The first molecular investigation of Besnoitia besnoiti infections among cattle in Mosul, Iraq.

BACKGROUND: Bovine besnoitiosis (elephant skin disease) caused by Besnoitia besnoiti is a costly endemic disease in the Middle East, Asia, and tropical and subtropical Africa and is also emerging as a significant problem in Europe. This study is aimed at determining the prevalence of B. besnoiti in blood and skin biopsies of cattle as well as evaluating the risk factors associated with the infection among cattle in Mosul, Iraq. METHODS AND RESULTS: To achieve this aim, four hundred and sixty apparently healthy cattle of different breeds, ages, and sexes were sampled from seven different locations in Mosul, Iraq. Blood and skin biopsies were carefully collected from each cattle, and these samples were subjected to molecular analysis. The detection of B. besnoiti was molecularly confirmed by the presence of 231 bp of ITS-1 in the rDNA gene of the protozoan. Besnoitia besnoiti DNA was present in 74 (16.09%; 95% CI = 13.01-19.72) and 49 (10.65%; 95% CI = 8.15-13.80) of the blood and skin biopsies, respectively, that were analyzed. Age, breed, and sex were significantly (p < 0.05) associated with the occurrence of B. besnoiti among cattle in the study area. CONCLUSIONS: Findings from this study will serve as baseline data in the epidemiology, prevention, and control of the protozoan among cattle in Iraq.

Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti.

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

Morphological and molecular characterization of Cystoisospora yuensis n. sp. and Cystoisospora rastegaievae (Protozoa: Eimeriidae) in amur hedgehogs, Erinaceus amurensis (Schrenk, 1859).

Twenty-four fecal samples were collected from captive amur hedgehogs (Erinaceus amurensis) in Zhengzhou, China. Based on morphological and molecular analysis, the overall prevalence of Cystoisospora was 62.5% (15/24). These samples contained two types of coccidian oocysts, including C. rastegaievae (50.0%, 12/24) and a new species named C. yuensis n. sp. (12.5%, 3/24). Sporulated oocysts (n = 30) of C. yuensis n. sp. are ovoid, (20.6 ± 1.4) µm × (20.9 ± 0.9) µm, with a shape index (length/width) of 1.0 and a smooth and bi-layered oocyst wall, 1.3 µm thick (outer layer 0.8 µm, inner 0.5 µm). A polar granule is present, but micropyle cap, micropyle, and oocyst residuum are absent. The sporocysts are ovoid-shaped, (9.3 ± 0.6) µm × (8.5 ± 1.1) µm, with a shape index (length/width) of 1.1. Stieda, substieda bodies, and refractile bodies are absent. Residuum is scattered and distributed around the entire sporocysts. At the 18S rRNA locus, C. yuensis n. sp. exhibited the highest identity to C. timoni (99.3%) from a slender-tailed meerkat. It has 98.0% identity at the 28S rRNA locus and 99.3% at the ITS locus. Based on morphological and molecular data, this isolate is a new species of Cystoisospora. Additionally, we have provided data on the prevalence of C. rastegaievae in China and sequences of the 18S rRNS, 28S rRNA, and ITS loci.

Molecular survey for cyst-forming coccidia (Toxoplasma gondii, Neospora caninum, Sarcocystis spp.) in Mediterranean periurban micromammals.

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.

Bovine besnoitiosis in an endemically infected dairy cattle herd in Italy: serological and clinical observations, risk factors, and effects on reproductive and productive performances.

