A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

Retroperitoneal perirenal pseudocyst in a Massese breed ram.

The macroscopic and microscopic features of a retroperitoneal perirenal pseudocyst in a 12-month-old ram without impairment of renal function are described. In humans and animals, uriniferous pseudocysts may be of traumatic origin, resulting from rupture of kidney, renal pelvis, or ureter, or congenital. Lymphatic pseudocysts may develop secondary to inflammatory obstruction of the hilar lymphatics after perinephritis or renal transplantation. In this case, histologic characteristics of the pseudocyst wall were suggestive of development from the parietal peritoneal layer encapsulating the kidney. This is the first case of retroperitoneal perirenal pseudocyst in a sheep.     

A follow-up of Beagle dogs intradermally infected with Leishmania chagasi in the presence or absence of sand fly saliva

In this study, we compare the development of infection and/or disease in Beagle dogs intradermally infected with Leishmania chagasi, in the presence or absence of Lutzomyia longipalpis saliva, with those of intravenously infected animals.Spleen samples of all the animals inoculated with parasites had positive polymerase chain reaction tests for Leishmania DNA. Positive spleen cultures for Leishmania were detected earlier (P < or = 0.018) and were more frequent (five out of the five animals) in intravenously infected animals than in the intradermally infected animals, in presence (two out of the six animals) or absence (three out of the five animals) of salivary gland lysate of L. longipalpis. Significant increase in serum antibodies against Leishmania was observed only in the intravenously infected group (P = 0.004). In addition, dogs with infection confirmed by isolation of amastigotes or detection of parasite DNA were, nevertheless, negative for anti-Leishmania antibodies up to 5 months or more after infection. Only animals of the intravenously infected group developed progressive decreases in hematocrit (Pearson r = -0.8076, P = -0.0026) and hemoglobin (Pearson r = -0.8403, P = 0.0012) during the infection period. No significant difference in the course of infection was observed between groups of intradermally infected animals. The data presented herein confirms that the intradermal inoculation of dogs with Leishmania produces an asymptomatic form of infection. It also fails to show an advantage in using L. longipalpis saliva as an infection-enhancing agent in experimental canine leishmaniasis.     

Serological response of guinea pigs to oily and aqueous inactivated vaccines containing a Brazilian isolate of the Bovine Viral Diarrhea Virus (BVDV)

Bovine Viral Diarrhea Virus (BVDV) is widespread in cattle in Brazil and research shows its large antigenic variability. Available vaccines are produced with virus strains isolated in other countries and may not be effective. In this study, inactivated vaccines containing the Brazilian BVDV-Ib IBSP11 isolate were developed and tested on 6 groups of 10 guinea pigs (Cavia porcellus). Animals in groups A and C received an aqueous vaccine (aluminum hydroxide); B and D groups received an oily vaccine (Montanide ISA50); Group E positive-control animals were given an imported commercial vaccine with BVDV-Ia Singer; Group F animals were sham vaccinated (negative control). Groups A, B and E received two doses, and Groups C and D, three, every 21 days. Twelve blood samples were taken, at 21-day intervals over 231 days, and evaluated for antibody titer through virus-neutralization (VN), using a homologous strain (IBSP11), and a heterologous strain (BVDV-Ia NADL). Most animals, 42 days following the first dose, seroconverted to both strains and, after the second dose, there was a significant increase of titers in all groups. The oily formulation induced greater response after the third administration. This increase was not observed with the aqueous vaccines, regardless of the virus used in the VN. Antibody decline was more rapid in animals that received aqueous vaccines. The results showed the importance of studying the influence of endemic strains of commercial vaccines, to improve the efficacy of BVD vaccination. Use of the endemic strain in vaccine formulation presented promising results, as well as the use of guinea pigs as a laboratory model.     

