Digital image analysis workflows for evaluation of cell behavior and tumor microenvironment to aid therapeutic assessment in high-risk neuroblastoma.

Digital pathology and artificial intelligence are promising emerging tools in precision oncology as they provide more robust and reproducible analysis of histologic, morphologic and topologic characteristics of tumor cells and the surrounding microenvironment. This study aims to develop digital image analysis workflows for therapeutic assessment in preclinical in vivo models. For this purpose, we generated pipelines that enable automatic detection and quantification of vitronectin and αvß3 in heterotopic high-risk neuroblastoma xenografts, demonstrating that digital analysis workflows can be used to provide robust detection of vitronectin secretion and αvß3 expression by malignant neuroblasts and to evaluate the possibility of combining traditional chemotherapy (etoposide) with extracellular matrix-targeted therapies (cilengitide). Digital image analysis added evidence for the relevance of territorial vitronectin as a therapeutic target in neuroblastoma, since its expression is modified after treatment, with a mean percentage of 60.44% in combined therapy tumors vs 45.08% in control ones. In addition, the present study revealed the efficacy of cilengitide for reducing αvß3 expression, with a mean αvß3 positivity of 34.17% in cilengitide treated material vs 66.14% in control and with less tumor growth when combined with etoposide, with a final mean volume of 0.04 cm3 in combined therapy vs 1.45 cm3 in control. The results of this work highlight the importance of extracellular matrix-focused therapies in preclinical studies to improve therapeutic assessment for high-risk neuroblastoma patients.

A novel time-saving multiplex PCR assay for detecting and discriminating the most common canine Babesia species in Europe.

In Europe, most cases of canine babesiosis are caused by Babesia canis, Babesia vogeli (large piroplasms) and Babesia vulpes (small piroplasm). Molecular diagnosis is recommended due to its high sensitivity. Species identification after sequencing allows applying a rapid and efficient treatment, leading to a better prognosis; however, it is expensive and time-consuming. Thus, the objective of the present study was to develop a time-saving multiplex polymerase chain reaction (PCR) for simultaneously detecting and discriminating between large and small forms without sequence analysis. A new multiplex PCR was designed and tested using blood samples from 79 dogs showing clinical signs compatible with babesiosis which were previously analysed using blood smears and molecular methods. Multiplex PCR successfully discriminated between both Babesia groups showing bands of 700 and 890 bp for B. canis/B. vogeli and B. vulpes, respectively. No significant differences in the results of both PCR were detected and a substantial agreement between protocols (κ = 0.64) was found. Our multiplex PCR represents a reliable tool for detecting infections by the major Babesia spp. in dogs from Europe. Since no sequence analysis is required for identifying the species involved, this PCR allows the rapid administration of an appropriate treatment, thus improving the survival rate of the infected animals. In addition, it will represent a helpful tool for unravelling the real prevalence and distribution of B. vulpes and its implication in clinical cases.

Tick-borne pathogens in ticks from urban and suburban areas of north-western Spain: Importance of Ixodes frontalis harbouring zoonotic pathogens.

To identify the questing tick populations in urban and suburban areas from the city of Lugo (NW Spain), ticks were collected monthly by flagging. The presence of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum also was determined by polymerase chain reaction (PCR) and sequence analysis. Overall, 342 questing ticks were collected; the tick abundance was higher in suburban (95.9%) than in urban areas (4.1%). Ixodes frontalis was the most abundant (86.5%); 88.5% were larvae, 11.1% nymphs and 0.3% adults. All development stages of I. ricinus (7.3%) and adults of Rhipicephalus sanguineus sensu lato (5.8%) and Dermacentor reticulatus (0.3%) were found. Rickettsia spp. (31.9%) was more prevalent than Borrelia spp. (2.7%); no ticks were positive to A. phagocytophilum. Six Rickettsia species were identified (R. slovaca, R. monacensis, R. massiliae, R. raoultii, R. sibirica subsp. mongolitimonae and R. aeschielmanii); Candidatus Rickettsia rioja and two novel Rickettsia species also were detected. In addition, Borrelia turdi (1.8%) and B. valaisiana (0.9%) were identified in Ixodes ticks. This is the first report of R. slovaca in R. sanguineus s.l. and of R. monacensis, R. raoultii, R. slovaca, R. sibirica subsp. mongolitimonae and Ca. R. rioja in I. frontalis. Since most of the pathogens detected are zoonotic, their presence in these areas may have implications for public health.

Specificity of Stenurus (Metastrongyloidea: Pseudaliidae) infections in odontocetes stranded along the north-west Spanish coast.

