Microscopic detection and molecular characterization of Sarcocystis miescheriana in wild boars (Sus scrofa): first report from Greece.

The genus Sarcocystis includes protozoan parasites with an indirect life cycle. Sarcocystis spp. can infect various animal species and humans, causing sarcocystosis, a parasitosis of economic importance and zoonotic concern. Wild boars can act as intermediate hosts for Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis that infects humans by consumption of raw or undercooked infected swine meat. In the present study, the diaphragmatic muscle tissue of 123 wild boars hunted in Greece was examined to determine the frequency of Sarcocystis spp. The samples were examined by tissue compression and molecular techniques. Under light microscopy, 34 out of 123 (27.6%) wild boars tested positive for Sarcocystis spp., while a higher infection prevalence (75%) was revealed by multiplex PCR performed in 100 of the samples. The partial mtDNA cox1 gene (~ 1100 bp) of 20 samples tested positive for S. miescheriana by multiplex PCR was amplified and sequenced. Sarcocystis miescheriana was identified as the only species involved in these infections. This is the first study on the prevalence of Sarcocystis spp. in wild animals in Greece. Further, large-scale surveys are needed to assess the prevalence and species of this parasite in Greece and to design efficient control and preventive measures in a One Health perspective.

Identification of Sarcocystis spp. in wild boars (Sus scrofa) from Argentina.

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study is to identify Sarcocystis spp. in wild boar muscles from Argentina by light and transmission electron microscopy and molecular characterization. Muscle samples from diaphragm, tongue, masseter, intercostals, heart, and forelimbs of 240 wild boars were analyzed. Of the animals, 48.3% (116/240) were positive for sarcocysts by light microscopy, whereas 45.8% (110/240) were positive for Sarcocystis spp. by PCR targeting 18S rRNA fragment. These samples were subjected to a specific PCR for S. suihominis coxI gene, 3.6% (4/110) of which were weak positives. Unfortunately, sequence analysis was inconclusive. This could be related to a potentially low S. suihominis cyst load in the samples, or to an incomplete primer matching with the South American S. suihominis sequences. Seventeen individual sarcocysts were positive by PCR for the 18S rRNA fragment, whose sequences showed 99.75-100% identity with each other and with previously reported S. miescheriana sequences. A total of 21 cysts collected from 11 muscle samples and analyzed by TEM presented a cyst wall type compatible with S. miescheriana, and one cyst presented an ultrastructure compatible with S. suihominis. The latter came from a sample that also contained S. miescheriana cysts, indicating that the animal was co-infected. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in wild boars from South America.

Molecular identification of Sarcocystis species in diaphragm muscle tissue of European mouflon (Ovis gmelini musimon) from Austria.

Previous morphological studies suggested that mouflon may have sarcocysts similar to those of sheep. However, to date, no molecular-based studies of the species of Sarcocystis infecting mouflon have been done. The present study identified Sarcocystis species in diaphragm muscle samples from 20 European mouflon (Ovis gmelini musimon). Molecular identification using the cox1 sequence analysis was performed on sarcocysts excised from muscle tissue and on DNA from digested muscle samples. Both frequency and intensity of infection in mouflon were high with 19 of 20 animals testing Sarcocystis positive and > 50 cysts per gram of tissue recovered from 10 of the 19 Sarcocystis positive animals. Molecular analysis revealed dominant Sarcocystis tenella (18/19 animals) and Sarcocystis arieticanis (1/19 animals), whose known intermediate hosts are sheep. In addition, Sarcocystis capracanis, which is known to form sarcocysts in goats, was detected in two animals. The results of this study demonstrated the digestion method to be superior over the direct isolation of sarcocysts for the molecular identification of Sarcocystis species in a certain host. Future research of Sarcocystis diversity in wild ovine and caprine species is needed.

Time-resolved transcriptional profiling of Trichinella-infected murine myocytes helps to elucidate host-pathogen interactions in the muscle stage.

