Seasonal change in the number of Cryptosporidium parvum oocysts in water samples from the rivers in Hokkaido,Japan, detected by the ferric sulfate flocculation method

An epidemiological study was carried out in natural water supplies of Hokkaido, one of the largest dairy prefectures in Japan. To investigate the prevalence of Cryptosporidium parvum (C. parvum) oocysts water samples were collected from three rivers in the eastern area of Hokkaido from August 1999 to October 2001, and C. parvum oocysts were collected and purified by the ferric sulfate flocculation method. The oocysts were detected using the immunofluorescent assay test (IFAT) and 4', 6-diamidino-2-phenylindole (DAPI) staining. The seasonal change in the number of oocysts detected was observed. Oocysts increased in numbers from the late summer to the early autumn (from August to November), thereafter, they exhibited a trend to decrease until December, when no oocysts could be detected. The maximum number of oocysts detected in the three rivers was 3.50, 5.00 and 3.33 oocysts/l, respectively. The oocyst density in river water changed in relation to the season in 1999, 2000 and 2001. This report first cleared up the seasonal changes in C. parvum oocysts number in river water.     

Ontogeny of the shi drum Umbrina cirrosa (Linnaeus 1758), a candidate new species for aquaculture

The ontogeny of shi drum Umbrina cirrosa (Linnaeus 1758), a candidate new species for aquaculture, was studied throughout the entire larval phase. Geometric morphometric analysis revealed two clear inflection points (7.0 and 12.7 mm total length, TL) in the shape ontogeny of this species, separating the studied period into three phases of different allometric priorities. Spline graphs demonstrated that the major non‐uniform shape ontogeny correlated with the development of the fins, the anterior dorsal area of the body, the caudal peduncle, the eye and the mouth. Concerning the morphological features, shi drum larvae are characterized by an upward anterior bending of the notochord. The ontogeny of the fins began with the formation of the pectoral buds (2.9 mm TL), continued with the notochord flexion (4.3 mm TL, associated with the caudal fin development), the appearance of the pelvic buds, the first anal rays (4.5 mm TL) and the first dorsal rays (4.8 mm TL). Shi drum juveniles presented 25 vertebrae and the following dominant fin types: D XI,23, AII,6, VI,5, P17 and C17.     

Steroid-responsive neutropenia in a cat with progressive feline leukemia virus infection

An 8-month-old female domestic shorthair cat was presented to the Animal Medical Center with anorexia, lethargy, and mild gastrointestinal signs. A CBC revealed a profound neutropenia, and serologic testing with an in-house test kit (SNAP FIV/FeLV Combo, IDEXX) was positive for feline leukemia virus (FeLV) antigen. Serial hematologic examinations during hospitalization showed a persistent neutropenia with occasionally severe anemia and thrombocytopenia. Prednisolone administration afforded complete hematologic remission within 3 days. Four weeks after the premature discontinuation of prednisolone, the patient relapsed; however, complete and prolonged hematologic remission was achieved after prednisolone was re-induced. Bone marrow aspiration cytology was consistent with immune-mediated destruction of the mature myeloid cells. steroid-responsive (likely immune-mediated) cytopenias rarely occur in cats with progressive FeLV infection. Although only a few cases of FeLV-positive, severely neutropenic cats that responded to immunosuppressive therapy have been reported, this case highlights that a grave prognosis should not always be given to these FeLV-positive cats.     

A study on ghrelin and LH secretion after short fasting and on ghrelin levels at perioestrual period in dairy cattle

In two experiments, we studied (a) the changes of LH secretion in heifers under different feeding schedules and (b) total ghrelin concentration at oestrus in cows and heifers. In experiment one, synchronized heifers were allocated in three groups (R, regularly fed controls; F, fasted; and F‐F fasted‐fed). One day after the completion of the oestrous induction protocol, group F and F‐F animals stayed without feed for 24 hr; thereafter, feed was provided to R and F‐F cattle; 2 hr later, GnRH was administered to all animals. Blood samples were collected for ghrelin, progesterone, LH and cortisol concentrations. Fasting caused increased ghrelin concentrations in groups F and F‐F, while in response to GnRH, LH surge was significantly attenuated in groups F and F‐F compared to R. In experiment 2, lactating cows and heifers were used. On day 9 of a synchronized cycle, PGF2α was administered, and blood samples were collected twice daily until the third day after oestrus and analysed for progesterone, estradiol, ghrelin, glucose and BHBA concentrations. No difference was recorded between groups in steroids and BHBA concentrations. In comparison to mid‐luteal values, ghrelin concentrations significantly increased at perioestrual period in cows, but not in heifers. This study provides evidence that starving‐induced elevated ghrelin concentrations can have suppressing effect on LH secretion, even after ghrelin's restoration to basal values and that during oestrus, ghrelin secretion is differently regulated in cows and heifers, likely being independent from oestradiol concentrations. Further research is required to identify the determining factors that drive the different regulation of ghrelin secretion in cows and heifers.     

