Role of dense granule antigen 7 in vertical transmission of Neospora caninum in C57BL/6 mice infected during early pregnancy.

Neosporosis is a parasitic disease affecting the health of dogs and cattle worldwide. It is caused by Neospora caninum, an obligate intracellular apicomplexan parasite. Dogs are its definitive host, it mostly infects livestock animals, especially cattle that acts as intermediate host. It is necessary to have well-established models of abortion and vertical transmission in experimental animals, in order to determine basic control measures for the N. caninum infection. We evaluated the role of N. caninum dense granule antigen 7 (NcGRA7) in the vertical transmission of N. caninum using the C57BL/6 pregnant mouse model. We inoculated mice on day 3.5 of pregnancy with parental Nc-1 or NcGRA7-deficient parasites (NcGRA7KO). Post-mortem analyses were performed on day 30 after birth and the surviving pups were kept until day 30 postpartum. The number of parasites in the brain tissues of offspring from NcGRA7KO-infected dams was significantly lower than that of the Nc-1-infected dams under two infection doses (1 × 106 and 1 × 105 tachyzoites/mouse). The vertical transmission rates in the NcGRA7KO-infected group were significantly lower than those of the Nc1-infected group. To understand the mechanism by which the lack of NcGRA7 decreases the vertical transmission, pregnant mice were sacrificed on day 13.5 of pregnancy (10 days after infection), although parasite DNA was detected in the placentas, no significant difference was found between the two parasite lines. Histopathological analysis revealed a greater inflammatory response in the placentas from NcGRA7KO-infected dams than in those from the parental strain. This finding correlates with upregulated chemokine mRNA expression for CCL2, CCL8, and CXCL9 in the placentas from the NcGRA7KO-infected mice. In conclusion, these results suggest that loss of NcGRA7 triggers an inflammatory response in the placenta, resulting in decreased vertical transmission of N. caninum.

Transplacental transmission of Neospora caninum to lambs in successive pregnancies of naturally infected sheep in Southern Brazil.

This study aims to report the detection of N. caninum DNA in a newborn lamb (1) with neurological signs and congenital neosporosis and in a stillborn lamb (2), both born from the same ewe in a herd of Southern Brazil. The lambs were born during different pregnancies of a Suffolk ewe seropositive to N. caninum and seronegative to T. gondii. Histopathological lesions were observed only in the central nervous system of the lambs. The newborn lamb (1) showed mild and focal gliosis in the frontal lobe. In the hippocampal region of the stillborn lamb (2), lymphoplasmacytic perivascular cuffs and N. caninum cysts were observed in the cytoplasm of neurons and confirmed by IHC. PCR was performed using brain samples to detect the protozoa N. caninum and Toxoplasma gondii. The infection with N. caninum was confirmed in the newborn lamb (1) by PCR and in the stillborn lamb (2) by histopathology, immunohistochemistry, and PCR tests.

Risk factor analysis associated with Neospora caninum in dairy cattle in Western Brazilian Amazon.

Neospora caninum is considered to be one of the main causes of abortion among cattle. The present survey was conducted in the municipality of Rolim de Moura, Rondônia State, Brazil. A questionnaire that investigates the epidemiological aspects of neosporosis was used in the analysis of risk factors associated with the animal-level and herd-level prevalence in dairy cattle. A total of 416 bovine blood samples were collected from 30 farms, and N. caninum antibody levels were measured by Indirect Fluorescent Antibody Test (IFAT). Analysis of dairy cattle serum samples revealed the presence of anti-N. caninum antibodies to be 47.36% (n = 197). Risk factors associated with N. caninum infection were the management system and access locations of dogs. The results of the present survey indicated that infection of dairy cattle with N. caninum is widespread in the studied region of Western Amazon, which has implications for prevention and control of neosporosis in this region. Therefore, integrated control strategies and measures are recommended to prevent and control N. caninum infection in dairy cattle. In addition, direct contact between dairy cattle, dogs and wild animals, which can influence the epidemiology of neosporosis, should be investigated further.

Parasites in a hotspot: diversity and specificity patterns of apicomplexans infecting reptiles from the Socotra Archipelago.