Bovine besnoitiosis (Besnoitia besnoiti) is an emerging parasitic disease of cattle in Europe. This study reports a case of bovine besnoitiosis in a dairy farm housing 217 cattle in Italy. A serological screening was performed on the whole herd using the recommended approach of ELISA and confirmatory Western Blot. Seropositive animals were clinically examined to reveal symptoms and lesions of besnoitiosis. Risk factors and the effects of the parasite infection on reproductive and productive performances were evaluated. Histopathology and molecular analyses on tissues from a slaughtered cow affected by the chronic phase of the disease were carried out. An overall seroprevalence of 23.5%, which increased up to 43.5% considering only cows, was recorded. Clinical examination of 33 of the seropositive cows evidenced the presence of tissue cysts in at least one of the typical localizations (sclera, vulva, or skin) in 25 animals. Statistical analysis did not evidence any significative impact of the parasite infection on herd efficiency; however, a decrease of productive parameters was recorded in cows showing cutaneous cysts. Concerning the chronically affected cow, histopathology revealed B. besnoiti tissue cysts in the skin of the neck, rump, hind legs, eyelid and vulva, in the muzzle, in mucosal membranes of the upper respiratory tract, and in the lungs. Parasite DNA was detected also in masseter muscles, tonsils, mediastinal lymph nodes, liver, cardiac muscle, aorta wall, ovaries, uterus, and vulva. Bovine besnoitiosis continues to spread in the Italian cattle population. Breeders and veterinarians should be aware of this parasitic disease, and control programs should be developed based on surveillance through a diagnostic procedure including both clinical examination and laboratory tests.

Hammondia sp. oocysts shed by a Brazilian fox (Lycalopex vetulus) differ from Hammondia heydorni and Hammondia triffittae.

A Brazilian fox (Lycalopex vetulus) was rescued from a highway, and 16 days after maintained in captivity, the fox shed oocysts with sizes compatible with Hammondia sp. and Neospora caninum. DNA extracted from oocysts were initially tested in two PCRs targeting the internal transcribed spacer 1 (ITS-1) of the rDNA of Hammondia heydorni and the Nc-5 gene of N. caninum. A 270-bp product was visualized in the PCR for H. heydorni. No amplification was observed for N. caninum PCR. Since ITS-1-based PCR is not sufficient to differentiate Hammondia species derived from canids, oocyst DNA was examined using multilocus sequence analysis of five genetic fragments [intron 1 of the alpha tubulin gene (intron 1), internal transcribed spaces 1 and 2 (ITS-1 and ITS-2) of the rDNA, 28S rRNA gene (D2/D3 domain), and heat shock protein 70 (Hsp70)]. The Hammondia sp. oocyst from the Brazilian fox, referred here as H-FOXBR isolate, is closely related to H. heydorni and Hammondia triffittae, but differs from these parasites in three genetic markers (alpha tubulin gene, ITS-2, and 28S rRNA). As reported by other research groups, Hammondia spp. excreted by canids are genetically diverse and may encompass additional species besides H. heydorni and H. triffittae. In this study, we confirmed that H-FOXBR has significant genetic differences in comparison to H. heydorni and H. triffittae and may represent a separate species. Further studies are needed to identify the life cycle of this parasite and to characterize the parasite stages in the intermediate and definitive hosts.

Characterization of tabanid flies (Diptera: Tabanidae) in South Africa and Zambia and detection of protozoan parasites they are harbouring.

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

A serosurvey of selected cystogenic coccidia in Spanish equids: first detection of anti-Besnoitia spp. specific antibodies in Europe.

BACKGROUND: Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. RESULTS: Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. CONCLUSIONS: This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.

Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis.

Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures (Tms) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified Tms at 85.8°C and 88.6°C, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using qPCR-Tms analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

Seroprevalence and risk factors of Besnoitia besnoiti infection in Korean cattle - short communication.

Besnoitia besnoiti is an obligate intracellular parasite that is transmitted by direct contact or via mechanical transmission by flies as vectors. Besnoitiosis causes economic losses in the cattle industry and is regarded as a re-emerging disease in Europe. This study evaluated the seroprevalence of B. besnoiti in Korean cattle using a commercial ELISA kit. Among 558 serum samples, 19 (3.4%) tested seropositive for B. besnoiti. The statistically significant risk factors included age (≥ 2 years), sex (castrated males), and region (lower latitudes) (P < 0.05). The overall seroprevalence suggested a wide distribution of B. besnoiti infection in cattle reared in Korea. Thus, the practice of intensive cattle husbandry and the regionally different seroprevalence of B. besnoiti infection in cattle in Korea warrant routine monitoring and vector control to reduce economical losses due to bovine besnoitiosis in the country.

