with time, whereas concentration of protozoal DNA and abundance of methanogens and fungi decreased. Diet×time interactions were observed for bacterial and protozoal DNA and relative abundance of F. succinogenes and R. albus in SOL, as well as for protozoal DNA in LIQ. Bacterial diversity in SOL increased with time, but no changes were observed in LIQ. The incubated diet influenced all microbial populations, with the exception of total bacteria and fungi abundance in LIQ. Bacterial diversity was higher in MC-fed than in HC-fed fermenters in SOL, but no differences were detected in LIQ. Values of pH, daily production of volatile fatty acids and CH4 and isobutyrate proportions remained stable over the incubation period, but other fermentation parameters varied with time. The relationships among microbial populations and fermentation parameters were in well agreement with those previously reported in in vivo studies. Using 15N as a microbial marker or quantifying total microbial DNA for estimating microbial protein synthesis offered similar results for diets comparison, but both methods presented contrasting results for microbial growth in SOL and LIQ phases. The study showed that fermentation parameters remained fairly stable over the commonly used sampling period (days 8 to 14), but shifts in microbial populations were detected. Moreover, microbial populations differed markedly from those in the inocula, which indicates the difficulty of directly transposing results on microbial populations developed in Rusitec fermenters to in vivo conditions.

Influence of rumen contents’ processing method on microbial populations in the fluid and subsequent in vitro fermentation of substrates of variable composition

The in vitro batch culture technique is being increasingly used to study rumen fermentation, but the results are affected by several factors, being the source of the inoculum one of the most relevant. This work was conducted to assess the effects of different processing methods of ruminal contents on microbial populations in the obtained fluid, and its influence on fermentation parameters when the fluid was used as inoculum for in vitro incubations. Rumen contents were obtained from four rumen-fistulated sheep fed a 2:1 alfalfa hay:concentrate diet and subjected to the following treatments: SQ: squeezed through four layers of cheesecloth; FL: SQ treatment and further filtration through a 100-μm nylon cloth; STO: blended for three min at 230revmin−1 in a Stomacher® and further filtrations as in SQ. Microbial populations’ abundance and bacterial diversity in the ruminal fluids were analysed by quantitative PCR (qPCR) and automated ribosomal intergenic spacer analysis (ARISA), respectively. Three forages (alfalfa hay, grass hay and barley straw) were incubated in vitro, either alone or mixed with concentrate (1:1), using each of the ruminal fluids as inoculum. There were no differences between SQ and FL methods in any of the microbial populations analysed, but STO increased the relative abundance of Fibrobacter succinogenes and Ruminococcus albus (P<0.05) and decreased (P<0.05) the concentration of protozoal DNA compared with SQ. Bacterial diversity was not affected (P>0.05) by the processing methods. There were no interactions (P>0.05) between the processing method and the characteristics of the substrates for any of the fermentation parameters analysed. Compared with SQ, the STO method resulted in greater (P<0.05) methane production and ammonia-N concentrations in 8h incubations. After 24h of incubation, the use of STO inoculum increased (P<0.05) methane production and dry matter degradability compared with SQ, with no differences in the rest of the parameters. No differences between SQ and FL methods were detected in any parameter. The results show that stomaching the rumen contents prior to inoculation of in vitro cultures modified some microbial populations, but had only subtle effects on fermentation parameters.

Influence of forage type in the diet of sheep on rumen microbiota and fermentation characteristics

