The emergence of Brucella canis as a public health threat in Europe: what we know and what we need to learn.

The zoonotic bacteria, Brucella canis, is becoming the leading cause of canine brucellosis in Europe. In dogs, it causes reproductive problems as well as non-specific lameness or discospondilitis. In humans, B. canis can be origin of chronic debilitating conditions characteristic to its genus such as undulant fever, splenomegaly, and lymphadenopathy. Although B. canis shows some pathogenic characteristics similar to B. abortus and B. melitensis, it lacks surface O-polysaccharide, like nonzoonotic B. ovis. This review shows that host-B. canis interactions are still poorly understood, with many knowledge and capability gaps, causing relatively poor sensitivity and specificity of existing diagnostic tools. Currently, there is no vaccine for this rough Brucella species. Besides, antimicrobial therapy does not guarantee bacterial elimination, and infection relapses are frequently reported, increasing the risks of antibiotic resistance development. B. canis has been detected in dogs in almost all European countries which increased human exposure, but currently there is no systematic surveillance. Moreover, B. canis caused brucellosis is not included in Animal Health Law, and therefore there is no legal framework to tackle this emerging infectious disease. To map out the diagnostic strategies, identify risks for human infections and propose management scheme for infected pet and kennel dogs, we present current understanding of canine B. canis caused brucellosis, outline major knowledge gaps and propose future steps. To address and highlight challenges veterinary and public health services encounter in Europe, we developed two B. canis infection scenarios: of a single household pet and of a kennel dog in larger group.

Evaluation of a Lateral Flow Immunochromatography Assay (LFIA) for Diagnosis and Surveillance of Brucellosis in French Alpine Ibex (Capra ibex).

France has been officially free of bovine brucellosis since 2005. Nevertheless, in 2012, as the source of two human cases, a bovine outbreak due to B. melitensis biovar 3 was confirmed in the French Alpine Bargy massif, due to a spillover from wild, protected Alpine ibex (Capra ibex). In order to reduce high Brucella prevalence in the local ibex population, successive management strategies have been implemented. Lateral flow immunochromatography assay (LFIA) was thus identified as a promising on-site screening test, allowing for a rapid diagnosis far from the laboratory. This study compared a commercial LFIA for brucellosis diagnosis with the WOAH-recommended tests for small ruminants (i.e., Rose Bengal test (RBT), Complement fixation test, (CFT) and Indirect ELISA, (iELISA)). LFIA showed the same analytical sensitivity as iELISA on successive dilutions of the International Standard anti-Brucella melitensis Serum (ISaBmS) and the EU Goat Brucella Standard Serum (EUGBSS). Selectivity was estimated at 100% when vaccinated ibex sera were analyzed. When used on samples from naturally infected ibex, LFIA showed high concordance, as well as relative sensitivity and specificity (>97.25%) in comparison with RBT and CFT. This work shows high reliability and ensures a better standardization of LFIA testing for wild ruminants.

Molecular Investigations of Two First Brucella suis Biovar 2 Infections Cases in French Dogs.

Despite Brucella suis biovar 2's (BSB2) active circulation in wildlife, no canine infections have been reported. The present paper is the first to describe two cases of BSB2 infections in French dogs. The first case occurred in 2020 and concerned a 13-year-old male neutered Border Collie with clinical signs of prostatitis. The urine culture revealed the excretion of significant levels of Brucella in the sample. The second case concerned a German Shepherd with bilateral orchitis, in which it was possible to detect Brucella colonies following neutering. HRM-PCR and classical biotyping methods classified both isolated strains as BSB2, in contrast to expected B. canis, which is usually the etiological agent of canine brucellosis in Europe. The wgSNP and MLVA analyses highlighted the genetic proximity of two isolates to BSB2 strains originating from wildlife. No pig farms were present in the proximity of either dog's residence, ruling out potential spill over from infected pigs. Nevertheless, the dogs used to take walks in the surrounding forests, where contact with wildlife (i.e., wild boars or hares, or their excrements) was possible. These cases highlight the importance of adopting a One Health approach to control the presence of zoonotic bacteria in wild animals and avoid spillovers into domestic animals and, potentially, humans.

