Antonio Rodríguez-Bertos

Canine inflammatory mammary carcinoma: histopathology, immunohistochemistry and clinical implications of 21 cases.

Human inflammatory breast carcinoma (IBC) is the most malignant type of breast cancer with an extremely poor prognosis. The dog is the unique animal species in which spontaneous inflammatory mammary carcinoma (IC) has been reported, although it is not well documented. The purpose of this study was to characterize histopathologically and immunohistochemically the canine IC, considering associated clinical features. Twenty-one dogs diagnosed with IC and with known clinical and necropsy data were included in the study. Tissue samples from necropsies underwent a histopathological review and an immunohistochemical study (Ki-67, estrogen receptor (ER), progesterone receptor (PR), and P53 tumor suppressor protein). The histological study revealed several types of carcinomas (solid, tubular, papillary, and adenosquamous) and three lipid-rich carcinomas. All tumors were ER negative. Two histological patterns of neoplastic dermal infiltration were observed: tubular/papillary and sarcomatous-like. Dermal sarcomatous-like infiltration was significantly related to previous treatments with progestagens (p=0.006), primary type of IC (p=0.03), extreme local pain (p=0.02), reduced observation of emboli in dermal lymphatic vessels (p=0.01), and increased expression of p53 (p=0.001). PR expression was significantly higher in secondary post-surgical IC (p=0.04). The absence of PR was related to the existence of pulmonary metastases at necropsy (p=0.04). Canine primary IC is the most aggressive form of this disease with distinct histopathological and immunohistochemical characteristics. Progestins and endocrine-related mechanisms seem to be involved in canine IC development. Canine IC could serve as a spontaneous model for human IBC, particularly in studies concerned with new therapeutics approaches.

Experimental infection with a low virulence isolate of Neospora caninum at 70 days gestation in cattle did not result in foetopathy

The Nc-Spain 1H isolate of Neospora caninum, which was newly obtained from the brain of a congenitally asymptomatic infected calf, demonstrated a reduced in vitro tachyzoite yield and viability rate, as well as low virulence in mouse models. The objective of the present study was to determine the ability of this isolate to induce foetal death in a pregnant bovine model. For this purpose, 13 naïve pregnant heifers were divided into three groups and were experimentally challenged with either 107 tachyzoites of Nc-1 (group 1, n = 5), Nc-Spain 1H (group 2, n = 5) isolates or phosphate-buffered saline (group 3, n = 3) intravenously at 70 days of gestation. After inoculation, pregnancy was monitored and dams were sacrificed when foetal death was detected. The remaining animals were slaughtered at 45 days post-infection. Maternal and foetal samples were collected for examination by histology and parasite DNA detection. Parasitaemia, specific anti-N. caninum IgG and interferon γ responses were also studied. At 3–4 weeks after infection, foetal death was detected in 3 out of 5 Nc-1-infected dams. However, no evidence of foetal death was observed in either Nc-Spain 1H-infected or control groups during the period studied. The most severe histopathological lesions were observed in the placenta and foetal organs from Nc-1-infected cattle that exhibited foetal death. It was in these animals that N. caninum DNA was more frequently detected. Parasitaemia was observed in all Nc-1-infected dams and in only 3 out of 5 Nc-Spain 1H-infected animals. The magnitude of the immune response was significantly higher in the Nc-1-inoculated group than in the group inoculated with the Nc-Spain 1H isolate. These data reveal the reduced virulence of the Nc-Spain 1H isolate in cattle.

Influence of Neospora caninum infection in BALB/c mice during pregnancy in post-natal development.

The influence of Neospora caninum infection during pregnancy on the post-natal period has been poorly investigated. In a previous study, we suggested that infection with N. caninum during pregnancy could affect the normal post-natal development of the offspring. For this reason, in the present work we evaluated the influence of N. caninum infection in pregnant BALB/c mice at days 0, 7 and 14 of gestation (groups A, B and C, respectively) on the post-natal development of the offspring from birth to day 60 post-partum (PP). Morbidity and mortality, vertical transmission, and histopathological lesions were investigated. The humoral immune response (IgG) of pups was also evaluated. Results showed that infection with N. caninum during pregnancy had fatal consequences for pups, especially during mid-gestation (day 7). Infection provoked a delay in the general development of neonates, clinical signs compatible with neosporosis and severe histopathological lesions. A high mortality rate was found in all infected groups. A 69% of mortality rate was found in group A, a 100% in group B and a 46% in group C. Necrotizing encephalitis and multifocal hepatocellular necrosis were the most severe lesions found. All neonates, except four animals from group C, had antibodies against N. caninum but the immune response was not sufficient to control parasite infection. We have demonstrated that extension of the observation period after N. caninum infection permits a more accurate study of vertical transmission, the major route of parasite transmission, and mortality rates. We propose that infection at mid-gestation (day 7) in BALB/c mice and its study during the post-natal period constitutes a valuable experimental model for testing new chemotherapeutic agents and vaccines designed to protect against congenital neosporosis, in order to select effective protocols before its use on bovine.