Phage Therapy and Photodynamic Therapy: Low Environmental Impact Approaches to Inactivate Microorganisms in Fish Farming Plants

Owing to the increasing importance of aquaculture to compensate for the progressive worldwide reduction of natural fish and to the fact that several fish farming plants often suffer from heavy financial losses due to the development of infections caused by microbial pathogens, including multidrug resistant bacteria, more environmentally-friendly strategies to control fish infections are urgently needed to make the aquaculture industry more sustainable. The aim of this review is to briefly present the typical fish farming diseases and their threats and discuss the present state of chemotherapy to inactivate microorganisms in fish farming plants as well as to examine the new environmentally friendly approaches to control fish infection namely phage therapy and photodynamic antimicrobial therapy.     

Cloning and expression of chicken anemia virus VP3 protein in Escherichia coli

Purification of chicken anemia virus (CAV) VP3 protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. CAV particle was obtained from infected liver of chicken and DNA was extracted. The VP3 protein gene was amplified from the extracted DNA by polymerase chain reaction (PCR) and cloned. The recombinant expression construct (pTrc-VP3) was identified by PCR and sequencing analysis. Expression of VP3 protein with a molecular mass of approximately 21kDa was confirmed by Western blotting analysis with CAV-specific antibodies. The in vitro expressed VP3 protein was purified to near homogeneity by elution from the gel, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP3 protein was recognized by CAV antibodies in a Western blotting assay. This finding indicates that recombinant VP3 expressed in the pTrcHis2 vector system can be used as antigen to detect anti-CAV antibodies.     

Recreational fisheries in Portofino Marine Protected Area,Italy: Some implications for the management

Since the 1970s, recreational fishing has become a mass hobby in Italy, reaching a large number of people, who, using modern equipment, increased their harvesting capacity, provoking serious conflicts with the professional fisheries. Recreational fishing is strictly regulated inside Italian Marine Protected Areas (MPAs) and is generally allocated to local residents, mainly to reduce the tensions caused by limitations of access to the resources. The aim of this study was to provide an analysis of recreational fishing activities within the Portofino MPA (Mediterranean Sea), to assess the possible impact on the local fish stocks and to plan potential management actions. Furthermore, some serious inconsistencies on the minimum lengths of fish caught as defined by law relative to the minimum size of first reproduction are discussed. Since 2014, all anglers exploiting the Portofino MPA have been obliged to fill out a logbook. The compilation of these was not completely satisfactory, when compared with a group of reliable anglers whose catches were accurately monitored, but allowed for estimated yields of about 24 kg/angler/yr. After 15 years of protection, the fish biomass has increased within the Portofino MPA, generating a positive spillover effect. At the same time, it has attracted many anglers whose gross harvesting was estimated at about 8 t/year, representing about the 8% of the total yield of the local small‐scale fishery.     

Weak effects of habitat type on susceptibility to invasive freshwater species: an Italian case study

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Genome sequencing analysis of Brazilian chicken anemia virus isolates that lack MSB-1 cell culture tropism

Specific amino acid (aa) substitutions in VP1, VP2 and VP3 genes were reported as a distinctive feature of the American CIA-1 strain, characterized as having a variable rate of growth and tropism for different MSB-1 cell sublines [Renshaw RW, Soiné C, Weinkle T, O'Connell PH, Ohashi K, Watson S, et al. A hypervariable region in VP1 of chicken anemia virus mediates rate of spread and cell tropism in tissue culture. J Virol 1996;70(12):8872-8]. DNA sequencing of 878 nucleotides from twelve Brazilian CAV, eight of which tested for in vitro isolation in three different sources of MDCC-MSB1 cell line and identified as lacking capacity to propagate in any of these cells, were compared to sequence data available for CAV strains propagated or not in cell culture. Alignment of the deduced aa resulted in a lack of singled out amino acid substitutions in the partial genomic sequences of Brazilian isolates that would entirely contrast them to viruses propagated in MSB-1 cells, indicating that the combined VP1, VP2 and VP3 substitutions observed may not entirely account as sole determinants of CAV isolation and propagation in MDCC-MSB-1 cells.