Sciatic-Vagal Nerve Stimulation by Electroacupuncture Alleviates Inflammatory Arthritis in Lyme Disease-Susceptible C3H Mice.

Lyme disease is caused by Borrelia burgdorferi, and the pathogenesis of the disease is complex with both bacterial and host factors contributing to inflammatory responses. Lyme disease affects different organs including joints and results in arthritis. Immune responses stimulated by B. burgdorferi through toll-like receptors cause infiltration of leukocytes, which produce inflammatory cytokines and facilitate spirochete clearance. However, arthritic manifestations and chronic fatigue syndrome-like symptoms persist long after completion of antibiotic treatment regimens in a significant number of patients. To counter the effects of inflammation, treatment by non-steroidal anti-inflammatory drugs, hydroxychloroquine, or synovectomy to eradicate inflammatory arthritis in the involved joint could be employed; however, they often have long-term consequences. Acupuncture has been used for a long time in Asian medicine to diminish pain during various ailments, but the effects and its mechanism are just beginning to be explored. Control of inflammation by neuronal stimulation has been exploited as a systemic therapeutic intervention to arrest inflammatory processes. Our objective was to determine whether activation of the sciatic-vagal network by electroacupuncture on ST36 acupoint, which is used to control systemic inflammation in experimental models of infectious disorders such as endotoxemia, can also alleviate Lyme arthritis symptoms in mice. This aim was further strengthened by the reports that sciatic-vagal neuronal network stimulation can lead to dopamine production in the adrenal medulla and moderate the production of inflammatory factors. We first assessed whether electroacupuncture affects spirochete colonization to attenuate Lyme arthritis. Interestingly, bioluminescent B. burgdorferi burden detected by live imaging and qPCR were similar in electroacupuncture- and mock-treated mice, while electroacupuncture induced a lasting anti-inflammatory effect on mice. Despite the discontinuation of treatment at 2 weeks, the simultaneous decrease in neutrophils in the joints and inflammatory cytokine levels throughout the body at 4 weeks suggests a systemic and persistent effect of electroacupuncture that attenuates Lyme arthritis. Our results suggest that electroacupuncture-mediated anti-inflammatory responses could offer promising healthcare benefits in patients suffering from long-term Lyme disease manifestations.

The Use of Flocked Swabs with a Protective Medium Increases the Recovery of Live Brucella spp. and DNA Detection.

Brucellosis is a worldwide zoonosis caused by bacteria from the genus Brucella. Once established, it is very hard to eradicate this disease, since it contaminates animals, the environment, and humans, causing problems for veterinary and public health as well as wildlife protection programs. Swabs are used for sampling in bacteriological and/or molecular diagnostics, from seropositive animals with disease symptoms, from genitalia or tissue lesions, as well as from contaminated environments. The aim of this study was to compare main of the commercially used swab types for sampling and diagnostics of Brucella spp. and determine the optimal storage conditions and time frame for testing. To achieve this, we tested bacterial and molecular methods for detection of Brucella abortus, Brucella melitensis, and Brucella suis using nine swab types, all with different tip materials, treated immediately after spiking, after 72 h at +4°C, and after 72 h at -20°C. Flocked swabs showed the highest capacity to preserve bacterial viability and DNA quality, regardless the storage conditions. Flocked swabs immersed in a protective medium provided the best conditions for Brucella survival in all three storage conditions. At the same time, the efficacy of quantitative PCR (qPCR) detection for all swabs, including the positive control, was above 50%, irrespective of the storage conditions, while bacterial survival was significantly lowered when swabs were kept at +4°C or -20°C for 72 h (48.2% and 27.5%, respectively). Compared to the positive control and other types, the flocked swabs maintained higher reproducibility regarding their capacity to preserve live bacteria in all three storage conditions. IMPORTANCE In order to protect public and veterinary health from highly zoonotic bacteria such as members of the genus Brucella and prevent their dissemination into the environment, direct diagnostics are of utmost importance. However, in addition to the highly specific diagnostic tests, the sampling methods, time necessary for specimens to reach the laboratories, and transport conditions are important factors to consider in order to increase the sensitivity of performed tests, especially bacterial culturing and qPCR. This paper shows how different swab types and storage conditions influence classical bacteriological diagnostics of the most prevalent Brucella species - B. melitensis, B. abortus, and B. suis - but have little impact on molecular methods. The presented results highlight (i) the choice of swab regarding the storage and transport conditions, (ii) the importance of immediate swab treatment upon sampling, and (iii) that molecular methods do not depend on storage conditions, unlike classical bacteriological isolation.