Parasites extracted from the lungs and the pterygoid sinus complex of 6 species of odontocetes stranded along the north-west Spanish coast (Northeast Atlantic) between 2009 and 2019 were morphologically identified. The samples belonged to 14 specimens, including 3 harbour porpoises, Phocoena phocoena, 6 short-finned pilot whales, Globicephala macrorhynchus, 1 long-finned pilot whale, Globicephala melas, 1 Risso's dolphin, Grampus griseus, 1 striped dolphin, Stenella coeruleoalba and 2 bottlenose dolphins, Tursiops truncatus. All animals (14/14) were infected by nematodes of the genus Stenurus spp.; moreover, two of them presented a mixed lung nematode infection by Stenurus spp. and Halocercus spp., and another two a mixed infection by Stenurus spp. and the trematode Nasitrema spp. in the pterygoid sinuses. The morphological characterization of the Stenurus specimens revealed the existence of three different species: Stenurus minor, present in the pterygoid sinuses of harbour porpoises with a mean intensity of 43.0 ± 9.0; Stenurus globicephalae, in the pterygoid sinuses of pilot whales and the Risso's dolphin (370.3 ± 579.4); and Stenurus ovatus infecting bottlenose and striped dolphins' lungs (47.7 ± 76.5). This is the first citation of S. minor and S. ovatus in odontoceti from the Galician coast. Nematodes of the genus Stenurus are frequent in odontocetes stranded along the north-west Spanish coast. A clear host-parasite association was observed between S. minor and the Phocoenidae family, between S. globicephalae and the subfamily Globicephalinae and between S. ovatus and subfamily Delphininae. Different trophic position and niche segregation may lead to different patterns of specificity.

Traumatic myiasis by Wohlfahrtia magnifica in sheep flocks from southeastern Spain: prevalence and risk factors.

Wohlfahrtia magnifica (Diptera: Sarcophagidae) is an obligate myiasis-causing fly of livestock that is widely distributed throughout the Mediterranean basin. A total of 73 683 sheep from 122 flocks in Albacete Province (southeastern [SE] Spain) were examined to detect active traumatic myiasis. The influence of several individual and flock factors on the prevalence was also assessed. The overall flock prevalence of traumatic myiasis was 95.9%, with an individual prevalence of 7.1%. Wohlfahrtia magnifica was identified in all of the positive animals except one that was infested by Lucilia sericata (Diptera: Calliphoridae). Adults of both genders showed a higher risk of infestation than juveniles, whereas males of all ages displayed an increased risk of infestation than females of all ages; the existence of a lambing building on the farm, poor husbandry facilities and the location area were also identified as risk factors. Prevalence was highest in the summer, and the fly season lasted from March to November. Genitalia were the main location of infestation in males (94.4%) and females (76.3%). Our results reveal that traumatic myiasis by W. magnifica is widespread in southeastern Spain, hence preventive measures minimizing the attractiveness of sheep to gravid flies are necessary to avoid health and welfare problems and significant production losses in sheep flocks.

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

Cytokine expression in bovine PBMC cultures stimulated with Hypoderma lineatum antigens.

Hypoderma antigens are involved in host inflammation and immune response, conditioning larvae survival. In this study, peripheral blood mononuclear cell (PBMC) cultures from Hypoderma sensitized and unsensitized cattle were performed to determine the effect of H. lineatum antigens and incubation time (18, 24, 48 h) on IFN-γ, TNF-α, IL-10 and IL-4 mRNA gene expression determined by RT-qPCR. TNF-α and IL-4 gene expression were higher in Hypoderma previously sensitized PBMCs, suggesting that a mixed Th1/Th2 response may play a significant role in host defence reactions against Hypoderma exhibited by previously infested cattle. Incubation time had a significant effect on IL-10 and TNF-α gene expression, which decreased over time. Regarding to H. lineatum antigens, the crude larval extract and the purified fraction hypodermin B (HB) produced a significant reduction of the mRNA expression levels of the proinflammatory cytokine, IFN-γ; moreover, the HB had a stimulating effect on the mRNA gene expression of the anti-inflammatory cytokine IL-10, demonstrating that the parasite would modulate the host defence mechanisms by avoiding harmful immune responses that would limit its survival into the host tissues.

Oral rehabilitation with dental implants in patients with recessive dystrophic epidermolysis bullosa: A retrospective study with 2-15 years of follow-up.