Although parasites represent a major component of biodiversity, they remain poorly assessed, especially in remote regions. In this study, we screened 461 reptiles from Socotra, the largest and most biologically diverse archipelago in Arabia. Using 18S rRNA primers, we detected various apicomplexan parasites, namely haemogregarines, sarcocystids and eimeriids. Haemogregarines were the most common and genetically diverse, followed by sarcocystids (genus Sarcocystis) and eimeriids (genera Isospora and Lankesterella). All were related to parasites of other reptiles, including species from Arabia, Northern Africa and Asia. Like their 29 endemic reptile hosts, almost all Socotran parasites presented high genetic divergence and ecological differences from those found elsewhere, and probably represent undescribed endemic species. Among hosts, skinks were the most parasitized, which contrasted with similar studies from other areas, probably due to their more generalist diet and habitat use. As expected due to its high species richness, geckos harboured the highest parasite diversity in the archipelago. Parasite diversity also seemed to be correlated to island size, as the largest island harboured most haplotypes. This study emphasizes the importance of screening parasites in wild hosts from remote regions and of considering host ecology to understand disease transmission across taxa.

Effects of probiotic and Bidens pilosa on the performance and gut health of chicken during induced Eimeria tenella infection.

AIM: In this study, we have examined the individual and combined protective mechanism of probiotic and Bidens pilosa on the performance and gut health of chickens during Eimeria tenella infection over a 29-day experimental trial. METHODS AND RESULTS: A total of one hundred and fifty 1-day-old chickens were equally distributed into five treatment groups with three biological replicates: two groups were allocated as control groups (control group untreated unchallenged, CG and control positive untreated challenged, CPG) and three groups were fed diets with probiotic (PG), B. pilosa (BPG) and probiotic + B. pilosa (PG + BPG) and challenged with E. tenella. Birds of all groups were assessed for pre and post-infection body weights, oocysts shedding, caecal lesion scores and mRNA expression levels of apoptosis related proteins (Bcl-2, Bax and caspase-3), antioxidant enzymes (CAT and SOD 1), pro-inflammatory cytokines (IL-6 and IL-8) and tight junction proteins (CLDN 1 and ZO 1). Our results revealed that during infection (day 21-29), E. tenella challenged chickens significantly decreased the body weight compared with uninfected control chickens; however, there was no significant effect on body weight of chickens fed with probiotic, B. pilosa and probiotic + B. pilosa was observed. Eimeria tenella challenged untreated birds increased (P < 0·05) oocysts shedding, destructive ratio of caeca and mortality as compared to treated challenged birds. CPG group up-regulated the mRNA expression levels of anti-apoptosis protein Bcl-2 while down-regulated the pro-apoptosis protein Bax relative to PG, BPG and PG + BPG groups. Moreover chickens fed probiotic, B. pilosa and probiotic + B. pilosa diets enhanced the activities of antioxidant enzymes, pro-inflammatory cytokines and tight junction proteins with the comparison of control positive untreated challenged chickens. CONCLUSION: These findings elaborated that feed supplementation of probiotic and B. pilosa (individually or in combination) appeared to be effective in inhibiting the occurrence of disease and decreasing the severity of Eimeria infection in chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study explained the underlying anti-coccidial mechanism in which probiotic and B. pilosa (individually and/or in combination) improve the performance of chicken and protect against gut inflammatory responses caused by E. tenella.

Risk factor analysis associated with Neospora caninum in dairy cattle in Western Brazilian Amazon / Análises de fatores de risco associado a Neospora caninum em gado leiteiro na Amazônia Ocidental Brasileira

Abstract Neospora caninum is considered to be one of the main causes of abortion among cattle. The present survey was conducted in the municipality of Rolim de Moura, Rondônia State, Brazil. A questionnaire that investigates the epidemiological aspects of neosporosis was used in the analysis of risk factors associated with the animal-level and herd-level prevalence in dairy cattle. A total of 416 bovine blood samples were collected from 30 farms, and N. caninum antibody levels were measured by Indirect Fluorescent Antibody Test (IFAT). Analysis of dairy cattle serum samples revealed the presence of anti-N. caninum antibodies to be 47.36% (n = 197). Risk factors associated with N. caninum infection were the management system and access locations of dogs. The results of the present survey indicated that infection of dairy cattle with N. caninum is widespread in the studied region of Western Amazon, which has implications for prevention and control of neosporosis in this region. Therefore, integrated control strategies and measures are recommended to prevent and control N. caninum infection in dairy cattle. In addition, direct contact between dairy cattle, dogs and wild animals, which can influence the epidemiology of neosporosis, should be investigated further.