Lessons Learned for Pathogenesis, Immunology, and Disease of Erythrocytic Parasites: Plasmodium and Babesia.

Malaria caused by Plasmodium species and transmitted by Anopheles mosquitoes affects large human populations, while Ixodes ticks transmit Babesia species and cause babesiosis. Babesiosis in animals has been known as an economic drain, and human disease has also emerged as a serious healthcare problem in the last 20-30 years. There is limited literature available regarding pathogenesis, immunity, and disease caused by Babesia spp. with their genomes sequenced only in the last decade. Therefore, using previous studies on Plasmodium as the foundation, we have compared similarities and differences in the pathogenesis of Babesia and host immune responses. Sexual life cycles of these two hemoparasites in their respective vectors are quite similar. An adult Anopheles female can take blood meal several times in its life such that it can both acquire and transmit Plasmodia to hosts. Since each tick stage takes blood meal only once, transstadial horizontal transmission from larva to nymph or nymph to adult is essential for the release of Babesia into the host. The initiation of the asexual cycle of these parasites is different because Plasmodium sporozoites need to infect hepatocytes before egressed merozoites can infect erythrocytes, while Babesia sporozoites are known to enter the erythrocytic cycle directly. Plasmodium metabolism, as determined by its two- to threefold larger genome than different Babesia, is more complex. Plasmodium replication occurs in parasitophorous vacuole (PV) within the host cells, and a relatively large number of merozoites are released from each infected RBC after schizogony. The Babesia erythrocytic cycle lacks both PV and schizogony. Cytoadherence that allows the sequestration of Plasmodia, primarily P. falciparum in different organs facilitated by prominent adhesins, has not been documented for Babesia yet. Inflammatory immune responses contribute to the severity of malaria and babesiosis. Antibodies appear to play only a minor role in the resolution of these diseases; however, cellular and innate immunity are critical for the clearance of both pathogens. Inflammatory immune responses affect the severity of both diseases. Macrophages facilitate the resolution of both infections and also offer cross-protection against related protozoa. Although the immunosuppression of adaptive immune responses by these parasites does not seem to affect their own clearance, it significantly exacerbates diseases caused by coinfecting bacteria during coinfections.

Pathogenesis of Borrelia burgdorferi and Babesia microti in TLR4-Competent and TLR4-dysfunctional C3H mice.

Toll-like receptors (TLRs) are a class of membrane-spanning proteins of host cells. TLR2 and TLR4 are displayed on the surface of macrophages, neutrophils and dendritic cells and recognise structurally conserved microbial signatures defined as Pathogen associated molecular patterns (PAMPs). C3H mice are susceptible to tick-borne pathogens; Lyme disease causing Borrelia burgdorferi that manifests arthritis and carditis and Apicomplexan protozoan, Babesia microti (Bm) that causes significant parasitemia associated with erythrocytopenia and haemoglobinuria. B. burgdorferi lacks typical TLR4 ligand lipopolysaccharides (LPS) and Bm TLR ligand(s) remain unknown. Only Borrelia lipoproteins that signal through TLR2 are established as PAMPs of these pathogens for TLR2/TLR4. Infection of C3H mice with each pathogen individually resulted in increase in the percentage of splenic B, T and FcR+ cells while their co-infection significantly diminished levels of these cells and caused increased B. burgdorferi burden in the specific organs. The most pronounced inflammatory arthritis was observed in co-infected C3H/HeJ mice. Parasitemia levels and kinetics of resolution of Bm in both mice strains were not significantly different. Transfected HEK293 cells showed pronounced signalling by B. burgdorferi through TLR2 and to some extent by TLR4 while Bm and infected erythrocytes did not show any response confirming our results in mice.

