Pathogenesis and transmissibility of highly (H7N1) and low (H7N9) pathogenic avian influenza virus infection in red-legged partridge (Alectoris rufa)

ABSTRACT: An experimental infection with highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) was carried out in red-legged partridges (Alectoris rufa) in order to study clinical signs, gross and microscopic lesions, and viral distribution in tissues and viral shedding. Birds were infected with a HPAIV subtype H7N1 (A/Chicken/Italy/5093/1999) and a LPAIV subtype H7N9 (A/Anas crecca/Spain/1460/2008). Uninoculated birds were included as contacts in both groups. In HPAIV infected birds, the first clinical signs were observed at 3 dpi, and mortality started at 4 dpi, reaching 100% at 8 dpi. The presence of viral antigen in tissues and viral shedding were confirmed by immunohistochemistry and quantitative real time RT-PCR (qRRT-PCR), respectively, in all birds infected with HPAIV. However, neither clinical signs nor histopathological findings were observed in LPAIV infected partridges. In addition, only short-term viral shedding together with seroconversion was detected in some LPAIV inoculated animals. The present study demonstrates that the red-legged partridge is highly susceptible to the H7N1 HPAIV strain, causing severe disease, mortality and abundant viral shedding and thus contributing to the spread of a potential local outbreak of this virus. In contrast, our results concerning H7N9 LPAIV suggest that the red-legged partridge is not a reservoir species for this virus.     

Usefulness of estimated surface area of damaged skin as a proxy of mite load in the monitoring of sarcoptic mange in free-ranging populations of Iberian wild goat, Capra pyrenaica

In this study we examined the influence of several factors, like the surface of skin with lesions attributable to Sarcoptes scabiei, the time of year and host sex and age on variations of mite load in Iberian wild goats, Capra pyrenaica, from the Sierra Nevada Natural Space, southern Spain. As well, the surface area of skin with scabietic lesions estimated during field surveys were compared with measurements taken in the laboratory and the potential for using categories based on surface areas estimated during monitoring and management programs was analyzed. The surface area of the skin lesions measured in the laboratory and the time of year were the main factors affecting mite load. Results also revealed discrepancies between the surface area of lesions estimated in the field and those measured in the laboratory. The highest error percentages were associated with lesions ranging between 25 and 75% of host skin surface area. By reducing the number of categories of mange infestation (from 5 to 3) the proportion of cases correctly classified using data from field surveys increases. Our analyses suggest that the estimated surface areas of scabietic lesions is a useful relative index of mite abundance and/or intensity of parasitation in Iberian wild goat. Therefore, we conclude that revised classifications should be used in the monitoring of sarcoptic mange in wild populations, since data can be obtained in an inexpensive, fast and non-invasive fashion.     

In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1 h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at −1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.     

Viability and nematophagous activity of the freeze-dried fungus Arthrobotrys robusta against Ancylostoma spp. infective larvae in dogs

Viability and in vitro and in vivo activities of freeze-dried conidia of the predatory fungus Arthrobotrys robusta (I-31) were evaluated against infective larvae (L(3)) of Ancylostoma spp. in dogs. A. robusta conidia were lyophilized and stored at 4°C for a month. Freeze-dried conidia were diluted to 1×10(3)conidia/ml and tested in vivo. The treated group consisted of a solution containing conidia (1ml) and 1000 Ancylostoma spp. (L(3)) placed on Petri dishes plated with 2% water-agar (2% WA), at 25°C, in the dark for 10 days. The control group consisted of 1000 Ancylostoma spp. L(3), plated on 2% WA. After 10 days, Ancylostoma spp. L(3) from both the treated and the control groups were recovered and counted. The in vivo test was performed on two dogs by administering a single oral dose of freeze-dried conidia (1.5×10(5)) in aqueous solution to one animal and only water to the other. Fecal samples were collected at 12, 24 and 48h after the treatments, plated 2% WA plates and incubated at 25°C for 15 days. A thousand Ancylostoma spp. L(3) larvae were spread on these plates. At day 15, infective L(3) recovered from the treated and control groups were counted. In the in vitro test, A. robusta was able to survive the freeze-drying process, grow in the plates, form traps and capture Ancylostoma spp. L(3). There was a 75.38% decrease in the number of infective larvae recovered from the treated group. The in vivo test showed that freeze-dried A. robusta conidia survived the passage through the gastrointestinal tract of the treated dog, was able to grow in the plates and capture Ancylostoma spp. L(3), reducing the number of recovered L(3) (p<0.01). Freeze-drying can be an alternative method for conservation of conidia of nematophagous fungi.     

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.     