Resumo A infecção por Neospora caninum é considerada uma das principais causas de aborto entre bovinos. Esta pesquisa foi realizada no município de Rolim de Moura, estado de Rondônia, Brasil. Um questionário que investiga os aspectos epidemiológicos da neosporose foi utilizado na análise dos fatores de risco associados à prevalência em animais e em rebanhos. Um total de 416 amostras de sangue bovino foi colhido em 30 fazendas, e os níveis de anticorpos de N. caninum foram mensurados pela reação de Imunofluorescência Indireta (RIFI). A análise das amostras mostrou prevalência de anticorpos contra N. caninum de 47,36% (n = 197). Os fatores de risco associados à infecção por N. caninum foram o sistema de manejo e os locais de acesso dos cães. Os resultados da presente pesquisa indicam que a infecção de bovinos leiteiros com N. caninum está disseminada na região estudada da Amazônia Ocidental, o que tem implicações para a prevenção e controle da neosporose nessa região. Portanto, estratégias e medidas de controle integrado são recomendadas para prevenir e controlar a infecção por N. caninum em gado leiteiro. Além disso, o contato íntimo entre gado leiteiro, cães e animais selvagens, pode influenciar a epidemiologia da neosporose e deve ser investigada mais detalhadamente.

Identical 18S rRNA haplotypes of Hepatozoon canis in dogs and foxes in Brandenburg, Germany.

Hepatozoon canis is a blood parasite of the suborder Adeleorina infecting wild and domestic canids. Transmission occurs by oral uptake of Rhipicephalus sanguineus sensu lato vector ticks infected with H. canis, but vertical transmission is also assumed to be possible. In German foxes, a high prevalence of H. canis has previously been reported despite the fact that R. sanguineus s.l. is not endemic. In the absence of knowledge about local transmission pathways, foxes should be considered to be possible reservoirs of H. canis and contribute to infection of domestic dogs. The present study aimed to determine how often foxes and dogs are infected in Brandenburg (Germany) and if identical or different H. canis 18S rRNA haplotypes are found in these host species. Hepatozoon spp. were detected by PCR in 46/1050 (4.4 %) of dog blood and 176/201 (77.6 %) of fox spleen samples from Brandenburg. Sequencing of 19 dog and 56 fox samples identified all as H. canis. For nine positive dogs, owners stated that they had never left Germany suggesting that autochthonous transmission occurs not only in foxes but also in dogs. Sequences for seven of these possible autochthonous cases were obtained and six were identical to the predominant haplotype found in the foxes. Haplotype network analysis confirmed that many dogs, including some without travel history, carried the same or very similar 18S rRNA haplotypes as the foxes suggesting that both hosts participate in the same epidemiological cycle.

Absence of Hemolivia mauritanica (Apicomplexa: Haemogregarinidae) in natural populations of Testudo graeca in Morocco.

During spring 2018, we captured 101 spur-thighed tortoises, Testudo graeca, from seven localities in central Morocco. All tortoises were examined for the presence of blood parasites Hemolivia mauritanica and Hyalomma aegyptium ticks, the known vectors. We looked for H. mauritanica infection by examination of blood smears and by genetic analysis with PCR using extractions from both tortoises and ticks. On all tortoises collected, 71.29% were infested with at least one tick, with a mean infestation intensity of 7.12 ticks/tortoise and maximum of 15.55 ticks/tortoises in Had Draa locality (Essaouira region). Although the definitive host is present and abundant in all tortoise populations, all blood samples were Hemolivia-negative. Our results support and confirm the finding of studies previously conducted in other populations of Morocco and indicate that H. mauritanica has a narrower distribution range than its tick vector.

Endogenous transplacental transmission of Neospora caninum in successive generations of congenitally infected goats.

Neospora caninum is a protozoan that is considered an important agent of reproductive disorders in ruminants worldwide, and vertical transmission is the main form of infection and maintenance of neosporosis in herds. In goats, there have been no studies that have evaluated the transmission of N. caninum between successive generations. Thus, the objective of this study was to evaluate, through IFAT and PCR, the endogenous transplacental transmission of N. caninum in up to five generations of six families of dairy goats naturally infected by the parasite and whether it was possible for dairy goats to become free of infection over successive generations. Ninety-five serum samples from positive animals and 75 samples from negative animals were analyzed for N. caninum. Of the 95 samples analyzed, 93 contained anti-N. caninum antibodies (97.8 %). Titers of anti-N. caninum antibodies varied (increasing or decreasing) in the offspring; however, with an increase in the number of the goat generations, the offspring tended to have lower titers (p = 0.021) at the day of birth. Reproductive disorders such as abortions, stillbirth or fetal retention occurred at a rate of 10.4 % and were not influenced by the mother's titer of anti-N. caninum antibodies at the day of parturition or abortion. The results showed that infection by N. caninum persists throughout generations in congenitally infected goats.