Evaluation of immunogenicity and protection of the Mic1-3 knockout Toxoplasma gondii live attenuated strain in the feline host.

Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii. Up to a third of the global human population is estimated to carry a T. gondii infection, which can result in severe complications in immunocompromised individuals and pregnant women. Humans and animals can become infected by ingesting either tissue cysts containing T. gondii bradyzoites, from raw or undercooked meat, or sporulated oocysts from environmental sources. T. gondii oocysts are released in the faeces of cats and other felids, which are the parasite's definitive hosts, leading to environmental contamination. Therefore, vaccination of the feline host against T. gondii is an interesting strategy to interrupt the parasitic life cycle and subsequently limit contamination of intermediate hosts. With this goal in mind, we tested in cats, an attenuated live strain of T. gondii deleted for the Mic1 and Mic3 genes (Mic1-3KO) that was previously shown to be an efficient vaccine candidate in mouse and sheep models. Subcutaneous or oral vaccination routes induced a high specific antibody titer in the cat sera, indicating that the Mic1-3KO strain is immunogenic for cats. To assess protection induced by the vaccine candidate strain, we followed oocysts shedding by vaccinated cats, after oral challenge with a T. gondii wild-type strain. Surprisingly, a high antibody titer did not prevent cats from shedding oocysts from the challenge strain, regardless of the vaccination route. Our results show that the Mic1-3KO vaccine candidate is immunogenic in the feline host, is well tolerated and safe, but does not confer protection against oocysts shedding after natural infection with wild type T. gondii. This result highlights the particular relationship between T. gondii and its unique definitive host, which indicates the need for further investigations to improve vaccination strategies to limit environmental and livestock contaminations.

Toxoplasma gondii in beef consumed in France: regional variation in seroprevalence and parasite isolation.

In France, the consumption of cattle and sheep meat appears to be a risk factor for infection of pregnant women with Toxoplasma gondii. Several nation-wide surveys in France have investigated the prevalence of T. gondii in sheep and pig meat, but little is known at present about the prevalence of the parasite in beef. The main objective of the present cross-sectional survey was to estimate the seroprevalence of T. gondii infection in beef consumed in France. A secondary objective was to attempt to isolate T. gondii from cattle tissues and to study the geographical and age variations of this seroprevalence. The overall estimate of seroprevalence of T. gondii in bovine carcasses (n = 2912), for a threshold of 1:6 was 17.38%. A strong age effect was observed (p < 0.0001) with a seroprevalence of 5.34% for calves (<8 months) and 23.12% for adults (>8 months). Seroprevalence estimates given by area of birth and area of slaughtering for adults showed that the areas with the highest seroprevalence were not the same between these two variables. Only two strains, corresponding to genotype II, were isolated from heart samples, indicating that there is a limited risk of human infection with T. gondii, which needs to be correlated with the food habit of consuming raw or undercook (bleu or saignant) beef. However, new questions have emerged, especially concerning the isolation of parasites from beef and the precise role of bovines, generally described as poor hosts for T. gondii, in human infection.

TITLE: Toxoplasma gondii dans la viande bovine consommée en France : variation régionale de la séroprévalence et isolement de parasites. ABSTRACT: En France, la consommation de viande bovine et ovine apparaît comme un facteur de risque pour la contamination des femmes enceintes par Toxoplasma gondii. Plusieurs enquêtes nationales ont été réalisées afin de déterminer le niveau de contamination par T. gondii de la viande ovine et porcine, en France, mais très peu est encore connu quant à la prévalence du parasite dans la viande bovine. La présente enquête transversale avait pour objectif principal d'estimer la séroprévalence de l'infection à T. gondii dans la viande bovine consommée en France, ainsi que d'isoler T. gondii à partir de tissus de bovins et d'étudier, à titre d'objectif secondaire, les variations géographiques et d'âge de cette prévalence. L'estimation globale de la séroprévalence de T. gondii dans les carcasses de bovins (n = 2912) était de 17,38 % (pour un seuil de dilution à 1:6). Un effet significatif de l'âge a été observé (p < 0,0001) avec une séroprévalence de 5,34 % pour les veaux (<8 mois) et de 23,12 % pour les adultes (>8 mois). Les estimations de séroprévalence données par zone de naissance et par zone d'abattage pour les adultes montrent que les zones de séroprévalence les plus élevées n'étaient pas les mêmes pour ces deux variables. Seulement deux souches, de génotype II, ont été isolées à partir d'échantillons de cœurs, soulignant que le risque d'infection humaine est limité, mais doit être corrélé avec les habitudes de consommation alimentaire de la viande bovine peu/pas cuite (bleu ou saignante). Cependant, de nouvelles questions se posent, notamment en ce qui concerne l'isolement du parasite à partir de la viande bovine, ainsi que le rôle précis des bovins, généralement décrits comme des hôtes médiocres pour T. gondii, dans la contamination humaine.