Chronic Cystoisospora belli infection in an immunocompetent Myanmar refugee - microscopy is not sensitive enough.

BACKGROUND: Cystoisosporiasis is an opportunistic infection seen more commonly in patients with acquired immunodeficiency syndrome. Although uncommon, Cystoisospora infection can occur in immunocompetent individuals but tend to be benign and self-limiting. Chronic infection however, has been described but diagnosis can often be challenging and requires a high clinical index of suspicion. CASE PRESENTATION: We present a case of delayed diagnosis of Cystoisospora belli (C. belli) in an immunocompetent 28-year-old refugee from Myanmar. She had a history of chronic diarrhea where exhaustive investigations over many years failed to reveal a diagnosis. Cystoisospora belli cysts were finally detected in stool 4 years after investigation commenced, and PCR testing on stored colon biopsies amplified a molecular product with 99 % sequence homology to C. belli. The patient improved promptly with trimethoprim-sulfamethoxazole treatment. CONCLUSION: In the appropriate clinical context we suggest molecular testing for C. belli or an empirical therapeutic trial.

Characterization of an outbreak of emerging bovine besnoitiosis in southwestern Spain.

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avileña beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

Parasitic Infection of the Gallbladder: Cystoisospora belli Infection as a Cause of Chronic Abdominal Pain and Acalculous Cholecystitis.

Herein we describe two cases of Cystoisospora belli infection of the gallbladder in patients with chronic abdominal pain and review the published literature to date. C. belli is an intracellular protozoan parasite that typically infects the small bowel of immunocompromised hosts. Little is known of the significance of C. belli infection of the gallbladder at this point as only four cases have been reported as yet, only one of which occurred in an immunocompetent patient. It is often treatable with antibiotics, and the patient's immune status, including HIV testing, should be investigated. Neither of the patients at our institution was found to be immunocompromised, and HIV-1/2 antibody testing was non-reactive in both.

Epidemiologic study on Besnoitia besnoiti infection in dairy herds in Jordan.

Besnoitia besnoiti is an apicomplexan parasite and the causative agent of bovine besnoitiosis which is considered as a re-emergent disease in Europe. A cross-sectional serological study was conducted to determine the seroprevalence and to identify risk factors associated with B. besnoiti infection in 68 dairy herds (n = 806 cows) in Jordan during the period from January to June 2007 and the spring of 2014. Data regarding herd's management was obtained by filling questionnaires through personal interviews with farmers. An indirect ELISA test was used to detect antibodies against B. besnoiti. Chi-square analysis and multivariable logistic regression model were used to identify risk factors associated with seropositivity to B. besnoiti. At the individual cow and herd level, the true prevalence of seropositive animals was 6 and 28.7 %, respectively. Cows between 2 and 6 years of age had significantly higher seroprevalence of B. besnoiti than other age groups. The highest seroprevalence of B. besnoiti was found in Zarqa and Irbid governorates. Multivariable logistic regression model identified that exchanging visits by farm workers to neighboring farms as a risk factor for seropositivity to B. besnoiti, while smaller herd size and twice a day farm cleaning using sweeping and water hosing were identified as protective factors. This is the first study that investigated the seroprevalence of B. besnoiti infection in dairy herds in Jordan. Further studies are warranted to explore the clinical manifestation of B. besnoiti infection as well as to identify the possible presence of other Besnoitia species and definitive hosts for the parasite.