BACKGROUND: Epidermolysis bullosa (EB) comprises a group of hereditary disorders characterized by mechanical fragility of the skin and mucous membranes, with the development of blisters and vesicles in response to minimum tissue friction. Recessive dystrophic epidermolysis bullosa (RDEB) with generalized involvement is the most common subtype in the oral cavity. The present study was carried out to investigate dental implant survival, peri-implant tissue condition, patient satisfaction, and the impact of treatment upon the quality of life of patients with RDEB rehabilitated with implants and full-arch implant-supported prostheses. MATERIAL AND METHODS: Thirteen patients with RDEB underwent dental implant treatment between September 2005 and December 2016. A retrospective study was made to analyze implant survival, peri-implant tissue health and patient satisfaction. RESULTS: A total of 80 implants were placed (42 in the maxilla and 38 in the mandible) in 13 patients between 20-52 years of age and diagnosed with RDEB. All the implants were rehabilitated on a deferred basis with 20 full-arch prostheses. Fifteen fixed prostheses and 5 implant-supported overdentures were placed. The implant survival rate was 97.5% after a mean follow-up of 7.5 years after prosthetic loading. Fifty percent of the implants showed mucositis at the time of evaluation. Probing depth was maintained at 1-3 mm in 96.2% of the implants, and bleeding upon probing was observed in 67.5% of the implants. There was a high prevalence of bacterial plaque (85%). CONCLUSIONS: The treatment of edentulous patients with RDEB by means of implants and implant-supported prostheses is predictable as evidenced by the high success rate, and improves patient self-esteem and quality of life.

A tooth whitening and chemical abrasive protocol for the treatment of developmental enamel defects.

This clinical report describes a chemical abrasive protocol and whitening techniques to quantify modifications to the color and volume produced when eliminating white spots associated with developmental defects in enamel. Chemical (oxidative-erosive) abrasive treatment (whitening + 6.6% hydrochloric acid) resolves white spots up to a depth of 0.2 mm. At greater depths, infiltration with 16% hydrochloric acid followed by composite resin infiltration is recommended.

Temporal and spatial spread of Hypoderma actaeon infection in roe deer from peninsular Spain determined by an indirect enzyme-linked immunosorbent assay.

The host switching of Hypoderma actaeon (Diptera: Oestridae), a specific parasite of red deer (Cervus elaphus), towards roe deer (Capreolus capreolus) has been recently reported in Spain. To provide information about the temporal and spatial spreading of H. actaeon infection in roe deer, 244 serum samples from animals hunted in Spain between 2013 and 2018 were analysed by an indirect enzyme-linked immunosorbent assay. The overall seropositivity was 13.9%. Seropositivity was higher in continental (27.7%) and mountainous (12%) areas from central Spain, followed by southern-Mediterranean (11.2%) and northern-oceanic regions (3.5%). Differences were significant between central-continental and northern-oceanic regions (P = 0.003). No differences were found according to the sex and age of roe deer (P > 0.05). In 2013, all seropositive animals were concentrated in two distant areas in central and southern Spain, suggesting that the host switch could have occurred independently in both regions. Changes in the pattern of distribution of red deer and roe deer could have favoured the spreading of this myiasis towards roe deer, indicating that roe deer may become infested by H. actaeon in areas where both cervids coexist at high densities.

Assessment of the capacity of a pyrophosphate-based mouth rinse to inhibit the formation of supragingival dental calculus. A randomized double-blind placebo-controlled clinical trial.

BACKGROUND: This study aimed to analyze the efficacy of an anti-calculus mouth rinse and its possible adverse effects on the mucosa and teeth. MATERIAL AND METHODS: This randomized double-blind placebo-controlled clinical trial included 40 patients with treated and managed periodontal disease, all with a history of rapid calculus formation. Patients used a pyrophosphate-based test mouth rinse (B) or a placebo (A). A range of parameters were measured for: saliva (saliva flow, pH and chemical composition); calculus (Volpe-Manhold [V-M] index, weight, and volume); adverse effects on mucosa and teeth; and the patients' subjective perceptive of mouth rinse efficacy. RESULTS: the test mouth rinse B produced reductions in urea, uric acid, and phosphorous, calcium, saliva flow, and increases in pH. V-M index and calculus weight decreased after using the test mouth rinse. Calculus volume decreased with both mouth rinses. No changes to the mucosa or teeth were observed. Patients perceived that the test mouth rinse was more effective. CONCLUSIONS: The test/B and placebo mouth rinses both modified certain parameters in saliva composition, particularly reductions in urea, uric acid, and phosphorous. Calcium tended to increase after using the test-B mouth rinse. The results did not demonstrate the anticalculus efficacy of the pyrophosphate-based mouth rinse or positive effects on saliva flow or composition. This field requires further research, as no product has been developed that prevents calculus formation completely.

Occurrence of Borrelia and Borreliella species in Ixodes ricinus collected from roe deer in northwestern Spain.