Protozoan Parasite Babesia microti Subverts Adaptive Immunity and Enhances Lyme Disease Severity.

Lyme disease is the most prominent tick-borne disease in the United States. Co-infections with the tick-transmitted pathogens Babesia microti and Borrelia burgdorferi sensu stricto are becoming a serious health problem. B. burgdorferi is an extracellular spirochete that causes Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Testing of donated blood for Babesia species is not currently mandatory due to unavailability of an FDA approved test. Transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. Infection of C3H/HeJ mice with B. burgdorferi and B. microti individually results in inflammatory Lyme disease and display of human babesiosis-like symptoms, respectively. Here we use this mouse model to provide a detailed investigation of the reciprocal influence of the two pathogens on each other during co-infection. We show that B. burgdorferi infection attenuates parasitemia in mice while B. microti subverts the splenic immune response, such that a marked decrease in splenic B and T cells, reduction in antibody levels and diminished functional humoral immunity, as determined by spirochete opsonophagocytosis, are observed in co-infected mice compared to only B. burgdorferi infected mice. Furthermore, immunosuppression by B. microti in co-infected mice showed an association with enhanced Lyme disease manifestations. This study demonstrates the effect of only simultaneous infection by B. burgdorferi and B. microti on each pathogen, immune response and on disease manifestations with respect to infection by the spirochete and the parasite. In our future studies, we will examine the overall effects of sequential infection by these pathogens on host immune responses and disease outcomes.

Analysis of host cell binding specificity mediated by the Tp0136 adhesin of the syphilis agent Treponema pallidum subsp. pallidum.

BACKGROUND: Syphilis affects approximately 11 million people each year globally, and is the third most prevalent sexually transmitted bacterial infection in the United States. Inability to independently culture and genetically manipulate Treponema pallidum subsp. pallidum, the causative agent of this disease, has hindered our understanding of the molecular mechanisms of syphilis pathogenesis. Here, we used the non-infectious and poorly adherent B314 strain of the Lyme disease-causing spirochete, Borrelia burgdorferi, to express two variants of a known fibronectin-binding adhesin, Tp0136, from T. pallidum SS14 and Nichols strains. Using this surrogate system, we investigated the ability of Tp0136 in facilitating differential binding to mammalian cell lines offering insight into the possible role of this virulence factor in colonization of specific tissues by T. pallidum during infection. PRINCIPAL FINDINGS: Expression of Tp0136 could be detected on the surface of B. burgdorferi by indirect immunofluorescence assay using sera from a secondary syphilis patient that does not react with intact B314 spirochetes transformed with the empty vector. Increase in Tp0136-mediated adherence of B314 strain to human epithelial HEK293 cells was observed with comparable levels of binding exhibited by both Tp0136 alleles. Adherence of Tp0136-expressing B314 was highest to epithelial HEK293 and C6 glioma cells. Gain in binding of B314 strain expressing Tp0136 to purified fibronectin and poor binding of these spirochetes to the fibronectin-deficient cell line (HEp-2) indicated that Tp0136 interaction with this host receptor plays an important role in spirochetal attachment to mammalian cells. Furthermore, preincubation of these cell lines with fibronectin-binding peptide from Staphylococcus aureus FnbA-2 protein significantly inhibited binding of B314 expressing Tp0136. CONCLUSIONS: Our results show that Tp0136 facilitates differential level of binding to cell lines representing various host tissues, which highlights the importance of this protein in colonization of human organs by T. pallidum and resulting syphilis pathogenesis.