Influence of adjuvant and antigen dose on protection induced by an inactivated whole vaccine against Neospora caninum infection in mice

In this study, the protection afforded by a Neospora caninum inactivated vaccine formulated with three different adjuvants (water-in-oil emulsion, aluminum hydroxide with CpG oligodeoxynucleotides and aluminum hydroxide with ginseng extract) and three different parasite doses (10(5), 5 × 10(5) or 10(6) inactivated whole tachyzoites) was evaluated using a mouse model. Mice were immunized subcutaneously twice at three-week intervals with inactivated Nc-Spain 1H tachyzoites and challenged by intraperitoneal inoculation with 10(6) live Nc-1 tachyzoites. The efficacy of the immunization was evaluated in non-pregnant BALB/c mice on days 1 and 5 (acute infection phase) and days 14 and 30 (chronic infection phase) post-challenge. The results showed the ability of water-in-oil emulsion combined with inactivated 5 × 10(5) tachyzoites to induce protection against neosporosis during the chronic stage, limiting parasite multiplication in the brain. Aluminum hydroxide-ginseng extract and inactivated tachyzoites reduced the number of parasites circulating in the blood during acute phase but failed to limit the establishment of chronic infection. On the other hand, a dose-effect was observed in groups vaccinated with aluminum hydroxide-ginseng extract in which the lesion severity increased as the inactivated tachyzoite dose. This study demonstrates that efficacy can significantly vary depending on the adjuvant, the dose of antigen and the phase of N. caninum infection in which the vaccine is tested.

Evidence of Leishmania infantum Infection in Rabbits (Oryctolagus cuniculus) in a Natural Area in Madrid, Spain

Leishmaniasis is one of the most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.

Evaluation of the performance of cellular and serological diagnostic tests for the diagnosis of tuberculosis in an alpaca (Vicugna pacos) herd naturally infected with Mycobacterium bovis

Tuberculosis (TB) in llamas and alpacas has gained importance in recent years since they are imported into the European Union mainly for serving as pets and for production of natural fibre. The intradermal tuberculin test has been widely used for diagnosis of TB in these species showing lack of sensitivity (Se) although little information has been previously reported evaluating the effect on its performance of different PPD inoculation sites and time of readings. Moreover, different cost-effective serological assays have been developed in the recent years for TB diagnosis in camelids obtaining a variety of results and, for this reason, new assays still being developed. The main objectives of this study were: (1) to evaluate the performance of the intradermal tuberculin test using different inoculation sites (axillary, prescapular and cervical) and times of reading (72 and 120 h) and (2) to test a novel serological assay based on MPB83 antigen in a Mycobacterium bovis naturally infected alpaca herd in Spain. In regards to skin test, single intradermal tuberculin (SIT) test at the prescapular site and reading at 72 h showed the highest proportion of test-positive-culture positive animals among all culture positive animals (T+/C+), ranging from 53.8% (95% CI, 37.2-69.9) to 80% (95% CI, 44.4-97.5) using a more stringent interpretation than typically prescribed although, in general, low T+/C+ was achieved using both SIT and single comparative intradermal tuberculin (SCIT) tests alone. T+/C+ of the serological assay increased using samples collected 15-30 days after PPD injection [76.9% (95% CI, 60.7-88.9) - 100% (95% CI, 69.2-100)]. The best results of T+/C+ were obtained applying in parallel the most sensitive SIT test and serology using samples collected 15-30 days after PPD inoculation [90% (95% CI, 55.5-99.7)-100% (95% CI, 69.2-100)]. Therefore implementation of serology in parallel with the most sensitive skin test could maximize the detection of infected animals.

