Agrobacterium-mediated transformation of Ceratocystis albifundus.

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

Diffuse type testicular seminoma in a stallion.

A 7-year-old stallion with progressive left testicular enlargement was presented. Grossly, the excised testicle measured 25 × 15 × 12 cm and weighed 3.7 kg. It was multinodular with a gray-white surface; however, the right testis was normal. Histologically, the neoplastic cells were disseminated diffusely in the tumoral stroma with a minimal fibrovascular stroma. Neoplastic cells were round to polygonal with abundant eosinophilic cytoplasm and large round to oval vesicular or hyperchromatic nucleous with a single prominent nucleolus. Immunohistochemically, the neoplastic cells were positive nuclear immunostaining for C-KIT and negative for OCT3/4. According to gross, histopathological, and immunohistochemical characteristics, the diffuse type of seminoma was diagnosed. Nine months later, the follow-up observation of the case showed that the tumor had no recurrence and metastasis.

Seroprevalence of Neospora caninum infection in free ranging chickens (Gallus domesticus).

Recently chickens are considered as an important intermediate hosts for Neospora caninum. Free range chickens expose to infection with N. caninum oocysts because they feed from the ground therefore they could be a good index of the environmental contamination. We studied N. caninum infection in free range chickens by serological. One hundred and fifty chickens purchased from five regions from Fars province and their blood were used for serological testing. Antibodies to N. caninum were found in 26 (17.33 %) of 150 serum samples by MAT. This study is the first to describe the presence of antibodies to N. caninum in chicken in Iran. These serological results indicate a widespread exposure of free range chickens to N. caninum in south of Iran.

Suspension culture of Neospora caninum by Theileria annulata-infected cell line.

There are some limiting aspects of scaling up the Neospora caninum tachyzoites in continuous cell lines, particularly as related to the absence of surface attachment. In this study, suspension cell culture of Theileria annulata-infected lymphoblastoid (TIL) was used as a host cell for the continous production of N. caninum tachyzoites. The numbers of free tachyzoites in the medium supernatant were showed regularly increased up to the day 6 post-cultivation. Transmission electron microscopy demonstrated that N. caninum tachyzoites invaded the TIL cells and multiplied intracellularly. This showed that the tachyzoites were successfully proliferated in TIL cells and were released in complete Dulbecco's modified Eagle's medium. This is a successful report of in vitro cultivation of N. caninum tachyzoites achieved by using suspension host cell culture.

First Report of Hyphodermella rosae Causing Dry Fruit Rot Disease on Plum in Iran.

Plum (Prunus domestica) and peach (P. persica) are widely grown, often in alternate rows with citrus, in the Mazandaran Province of Iran. In June 2011, a dry fruit rot of plum was observed in several production regions in Mazandaran Province (35°47'N, 50°34'E). Initial symptoms at pit-hardening stage appeared as dark brown, circular, necrotic spots from 2 to 5 cm in diameter. They later developed into a dry fruit rot. Severe symptoms occurred during June and July when warm weather (temperature around 28°C) and high relative humidity (RH) (>85%) were present. Marketable yield losses reached 50% to almost 100% in many orchards. To isolate the causal organism, symptomatic fruits were surface disinfested for 1 min in 0.5% active chlorine, washed thoroughly with sterile distilled water, and segments were plated on potato dextrose agar (PDA) amended with 50 mg/liter of streptomycin sulfate and incubated at 25°C for 3 days. The fungus Hyphodermella rosae (Bresadola) Nakasone was consistently isolated (37 isolates from 79 samples) and identified on the basis of morphological characteristics on PDA. Basidiomata were effuse, resupinate, 15 × 10 mm, crustaceous, tubercules small with apical bristles, and light orange to grayish orange. Subhymenium was up to 30 µm thick, composed of vertically arranged, short-celled, nonagglutinated hyphae; subhymenial hyphae were 3 to 4 µm in diameter. Basidiospores were ellipsoid, 7.5 to 8.5 × 4.5 to 5.5 µm (100 determination), and their cell walls were thin, hyaline, and smooth (1). Genomic DNA was extracted from mycelium with a DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's directions and grown on potato dextrose broth for 4 days at 28°C. The rDNA region was amplified with the primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'- GGAAGTAAAAGTCGTAACAA-3') (4) and the PCR product was sequenced. Nucleotide BLAST analysis of the amplified 627-bp fragment confirmed a 99% similarity with the sequence of H. rosae (GenBank Accession No. JN593086). A pathogenicity test was conducted with isolate MA4099 by placing 5-day-old mycelial plugs grown on PDA at the surface of healthy fruit (n = 6) incubated under >85% RH at 25°C for at least 4 days until the appearance of symptoms, which were similar to those displayed under orchard conditions. Control fruits, inoculated with blocks of PDA plugs, remained intact and symptomless. Reisolation from inoculated fruit samples consistently yielded the inoculated fungus, completing Koch's postulates. The genus Hyphodermella has been reported to be causing wood rot on apricot (2) and sweet and sour cherry (3). To our knowledge, this is the first report of H. rosae causing dry fruit rot on a stone fruit species in the world. References: (1) K. K. Nakasone. Mycologie, 29:231, 2008. (2) J. M. Ogawa et al. Diseases of Apricot (Prunus armeniaca L.). The American Phytopathological Society, St. Paul, MN, 2003. (3) J. K. Uyemoto et al. Diseases of Sweet Cherry (Prunus avium L.) and Sour Cherry (P. cerasus L.). IS-MPMInet, http://www.ismpminet.org/resources/common/comment/cherry.asp , accessed June 2012. (4) T. J. White et al. Page: 315 in: PCR Protocols: A Guide to Methods and Application. M.A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

Microscopic evaluation of renal changes in experimental canine visceral leishmaniosis after chemo- and immunotherapy.

Visceral Leishmania (VL) with diverse clinical manifestation is prevalent and remains a major public health problem in Iran. This study was performed in Ahwaz, Khozestan province southwest to increase immune system and to reduce of the renal lesions. Treatment of dogs with visceral leishmaniosis is basically the same as the treatment of human. However, cure is not usually achieved, leaving the sacrifice of animal as the only feasible choice. The goal of this work was to test the therapeutic efficacy of N-methyl glutamic antimoate (glucnime), Mycobacterium vaccae adjuvant (SRL 172), alone and in association with L. major promastigote and the latter compound in association to glucantime, in dog with visceral leishmaniasis. In this trial 18, mixed bred dogs with different ages, receiving amastigte promastigote of L. infantum intravenously were used. They were monitored for 6 months. Serologic assays (Elisa, Dot and IFAT) were performed on blood samples of each animal. The animals were divided into six groups, each having 3 dogs: Group 1: receiving 100 mg kg(-1) day(-1) Glucantime for 30 days, IM. Group 2: Receiving 3 mg dog(-1) (0.1 mL) of Mycobacterium vaccae adjuvant suspension intradermaly. Group 3: receiving L. major promastigote plus M. vaccae adjuvant each of them 0.1 mL intradermaly by one month intervals for 3 months. Group 4: receiving Glucantime in association L. major promastigote plus M. vaccae adjuvant with previous doses. Group 5: Receiving no treatment. Group 6: was control group with no infection and treatment. In microscopic evaluation following lesions have been shown in kidney: Chronic, interstitial nephritis, sever glomerulosclerosis, membranoproliferative glomerulonephritis and also non suppurative nephritis were the lesions in 5 groups. The prescription of Mycobacterium vaccae adjurant was able to reduce the number of parasites in the macrophages of liver and spleen in this round of treatment.