Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011--review and recommendations for surveillance

Since 2006, the members of the molecular epidemiological working group of the European "EPIZONE" network of excellence have been generating sequence data on avian influenza and avian paramyxoviruses from both European and African sources in an attempt to more fully understand the circulation and impact of these viruses. This review presents a timely update on the epidemiological situation of these viruses based on sequence data generated during the lifetime of this project in addition to data produced by other groups during the same period. Based on this information and putting it all into a European context, recommendations for continued surveillance of these important viruses within Europe are presented.     

Avian conservation priorities in a top-ranked biodiversity hotspot

Rio de Janeiro state in Brazil has one of the most diverse and most endangered avifaunas in the continental Americas. Many of these endangered birds are endemic to the Atlantic Forest biodiversity hotspot, and some even endemic to Rio de Janeiro itself. As with all other forested hotspots, little original forest remains. Much of that is outside formal protected areas and faces the risk of deforestation. These factors create special circumstances for setting conservation priorities — ones common to hotspots in general — but typically not to many conservation priority setting exercises.We mapped the distribution of the remaining habitat for the 189 birds in Rio de Janeiro state that are officially endangered and/or endemic to the Atlantic Forest. Using those habitat maps, we calculated the amount of habitat currently within protected areas for each species. We then prioritized all non-protected parts of the state for their avian conservation value and their potential contribution to a comprehensive protected area system. This analysis identified 10% of the remaining unprotected part of the state as the highest priority for avian conservation. We further highlight specific locations where conservation actions could create a more comprehensive protected area system for the avifauna of Rio de Janeiro state.     

Evidence to support horses as natural intermediate hosts for Sarcocystis neurona

Opossums (Didelphis spp.) are the definitive host for the protozoan parasite Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). Opossums shed sporocysts in feces that can be ingested by true intermediate hosts (cats, raccoons, skunks, armadillos and sea otters). Horses acquire the parasite by ingestion of feed or water contaminated by opossum feces. However, horses have been classified as aberrant intermediate hosts because the terminal asexual sarcocyst stage that is required for transmission to the definitive host has not been found in their tissues despite extensive efforts to search for them [Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001b. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol. 95, 89-131]. In a 4-month-old filly with neurological disease consistent with EPM, we demonstrate schizonts in the brain and spinal cord and mature sarcocysts in the tongue and skeletal muscle, both with genetic and morphological characteristics of S. neurona. The histological and electron microscopic morphology of the schizonts and sarcocysts were identical to published features of S. neurona [Stanek, J.F., Dubey, J.P., Oglesbee, M.J., Reed, S.M., Lindsay, D.S., Capitini, L.A., Njoku, C.J., Vittitow, K.L., Saville, W.J., 2002. Life cycle of Sarcocystis neurona in its natural intermediate host, the raccoon, Procyon lotor. J. Parasitol. 88, 1151-1158]. DNA from schizonts and sarcocysts from this horse produced Sarcocystis specific 334bp PCR products [Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Greiner, E.C., MacKay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J. Parasitol. 85, 221-228]. Restriction fragment length polymorphism (RFLP) analysis of these PCR products showed banding patterns characteristic of S. neurona. Sequencing, alignment and comparison of both schizont and sarcocyst DNA amplicons showed 100% identity. Although Koch's postulates have not been demonstrated in this case study, the finding of mature, intact S. neurona schizonts and sarcocysts in the tissues of this single horse strongly suggests that horses have the potential to act as intermediate hosts. Further studies are needed to demonstrate Koch's postulates with repeated transfer of S. neurona between opossums and horses.     

Seroprevalence of antibodies against Leishmania spp among dogs in the United States

OBJECTIVE: To determine seroprevalence of antibodies against Leishmania spp among dogs other than Foxhounds in the United States. DESIGN: Cross-sectional study. SAMPLE POPULATION: 957 serum samples from dogs throughout the United States submitted between January 2000 and August 2001 to the Diagnostic Center for Population and Animal Health at Michigan State University for serologic testing for tick-borne diseases. PROCEDURE: Samples were tested for antibodies against Leishmania spp with an immunofluorescent antibody (IFA) assay. Samples with positive results were submitted to the Centers for Disease Control and Prevention for confirmatory testing. RESULTS: Results of the IFA assay were negative for 939 of 957 samples. For 16 samples, titers were from 1:16 to 1:64, and titers in these dogs were considered likely to be a result of cross-reactivity with antibodies directed against other organisms. For the remaining 2 samples, the titers were > or = 1:128. One of these samples was from a blood donor dog that had never had any clinical signs of leishmaniasis. Follow-up samples from both dogs also had Leishmania IFA titers > or = 1:128. Both dogs had antibodies against Trypanosoma cruzi, as determined with a radioimmunoprecipitation assay. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the seroprevalence of antibodies against Leishmania spp in dogs in the United States was low. However, results further suggested that leishmaniasis may not be limited to Foxhounds in the United States.     

ULTRASONOGRAPHIC ANATOMY OF THE NORMAL CANINE CALCANEAL TENDON

To provide a series of detailed ultrasonographic images of the canine calcaneal tendon, greyhound cadaver pelvic limbs were scanned with a high-resolution transducer, and images compared with dissected specimens. The three components of the calcaneal tendon are the tendons of insertion of the gastrocnemius, superficial digital flexor, and a conjoined tendon formed by tendons from the biceps femoris, semitendinosus, and gracilis. Each of these three tendons was visible in transverse ultrasound images, each measuring 2.4-3.2-mm thick at mid-calcaneal tendon. Improved understanding of the anatomy of the calcaneal tendon will support clinical ultrasonography of this region.     