Babesia microti Infection Changes Host Spleen Architecture and Is Cleared by a Th1 Immune Response.

Babesia microti is a malaria-like parasite, which infects ∼2000 people annually, such that babesiosis is now a notifiable disease in the United States. Immunocompetent individuals often remain asymptomatic and are tested only after they feel ill. Susceptible C3H/HeJ mice show several human-like disease manifestations and are ideal to study pathogenesis of Babesia species. In this study, we examined parasitemia of B. microti at different time points and assessed its impact on hemoglobin levels in blood, on spleen pathology and overall immune response in C3H/HeJ mice. Peak parasitemia of 42.5% was immediately followed by diminished hemoglobin level. Parasitemia at 21 days of infection was barely detectable by microscopy presented 5.7 × 108 to 5.9 × 109B. microti DNA copies confirming the sensitivity of our qPCR. We hypothesize that qPCR detects DNA released from recently lysed parasites or from extracellular B. microti in blood, which are not easily detected in blood smears and might result in under-diagnosis of babesiosis in patients. Splenectomized patients have been reported to show increased babesiosis severity and result in high morbidity and mortality. These results emphasize the importance of splenic immunity in resolution of B. microti infection. Splenomegaly in infected mice associated with destruction of marginal zone with lysed erythrocytes and released B. microti life forms in our experiments support this premise. At conclusion of the experiment at 21 days post-infection, significant splenic B and T cells depletion and increase in macrophages levels were observed in B. microti infected mice suggesting a role of macrophage in disease resolution. Infected mice also showed significantly higher plasmatic concentration of CD4 Th1 cells secreted cytokines such as IL-2 and IFN-γ while cytokines such as IL-4, IL-5, and IL-13 secreted by Th2 cells increase was not always significant. Thus, Th1 cells-mediated immunity appears to be important in clearance of this intracellular pathogen. Significant increase in IL-6 that promotes differentiation of Th17 cells was observed but it resulted in only moderate change in IL-17A, IL-17F, IL-21, and IL-22, all secreted by Th17 cells. A similar immune response to Trypanosoma infection has been reported to influence the clearance of this protozoan, and co-infecting pathogen(s).

Age-Related Differential Stimulation of Immune Response by Babesia microti and Borrelia burgdorferi During Acute Phase of Infection Affects Disease Severity.

Lyme disease is the most prominent tick-borne disease with 300,000 cases estimated by CDC every year while ~2,000 cases of babesiosis occur per year in the United States. Simultaneous infection with Babesia microti and Borrelia burgdorferi are now the most common tick-transmitted coinfections in the U.S.A., and they are a serious health problem because coinfected patients show more intense and persisting disease symptoms. B. burgdorferi is an extracellular spirochete responsible for systemic Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Immune status and spleen health are important for resolution of babesiosis, which is more severe and even fatal in the elderly and splenectomized patients. Therefore, we investigated the effect of each pathogen on host immune response and consequently on severity of disease manifestations in both young, and 30 weeks old C3H mice. At the acute stage of infection, Th1 polarization in young mice spleen was associated with increased IFN-γ and TNF-α producing T cells and a high Tregs/Th17 ratio. Together, these changes could help in the resolution of both infections in young mice and also prevent fatality by B. microti infection as observed with WA-1 strain of Babesia. In older mature mice, Th2 polarization at acute phase of B. burgdorferi infection could play a more effective role in preventing Lyme disease symptoms. As a result, enhanced B. burgdorferi survival and increased tissue colonization results in severe Lyme arthritis only in young coinfected mice. At 3 weeks post-infection, diminished pathogen-specific antibody production in coinfected young, but not older mice, as compared to mice infected with each pathogen individually may also contribute to increased inflammation observed due to B. burgdorferi infection, thus causing persistent Lyme disease observed in coinfected mice and reported in patients. Thus, higher combined proinflammatory response to B. burgdorferi due to Th1 and Th17 cells likely reduced B. microti parasitemia significantly only in young mice later in infection, while the presence of B. microti reduced humoral immunity later in infection and enhanced tissue colonization by Lyme spirochetes in these mice even at the acute stage, thereby increasing inflammatory arthritis.