Skin and subcutaneous mycoses in tilapia (Oreochromis niloticus) caused by Fusarium oxysporum in coinfection with Aeromonas hydrophila

Subcutaneous mycoses in freshwater fish are rare infections usually caused by oomycetes of the genus Saprolegnia and some filamentous fungi. To date, Fusarium infections in farmed fish have only been described in marine fish. Here, we report the presence of Fusarium oxysporum in subcutaneous lesions of Nile tilapia (Oreochromis niloticus). Histopathologic evaluation revealed granuloma formation with fungal structures, and the identity of the etiological agent was demonstrated by morphological and molecular analyses. Some of the animals died as a result of systemic coinfection with Aeromonashydrophila

Experimental Lactococcus garvieae infection in zebrafish and first evidence of its ability to invade non-phagocytic cells.

Zebrafish has been used for studying infections and host-pathogen interactions in different bacterial fish pathogens. In the present study we evaluated the ability of Lactococcus garvieae to infect zebrafish when inoculated intraperitoneally with 2 × 10(7)UFC of this pathogen. L. garvieae can colonize and invade zebrafish at multiple anatomical sites causing a lethal acute septicemic infection with clinical signs and lesions consistent with those observed in lactococcosis outbreaks. Immunohistochemical studies showed the presence of L. garvieae into macrophages as well as into non-phagocytic zebrafish cells of liver (hepatocytes). The internalization capacity showed by L. garvieae in zebrafish cells was confirmed in the rainbow trout cell line RTG-2. Our results provide the first evidence that L. garvieae is able to invade non-phagocytic host cells.

Application of a specific quantitative real-time PCR (qPCR) to identify Leishmania infantum DNA in spleen, skin and hair samples of wild Leporidae

The aim of this study was to compare a quantitative real-time PCR (qPCR) validated for the detection of Leishmania infantum in dogs with a nested PCR but in wild Leporidae. Additionally, L. infantum results from indirect immunofluorescent antibody test (IFAT) and in vitro culture were also compared with qPCR. Different samples (spleen, skin and hair) recovered from 224 European rabbits and 70 Iberian hares from two green areas of Madrid Council were analysed for the detection of L. infantum. The presence of Leishmania kDNA was detected by qPCR in 58 out of 221 (26.24%), 162 out of 203 (79.8%) and 22 out of 33 (66.67%) analysed rabbits on spleen, skin and hair samples, respectively; and in 7 out of 69 (10.14%), 39 out of 70 (55.71%) and 17 out of 32 (53.13%) test hares on spleen, skin and hair samples, respectively. The qPCR in all test samples resulted to be more sensitive than nested PCR, with a limit of detection of 1.43 fg/reaction (0.039 parasites) for L. infantum genomic DNA. Additionally, the percentage of positive animals detected by qPCR in at least two out of three samples (n=221 rabbits and 70 hares) tested was higher than those detected by IFAT (n=190 rabbits and 61 hares) and isolation (n=75 rabbits and 20 hares). The highest level of agreement was obtained by nested PCR on spleen/skin (89%/83%) samples and qPCR on spleen samples (81%), followed by IFAT (48%) and qPCR on skin (32%) samples. Our results demonstrate this qPCR is a suitable method for detecting L. infantum DNA in different samples suggesting hair could be considered an adequate sample for direct, reliable and non-invasive diagnosis of L. infantum in wild animals.

LPS and inactivated Propionibacterium acnes elicit a partially protective response in primary infections of Heligmosomoides polygyrus.

Intestinal helminth infections are common and of paramount economic importance in domestic animals. Available chemotherapy is limited and anthelmintic resistance is widespread in some hosts. This scenario favors the exploration of alternative methods of control, among them immune modulators. The effect of Escherichia coli LPS+Propionibacterium acnes on a primary infection of Heligmosomoides polygyrus (Trichostongyloidea) in mice has been tested. Nematode infection induced a rise of specific IgG1, both serum and intestinal, and a significant reduction in the unspecific (ConA) lymphoproliferative response. Treatment with the immune modulator (days -2, 0, 7 and 14 post infection) elicited an apparent delay of larval intramucosal development. Moreover cumulative nematode egg shedding in treated mice was significantly lower (p=0.0041). Preliminary results point toward the interest of immune modulators to control intestinal helminths.