Chronotypology and melatonin alterations in minimal hepatic encephalopathy

Background  

"Minimal (subclinical) hepatic encephalopathy" is a term that describes impairment of every day life activities in cirrhosis patients without clinical neurologic abnormalities. Melatonin diurnal pattern disruption and metabolic changes due to liver insufficiency can affect the human biologic clock. Our study was conducted to measure plasma melatonin levels in an attempt to correlate plasma melatonin abnormalities with liver insufficiency severity, and describe chronotypology in cirrhosis patients with minimal encephalopathy.     

Persistence and efficacy of spinosad on wheat,maize and barley grains against four major stored product pests

The insecticidal and residual effect of spinosad on wheat, maize and barley grain was evaluated in the laboratory against adults of Sitophilus oryzae (F.) (Coleoptera: Curculionidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), Tribolium confusum (DuVal) (Coleoptera: Tenebrionidae), Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) as well as against larvae of T. confusum. Spinosad was applied as a solution to 2 kg lots of each commodity at three concentrations, 0.1, 0.5 and 1 ppm, and the treated grain quantities were kept at 25 °C and 65% RH. Samples were taken from each concentration-commodity combination at the day of storage and every 30 d for 6 consecutive months (6 bioassays). The test species were exposed for 14 d to the samples and mortality and reproduction were assessed over this exposure interval. With the exception of T. confusum, 1 ppm of spinosad was highly effective against the remainder of the tested species and provided protection for a period of storage at least 4 months. Although in general, spinosad performance was not very much affected by the grain type, efficacy on maize was less stable over the 6-month period of storage and declined sooner compared to the other commodities. Spinosad almost suppressed progeny production of R. dominica during the storage period, but did not suppress progeny of the other species, since progeny were recorded even 30 d post application especially with the lowest of the tested concentrations. The results of this study indicated that spinosad may provide suitable protection for 6 months against S. oryzae or R. dominica, but is not suitable for long-term protection against T. confusum or C. ferrugineus.     

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido,Japan

The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.     

A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples. To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD=1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD=126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.     

Exploitation of the chloroplast trnL (UAA) intron polymorphisms for the authentication of plant oils by means of a lab-on-a-chip capillary electrophoresis system

Methods to discriminate plant oils facilitate the detection of either deliberate or accidental adulteration. To this direction, the variability in length among plant species of the chloroplast trnL intron was exploited for the authentication of edible and cosmetic plant oils, with an extra emphasis on olive oil. The methodology was based on the combinatorial use of a PCR assay with a capillary electrophoresis system such as the lab-on-a-chip technology. Application of the assay on DNA extracted from different oil producing plant species, including olive oil and sesame oil, indicated the ability of the trnL intron to be used as an analytical target. Furthermore, this assay could be used for the detection of adulteration of olive oil with various other plant oils, with the exception of avocado and sesame oil.     

Response of ungrafted rootstocks and rootstocks grafted with wine grape varieties (Vitis sp.) to ‘lime-induced chlorosis’

The ungrafted rootstocks 41B, 1103P, 110R and 140Ru, the grafted combinations of 41B, 1103P and 110R with Xinomavro (one of the most important red wine grape varieties in Greece), as well as those of 1103P, 110R and 140Ru with Chardonnay, were evaluated for 'lime-induced chlorosis' tolerance by growing them with a) basic nutrient solution (BNS), b) BNS + 10 mM bicarbonate, c) BNS without iron (Fe) and d) BNS without zinc (Zn), in hydroponics. The ungrafted 140Ru followed by 41B under high bicarbonate presented the lowest degree of chlorosis; however only 41B presented non-differentiated biomass production and root/shoot ratio. Chlorotic symptoms in combination with plant growth parameters should be used as a tool for grapevine rootstock lime-tolerance screening whereas leaf Fe concentration and root ferric chelate reductase (FCR) activity should not. Lime-stress conditions affected plant mineral nutrition by depressing leaf nitrogen (N), phosphorus (P), calcium (Ca), magnesium (Mg) and increasing potassium (K), and zinc (Zn).