BACKGROUND: Parasites of the genus Trichinella are the pathogenic agents of trichinellosis, which is a widespread and severe foodborne parasitic disease. Trichinella spiralis resides primarily in mammalian skeletal muscle cells. After invading the cells of the host organism, T. spiralis must elude or invalidate the host's innate and adaptive immune responses to survive. It is necessary to characterize the pathogenesis of trichinellosis to help to prevent the occurrence and further progression of this disease. The aims of this study were to elucidate the mechanisms of nurse cell formation, pathogenesis and immune evasion of T. spiralis, to provide valuable information for further research investigating the basic cell biology of Trichinella-infected muscle cells and the interaction between T. spiralis and its host. METHODS: We performed transcriptome profiling by RNA sequencing to identify global changes at 1, 3, 7, 10 and 15 days post-infection (dpi) in gene expression in the diaphragm after the parasite entered and persisted within the murine myocytes; the mice were infected by intravenous injection of newborn larvae. Gene expression analysis was based on the alignment results. Differentially expressed genes (DEGs) were identified based on their expression levels in various samples, and functional annotation and enrichment analysis were performed. RESULTS: The most extensive and dynamic gene expression responses in host diaphragms were observed during early infection (1 dpi). The number of DEGs and genes annotated in the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases decreased significantly in the infected mice compared to the uninfected mice at 3 and 7 dpi, suddenly increased sharply at 10 dpi, and then decreased to a lower level at 15 dpi, similar to that observed at 3 and 7 dpi. The massive initial reaction of the murine muscle cells to Trichinella infection steadied in the later stages of infection, with little additional changes detected for the remaining duration of the studied process. Although there were hundreds of DEGs at each time point, only 11 genes were consistently up- or downregulated at all 5 time points. CONCLUSIONS: The gene expression patterns identified in this study can be employed to characterize the coordinated response of T. spiralis-infected myocytes in a time-resolved manner. This comprehensive dataset presents a distinct and sensitive picture of the interaction between host and parasite during intracellular infection, which can help to elucidate how pathogens evade host defenses and coordinate the biological functions of host cells to survive in the mammalian environment.

Effects of Trichinella spiralis and its excretory/secretory products on autophagy of host muscle cells in vivo and in vitro.

Trichinella spiralis (T. spiralis) is a widely distributed pathogenic microorganism that causes trichinellosis, a disease that has the potential of causing severe harm to their host. Numerous studies have demonstrated that autophagy can be triggered by microbial infection, such as bacteria, viruses, protozoa, and parasitic helminths. However, it's still unknown whether autophagy can facilitate host resistance to T. spiralis infection. The present study examined the role of autophagy in striated muscle cell transformation following infection with T. spiralis in BALB/c mice. Transmission electron microscopy (TEM) was used to detect the production of the host diaphragm autophagosome after T. spiralis infection, and changes in the protein and transcriptional levels of autophagic marker proteins were also detected. The significance of autophagy in T. spiralis infection, namely inhibition of T. spiralis growth, was preliminarily evaluated by conducting in vivo experiments using autophagy inhibitors. Besides, we studied the effect of excretory-secretory products (ES) of T. spiralis on autophagy of C2C12 myoblasts. The changes in protein and gene expression levels in autophagy-related pathways in vitro and in vivo were measured as further evidence. The results showed that T. spiralis infection induced autophagy in the host muscle cells. Meanwhile, ES inhibited autophagy of myoblasts in vitro, but this did not affect the cell viability. The upregulation and downregulation of autophagy-related factors in skeletal muscle cells may indicate an adaptive mechanism providing a comfortable niche for the parasite.

Sarcocystis spp. diversity in the roe deer (Capreolus capreolus) from Lithuania and Spain.

