Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status

Due to the diminished use of growth-promoting antibiotics in the European Union, Clostridium perfringens induced necrotic enteritis and subclinical disease have become important threats to poultry health. A study was set up to genotypically and phenotypically characterise C. perfringens isolates from poultry flocks with different health status. Animals from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of animals suffering from necrotic enteritis were analysed. A total of 27 isolates was obtained from 23 broiler flocks without clinical problems and 36 isolates were obtained from 8 flocks with clinical problems. Using PFGE typing, high genetic diversity was detected between isolates from different flocks. Isolates derived from flocks where disease outbreaks occurred were clonal within each flock, but each flock harboured a different clone. All isolates were of toxin type A. Isolates from 5 out of 35 PFGE types carried the cpb2 gene, encoding the beta2 toxin, and isolates from 2 out of 35 PFGE types harboured the cpe gene, encoding the enterotoxin. In vitro alpha toxin production for all isolates was quantified by enzyme-linked immunosorbent assay. It was shown that in vitro alpha toxin production of C. perfringens isolates from diseased flocks was not higher than in vitro alpha toxin production from isolates derived from healthy flocks.     

Polymorphism analysis of BMPR1B gene by forced RFLP and PCR-SSCP techniques and expression of the mutation in introgressed sheep

The present study was conducted to screen Kashmir valley sheep with history of prolificacy for the presence of FecB mutation. Forced polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and single strand conformation polymorphism (SSCP) techniques were employed to detect any polymorphism present in bone morphogenetic protein receptor type 1B (BMPR1B) gene. Further, it was aimed at introgressing the FecB mutation into nonprolific noncarrier sheep. A 140-bp fragment of BMPR1B gene was amplified from isolated genomic DNA and subjected to forced RFLP with restriction enzyme AvaII. Three different RFLP patterns were identified. SSCP analysis showed one-to-one correspondence with RFLP patterns. Sequencing of the samples showing different patterns revealed that the wild (+) and mutant (B) alleles were different by a single nucleotide substitution in the form of A109G from wild to mutant allele. It led to change in amino acid from Glutamine (Q) to Arginine (R) from wild to mutant allele. The mutation was only detected in NARI-Suwarna and their crosses; all Kashmir valley sheep with prolific history lacked it. The + allele was abundant in the studied population. The FecB mutation was introgressed in nonprolific noncarrier sheep by crossing ewes with NARI-Suwarna rams possessing the mutation. First generation crossing produced heterozygous (B+) progeny. Some of the F₁ heterozygous ewes gave birth to twins when mated to unrelated NARI-Suwarna rams. It showed that FecB mutation was successfully expressing in those crosses.     

Molecular detection of Theileria spp. in sheep and vector ticks in the North Khorasan Province, Iran

A survey was carried in North Khorasan Province, Iran in 2010–2011, designed to identify Theileria spp. infections of both sheep and ticks. The tick species were also examined. Ninety sheep from different flocks were clinically examined, and blood samples and ixodid ticks were collected. Light microscopy of blood smears revealed Theileria spp. infection in 37 (41.1 %), while 74 (82.2 %) of blood samples were positive using semi-nested PCR. Theileria ovis, Theileria lestoquardi, and mixed infection were detected in 63/90 (70 %), 5/90 (5.5 %), and 6/90 (6.6 %) of samples, respectively. Of the 434 ticks that were collected, the most prevalent species was Rhipicephalus turanicus (69.3 %) followed by Hyalomma marginatum turanicum (18.4 %), Dermacentor marginatus (6.4 %), and Rhipicephalus bursa (5.7 %). The ticks were separated into 42 tick pools, and the salivary glands were dissected out in 0.85 % (w/v) saline under a stereomicroscope and examined using semi-nested PCR. Three pools of H. marginatum turanicum salivary glands were infected with T. ovis and T. lestoquardi, and one pool of R. turanicus was infected with T. ovis. Based on these results, it is concluded that the prevalence of T. ovis is higher than T. lestoquardi and that H. marginatum turanicum and R. turanicus are likely vectors of T. lestoquardi and T. ovis in this area.     