Four ruminally cannulated sheep were used in a cross-over design to assess the changes in rumen fluid microbial populations during the feeding cycle as affected by the type of forage (FOR) in the diet. The two experimental diets contained 70% of either alfalfa hay (AL) or grass hay (GR) as FOR and 30% of concentrate. Sheep were fed the diets twice daily and samples from rumen fluid were taken at 0, 4 and 8h after the morning feeding. Ruminal pH, lactate concentrations and xylanase activity were not affected (P>0.05) by FOR, but concentrations of NH3–N and total volatile fatty acid (VFA), and carboxymethylcellullase (CMCase) and amylase activities were greater (P<0.05) for AL compared with GR diet. Total protozoa numbers determined by microscopic counting were higher (P<0.05) in AL-fed sheep than in sheep fed the GR diet; in contrast, concentrations of protozoal DNA determined by quantitative real-time PCR tended (P=0.06) to be higher in GR-fed sheep, and no correlation (P>0.05) between protozoal numbers and total protozoal DNA concentrations was detected. Sheep fed GR had higher (P<0.001–0.05) relative abundance of Fibrobacter succinogenes, Ruminococcus flavefaciens and fungi than did AL-fed sheep, but bacterial DNA concentrations and relative abundance of Ruminococcus albus and methanogenic archaea were unaffected (P>0.05) by FOR. Postprandial changes of DNA concentrations of all determined microbial populations were similar for the two diets. Total bacterial and protozoal DNA concentrations decreased (P<0.05) at 4h post-feeding but recovered before-feeding values at 8h post-feeding. No correlations (P>0.05) were found between CMCase and xylanase activities and either the concentration of total bacterial DNA or the relative abundance of the three cellulolytic bacteria, but xylanase activity was positively correlated (P<0.05) with both protozoa numbers and protozoal DNA concentration. Although the postprandial evolution of fermentation parameters and microbes were similar for both forages, sheep fed the lower quality forage showed higher abundance of some cellulolytic bacteria and fungi, which could be interpreted as an adaptation to digest a more fibrous and complex forage.

Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets varying in type of forage as monitored by real-time PCR and automated ribosomal intergenic spacer analysis1

Four ruminally cannulated sheep were used in a crossover design to assess the postprandial changes of fiber-degrading microbes in the solid phase of the rumen of sheep fed 2 high-forage diets. The diets had forage:concentrate ratio of 70:30 (DM basis) and either alfalfa (Medicago sativa) hay (AL) or grass hay (GR) as forage (FOR). Sheep were fed twice daily, and samples from solid rumen digesta were taken at 0, 4, and 8 h after the morning feeding. Postprandial changes of DNA concentrations of all determined microbial populations were similar for the 2 diets. Samples taken at 4 h after feeding had lesser (P < 0.05) concentrations of total bacterial DNA determined with real-time PCR and bacterial diversity and greater (P < 0.05) protozoal DNA concentrations, relative abundance of fungal, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus DNA compared with those taken at 0 and 8 h. No effect (P = 0.41 to 0.76) of FOR was detected either on concentrations of bacterial and protozoal DNA or the relative abundance of the 2 Ruminococcus DNA, but GR diet promoted greater (P < 0.001) relative abundance of F. succinogenes and fungal DNA compared with AL diet. Fibrobacter succinogenes was the most abundant (P < 0.05) of the 3 cellulolytic bacteria for both diets, with no differences (P < 0.05) between the 2 Ruminococcus species. Rumen pH and carboxymethylcellulase, Avicelase, and amylase activities were not affected (P = 0.15 to 0.69) by FOR, but xylanase activity was greater (P = 0.01) for GR diet. The influence of FOR on microbial communities in ruminal solid digesta was more evident in the first hours after feeding than at later times after feeding, which highlights the influence of sampling time when investigating dietary effects on rumen function and microbial populations.

Effect of diet and absence of protozoa on the rumen microbial community and on the representativeness of bacterial fractions used in the determination of microbial protein synthesis1