The roe deer (Capreolus capreolus) has been identified as an intermediate host for six known Sarcocystis species, S. capreolicanis, S. entzerothi, S. gracilis, S. linearis, S. oviformis, and S. silva. In this study, we identified Sarcocystis species in the diaphragm and tongue muscles from the Lithuanian and Spanish roe deer, respectively, on the basis of a microscopic examination and DNA analysis. A total of 43 and 27 sarcocysts were isolated and characterized from the Lithuanian and Spanish roe deer, respectively. Overall six Sarcocystis species were identified in roe deer from Lithuania, and only three of them, S. gracilis, S. linearis, and S. silva were found to have infecting animals from Spain. The current paper represents first molecular results of Sarcocystis species in the Spanish roe deer. Furthermore, transmission electron microscopy examination revealed specific wall structure of sarcocysts studied, S. linearis was characterized by ribbon-like villar protrusions (vp) (type 8a), and S. oviformis was distinguished by elongated vp resembling spades or mushroom-like structures (type 39). Based on 18S rDNA and cox1 sequences, Sarcocystis species from the roe deer showed considerable intraspecific genetic variability. However, similar values of intraspecific genetic variation were estimated at both genes analysed. The highest variability was observed for S. capreolicanis and S. linearis in both genes and for S. silva at cox1. Consequently, the level of genetic variability of Sarcocystis from the roe deer varied depending on species rather than on gene analysed or geographical area.

Histopathological Survey on Sarcocystis Species Infection in Slaughtered Cattle of Zabol-Iran

Objective: Sarcocystosis is an important zoonotic protozoal disease with worldwide distribution and wide range of hosts. The aim of the present study was to determine the intensity of Sarcocystis spp. infection and to show histopathological features of their cystic lesions in slaughtered cattle of Zabol- Iran. Methods: From April to September 2018, 500 tissue samples from esophagus, heart, diaphragm, tongue and masticatory muscles were prepared from 100 slaughtered cattle. All samples were fixed in 10% neutral buffered formalin and routine tissue processing protocol was performed. Results: The microscopic results showed that 92.2% of specimens had thin-walled cysts of S. cruzi and 14% had thick-walled Sarcocystis (S. hirsuta and S. hominis) but macrocyst was only observed in one cattle. The positivity rate of thin walled cysts was 58.8% for heart, 13.9% for masticatory muscles, 10.2% for tongue, 9.3% for esophagus and 7.8% for diaphragm. The positivity rate of thick walled cysts was 32.8% for esophagus, 28.6% for tongue, 22.9% for heart, 15.7% for masticatory muscles and 0% for diaphragm, which could represent either S. hominis or S. hirsuta. The most infected tissue was heart and the least infected tissue was diaphragm. Thin walled cysts (S. cruzi) were mostly found in heart and were less found in diaphragm. However, thick-walled cysts (S. hirsuta and S. hominis) were mostly detected in esophagus. No thick-walled cysts were found in diaphragm muscle. Conclusion: A high positivity rate of sarcocystosis in slaughtered cattle in Zabol abattoir revealed heavily environmental contamination of Sistan region by this important parasitic disease.

Molecular characterization of three Sarcocystis spp. from wild sika deer (Cervus nippon yesoensis) in Hokkaido, Japan.