Experimental caprine coccidiosis caused by Eimeria arloingi: Morphopathologic and electron microscopic studies

The progressive morphohistopathologic changes, distribution pattern of lesions and ultrastructural characteristics in Eimeria arloingi infection were investigated in experimentally infected kids. The 18 newborn animals allocated to 3 equal groups. Two of groups, A, B were inoculated with a single dose of 1 × 10³ and1 × 10⁵ sporulated oocysts of E. arloingi, respectively. At 7, 14, 21, 28, 35, and 42 days postinoculation (DPI), 1 kid from each group was necropsied for pathologic and ultrastructural studies. Progressive lesions were present at 21, 28, 35 and 42 DPI in the jejunum, ileum, cecum with fewer in the duodenum and proximal colon. The oocysts shedding begin between 16 to 18 DPI. Grossly, minimal changes were observed at 21 DPI as few whitish plaques or nodules and advanced lesions at 42 DPI as pseudoadenomatous pattern in the mucosa and a cerebriform pattern on the serosal surface of jejunum and ileum. Early histopathologic lesions due to schizogony phase were including presence of intracytoplasmic developmental stages of the parasite such as trophozoites, immature to mature schizonts and mild infiltration of inflammatory cells. In late lesions due to various stages of gametogony, the histological pattern was mainly remarkable hyperplasia of the villi and crypts epithelial cells, eventually developed into papillary projections of reactive epithelium. The mesenteric lymph nodes showed a few numbers of large schizonts in the cortical lacteals. This study showed E. arloingi as a highly pathogenic species for kids, the incubation period was 16–18 days and the main target organ was jejunum with characteristic morphohistopathologic lesions.     

In vitro and in vivo anthelmintic activity of Euphorbia helioscopia L

The anthelmintic potentials of the aqueous and methanol extracts of Euphorbia helioscopia were investigated. In folklore medicine, E. helioscopia (Euphorbiaceae) is used in the treatment of various gastrointestinal ailments and diseases. A worm motility inhibition (WMI) assay and egg hatch assay (EHA) were used for in vitro assessment, and a faecal egg count reduction (FECR) assay was used for an in vivo study. The in vitro study revealed anthelmintic effects of crude methanolic extracts of E. helioscopia on live Haemonchus contortus worms as evident from their paralysis and/or death at 8h after exposure, different concentrations (12.5mgml(-1), 25mgml(-1) and 50mgml(-1)) of aqueous and methanolic extracts were used against H. contortus which exhibited dose-dependent anthelmintic effects on H. contortus. Aqueous and methanolic extracts of aerial parts (stem, leaves and flowers) of E. helioscopia were found to have very low percent inhibitory egg hatching as compared to levamisole. It is concluded that the entire plant of E. helioscopia possesses significant anthelmintic activity and could be a potential alternative for treating cases of helminth infections in ruminants.     

Potential for using Datura alba leaf extracts against two major stored grain pests, the khapra beetle Trogoderma granarium and the rice weevil Sitophillus oryzae

Various plants are well known for their insecticidal activity and their use was maintained for millennia throughout all the agricultural regions of the world. In a current context, the use of Botanical insecticides represents one of the best alternatives to chemicals for the development of environmental-friendly strategies for stored grain pest control. Datura alba Nees is a plant found extensively in the warmer regions of the world and it is used as a medicinal plant. This study aims to assess the contact toxicity and the trans-generational effect of D.?alba leaf extract (DLE) against two important insect pests on stored rice, Trogoderma granarium and Sitophilus oryzae, under laboratory conditions. Filter papers were soaked in three DLE concentration solutions and in two control treatments: water and acetone. The survivor specimens (F₀) were transferred to a new untreated feeding substrate and the population build up of the two following generations (F₁ and F₂) were counted after 30 and 60?days, respectively. The highest DLE concentration (2.5?%) induced the significantly highest mortality with 33.5 and 45?% mortality in T.?granarium and S.?oryzae after 7?days of exposure, respectively. The DLE long-term effect toward both tested species was also proved by the high demographic decrease in the F₂ generations, when compared to control groups. This study is the first step toward establishing a scientific basis for the effective application of D.?alba plant materials as biorational tools to control stored grain pests.     

