In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR).

Poultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p < 0.05). After 96 hpi, E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p < 0.05). Our results show that MDBK monolayers could be used for in vitro research aimed to study E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

Morphological and molecular characterization of Isospora amphiboluri (Apicomplexa: Eimeriidae), a coccidian parasite, in a central netted dragon (Ctenophorus nuchalis) (De Vis, 1884) in Australia.

An Isospora species, Isospora amphiboluri, originally described by Canon in 1967 and later by McAllister et al. (1995), was isolated from a central netted dragon (Ctenophorus nuchalis) housed at a wildlife rehabilitation centre in Perth, Western Australia. Sporulated oocysts of Isospora amphiboluri (n = 30) are spherical, 24.2 (26.5-23.0) µm in length and 23.9 (22.4-25.9) µm in width, with a shape index of 1.01. The bilayered oocyst wall is smooth and light-yellow in color. Polar granule, oocyst residuum and micropyle are absent. The sporocysts are lemon-shaped, 15.7 (15.2-18.0) × 10.2 (8.9-11.2) µm, with a shape index (length/width) of 1.53. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda half-moon-shaped. Each sporocyst contains four vermiform sporozoites arranged head to tail. The sporozoites are 11.7 (9.9-16.2) × 3.0 (2.4-3.5) µm, with a shape index (length/width) of 3.87. A sporocyst residuum is present. Sporozoites contain a central nucleus with a finely distributed granular residuum. Comparison of oocyst measurements and their features with other valid Isospora species from hosts in the Agamid family confirmed that this Isospora species is Isospora amphiboluri. Molecular characterization of I. amphiboluri at the 18S rRNA and MTCOI loci showed the highest similarity with I. amphiboluri from the central bearded dragon, 99.8% and 99.7% respectively. This is the first report of I. amphiboluri from a central netted dragon in Australia.

Exploring the impact of cattle on human exposure to malaria mosquitoes in the Arba Minch area district of southwest Ethiopia.

BACKGROUND: The success of indoor interventions that target mosquitoes for malaria control is partially dependent on early evening and outdoor biting behaviours of mosquito vectors. In southwest Ethiopia, people and cattle live in proximity, which calls to investigate whether the presence of cattle increase or decrease bites from malaria mosquito vectors. This study assessed both host-seeking and overnight activity of malaria mosquito vectors given the presence or absence of cattle in Chano Mille village, Arba Minch district, Ethiopia. METHODS: Anopheles species density and activity time was compared when a calf was: (i) placed inside; (ii) 1 m away from; or (iii) absent from a tent with a human volunteer resting insides using hourly human landing catches (HLC) conducted from 18:00-0:00 h for 3 months. This trial was performed close to the shore of the Lake Abaya to minimize the interference of other animals on mosquito movement. The overnight activity of malaria vectors was assessed within a Chano village from 18:00-6:00 h with collections carried out both indoors and outdoors by HLC. Generalized estimating equations were used to statistically assess differences. RESULTS: Anopheles pharoensis was significantly more prevalent when a calf was present either inside (42%, P < 0.001), or adjacent to (46%, P = 0.002) a tent relative to a tent without a calf present. The presence of a calf did not affect densities of the primarily anthropophilic species A. gambiae (s.l.), or An. tenebrosus. Anopheles gambiae (s.l.) (P < 0.001) and An. pharoensis (P = 0.015) both had a tendency for early evening biting between 19:00 h and 20:00 h. Anopheles gambiae (s.l.) was mainly biting humans outdoors in the village. CONCLUSIONS: The presence of calves within and close to human dwellings acts to draw malaria mosquitoes toward the human occupant with the potential to increase their risk of malaria. Hence, deployment of cattle far from human residence could be recommended to reduce human exposure. Outdoor and early evening biting could threaten the success of current indoor-based interventions. Hence, tools could be designed to reduce this threat.

Isospora svecica sp. n. (Apicomplexa: Eimeriidae), a new species of coccidium from the white-spotted bluethroat Luscinia svecica cyanecula (Aves: Passeriformes: Muscicapidae).

Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, Isospora svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1-3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 µm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 µm, with a thin, colourless wall. A conspicuous knob-like Stieda body was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including coccidia, a highly prevalent parasitic protist group in all vertebrate classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several "Isospora sp." that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid birds, and represents the first coccidian species described from Luscinia svecica.

Morphological and molecular characterisation of Isospora butcherae n. sp. in a silvereye (Zosterops lateralis) (Latham, 1801).