Ambulatory esophageal pHmetry in healthy dogs with and without the influence of general anesthesia

This research aimed to determine the value of esophageal pH in awake and anesthetized dogs, to evaluate the esophageal pH value in awake dogs, in different body positions, as well as to study the occurrence of gastroesophageal reflux episodes in these positions. Thus, 40 healthy male and female adult dogs with mean body weight of 15.5 ± 4.6 kg were used. Esophageal pHmetry was conducted by inserting a catheter through the oropharynx in 30 dogs (stage 1) anesthetized with acepromazine, propofol and isoflurane, submitted to elective ovariosalpingohysterectomy. In addition, 8-h esophageal pHmetry was carried out transnasally in 10 awake dogs (stage 2), allowed to move and change body positions (lateral and sternal decubitus, and standing position), which were recorded. The mean esophageal pH value was lower (p < 0.01) in the anesthetized dogs (7.3 ± 0.82) than in the awake dogs (8.2 ± 0.3). Only four anesthetized dogs (13.33%) suffered reflux episodes. Reflux was not observed in the awake dogs and no esophageal pH differences were found between the body positions studied. Compared to the alert state, general anesthesia in dogs submitted to the previously mentioned anesthesia protocol causes esophageal pH reduction and predisposes to the occurrence of gastroesophageal reflux episodes. Transnasal pHmetry of 8 h in healthy awake dogs reveals that the esophageal pH value is alkaline and does not vary according to body position. In these animals, decubitus position is not a determining factor for reflux episodes to occur.     

Negative effect of the arthropod parasite, Sarcoptes scabiei, on testes mass in Iberian ibex, Capra pyrenaica

Testes mass is a key factor in male reproductive success and is potentially exposed to so-called 'parasitic castration'. This is the result of the direct destruction or alteration of reproductive cell lineages (parasitic castration sensu stricto), or the indirect detrimental effects - for example, via body condition - on the ability of progenitors to produce or rear offspring (parasitic castration sensu lato). There are enormous gaps in our knowledge on the effects of parasites on the testes of wild mammals and in an attempt to rectify this dearth of data we examined the relationship between the skin parasite Sarcoptes scabiei and testes mass in Iberian ibex Capra pyrenaica. We considered data from 222 males that were culled in the population from the Sierra Nevada in Spain. Our results provide evidence that sarcoptic mange is associated with reduced size-corrected testes mass in Iberian ibex which supports the hypothesis that parasitism is a determining factor in gonad plasticity in male mammals. We discuss several hypothetical causes of this relationship and highlight the need to deepen the sub-lethal effects of pathogens if we are to accurately understand their modulator effects on host population dynamics.     

Paenibacillus larvae enolase as a virulence factor in honeybee larvae infection

Paenibacillus larvae is a gram-positive spore-forming bacteria, causative agent of American Foulbrood (AFB), a severe disease affecting larvae of the honeybee Apis mellifera. In an attempt to detect potential virulence factors secreted by P. larvae, we identified an enolase among different secreted proteins. Although this protein is a cytosolic enzyme involved in glycolytic pathways, it has been related to virulence. The aim of the present work was to evaluate its role during the infection of honeybee larvae. Toxicity assays showed that enolase was highly toxic and immunogenic to honeybee larvae. Its production was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. P. larvae enolase production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyze milk proteins as described for P. larvae, suggesting that could be involved in larval degradation, maybe through the plasmin(ogen) system. These results suggest that P. larvae enolase may have a role in virulence and could contribute to a general insight about insect-pathogen interaction mechanisms.     

Comparative analysis of destruction of the infective forms of Trichuris trichiura and Haemonchus contortus by nematophagous fungi Pochonia chlamydosporia; Duddingtonia flagrans and Monacrosporium thaumasium by scanning electron microscopy

The present study aimed to demonstrate by scanning electron microscopy (SEM) the in vitro predatory activity of nematophagous fungi Pochonia chlamydosporia (VC1 and VC4 isolates) Duddingtonia flagrans (AC001 isolate) and Monacrosporium thaumasium (NF34a isolate) on eggs of Trichuris trichiura and infective larvae (L3) of Haemonchus contortus. The work was divided into two experimental tests (A and B). In tests A and B, the predatory activity of nematophagous fungi P. chlamydosporia, D. flagrans and M. thaumasium on eggs of T. trichiura and H. contortus L3 was observed. After 6 h, in test A, isolates P. chlamydosporia (VC1 and VC4) had a role in destroying eggs of T. trichiura. For fungi D. flagrans and M. thaumasium the ovicidal activity on T. trichiura eggs was not observed. Test B showed that D. flagrans (AC001) and M. thaumasium (NF34a) were capable of predating H. contortus L3, but no predation by the fungus P. chlamydosporia was seen. These fungi can offer potential for the biological control of nematodes.     

Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.