Diaphragm samples from 65 hunted sika deer (Cervus nippon yesoensis) from Hokkaido, Japan were examined for the presence of sarcocysts based on histological sections. Morphologically, the detected sarcocysts grouped into three types: (Type 1) 108.0-305.0 µm in width, thick-walled (4.3-7.0 µm) with tombstone-like protrusions; (Type 2) 25.0-69.5 µm in width, thick-walled (3.8-8.0 µm) with finger-like protrusions; and (Type 3) 22.5-55.0 µm in width, thin-walled (under 1 µm) with no visible protrusions under light microscopy. All samples contained at least one sarcocyst type, and multiparasitism was apparent in 58 samples. Morphologically, Type 1 sarcocysts were found in 19 (29.2%) samples, Type 2 in 62 (95.4%) samples, and Type 3 in 60 (92.3%) samples. The sarcocysts were collected using laser microdissection, the DNA extracted from them was PCR-amplified, and their 18S ribosomal RNA and cytochrome c oxidase subunit 1 genes were sequenced. Phylogenetic analysis showed that, for both genes, each morphological sarcocyst type (Types 1, 2, and 3) aligned most closely with S. silva/S. truncata, S. tarandi/S. elongata, and S. pilosa, respectively. Based on the sequence identities between taxa and the molecular information for sarcocysts in C. nippon centralis, the sarcocyst types were presumed to be S. truncata-like (Type 1), S. tarandi-like (Type 2), and S. pilosa (Type 3). The phylogenetic analyses based on the present comprehensive molecular characterization of three Sarcocystis spp. from C. nippon yesoensis in Hokkaido suggest that canids (e.g., wild foxes) may be the definitive hosts for S. pilosa, and felids (or unknown species) the definitive hosts for the other two species.

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Prevalence and molecular characterisation of Sarcocystis miescheriana and Sarcocystis suihominis in wild boars (Sus scrofa) in Italy.

A sample of the diaphragm was collected from each of 100 wild boars legally hunted in the Val Grande National Park in north-western Italy and examined for the presence of Sarcocystis infection by histological and molecular methods. In histological sections, thick-walled sarcocysts consistent with those of Sarcocystis miescheriana were detected in 32 wild boars. Genomic DNA extracted from diaphragm samples was initially subjected to PCR amplification of the internal transcribed spacer 1 (ITS1) region, and 97 wild boars were found to harbour a Sarcocystis infection at this screening. Selected DNA samples were then subjected to PCR amplification and sequencing of the ITS1 region and the 18S and 28S ribosomal RNA (rRNA) genes of the nuclear ribosomal DNA unit, while all positive samples were subjected to PCR amplification of the mitochondrial cytochrome c oxidase subunit I (cox1) gene. S. miescheriana was identified in 97 wild boars (97%), while the zoonotic Sarcocystis suihominis was identified in one wild boar (1%), which also harboured S. miescheriana. Intra-specific sequence variation was found in all four DNA regions of S. miescheriana examined and in the 18S rRNA gene and ITS1 region of S. suihominis. The partial cox1 gene was amplified and sequenced from 72 isolates of S. miescheriana, yielding 43 haplotypes with pairwise sequence identities of 97.6-99.9%. These haplotypes were 79.1-79.8% identical with the cox1 sequence of S. suihominis. Phylogeny based on cox1 sequences placed S. miescheriana and S. suihominis as sister species within a clade comprising mainly Sarcocystis spp. of ruminants with felids as known or presumed definitive hosts. The same was true for the phylogeny based on 18S rRNA gene sequences.

Isolation of viable Toxoplasma gondii from organs and Brazilian commercial meat cuts of experimentally infected pigs.

The present study evaluated the distribution and viability of Toxoplasma gondii tissue cysts in the organs and Brazilian commercial cuts of experimentally infected pigs. The pigs were infected with 3 × 103 oocysts of the T. gondii isolate TgCkBr57 (Type BrII). Mouse bioassays were performed on the brain, retina, tongue, diaphragm, and heart as well as the following muscle cuts: loin (longissimus), coppa (longissimus, spinalis dorsi, rhomboideus), tenderloin (psoas major), outside flat (biceps femoris), topside (semimembranosus), and top sirloin (gluteus medius). Toxoplasma gondii was isolated from the coppa, heart, diaphragm, and tongue of three pigs; from the tenderloin, outside flat, and brain of two pigs; and from the top sirloin and loin of one pig. Thus, the viability of T. gondii cysts was observed in all of the organs and cuts evaluated (except for the topside and retina), demonstrating the broad distribution of this parasite in pig organs and commercial meat cuts, and the importance of this species as a source of human infection.