Effect of lymphocytes and broncho-alveolar washing cells on the replication of bovine herpesvirus type 1 in tracheal organ cultures

The daily addition of lymphocytes collected from a calf between 7 and 11 days after experimental infection with bovine herpesvirus type 1 (BHV-1) to bovine fetal tracheal organ cultures after infection with BHV-1 did not inhibit virus replication. The daily addition of normal lymphocytes, together with a low concentration of serum antibody against BHV-1, had a slight viral inhibitory effect which was believed to be due to antibody-dependent cell-mediated cytotoxicity. The addition of broncho-alveolar washing (BAW) cells, collected before infection or 30 days after infection of a calf with BHV-1, together with lymphocyte culture supernatant, to tracheal organ cultures immediately after infection with BHV-1 produced some inhibition of virus replication. Virus replication was markedly inhibited when BAW cells collected from the calf 18 days after infection were used in a similar manner.     

Viability of Sarcocystis neurona sporocysts after long-term storage

The effect of long-term storage on the viability and infectivity of Sarcocystis neurona sporocysts was investigated. S. neurona sporocysts were harvested from the small intestine of Virginia opossums from 1996 to 2002 and stored at 4 degrees C. Viability of sporocysts was assessed by propidium iodide (PI) exclusion assay, in vitro excystation and development in tissue cultures, and bioassay in gamma-interferon gene knockout (gamma-IFN-KO) mice. The rate of excystation was apparently unaffected by long-term storage; sporocysts retained their ability to excyst after 7 years of storage at 4 degrees C. However, the ability of sporocysts to exclude PI stain, to invade and proliferate in cells in vitro, and to cause disease and lesions in gamma-IFN-KO mice appeared to decline as sporocysts age. The results demonstrated that sporocysts of S. neurona were able to survive and maintain moderate to high viability for up to 7 years when stored in phosphate buffered saline and Hank's balanced salt solution containing antibiotic-antimycotic mixture at 4 degrees C.     

Prevalence of and risk factors associated with the presence of Sarcocystis neurona sporocysts in opossum (Didelphis virginiana) from Michigan: a retrospective study

From April 1996 to December 2002 the prevalence of Sarcocystis neurona sporocysts in North American opossum (Didelphis virginiana) in Southern Michigan was estimated. Sporocysts of S. neurona were found in intestinal scrapings from 31 (15%) of 206 examined opossum. The frequency of infection was higher in adult animals (26/206; 12.6%) and females (19/206; 9.2%) than in juveniles (5/206; 2.4%) and males (12/206; 5.8%). Also, prevalence of S. neurona sporocysts in opossums in relation to factors such as age, sex, season, body condition, presence of concomitant infection, and presence of young in the pouch of females was studied in detail over the course of the year, 2002. Univariate analyses identified the following factors as being associated with the presence of S. neurona sporocysts in opossums: (i) for age, adult (odd ratio [OR] = 2.074, P = 0.0005); (ii) for sex, female (OR = 7.016, P = 0.0119); (iii) for season, summer (OR = 7.917, P = 0.0032) and spring (OR = 4.071, P = 0.1063); (iv) for body condition, poor (OR = 3.50, P = 0.1200) and good (OR = 1.167, P = 0.8637); (v) for the presence of concomitant infection (OR = 23.056, P = 0001), and (vi) for the presence of young in the pouch of females (OR = 40.083, P = 0.0001). Multivariate logistic-regression analyses selected the following factors as being significantly associated with presence of S. neurona sporocysts in opossums: (i) for the presence of concomitant infection (OR = 8.722, P = 0.0160) and (ii) for the presence of young in the pouch of females (OR = 31.915, P = 0.0065). The prevalence of S. neurona sporocysts in D. virginiana suggests that this opossum may constitute an ample reservoir of infection to other animals in the northern United States.     

Anti-leishmanial and immunomodulatory activities of extracts from Portulaca hirsutissima and Portulaca werdermannii

Ethyl acetate and chloroform extracts from aerial parts of Portulaca werdermannii and P. hirsutissima were tested in lymphoproliferation assays and axenic cultures of Leishmania amazonensis and Trypanosoma cruzi. Both extracts of P. werdermannii and P. hirsutissima had a potent inhibitory activity on lymphocyte proliferation. On the contrary, only the chloroformic extract of both plants inhibited L. amazonensis growth, without effect on T. cruzi cultures. These results indicate these Portulaca species as potential sources of new active molecules for the treatment of leishmaniasis and immune-mediated pathologies.     

Impact of sourdough on buckwheat flour, batter and bread: Biochemical, rheological and textural insights

In this study, the impact of sourdough fermentation on the biochemical, rheological and bread-making performances of buckwheat flour was investigated. In order to assess the effects of the solely acidification, a chemically acidified batter with the same pH of the sourdough was prepared. Extensive hydrolysis of the globulin fraction and release of small polypeptides occurred upon fermentation. A major reduction in the extent of interactions was observed in the sourdough, mainly due to the modification of the major structural components of buckwheat during the fermentation. The hydrolysis of proteins and, possibly, starch was in turn responsible for the major decrease in elasticity observed in the sourdough. In the batters destined for bread-making, the presence of acids was the major cause for reduced elasticity and increased strength, which could be related to the enhanced water-holding capacity of the proteins and/or protein/starch complexes. The addition of sourdough induced dramatic inhibition of the CO₂ production by the baker’s yeasts during proofing, resulting in lower volume and harder crumb of the sourdough bread. On the other hand, the solely acidification induced hardening of the starch gel upon cooking, which was responsible for lower volume and irregular crumb grain in buckwheat bread.