In vitro efficacy of nitro- and bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoitia besnoiti infection in Vero cells

Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattl

Isolation of Besnoitia besnoiti from infected cattle in Portugal

Abstract Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti. Keywords: Besnoitia besnoiti; Bovine besnoitiosis; Portugal; Cyst; In vitro cell cultur

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany

Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for g-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK21, KH-R). The best growth of tachyzoites was observed in BHK21 cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK21 and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell linesmay be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany

Rastreos del grupo de lucha contra la equinococosis-hidatidosis en los municipios de Elvas y Alandroal¿

The activity of this multidisciplinary Group is running in north-east Alentejo, an area where there are high prevalence level of parasitism by Echinococcus granulosus. This factor, lead to the first of survey in 1991 and the establishment of a multidisciplinary group fighting Hydatidosis- Echinococcosis on a regular basis in 1998. The group develops different activities: Surveys in humans (epidemiological questionnaire, blood collection and abdominal ultrasound survey); health education (main talks for rural citizens, distribution of information in paper format, individual interviews with the target populations, collection of epidemiological data); close interaction/ collaboration with the Hospital de Santa Luzia de Elvas (serological analysis of collected sera; Hydatidological consultation and surgery). 18 rural population areas were surveyed, representing 7,8% of the population. The activity of this multidisciplinary group aims to develop activity in the definitive host and livestock having as a main goal not only the control but yet the eradication of the parasite

Mini-FLOTAC for the diagnosis of Eimeria infection in goats: An alternative to McMaster

Caprine coccidiosis is responsible for remarkable economic losses. Diagnosis must therefore take into account a number of epidemiological and clinical factors supported by laboratory investigations. The detection of Eimeria oocysts and the determination of oocysts per gram of faeces (OPG) require a trustworthy oocyst count technique. Mini-FLOTAC is a new apparatus from the FLOTAC family, which was designed for optimal examination of faecal sample suspensions in each flotation chamber (total volume = 2 mL) and which permits a maximum magnification of 400x. Faecal samples from sixteen goats reared in an intensive goat farm were qualitatively (simple flotation) and quantitatively analyzed. A comparison between McMaster (McM) and Mini-FLOTAC was carried out, by performing 288 different readings. Eimeria arloingi, E. caprovina and E. ninakohlyakimovae were the most prevalent species. The mean OPG detected by Mini-FLOTAC was higher than the values observed with McMaster techniques (P0.05) but lower than those resulted from McM 0.3 mL. Thereby, the present study suggests that the Mini-FLOTAC is a promising technique for detecting and counting Eimeria spp. in goat faeces

Mini-FLOTAC for the diagnosis of Eimeria infection in goats: an alternative to McMaster

Caprine coccidiosis is responsible for remarkable economic losses. Diagnosis must therefore take into account a number of epidemiological and clinical factors supported by laboratory investigations. The detection of Eimeria oocysts and the determination of oocysts per gram of faeces (OPG) require a trustworthy oocyst count technique. Mini-FLOTAC is a new simple and easy-to-use apparatus from the FLOTAC family. The present study was aimed at investigating whether Mini-FLOTAC could be an alternative to McMaster for the diagnosis of Eimeria spp. in goats. Faecal samples from 16 goats reared in an intensive goat farm were qualitatively (simple flotation) and quantitatively analyzed. A comparison between McMaster (McM 1 mL and McM 0.3 mL) and Mini-FLOTAC was carried out, by performing 288 different readings (6 replicates for each of the 3 techniques). Eimeria arloingi, Eimeria caprovina and Eimeria ninakohlyakimovae were the most prevalent species. The mean (and median) OPG detected by Mini-FLOTAC was higher than the values observed with McMaster techniques (P < 0.05). Mini-FLOTAC also produced coefficient of variations similar to those resulted from McM 1 mL (19.4 versus 17.5; P > 0.05) but lower than those resulted from McM 0.3 mL. The findings of the present study suggest that the Mini-FLOTAC is a promising technique for detecting and counting Eimeria spp. in goat faeces

Immunodiagnosis of Besnoitia besnoiti infection by ELISA and Western blot

Abstract Besnoitia besnoiti, an obligate intracellular apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may dramatically affect body condition and lead to irreversible infertility in males, resulting in important economical losses in livestock production. Identification of serologically positive animals is of major relevance to elaborate appropriate measures of control. While identification of clinical cases is relatively easy to carry out, the finding of subclinical forms of infection is more difficult, thus serology is considered as an appropriate diagnostic tool. In view to improve and validate immunodiagnosis, we evaluated an enzyme-linked immunosorbent assay (ELISA), complemented with a Western blot (both using a somatic B. besnoiti-tachyzoite antigen) to detect anti-B. besnoiti antibodies in bovine sera. The comparative evaluation of the 2 methods, using 13 sera from animals affected by the chronic phase of besnoitiosis and 10 asymptomatic carriers, yielded a diagnostic sensitivity of 87% for ELISA and 91% for Western blot analyses. Specificity was tested with sera from animals with confirmed Toxoplasma gondii (n = 5) and Neospora caninum (n = 12) infection, and with 64 negative sera from either an endemic or a non-endemic area. The ELISA specificity ranged between 96.4% and 98%, the Western blot specificity between 96.4% and 100%. The present study demonstrated that ELISA and Western blot, using in vitro generated somatic B. besnoiti antigen, is a useful tool combination to reliably detect animals that have been exposed to B. besnoiti infection, including both asymptomatic and symptomatic courses of disease

Insights into hookworm prevalence in Asia: A systematic review and meta-Analysis

Introduction: Hookworm infections are neglected tropical diseases of humans and animals worldwide. A meta-Analysis and systematic review was designed to evaluate the prevalence of hookworm infection in animal and human hosts in Asia until July 2018. Methods: The available online articles of five English databases (PubMed, Scopus, Science Direct, Web of Science and Google Scholar) were explored. Results: The most parasitized carnivores were jackal (48, CI: 4 to 91), followed by dog (41, CI: 29 to 53), cat (26, CI: 14 to 38) and the red fox (19, CI: 13 to 24). The weighted prevalence of Ancylostoma braziliensis, A. caninum, A. ceylanicum, A.Tubaeforme and Uncinaria stenocephala isolated from different canids were found to be 27 (CI: 21 to 33), 23 (CI: 7.0 to 53), 24 (CI: 12 to 35), 44 (CI: 37 to 51) and 37 (CI: 18 to 55), respectively. In total, 98 records were obtained for human hookworms from 3209 760 examined individuals and the calculated weighted prevalence in this population was 19 (CI: 17 to 20). Conclusions: These findings highlight a desirable ecological milieu for parasite survival and transmission in such territories, which implicates revisiting control programs and public health infrastructures in those areas. © 2020 The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected]

Prevalence of Babesia microti-like infection in red foxes (Vulpes vulpes) from Portugal

Prevalence of Babesia microti-like infection in red foxes (Vulpes vulpes) from Portugal Abstract The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7–78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0–6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p < 0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p = 0.219) or age groups (<2 years vs. ≥2 years) (p = 1.0). This is the first report of the B. microti-like piroplasm in foxes from Portugal as well as the first report on detection by PCR and genotyping of B. canis in a red fox worldwide. A natural cycle of the B. microti-like piroplasm is suggested in red fox populations based on the high prevalence of the protozoan. Red foxes might be a reservoir of the B. microti-like piroplasm and a source of infection to dogs