Pathological and molecular identification of porcine cysticercosis in Maharashtra, India.

Porcine cysticercosis, caused by metacestodes of Taenia solium is an important emerging zoonotic disease with public health and economic significance. Pigs acquire the disease through consumption of Taenia solium eggs excreted by human tapeworm carriers. The present study was conducted to investigate the prevalence of porcine cysticercosis in Nagpur and Mumbai region of Maharashtra, India by P/M examination of carcasses followed by histopathology of affected organs in infected animals and molecular identification of cysts for confirmation. Out of 1000 pigs examined during slaughter, three pigs were found to be heavily affected with T. solium cysts giving a prevalence of 0.3%. Histological section of brain in infected animals revealed marked vascular congestion of meninges, mild neuronal degeneration, perivascular cuffing and gliosis while the liver showed the infiltration of mononuclear cell, predominantly eosinophils throughout the parenchyma. Some degree of calcification was observed in the cysts lodged in liver while calcification was not evident in case of cysts lodged in brain, tongue, diaphragm and skeletal muscle. Molecular identification by PCR using two sets of oligonucleotide primers against LSU rRNA gene and Mt-Cox1 gene of T. solium confirms the cysts to be that of T. solium. The molecular diagnostics methods have been considered for validation in conjunction with P/M inspections, parasitological and histopathological examinations. The study confirms the presence of porcine cysticercosis in the two regions and demands proper sanitary measures to minimize the risk of infection from zoonoses and food safety point of view.

High prevalence of Sarcocystis spp. infections in cattle (Bos taurus) from central China.

Myocardium and diaphragm samples of cattle (n = 521) from HeNan Province (China) were screened for Sarcocystis sarcocysts by histological examination, pepsin digestion, and molecular assays. Morphology and molecular assays were used for identification. The prevalence of Sarcocystis infection in cattle was 41.5% (216/521). Histological examination identified sarcocysts in the myocardium (49.4%, 200/405) and diaphragm (13.8%, 16/116) of cattle. Two species were identified, namely S. cruzi (41.3%, 215/521) and S. hominis (0.2%, 1/521). The findings of the present study indicate a high prevalence of S. cruzi infection in cattle from central China.

Molecular identification of Sarcocystis lutrae (Apicomplexa: Sarcocystidae) in muscles of five species of the family Mustelidae.

Carnivores usually act as definitive hosts of Sarcocystis species. However, the number of reports on sarcocyst formation in musculature of predators is on the increase. In the present study, muscle samples of 68 mustelids collected in Lithuania were examined for sarcocysts of Sarcocystis species. Sarcocysts were detected in diaphragm, tongue and limb muscles of ten animals (14.7%) but were not discovered in the heart. Based on 18S rDNA, 28S rDNA, cox1 and ITS1 sequence analysis, Sarcocystis lutrae was identified in three American minks (Neovison vison), two beech martens (Martes foina), three Eurasian badgers (Meles meles), one Eurasian otter (Lutra lutra) and one European polecat (Mustela putorius). The intraspecific variability of this Sarcocystis species was determined only in ITS1 region. Based on the phylogenetic analysis, no clear separation of S. lutrae by intermediate hosts or geographical locations was established. This paper represents the first identification of S. lutrae in the American mink, the beech marten and the European polecat. Current results indicate that S. lutrae is a common species in the muscles of various European mustelids.

Tongue has higher larval burden of Trichinella spp. than diaphragm in wolverines (Gulo gulo).