Dynamics of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis is caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. This disease progresses in two sequential phases: a febrile acute phase with oedemas and respiratory disorders, and a chronic phase characterized by the presence of subcutaneous tissue cysts and skin lesions. Serious consequences of the infection are poor body condition, sterility in bulls and eventual death. The role of host/parasite-dependent factors, which play a major role in the pathogenesis of the disease, is not yet fully elucidated. Isolate/strain virulence, parasite stage, dose and the route of parasite inoculation were studied under different experimental conditions, which make it difficult to compare the results. Data on host-dependent factors obtained from naturally infected cattle showed that (i) the seroprevalence of infection is similar in both sexes; (ii) seropositivity increases with age; (iii) both beef and dairy cattle are susceptible to the infection; and (iv) the cell-mediated immune response is likely to play a major role because a T cell response has been observed around several tissue cysts. Whether colostral antibodies are protective and to what extent the humoral immune response might reflect the disease/protection status require further research. Thus, a well-established experimental bovine model could help to clarify these important questions. The dynamics of B. besnoiti infection in cattle and available knowledge on relevant factors in the pathogenesis of the infection are reviewed in the present work.

A review on bovine besnoitiosis: a disease with economic impact in herd health management, caused by Besnoitia besnoiti (Franco and Borges, ).

Bovine besnoitiosis is caused by the largely unexplored apicomplexan parasite Besnoitia besnoiti. In cows, infection during pregnancy often results in abortion, and chronically infected bulls become infertile. Similar to other apicomplexans B. besnoiti has acquired a largely intracellular lifestyle, but its complete life cycle is still unknown, modes of transmission have not been entirely resolved and the definitive host has not been identified. Outbreaks of bovine besnoitiosis in cattle were described in the 1990s in Portugal and Spain, and later several cases were also detected in France. More cases have been reported recently in hitherto unaffected countries, including Italy, Germany, Switzerland, Hungary and Croatia. To date, there is still no effective pharmaceutical compound available for the treatment of besnoitiosis in cattle, and progress in the identification of novel targets for intervention through pharmacological or immunological means is hampered by the lack of molecular data on the genomic and transcriptomic level. In addition, the lack of an appropriate small animal laboratory model, and wide gaps in our knowledge on the host-parasite interplay during the life cycle of this parasite, renders vaccine and drug development a cost- and labour-intensive undertaking.

Prevalence and geographic distribution of Besnoitia besnoiti infection in cattle herds in Portugal.

Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti is considered an emergent disease in Europe. This study aimed to determine the prevalence and geographic distribution of B. besnoiti in cattle herds in continental Portugal and to identify potential spatial clustering of infection. A stratified two-stage cross-sectional serological survey was carried out between March 2012 and May 2013 with the five administrative NUTS II regions, Norte, Centro, Lisboa, Alentejo, and Algarve, as the stratification level. Sera from 391 herds in 220 parishes and 83 municipalities were analyzed by a serial testing strategy, with the modified agglutination test (B-MAT) as the first screening assay and the immunofluorescent antibody test (IFAT) as the confirmatory test. Within-herd prevalence of positive herds varied between 0.7 and 72.4% and was ≥10.3% in half of the infected herds. Using a Bayesian approach, the true prevalence of B. besnoiti in cattle herds was determined to be 5.1% (confidence interval (CI), 3.1-7.8%) and the mean within-herd prevalence of positive herds was 33.0% (CI, 20.3-46.0%). The sensitivity and specificity of the B-MAT were estimated to be 96.9% (CI, 93.7-98.8 %) and 99.7% (CI, 99.6-99.8%), whereas those of the IFAT were 89.6% (CI, 86.0-92.5%) and 99.7% (CI, 98.5-99.9%), respectively. Spatial scan statistics analysis identified one spatial cluster covering the majority of the Alentejo region. Seropositive herds were detected for the first time outside Alentejo, in the region Centro and in the northeast of Portugal. Further epidemiological research is needed to identify eco-biological factors, which could explain the geographic clustering of B. besnoiti in Portugal.

No detection of Besnoitia besnoiti DNA in the semen of chronically infected bulls.