Hepatozoon canis in hunting dogs from Southern Italy: distribution and risk factors.

Hepatozoon canis is a hemoprotozoan organism that infects domestic and wild carnivores throughout much of Europe. The parasite is mainly transmitted through the ingestion of infected ticks containing mature oocysts. The aims of the present survey were to determine the prevalence of H. canis in hunting dogs living in Southern Italy and to assess potential infection risk factors. DNA extracted from whole blood samples, collected from 1433 apparently healthy dogs living in the Napoli, Avellino, and Salerno provinces of Campania region (Southern Italy), was tested by a quantitative real-time polymerase chain reaction (qPCR) assay to amplify H. canis. Furthermore, the investigated dog population was also screened by qPCR for the presence of Ehrlichia canis, a major tick-borne pathogen in Southern Italy, in order to assess possible co-infections. Two hundred dogs were H. canis PCR-positive, resulting in an overall prevalence of 14.0% (CI 12.2-15.9). Breed category (P < 0.0001), hair coat length (P = 0.015), and province of residence (P < 0.0001) represented significant risk factors for H. canis infection. The presence of H. canis DNA was also significantly associated with E. canis PCR positivity (P < 0.0001). Hunting dogs in Campania region (Southern Italy) are frequently exposed to H. canis, and the infection is potentially associated with close contact with wildlife. Further studies are needed to assess the pathogenic potential of H. canis, as well as the epidemiological relationships between hunting dogs and wild animal populations sharing the same habitats in Southern Italy.

First highly sensitive and specific competitive ELISA for detection of bovine besnoitiosis with potential as a multi-species test.

Serological cross-reactions represent a serious problem in some currently available tests to diagnose Besnoitia infections in many species including cattle, caribou and donkeys. False-positive results are due to the low positive-predictive value of these serological tests for besnoitiosis. These tests therefore have clear limitations if large herds are screened in areas with low prevalence, since increased numbers of false-positive reactions require confirmatory testing by alternative serological methods, e.g. immunoblotting, which are time-consuming and create extra costs. To overcome this problem, we aimed to develop a highly sensitive and specific competitive ELISA (cELISA) using a panel of 12 monoclonal antibodies raised against the tachyzoite stage of Besnoitia besnoiti. A cELISA set up with one of these antibodies (Bb-cELISA1) was screened with a large panel of B. besnoiti-positive bovine sera to estimate the diagnostic sensitivity of the test. Sera from herds with Neospora caninum- or Sarcocystis spp.-infected cattle were used to estimate its diagnostic specificity. Relative to a reference standard, which combined the results obtained in a previously established highly sensitive and specific ELISA, in the immunofluorescence antibody test and in B. besnoiti tachyzoite and bradyzoite immunoblots, the new Bb-cELISA1 revealed a diagnostic sensitivity of 99.2% (95% confidence interval: 97.1-99.9%) and a diagnostic specificity of 99.9% (95% confidence interval: 97.7-100%). This novel assay was tested on a variety of proven Besnoitia-positive sera from other species, including B. besnoiti-infected cats, rabbits or Besnoitia bennetti-infected donkeys or Besnoitia tarandi-infected caribou. The results obtained with the new Besnoitia-cELISA for these animal species also corresponded almost perfectly with those of the reference tests, which included immunoblot and immunofluorescence antibody tests. In conclusion, the novel Besnoitia-cELISA represents a valuable tool for the diagnosis and control of bovine besnoitiosis and for studies on the epidemiology of Besnoitia infections in a variety of host species, including naturally exposed wildlife and experimental hosts.

Complete sporogony of the blood parasite Haemoproteus nucleocondensus in common biting midges: why is its transmission interrupted in Europe?