Trichinella is an important zoonotic parasite found in a range of wildlife species harvested for food and fur in Canada. We compared larval intensity from tongue and diaphragm, the best predilection sites in other animal species, from naturally infected, wild wolverines (Gulo gulo) (n = 95). Muscle larvae of Trichinella spp. were recovered by the pepsin/HCl artificial digestion method (gold standard) using double separatory funnels, and species were identified using multiplex PCR. Prevalence was 83% (79/95). Of those positive for Trichinella spp. (n = 79), 76 (96.2%) were detected in both tissues, 2 (2.5%) were positive only on diaphragm, and 1 (1.3%) only on tongue. A total of 62 of 79 wolverines (78.5%) had higher larval burden in tongue than in diaphragm, whereas 17 wolverines (21.5%) had higher larval burden in diaphragm. The predilection site (higher larval burden) of Trichinella spp. larvae did not vary significantly between juvenile and adult wolverines (P = 0.2), between male and female wolverines (P = 0.9), and among wolverines classified as having low and high larval intensities overall (P = 0.2). Trichinella T6 was the predominant genotype (63 of 79; 80%), followed by T. nativa (T2) (6 of 79; 8%). Mixed infections of T2 and T6 were observed in 9 of 79 (12%) wolverines. Larval intensity of Trichinella T6 was higher in tongues than diaphragms. No statement can be made for T2 due to insufficient T2 positive samples. In conclusion, tongues are a better site for sampling than diaphragms in future surveys of Trichinella larval intensity in wolverines; however, either tissue is suitable for prevalence studies.

Morphological and molecular identification of Sarcocystis spp. from the sika deer (Cervus nippon), including two new species Sarcocystis frondea and Sarcocystis nipponi.

Diaphragm muscles of 25 sika deer (Cervus nippon) farmed in Lithuania were examined for sarcocysts of Sarcocystis species. Two new Sarcocystis species, Sarcocystis frondea and Sarcocystis nipponi, were observed using light microscopy (LM) and transmission electron microscopy (TEM) and characterized by 18S ribosomal DNA (rDNA) and subunit I of cytochrome c oxidase (cox1) sequence analyses. By LM, sarcocysts of S. frondea and S. nipponi were ribbon-shaped and had finger-like sarcocyst wall protrusions, respectively. Under TEM, protrusions of S. frondea were about 9 × 1-1.5 µm, filled with clearly visible electron-dense substance and microtubules, type 39-like. Whereas, protrusions (about 9 × 0.2 µm) of S. nipponi arose from dome-shaped bases were filled with microtubules extending to the ground substance layer, type 9o-like. Moreover, three known Sarcocystis spp., Sarcocystis entzerothi, Sarcocystis ovalis, and Sarcocystis truncata previously described in other cervids as intermediate hosts, were characterized in sika deer. The cox1 was more suitable than 18S rDNA delimitating closely related Sarcocystis species from cervids. The phylogenetic results suggest that scavenger birds could be definitive hosts of S. frondea. According to the summarized morphological data on Sarcocystis found in the sika deer, such host should harbor at least nine different Sarcocystis species.

[The surgery of diaphragmatic hydatidosis and their complications]. / La chirurgie des hydatidoses diaphragmatiques et leurs complications.

Primary or secondary diaphragmatic echinococcosis is rare, accounting for 1% of the thoracic locations. They may be operative discovery or by their complication, hence a variable symptomatology making this localization a particular entity. The thoracic and abdominal CT allows a complete assessment. Surgery remains the only therapeutic approach. In complicated forms an additional surgery is required for complete care. The prognosis is generally good apart from the risk of recurrence. Through a series of 4 operated patients, we focus on the clinical and therapeutic features of this pathology and its complications.

IL-4/5 signalling plays an important role during Litomosoides sigmodontis infection, influencing both immune system regulation and tissue pathology in the thoracic cavity.