Accurate estimates of microbial synthesis in the rumen are vital to optimize ruminant nutrition. Liquid- (LAB) and solid-associated bacterial fractions (SAB) harvested from the rumen are generally considered as microbial references when microbial yield is calculated; however, factors that determine their composition are not completely understood. The aim of this study was to evaluate the effect of diet and absence or presence of rumen protozoa on the rumen microbial community. It was hypothesized that these treatments could modify the composition and representativeness of LAB and SAB. Twenty twin lambs (Ovis aries) were used; one-half of the twins were kept protozoa-free, and each respective twin sibling was faunated. At 6 mo of age, 5 animals from each group were randomly allocated to the experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain. After 15 d of adaptation to the diet, animals were euthanized, rumen and abomasum contents were sampled, and LAB and SAB isolated. The presence of protozoa buffered the effect of diet on the rumen bacterial population. Faunated animals fed alfalfa hay had a greater abundance of F. succinogenes, anaerobic fungi and methanogens, as well as an enhanced rumen bacterial diversity. Cellulolytic bacteria were more abundant in SAB, whereas the abomasal abundance of most of the microorganisms studied was closer to those values observed in LAB. Rumen and abomasal samples showed similar bacterial DNA concentrations, but the fungal and protozoal DNA concentration in the abomasum was only 69% and 13% of that observed in the rumen, respectively, suggesting fungal and protozoal sequestration in the rumen or possible preferential degradation of fungal and protozoal DNA in the abomasum, or both. In conclusion, absence of protozoa and type of diet extensively modified the chemical composition of LAB and SAB as a consequence of changes in the microbial composition of these fractions.

Amoebic Liver Abscess: An Appraisal

Amoebic Liver Abscess: An Appraisal

Experimental Infection of Turkeys and Chickens with a Clonal Strain of Tetratrichomonas gallinarum Induces a Latent Infection in the Absence of Clinical Signs and Lesions

The pathogenicity of a mono-eukaryotic culture of Tetratrichomonas gallinarum in specific pathogen free (SPF) chickens and turkeys was studied. Two experiments of identical design were performed: the first with SPF chickens and the second with commercial turkeys. Each experiment included three groups. Groups 1 and 2 each contained 12 infected and three in-contact birds. The birds in these groups were infected on the first day of life, either cloacally (group 1) or orally (group 2). Group 3 consisted of four control birds. Re-isolation of the parasite from cloacal swabs was performed to verify the excretion of T. gallinarum. The infected birds excreted trichomonads from the second day post-infection. Spread of the flagellate from infected to in-contact birds was detected after 5 days post-infection (dpi), based on the re-isolation of the protozoa. No clinical signs or deaths were recorded in chickens or turkeys. Three birds were killed at 4, 8, 14 and 21dpi and various tissues were collected for pathological examination. No gross lesions were noted. Protozoal DNA was demonstrated in the oesophagus, duodenum, jejunum, caecum, liver, lung, bursa of Fabricius and brain by polymerase chain reaction and in-situ hybridization. No antibodies were detected in the serum of infected birds by enzyme linked immunosorbent assay. Microscopical changes were only present in the caecum, where there was sloughing of the epithelium associated with the presence of numerous flagellates on the epithelial surface, within the crypts of Lieberkühn and in the lamina propria. These changes were found in caecal samples from infected and in-contact birds. These studies have demonstrated the rapid transmission of T. gallinarum between both turkeys and chickens and the establishment of a latent infection in both species.

Use of flow cytometry to separateLeucocytozoon caulleryigametocytes from avian blood

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.

Cross-sectional survey of Toxoplasma gondii infection in colony cats from urban Florence (Italy).

Cats are the key species in the epidemiology of Toxoplasma gondii infection, even if the proportion of subjects excreting oocysts is low. The aim of the present paper was to obtain information about seroprevalence, oocyst shedding rate and presence of T gondii DNA in faeces collected from an urban population of colony cats in Florence (Tuscany). Fifty European shorthair feral cats were examined for anti-T gondii specific antibodies by a modified agglutination test (MAT), and for oocysts by microscopic examination and for faecal protozoal DNA, by means of a nested polymerase chain reaction (n-PCR) protocol. Twenty-two out of 50 serum samples (44%) were MAT positive. T gondii oocysts were not detected in any of the examined faecal samples. Eight out of 50 faecal specimens (16%) were n-PCR positive and sequencing of the bands was specific for T gondii. Detection by combination of the two methods was higher than single techniques and enhanced the detection of T gondii up to 48%. Our results suggest that the use of MAT plus PCR in faeces may be the best choice for diagnosis of feline toxoplasmosis. Further studies to ascertain the real infectivity of the copro-PCR positive subjects are required.