Experimental Toxoplasma gondii infections in pigs: Humoral immune response, estimation of specific IgG avidity and the challenges of reproducing vertical transmission in sows.

Ten pregnant sows were experimentally inoculated per os with T. gondii in order to investigate vertical and galactogenic transmission of the parasite and the evolution and maturation of the specific IgG humoral response in the sows and piglets. Five seronegative sows received 104T. gondii (CZ isolate clone H3) sporulated oocysts during late-pregnancy (Exp. 1), three sows received 104 oocysts during mid-pregnancy (Exp. 2) and three sows from Exp. 1 (and two seronegative sows) were re-inoculated with 105 oocysts during a further pregnancy (late-pregnancy) (Exp. 3). Besides, six 4.5 week-old piglets inoculated per os with 5×103 oocysts were also included in the serological investigations. All animals seroconverted (PrioCHECK Toxoplasma Ab porcine ELISA, Prionics, Switzerland) by 2-3 weeks post inoculation (wpi) and remained seropositive for at least 38 weeks or until euthanasia. Four chronically infected sows from Exp. 1 and 2 were serologically monitored during a further pregnancy and no reactivation, but a decrease of the antibody levels was observed at farrowing (Exp. 4). In all experiments, the specific IgG-avidity was initially low, increased during the course of infection and after re-inoculations. An avidity index (AI) ≥40% could be used to rule out recent infections (<8 weeks) in most (15 of 16) animals. In some piglets (18.6% of 70) delivered by inoculated sows (Exp. 1 and 2), maternal antibodies were still detectable at 2 months (but not by 3 months) of age, with constant high avidity values, comparable to those of the dams at farrowing. In all experiments, the sows remained asymptomatic and delivered non-infected offspring at term. A total of 208 normal and 5 stillborn piglets delivered by the inoculated sows (Exp. 1-4) tested serologically negative before colostrum uptake. Placentas (n=88) from all sows and tissues (brain, liver, lung, heart, and masseter muscle) from 56 delivered piglets were analysed histopathologically and by real-time PCR for T. gondii with negative results. Colostrum and milk samples from all sows were negative by real-time PCR for T. gondii DNA. In addition, no seroconversion was observed in 16 piglets from seronegative dams that were transferred to infected dams one day after birth to detect a possible infection through colostrum or milk during the suckling period. Although vertical transmission of T. gondii was demonstrated in naturally infected pigs, many factors involved in the outcome of vertical transmission and congenital toxoplasmosis in pigs are still unknown.

Mini-FLOTAC for counting Toxoplasma gondii oocysts from cat feces--comparison with cell counting plates.

Oocysts of Toxoplasma gondii represent one of the most common environmental contaminants causing the zoonotic infection toxoplasmosis. The aim of the present study was to compare the Mini-FLOTAC device with traditional cell counting plates (Kova Slide) for the detection of T. gondii oocysts from feline feces. Two types of experiments were performed: (i) purified oocysts were counted in different dilutions and (ii) specific pathogen free T. gondii-negative cat feces was inoculated with numbers of purified oocysts and counting was performed directly from feces. Our analysis showed a thousand times higher sensitivity of Mini-FLOTAC (5 × 10(2) oocysts) compared to Kova Slide (5 × 10(5) oocysts). Also, when compared by McNemar's test, counting of the purified oocysts showed a higher sensitivity of Mini-FLOTAC compared to Kova Slide, for a dilution of 10(3) oocysts/ml (chi(2) = 6.1; P < 0.05). A better sensitivity was also found with Mini-FLOTAC in dilutions of 10(5) and 10(4) oocysts/ml, when counted from feces (chi(2) = 4.2 and 8.1, respectively, P < 0.05). Our results show that Mini-FLOTAC is more sensitive than traditional methods of T. gondii oocysts detection and quantification is more accurate. Furthermore, Mini-FLOTAC simplicity and cost effectiveness allow it to be used with light microscopes in any laboratory or field conditions. We therefore recommend its use for regular screening. Further studies are needed to validate Mini-FLOTAC for the detection of oocysts in soil and water samples in field conditions.