A new Isospora (Apicomplexa:Eimeriidae) species is described from a silvereye (Zosterops lateralis) in Western Australia. Sporulated oocysts of this species are spherical, 24.2 (23.1-25.2) × 23.3 (22.8-23.9) µm, with a shape index (length/width) of 1.02, and with a smooth bi-layered oocyst wall, 1.2 µm thick (outer layer 0.9 µm, inner 0.3 µm). A polar granule is present, but the oocyst residuum and a micropyle are absent. The ovoid-shaped sporocysts are 16.1 (15.7-17.3) × 10.5 (15.7-17.3) µm and have a shape index of 1.53. A hemidome-shaped Stieda and a rectangular-shaped substieda body are present. A sporocyst residuum is present and composed of numerous granules of different sizes scattered among the sporozoites. The oocysts from this isolate are morphologically different from those of all known Isospora spp. This coccidian parasite was molecularly characterised at the 18S, 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, based on 1210 bp of sequence, this new isolate exhibited 99.9, 99.8, 99.7 and 99.5% similarity to I. sp. MAH-2013a (KF648870) from a superb starling (Lamprotornis superbus) in Canada, I. sp. MS-2003 (AY33157) from a Southern cape sparrow (Plocepasser mahali) in America, I. sp. Tokyo (AB75786) from Japan and I. sp. respectively. Further analysis of a subgroup of 300 bp long 18S sequences (n = 11), including I. anthochaerae and the other three Isospora characterised from birds in Western Australia, revealed that I. butcherae n. sp. exhibited 98.3% similarity to both I. sp. MAH-2013a (KF648870) and I. MS-2003 (AY33171). At the 28S locus, this new isolate exhibited 97.3% similarity with I. sp. MS-2003 from a California towhee (Melozone crissalis). At the COI locus, this new isolate exhibited 99.8% similarity to I. neochmiae from a red-browed finch. Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora butcherae n. sp. after Mrs. June Butcher for her lifelong dedication as a wildlife rehabilitator. Graphical abstract ᅟ.

Vector Competence of Anopheles kleini and Anopheles sinensis (Diptera: Culicidae) From the Republic of Korea to Vivax Malaria-Infected Blood From Patients From Thailand.

In total, 1,300 each of Anopheles kleini Rueda and Anopheles sinensis Wiedemann sensu stricto (s.s.) females (colonized from the Republic of Korea) and Anopheles dirus Peyton & Harrison (Thai strain) were allowed to feed on blood from Thai malaria patients naturally infected with Plasmodium vivax The overall oocyst infection rates for An. dirus, An. kleini, and An. sinensis s.s. were 77.4, 46.1, and 45.9%, respectively. The mean number of oocysts was significantly higher for An. dirus (82.7) compared with An. kleini (6.1) and An. sinensis s.s. (8.6), whereas the mean number of oocysts for An. kleini and An. sinensis s.s. was similar. The overall sporozoite infection rates for An. dirus, An. kleini, and An. sinensis s.s. dissected on days 14-15, 21, and 28 days post-feed were significantly higher for An. dirus (90.0%) than An. kleini (5.4%), whereas An. kleini sporozoite rates were significantly higher than An. sinensis s.s. (<0.1%). The overall sporozoite indices for positive females with +3 (100-1,000 sporozoites) and +4 (>1,000 sporozoites) salivary gland indices were significantly higher for An. dirus (85.7%), compared with An. kleini (47.1%). Only one An. sinensis s.s. had sporozoites (+2; >10-100 sporozoites). These results indicate that An. kleini is a competent vector of vivax malaria. Although An. sinensis s.s. develops relatively high numbers of oocysts, it is considered a very poor vector of vivax malaria due to a salivary gland barrier.

A new species of Choleoeimeria (apicomplexa: eimeriidae) from oustalet's chameleon, Furcifer oustaleti (Sauria: Chamaeleonidae).

One of three (33%) captive specimens of Oustalet's chameleon, Furcifer oustaleti (Mocquard) originally from Madagascar and housed at the Oklahoma City Zoological Park Herpetarium, Oklahoma County, Oklahoma, USA, was found to be passing an undescribed species of Choleoeimeria in its faeces. Oocysts of Choleoeimeria fischeri sp. n. were cylindroidal, 30.3 x 16.8 (28-34 x 15-18) microm, with a smooth, bilayered wall and a length/width ratio (L/W) of 1.8. A micropyle and oocyst residuum was absent but a fragmented polar granule was often present. Sporocysts were ovoidal, 9.6 x 8.0 (9-10 x 7-9) jm, with an L/W of 1.2. Stieda, sub-Stieda, and para-Stieda bodies were absent. The sporocyst residuum consists of large globules dispersed between sporozoites. Sporozoites were elongate, 8.6 x 2.9 (8-10 x 2-3) microm, with an elongate posterior refractile body. The new species represents the second coccidian described from this lizard.

Redescription of Eimeria dorcadis Mantovani, 1966 (Apicomplexa: Eimeriidae) from the dorcas gazelle (Gazella dorcas) in Saudi Arabia.

Eimeria dorcadis Mantovani, 1966 is redescribed from dorcas gazelle (Gazella dorcas (L.)) from Saudi Arabia. Oocysts were detected in 7 out of 22 faecal samples (32%) using floatation method. The sporulated oocysts are cylindrical, slightly flattened at the micropylar pole, measure in average 32 x 19 microm (27-36 x 16-24 microm), length/width ratio being 1.7 (1.5-2.1). Oocyst wall is 1.2 microm thick, smooth, double-layered; outer layer is slightly thicker, light blue in colour; inner layer brownish, with micropyle in the inner layer and apparently continual outer one, measures 2.2 microm, but lacks a micropylar cap. The sporocyst elongate-ellipsoidal, measures 14 x 8 microm (12-17 x 6-9 microm), length/width ratio being 1.8, with sporocyst residuum as circular compact, coarse, refractile granules. Stieda body is present, while substieda body is absent. Sporozoites banana-shaped, measure 11 x 2.5 microm, each with a large spheroidal refractile body at the wider pole. Sporulation time is 2-3 days at 25 +/- 2 degrees C.

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence.

The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.