Approximately 100 million people suffer from filarial diseases including lymphatic filariasis (elephantiasis), onchocerciasis (river blindness) and loiasis. These diseases are amongst the most devastating of the neglected tropical diseases in terms of social and economic impact. Moreover, many infection-induced immune mechanisms in the host, their relationship to disease-related symptoms and the development of pathology within the site of infection remain unclear. To improve on current drug therapies or vaccines, further studies are necessary to decipher the mechanisms behind filaria-driven immune responses and pathology development, and thus the rodent model of Litomosoides sigmodontis can be used to unravel host-filaria interactions. Interestingly, BALB/c mice develop a patent state (release of microfilariae, the transmission life-stage, into the periphery) when exposed to L. sigmodontis. Thus, using this model, we determined levels of host inflammation and pathology development during a L. sigmodontis infection in vivo for the first known time. Our study reveals that after 30days p.i., inflammation and pathology began to develop in infected wild type BALB/c mice between the lung and diaphragm, close to the site of infection - the thoracic cavity. Interestingly, infected IL-4Rα/IL-5-/- BALB/c mice had accentuated inflammation of the pleural lung and pleural diaphragm, and higher parasite burdens. Corresponding to the pleural inflammation, levels of IP-10, MIP-1α, MIP-1ß, MIP-2 and RANTES were significantly elevated in the thoracic cavity fluid of infected IL-4Rα/IL-5-/- mice compared with wild type controls. Moreover, upon L. sigmodontis antigen stimulation, IFN-γ and IL-17A secretions by cells isolated from draining lymph nodes of IL-4Rα/IL-5-/- mice were significantly elevated, whereas secretion of IL-5, IL-13 and IL-10 was reduced. Elevated filaria-specific IFN-γ secretion was also observed in spleen-derived CD4+ T cell co-cultures from IL-4Rα/IL-5-/- mice. In summary, this study unravels the essential role of IL-4/IL-5 signalling in controlling immunity against filarial infections and demonstrates the requirement of this pathway for the host to control ensuing pathology and inflammation.

Sequence variation in the B1 gene among Toxoplasma gondii isolates from swine and cats in Italy.

The evaluation of the genetic variations of Toxoplasma gondii among isolates of a wide variety of animal hosts can provide significant information for better understanding the epidemiology and population structure of the parasite in different geographical areas. The aim of this study was to provide information on T. gondii genetic diversity in host species living in central Italy, which could act as a potential source of human infection. Seventy-seven feline faecal samples, and 36 and 20 diaphragm pillar tissue samples from pigs and wild boars were collected in Umbria (central Italy). The samples were tested by a nested-PCR protocol amplifying an informative region within the B1 gene, a multi-copy genetic target, showing a good rate of variability. Thirty-six specimens (27.07%) belonging to 10 pigs, 13 wild boars and 13 cats, tested positive to the B1 nested-PCR screening. Of these, 23 good quality sequences (8 from wild boars, 5 from pigs, and 10 from cats) were analyzed. A comparison of the B1 DNA sequences showed that a single homogeneous nucleotide substitution (C/T) was present at position 31 in the isolates from pigs and wild boars compared with the sampled cats and other hosts (including humans) available in GenBank™. The present results suggest the existence of a T. gondii genetic diversity for swine host species, based on a SNP (C/T) of the B1 gene. Further studies are needed to draw more solid conclusions on the discriminatory power of the B1 target by collecting more swine samples from much broader geographical areas.

Gasterophilus intestinalis (Diptera: Oestridae) in the diaphragmatic muscle: An unusual finding.

Larval forms of the bot-fly Gasterophilus are obligate parasites commonly found in the gastrointestinal tract of equids, causing intestinal myiasis. Five species are reported so far in Italy, mostly observed during necroscopy, located in different portion of gastrointestinal tract of equids: G. intestinalis, G. nasalis, G. inermis, G. pecorum and G. haemorrhoidalis. An unusual finding of larval Gasterophilus intestinalis deeply inserted into the diaphragmatic muscle is here reported. Due to the uncommon localization, to the absence of clinical signs related to myiasis and subsequent uncertainty of species identity, identification was performed using an integrative taxonomical approach combining morphology with molecular tools for confirmatory reasons. This finding adds information on migration patterns of erratic larval forms in G. intestinalis, a feature of interest as gasterophiliasis is among the less studied intestinal myiasis of horses.