Gastric Cryptosporidiosis in Freshwater Angelfish (Pterophyllum Scalare)

A freshwater angelfish (Pterophyllum scalare) hatchery experienced variable levels of emaciation, poor growth rates, swollen coelomic cavities, anorexia, listlessness, and increased mortality within their fish. Multiple chemotherapeutic trials had been attempted without success. In affected fish, large numbers of protozoa were identified both histologically and ultrastructurally associated with the gastric mucosa. The youngest cohort of parasitized fish was the most severely affected and demonstrated the greatest morbidity and mortality. The protozoa were morphologically most consistent with Cryptosporidium. All of the protozoan life stages were identified ultrastructurally and protozoal genomic DNA was isolated from parasitized tissue viscera and sequenced. Histological, ultrastructural, genetic, and phylogenetic analyses confirmed this protozoal organism to be a novel species of Cryptosporidium.

10th Annual Congress of the German Association of Clinical Pharmacology

10th Annual Congress of the German Association of Clinical Pharmacology

Toxoplasmosis in a Woodchuck (Marmota Monax) and Two American Red Squirrels (Tamiasciurus Hudsonicus)

Toxoplasmosis was diagnosed in a woodchuck (Marmota monax) and 2 American red squirrels (Tamiasciurus hudsonicus). The woodchuck was euthanized by a wildlife rescue organization in New York after progressive clinical signs of head tilt, circling, and rapid weight loss. Necropsy examination revealed acute subdural hemorrhage over the right cerebral hemisphere. Histologic lesions included meningoencephalitis, myocarditis, and hepatitis. Protozoal cysts were present in affected and unaffected neuroparenchyma. The squirrels were found dead, emaciated, and moderately infested with fleas near a park in northern Indiana. In both squirrels, the lungs were consolidated with numerous nodules up to 2 mm in diameter. Histologically, pneumonia and encephalitis were associated with intracellular and free protozoa. Additional histologic lesions included multifocal lymphoplasmacytic encephalitis with intralesional protozoa in both squirrels. The protozoa were positive with Toxoplasma gondii-specific immunohistochemistry and had ultrastructural features consistent with T. gondii in both squirrels. A real-time polymerase chain reaction test using T. gondii-specific probes demonstrated protozoal DNA in the lung, brain, and kidney of the squirrels and in the brain and heart of the woodchuck. To the authors' knowledge, this is the first report of toxoplasmosis in woodchucks or American red squirrels. Because rodents are common near urban settlements, this finding underscores their role as important intermediate hosts for T. gondii.

DNA topoisomerases as targets for antiprotozoal therapy.

Diseases caused by parasitic protozoa present a health problem of immense magnitude, and there is an urgent need for safe and effective new therapies. DNA topoisomerases are clinically relevant targets for anti-cancer and anti-bacterial agents. Inhibitor studies on parasite topoisomerases have revealed that these enzymes have great promise as molecular targets for anti-parasitics, and have helped to dissect the basic biology of DNA topoisomerases in these organisms. This review provides a brief introduction to DNA topoisomerases and anti-topoisomerase drugs, and an overview of studies on protozoal DNA topoisomerases and their inhibitors.

Protozoal DNA triggers innate immunity

The recognition of protozoal DNA by immune cells could provide an important innate defense mechanism for control of parasitic disease. By culturing macrophages with DNA from Babesia bovis, Trypanosoma cruzi and T. brucei, L. Shoda et al. [(2001) Infect. Immun. 69, 2162–2171] found increased production of pro-inflammatory cytokines IL-12 and TNF-α, in addition to enhanced nitric oxide synthesis. In particular, unmethylated CpG dinucleotides were found to induce strongly macrophage production of these mediators, all three of which are associated with protective immunity against protozoan parasites. Protozoan DNA also has a strong mitogenic effect on B lymphocytes. SHK

DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide.

The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423-5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-alpha, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli > or = T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations, E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.