Bovine besnoitiosis is a chronic and debilitating disease observed in many European countries that may cause important economic losses in cattle. The recent widespread of the parasite in Europe had led the European Food Safety Authority to declare bovine besnoitiosis as a re-emerging disease in Europe. Many aspects of the epidemiology of bovine besnoitiosis such as the main routes of transmission are still unclear and need to be further studied. Among the different hypotheses, a sexual transmission has not yet been investigated. Therefore, the aim of this study was to evaluate the presence of Besnoitia besnoiti DNA in the semen of naturally infected bulls by using a highly sensitive method (real-time qPCR). Both pre-sperm and sperm fractions of 40 bulls, including seronegative (n = 11), seropositive subclinically (n = 17), and seropositive clinically (n = 12) infected animals, were collected by electroejaculation and analyzed by real-time qPCR. No B. besnoiti DNA was detected in 27 pre-sperm and 28 sperm fractions of the 40 examined bulls, suggesting that the transmission of B. besnoiti infection by the semen of chronically infected bulls is very unlikely.

Prevalence of Sarcocystis spp. and Hammondia spp. microcysts in esophagus tissue of sheep and cattle, emphasized on their morphological differences.

Sarcocystis and Hammondia are two obligatory protozoan parasites. These genera belong to cyst-forming coccidia group of the phylum Apicomplexa. They both need two different hosts to complete their life cycles. Felids and canids can act as definitive hosts, while herbivores, such as sheep and cattle, are the most important intermediate hosts. Reports verify that no important disease has been caused by Hammondia spp.; on the other hand, Sarcocystis spp. can cause some severe infectious disease in livestock industry such as abortion. Economic losses are another concern due to carcass condemnation during meat inspection in abattoirs and decrease in the quality and quantity of milk and wool production. Due to the Sarcocystis and Hammondia tissue cysts being similar, the distinction between these different genera is so important. In this study, the prevalence of Sarcocystis and Hammondia in the esophagus tissue of sheep and cattle slaughtered in one of the industrial abattoir in Iran was reported and an easy and rapid method for accurate diagnosing of Sarcocystis and Hammondia bradyzoites was explained.

Development of a protocol testing the ability of Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae) to transmit Besnoitia besnoiti (Henry, 1913) (Apicomplexa: Sarcocystidae).

Cattle besnoitiosis due to the cyst-forming coccidian parasite Besnoitia besnoiti has recently been reported in expansion in Europe since the end of the twentieth century. The B. besnoiti life cycle and many epidemiological traits are still poorly known. Hematophagous flies, including the worldwide-distributed Stomoxys calcitrans, could be mechanical vectors in the contamination of mouthparts after the puncture of cutaneous cysts or ingestion of infected blood. In this study, a protocol is presented to assess more deeply the role of S. calcitrans, reared in laboratory conditions, in parasite transmission. A preliminary trial showed that stable flies could transmit tachyzoites from bovine artificially parasite-enriched blood to B. besnoiti-free blood using glass feeders. Evidence of transmission was provided by the detection of parasite DNA with Ct values ranging between 32 and 37 in the blood recipient. In a second time, a B. besnoiti-infected heifer harboring many cysts in its dermis was used as a donor of B. besnoiti. An interruption of the blood meal taken by 300 stable flies from this heifer was performed. Immediately after the blood meal was interrupted, they were transferred to a glass feeder containing B. besnoiti-free blood from a non-infected heifer. Quantitative PCR and modified direct fluorescence antibody test (dFAT) were used to detect B. besnoiti DNA and entire parasites, respectively, in the blood recipient, the mouthparts, and the gut contents of S. calcitrans at two time intervals: 1 and 24 h after the interrupted blood meal. Parasite DNA was detected at both time intervals (1 and 24 h) in all samples (blood recipient, mouthparts, and gut contents of stable flies) while entire parasites by dFAT were only found in the abdominal compartment 1 h after the interrupted blood meal. Then, S. calcitrans were able to carry B. besnoiti from chronically infected cattle to an artificial recipient in the conditions of the protocol.