On the Current Situation of Glanders in Various Districts of the Pakistani Punjab

Glanders is a highly infectious and zoonotic disease of solipeds caused by Burkholderia mallei. Progressive loss of efficiency and fatal outcome resulted in massive economic losses, which forced veterinary authorities throughout the world to implement disease control measures; these measures included mass testing using the complement fixation test and/or malleinization, and the culling of positives. This led to the eradication of glanders from Western Europe and North America in the 1950s. However, in the last decade, the number of outbreaks in Asia and South America increased steadily, and glanders regained the status of a re-emerging transboundary disease. Pakistan has been an endemic country for the past 120 years, but concise data on the presence of disease are not available. A total of 533 serum samples were collected from draught equines, a suspected risk group for glanders, from various districts of Punjab in Pakistan. The complement fixation test and the highly sensitive Western blot technique were used for serodiagnosis. No animal (horse, mule, and donkey) was found to be positive for infection. Glanders seems to be restricted to remote, sporadic pockets of endemicity and may cause outbreaks after being introduced into naive populations by (asymptomatic) shedders.     

保护性耕作下西南旱三熟农田土壤水分动态及产量效应

以西南地区麦/玉/薯三熟制种植模式为研究对象,研究了不同保护性耕作模式下旱地土壤水分动态变化、水分利用效率、作物产量和经济效应。结果表明,不同保护性耕作对整个农田生态系统产量和水分利用效率具有显著影响。秸秆覆盖(TS)、地膜覆盖(TM)、秸秆覆盖+地膜覆盖(TSM)和垄作+秸秆覆盖+地膜覆盖(RSM)能改善7,10月份0—60cm土层土壤含水量及贮水量,而垄作(R)、垄作+秸秆覆盖(RS)和垄作+地膜覆盖(RM)的作用不明显。与平作(CK)相比,RSM,RM,RS,TSM,TM,TS和R分别使系统增产10.52%,6.11%,6.77%,4.59%,5.12%,3.15%,1.24%。这些处理对水分利用效率和降水利用效率的增幅分别为1.26%～11.08%,1.27%～10.57%。研究结果说明,从WUE、降水利用效率、产量效应、经济效益这4个方面来看,垄作+秸秆覆盖+地膜覆盖耕作模式综合效益最佳。     

Prevalence and tissue distribution of Besnoitia darlingi cysts in the Virginia opossum (Didelphis virginiana) in Michigan

Specimens of Virginia opossums (Didelphis virginiana) in Michigan were examined over 1 year to document the presence of Besnoitia darlingi cysts. Cyst morphology, prevalence, seasonal variation, and tissue sites of isolation were studied. Histology and ultrastructural features of the detected cysts and bradyzoites were consistent with B. darlingi. In the opossums, B. darlingi had intracellular tissue cysts. Tissue cysts had a mean diameter of 560 microm and were separated from the host tissue by a thick (5-20 microm) cyst wall. Overall prevalence of B. darlingi cysts in opossums was 10.9% (15/137). Variations in the prevalence were detected during spring (3/17; 17.6%), summer (10/34; 29.4%), and fall (2/60; 3.3%). No cysts were detected in the specimens examined during winter (0/26; 0%). Numerous B. darlingi cysts were detected in ears, conjunctiva, tongue, abdominal muscles, diaphragm, stomach, heart, liver, kidney, lung, and spleen. Cysts were detected mainly in adult female opossums that were debilitated. Ear was the most frequent organ from which the cysts were reported (10/15; 66.7%) when compared individually with other body tissues (P<0.05).     

Effect of temperature on lutein esterification and lutein stability in wheat grain

The creamy colour of many wheat-based end-products is conferred by endogenous lutein. During post-harvest storage of grain, free lutein may be converted in part to potentially more stable lutein mono- and di-fatty acid esters. This study investigates the synthesis of lutein esters and stability of free lutein and lutein esters over a wide range of temperatures in grain of a high lutein bread wheat, Triticum aestivum L. line DM5685*B12 and a durum, Triticum durum L cv Kamilaroi. Disappearance of free lutein and lutein esterification followed first order reaction rates. The maximum rate of lutein esterification was at ≈80 °C, however, the optimum temperature for maximum synthesis with minimum degradation was between 30 °C and 60 °C. No ester synthesis was observed at temperatures higher than 120 °C. The data are consistent with an enzyme participating in the esterification reaction. Lutein esters were found to be more stable than free lutein with a longer shelf life at 60 °C whilst at temperatures ≤40 °C, lutein degradation was minimal. This study provides new information on lutein ester formation and lutein stability that should be useful to grain handlers and food manufacturers seeking to optimise retention of lutein for the benefit of consumers.