Spatial epidemiology of Toxoplasma gondii infection in goats in Serbia.

A major risk factor for Toxoplasma gondii infection is consumption of undercooked meat. Increasing demand for goat meat is likely to promote the role of this animal for human toxoplasmosis. As there are virtually no data on toxoplasmosis in goats in Serbia, we undertook a cross-sectional serological study, including prediction modelling using geographical information systems (GIS). Sera from 431 goats reared in 143 households/farms throughout Serbia, sampled between January 2010 and September 2011, were examined for T. gondii antibodies by a modified agglutination test. Seroprevalence was 73.3% at the individual level and 84.6% at the farm level. Risk factor analysis showed above two-fold higher risk of infection for goats used for all purposes compared to dairy goats (P = 0.012), almost seven-fold higher risk for goats kept as sole species versus those kept with other animals (P = 0.001) and a two-fold lower risk for goats introduced from outside the farm compared to those raised on the farm (P = 0.027). Moreover, households/farms located in centre-eastern Serbia were found to be less often infected than those in northern Serbia (P = 0.004). The risk factor analysis was fully supported by spatial analysis based on a GIS database containing data on origin, serology, land cover, elevation, meteorology and a spatial prediction map based on kriging analysis, which showed western Serbia as the area most likely for finding goats positive for T. gondii and centre-eastern Serbia as the least likely. In addition, rainfall favoured seropositivity, whereas temperature, humidity and elevation did not.

Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia.

To contribute to the insight into the worldwide population structure of Toxoplasma gondii, we genetically characterized a total of eight strains isolated from intermediate hosts including humans, sheep and pigeons in Serbia. Although parasite DNA was detected in 28.2% (60/213) of the human samples from 162 patients serologically suspected of active toxoplasmosis, as well as in 5/7 seropositive pigeons and in 2/12 seropositive sheep examined, multilocus PCR-RFLP genotyping, using SAG1, 5'SAG2, 3'SAG2, GRA6, 5'GRA7 and 3'GRA7 as markers, was successful in only four human isolates (of which one was isolated from both the bronchoalveolar lavage fluid and blood samples of a single patient), one sheep and three pigeons. Of the eight isolates, five were type II (62.5%), one was type III, one was atypical, and one had a type I allele at GRA6 as the single locus genotyped. Although type II, as elsewhere in Europe, predominated, these results may suggest a higher genetic diversity of T. gondii in Serbia, reflecting local environmental contamination and also the geographical position of the country in South-East Europe.

Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis.

A sensitive real-time PCR technique was used to examine the distribution of Toxoplasma gondii in the blood and tissues of mice during acute and chronic infection. Groups of Swiss Albino mice, inoculated i.p. with 10(2) or 10(6) tachyzoites of the RH strain as a typical type-1 strain, or fed 10 cysts of the Me49 strain as a typical type-2 strain, were killed at different time points post-infection (p.i.), and blood and organs including the lungs, brain and liver were harvested for DNA extraction. Toxoplasma DNA was quantified by a real-time PCR targeted at the 529bp gene fragment, with a detection limit of a single parasite per g/ml of tissue. The results showed a strain- and dose-dependent spread of Toxoplasma. In infection with type-1 parasites, in case of a high infective dose, Toxoplasma DNA was detected within 24h p.i. in all analyzed tissues including the brain. Conversely, in case of a low infective dose, parasitaemia was undetectable early p.i., at a time when Toxoplasma DNA was detected in the tissues, but reached very high levels as infection progressed. With both infective doses, pre-death parasite burdens were higher in the blood than in the tissues, whereas the same loads in the lungs suggest that reaching these Toxoplasma burdens may be critical for survival. In infection with Me49 parasites, steady high parasite burdens were noted up to the end of the experiment at d42 only in the brain, parasitaemia was low but detectable throughout, and Toxoplasma DNA was completely cleared only from the liver. These data are important to better understand the pathogenesis of toxoplasmosis, and also as baseline data for the experimental evaluation of novel chemotherapeutics.