Haemoproteus species (Haemoproteidae) are widespread blood parasites and are transmitted by Culicoides biting midges and Hippoboscidae louse flies. Although these pathogens may cause morbidity or mortality, the vectors and patterns of transmission remain unknown for the great majority of avian haemoproteids. Haemoproteus nucleocondensus has been frequently reported in Europe in great reed warblers Acrocephalus arundinaceus after their arrival from African wintering grounds, but this infection has not been found in juveniles at the breeding sites. The factors that prevent its transmission remain unclear. This study was designed to test whether the sporogony of H. nucleocondensus (lineage hGRW8) can be completed in Culicoides impunctatus, one of the most abundant European biting midge species. Wild-caught females were infected with H. nucleocondensus from great reed warblers. Microscopic examination and PCR-based methods were used to detect sporogonic stages and to confirm species identity. This study showed that H. nucleocondensus completes sporogony in C. impunctatus, suggesting that there are no obstacles to its transmission from the point of view of vector availability and average temperature in Northern Europe. We discuss other ecological factors which should be considered to explain why the transmission of H. nucleocondensus and some other Southern origin haemosporidians are interrupted in North Europe.

The role of the American cockroach (Periplaneta americana) as transport host of Eimeria tenella to chickens.

The role of the American cockroach, Periplaneta americana as transport host for Eimeria tenella was evaluated. Twenty-four cockroaches were orally fed with sporulated oocysts of E. tenella. Their feces and digestive tract were examined for oocysts by sugar centrifugal flotation technique and PCR. Infectivity of the oocysts recovered from the digestive tract of infected cockroaches as well as from their feces was evaluated by orally inoculating them into Boris Brown chickens. E. tenella oocysts were found in the digestive tract and feces of infected cockroaches up to day 4 after ingestion of oocysts. Furthermore, oocysts that were recovered from the digestive tract and feces of cockroaches remained infective for 4 and 3 days after ingestion of oocysts, respectively. Presence of oocysts in the feces of chicken that had been inoculated with either digestive tract or feces of P. americana demonstrated the infectivity of E. tenella oocysts from digestive tract or feces, suggesting that P. americana may play a role in the transmission of E. tenella among chicken and between chicken flocks.

Seroprevalence of Neospora caninum infection and associated risk factors in dairy cattle in Serbia.

Neosporosis in cattle is a globally important disease, causing abortions and significant economic losses if epidemic abortions occur. In Serbia, however, studies of Neospora caninum infection in cattle are few and are based on limited samples and/or from limited areas. We thus performed a nationwide study to examine the seroprevalence of N. caninum infection in a sample of cows randomly selected from dairy farms in 12 epidemiological units from throughout Serbia, as well as the possible transmission risk factors. Sera from a total of 1496 cattle were tested by competitive ELISA, and N. caninum-specific antibodies were shown in 7.2% animals (95% confidence interval CI, 6.6-7.9%), ranging from 2.2 to 12% across the epidemiological units. At least one seropositive animal was detected on 10.7% (95% CI, 9.7-11.8%) of farms, with a range of 5.9-25.9%. Logistic regression analysis showed that the single risk factor for infection in individual animals was keeping cows in loose-stalls (OR = 3.31, 95% CI = 1.95-5.60, P < 0.001). Risk factors for the presence of infection on farms also included housing in loose-stalls (OR = 18.49, 95% CI = 5.40-63.36, P < 0.001), and herd size > 100 animals (OR = 24.08, 95% CI = 3.85-150.50, P = 0.001). In view of the relatively low prevalence of infection showed at both the individual and farm level, this is the perfect time to undertake appropriate preventive measures to improve animal health and reduce economic losses.

Viable Eimeria oocysts in poultry house litter at the time of chick placement.

The purpose of this study was to determine if Eimeria oocysts recovered from litter at the time of chick placement in commercial broiler houses contained oocysts that were infectious for chickens. Over 100 litter samples were collected from 30 poultry farms representing a total of 60 different broiler houses with 9 houses sampled more than once over 1.5 yr. The samples were collected just before the placement of newly hatched chicks and after an anticoccidial drug (ACD) or Eimeria vaccine (VAC) program, and processed for counting oocysts followed by Eimeria species determination using ITS1 PCR. Broiler chicks were inoculated with recovered Eimeria oocysts to determine if the litter oocysts were viable and capable of causing patent infection. At placement, E. maxima (Emax) oocysts were detected in 70 of 75 houses after ACD program and 46 of 47 houses after VAC program. Eimeria acervulina, E. praecox, and/or E. tenella (Eapt) were detected in 75 of 75 houses after ACD program and 47 of 47 houses after VAC program. Viability testing revealed that 33.0% of broiler houses contained viable Emax oocysts, while 46.9% contained viable Eapt oocysts. During VAC programs, the concentration of Emax oocysts at placement and the total number of Emax oocysts shed by chickens in viability studies showed a very strong correlation (r = 0.83). Likewise, during ACD programs, the concentration of Eapt oocysts at placement and the total number of Eapt oocysts shed by chickens in the viability study showed a strong correlation (r = 0.62). In general, Eimeria oocyst levels at placement and number of viable oocysts shed by chickens in the viability study were similar among houses on the same farm. However, the number of Eimeria oocysts shed in the viability studies was considerably less than expected based on the number of oocysts given. These data suggest that nearly 100% of all poultry houses contain Emax and Eapt oocysts at placement with 30 to 50% of the houses containing viable Eimeria oocysts, thus possibly representing a source of the protozoa to newly hatched chicks.

