Francisco Carmelo Almeida Rodríguez

Detection of African swine fever virus protein VP73 in tissues of experimentally and naturally infected pigs

African swine fever (ASF) is caused by a DNA virus that targets cells of the mononuclear phagocyte system (MPS). ASF virus (ASFV) antigens have been detected in tissue sections using purified polyclonal sera from hyperimmune pigs by immunofluorescence or immunoperoxidase techniques. These investigations have been performed to study the pathogenesis of the disease. In this respect, the use of immunoperoxidase techniques on paraffin-embedded tissue samples represents a considerable step forward in the study of the disease because it facilitates the identification of different cell populations and links the presence of viral antigen with cell and tissue lesions. However, monoclonal antibodies (MAbs) have not been used for the detection of ASFVspecific proteins in immunohistologic studies. The purpose of the present investigation was to study the distribution of ASFV protein VP73 in tissue sections of different organs from pigs experimentally infected with the moderately virulent ASFV isolate E75 and in naturally infected pigs with characteristic lesions of acute, subacute, and chronic ASF by both avidin-biotin-peroxidase and alkaline phosphatase-conjugated biotin-streptavidin immunohistochemical techniques using an MAb. VP73 distribution was also studied in asymptomatic seropositive pigs. Three 3-month-old miniature pigs weighing 12 kg each and free from parasitic and infectious diseases at the beginning of the study were inoculated intramuscularly with 10 50% hemadsorbing doses of the moderately virulent and hemadsorbent ASFV E75 isolate. Two miniature pigs were used as negative controls. Test animals were euthanized by exsanguination after azaperone and thiobarbital treatment at 10 and 12 days postinoculation (dpi). One of the pigs scheduled for euthanasia at 8 dpi was found dead at 7 dpi. In addition, 27 naturally infected Iberian pigs from southwestern Spain that had anti-ASFV antibodies detected in serum by an indirect enzyme-linked immunosorbent assay ELISA were also used. These pigs were euthanized by the Agricultural Ministry of Spain and the Junta of Andalucia between 1985 and 1991. Six Large White × Landrace pigs, which served as controls in a previous experience, were employed as negative controls in this study. Tissue samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin wax, and stained with hematoxylin and eosin. For the immunohistochemical analysis, 3-μm-thick tissue sections were predigested with protease

A pathological and immunohistochemical study of caprine pleuropneumonia induced by subspecies of Mycoplasma mycoides

In a population of 700 goats, 150 died; of these, 29 were necropsied. Ten of the 29 goats had pleuropneumonia. Mycoplasma mycoides subsp. mycoides (Large Colony) (MmmLC), Mycoplasma mycoides subsp. capri (Mmc), and Pasteurella multocida were isolated from five of the pleuropneumonic goats. Gross and microscopical lesions were typical of caprine pleuropneumonia (CPP), with bronchopneumonia, fibrinopurulent or fibrinonecrotic pleuropneumonia and dilatation of the interlobular septa and pleura. Immunohistochemical examination with antisera against MmmLC and Mmc showed mycoplasma antigens in all 10 goats with CPP. In all cases, both MmmLC and Mmc antigens were detected together. Mycoplasma antigens were present in the lumina of the airways and alveoli, mainly inside the cytoplasm of neutrophils and macrophages, but extracellular antigen was demonstrated in areas of necrosis. Pasteurella antigens were detected in four of the 10 animals with CPP. From the histological, immunohistochemical and microbiological results it was concluded that the two mycoplasmas, acting together, caused the pleuropneumonia, with P. multocida playing a subsidiary synergistic role.

INTESTINAL CRYPTOSPORIDIOSIS IN PIGEONS (COLUMBA LIVIA)

An intestinal disease in pigeons (Columba livia) from the Canary Islands characterized by diarrhea and body weight loss is described. Intestinal cryptosporidiosis was identified in three young pigeons. Cryptosporidia were associated with hyperplasia of the intestinal crypts and moderate inflammatory infiltration in lamina propria. This is the first report of cryptosporidiosis in pigeons.

On the Use of Path Relinking for the ρ – Hub Median Problem

The ρ – hub median problem is an NP hard location – allocation problem, which consists of finding p points to establish facilities and the assignment of the users to these points. A new evolutionary approach that has been very effective for solving optimisation problems is Path Relinking, an extension of Scatter Search that links solutions over neighborhood spaces. Path Relinking gives a framework for combining solutions that goes beyond the crossover mechanism of genetic algorithms, and introduces new fundamental principles, such as the use of systematic strategies instead of random strategies. In this paper, we present an application of Path Relinking to solve the p – hub median problem and compare its effectiveness with other classical techniques. This procedure provides high quality solutions in reasonable execution times and yields a significant improvement in the size of the problems that can be solved.

Debilitating cutaneous poxvirus infection in a Hodgson's grandala (Grandala coelicolor).

A case of cutaneous avian pox infection in a Hodgsons grandala (Grandala coelicolor) is described. The bird was emaciated and had nodules on the eyelids, bill, neck, legs, and toes. Eosinophilic intracytoplasmic inclusion bodies were visualized by light microscopy in epithelial cells of the cutaneous nodules. Electron microscopy revealed numerous pox virions in the inclusion bodies. This is the first report of cutaneous poxvirus infection in a Hodgsons grandala.

Immunohistopathological study of African swine fever (strain E-75)-infected bone marrow

A study was made of the action of African swine fever virus (ASFV) on the bone marrow of 12 miniature pigs inoculated intramuscularly with the moderately virulent ASFV isolate E75 and killed 2 to 12 days after infection. A sequential description is provided of the histological lesions of the bone marrow in the experimental animals, which developed haemorrhagic lesions from 6 days after inoculation onwards. Immunohistochemical techniques were used to demonstrate the viral protein VP73 and immunoglobulins (IgG and IgM) in formalin-fixed and paraffin wax-embedded samples of bone marrow tissue. The immunohistological results, platelet counts, viraemia, and anti-ASFV immunoglobulin titres all indicated that thrombocytopoiesis impairment by direct viral action plays a role in the progressive thrombocytopenia characteristic of infection by moderately virulent ASFV isolates.