Vertical transmission of Neospora caninum in bovine fetuses from a slaughterhouse in Brazil.

Neospora caninum is considered as one of the main causes of reproductive failure in cattle. Vertical transmission is the main route of infection in the bovine host and plays an important role in maintaining the parasite in the herd. Molecular detection of N. caninum is important to determine the occurrence of the disease and to evaluate the genetic diversity of the parasite. The present study aimed at assessing the vertical transmission of N. caninum using molecular techniques to detect the parasite in tissue samples from bovine fetuses collected in a slaughterhouse in the state of São Paulo, Brazil. Seventy fetuses and 70 blood samples from pregnant cows were collected in a slaughtering line. Fresh samples of heart and brain tissue from fetuses were analyzed using molecular assays. Serum samples from fetuses and cows were subjected to an indirect fluorescent antibody test (IFAT) to detect antibodies against N. caninum. Nested PCR targeting the internal transcriber 1 (ITS1) region of the protozoan organism was used in the molecular testing. From the total of fetuses examined, 71.42% were positive for N. caninum by PCR. A higher number of heart samples (47.1%) were positive for the parasite using this technique. Antibodies against the protozoa were detected in 12.9% of serum samples of cows; 2.8% of fetuses were seropositive for this pathogen. Our results show that vertical transmission of N. caninum occurs in cattle from this region of Brazil, and that the use of different diagnostic techniques contributes to successful diagnosis of congenital transmission of the parasite in cattle.

Evidence of reduced vertical transmission of Neospora caninum associated with higher IgG1 than IgG2 serum levels and presence of IFN-γ in non-aborting chronically infected cattle under natural condition.

Neospora caninum infection of cattle can be vertically transmitted, resulting in abortion or birth of infected calves. Vertical transmission occurs both in acutely or chronically infected cattle. There is little information on the immune response needed to prevent endogenous transplacental transmission, particularly from chronically infected cattle to their offspring in a natural environment. In this study, N. caninum seropositive pregnant cattle from three different farms with high avidity antibodies and low IgM titers were selected and their newborn colostrum-deprived calves were tested for anti-N. caninum antibodies. Based on these results, dams were grouped according to their congenital transmission status. The analysis of the immune profile of the chronically-infected pregnant cattle revealed that higher ratio between IgG1 and IgG2 anti-N. caninum serum titers and higher levels of systemic IFN-γ were associated with diminished vertical transmission rates, compared to dams with the opposite profile. Our results evidenced an association between the immune profile and vertical transmission in non-aborting chronically infected dams, and confirm that vertical transmission, even when not leading to abortion, is related to a defined immune profile. This is important information to accomplish successful vaccine development efforts.

Stomoxys calcitrans, mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit.

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re-emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re-examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non-infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.

Intranuclear coccidiosis in tortoises - discovery of its causative agent and transmission.

Intranuclear coccidiosis of testudines (known as TINC) is an emerging disease in chelonians. Although endogenous stages were repeatedly detected in various tissues, attempts to find the oocysts in faeces failed, leaving the question of the transmission and classification of the causative agent of TINC unresolved. We recorded small spherical oocysts (∼6-7 µm in diameter) of an eimeriid coccidium in faeces of a leopard tortoise (Stigmochelys pardalis). Sporulated oocysts were used for the experimental oral inoculation of juvenile coccidia-free tortoises representing 5 species (S. pardalis, Testudo graeca, T. hermanni, T. horsfieldii, and Geochelone sulcata). The oocysts' association with TINC was confirmed based on clinical signs, histopathological findings of intranuclear endogenous stages of the coccidium in many organs (including intestine), and by the partial 18S rDNA sequence analysis of the DNA isolated from organs of the experimentally infected animals and from a single naturally infected as well as from all experimentally infected tortoises. Breeding colonies of chelonians should be screened for this pathogen in order to prevent its further spread and unwanted introduction into endangered free-ranging chelonian populations.