Ixodes ricinus, comprising the predominant tick species in Europe, can transmit important human pathogens, including Borreliella spp., the causal agent of Lyme borreliosis. One hundred and seventy five roe deer hunted in two areas (plateau and mountain) of Galicia (northwest Spain) were examined for the presence of ticks; all roe deer were infested by I. ricinus. Nymphs (n = 1000), males (n = 1449) and females (n = 1000) of I. ricinus were analysed in pools of up to 10 ticks to detect both Borreliella and Borrelia DNA. The average number of I. ricinus per roe deer was similar in both areas, regardless of the life stage; although the percentage of Borreliella and Borrelia positive pools was higher in ticks collected from roe deer hunted in the plateau area, no significant differences were detected. Sequence analysis at the flagellin gene allowed the identification of four Borreliella species (Borreliella afzelii, Borreliella garinii, Borreliella lusitaniae and Borreliella valaisiana) and Borrelia miyamotoi in adult males; only B. valaisiana and B. miyamotoi were detected in nymphs and all females were negative. All Borreliella and Borrelia species found in roe deer were previously identified in questing I. ricinus collected in the same study area, although the prevalence was lower in the present study. The analysis of male I. ricinus ticks collected from roe deer gives a good estimation of Borreliella diversity in questing ticks.

Comparative in vitro study of the accuracy of impression techniques for dental implants: Direct technique with an elastomeric impression material versus intraoral scanner.

BACKGROUND: The aim of this study was to compare a conventional technique (elastomeric impression material - EIM) and a digital technique (scanner digital model - SDM) on a six-analog master model (MM) to determine which was the most exact. MATERIAL AND METHODS: Twenty impressions were taken of a master model (EIM) and twenty scanned impressions (SDM) (True Definition). A coordinate measuring machine (CMM) was used to measure the distances between adjacent analogues (1-2, 2-3, 3-4, 4-5, 5-6), intermittently positioned analogues (1-4, 3-6) and the most distal (1-6). Reference values were established from the master model, which were compared with the two impression techniques. The significance level was established as 5% (p<0.05). RESULTS: The precision of each technique was compared with MM. For adjacent analogues (1-2), no significant differences were found between EIM-MM (p=0,146). For intermittently positioned analogues (1-4), SDM did not show significant differences with MM (p=0.255). For the distance between distal analogues (1-6), significant differences were found between both techniques and MM (p=0.001). CONCLUSIONS: In a clinical situation with < three implants, EIM is more exact than SDM, but in cases of four implants SDM is more exact. For rehabilitations (> four implants), neither technique can be considered accurate although error falls within the tolerance limits established in the literature (30-150µm).

Lesser housefly (Fannia canicularis) as possible mechanical vector for Aleutian mink disease virus.

Flies are known vectors for a variety of infectious diseases in animals. In fur mink farming, one of the most severe diseases is Aleutian disease, which is caused by the Aleutian mink disease virus (AMDV). The presence of large fly populations is a frequent issue in mink farms; however, no studies assessing their role as AMDV carrier vectors have been conducted to-date. In order to determine the presence of AMDV in aerial flies from an infected mink farm, flies (n = 254) and environmental swab samples (n = 4) were collected from two farm barns. Fannia canicularis (L.) (Diptera: Muscidae) represented more than 99% of the fly population. One hundred and fifty specimens of this species were divided into pools of ten flies and analysed by qPCR, and positive samples were further sequenced. All fly pools and environmental samples tested positive for AMDV, and sequence analysis revealed identical genotypes in both types of samples. This is the first report of AMDV contamination in flies from mink farms, suggesting that F. canicularis may act as an AMDV vector. These results may be of interest for epidemiological studies and also for the improvement of control measures against this virus in mink farms.

Cryptosporidium species in post-weaned and adult sheep and goats from N.W. Spain: Public and animal health significance.

Application of molecular approaches has led to a significant progress on the knowledge of the epidemiology of Cryptosporidium spp. Nevertheless, molecular information on the occurrence of cryptosporidiosis in domestic small ruminants, especially in goats, are limited and restricted to the study of a modest number of isolates, mainly from diarrhoeic neonates. In order to determine the Cryptosporidium species present in healthy post-weaned and adult small ruminants from north-western Spain and to analyse a possible age-related distribution of species, faecal specimens were collected in sheep and goat farms without neonatal diarrhoea outbreaks the year before the sampling. Cryptosporidium spp. DNA was detected by SSU-rRNA PCR-RFLP, using restriction enzymes SspI, VspI and MboII. C. parvum and C. ubiquitum isolates were further characterized at the GP60 locus. Our results reveal that Cryptosporidium spp. is widely distributed in small ruminant farms (47.4-50.0%), although its prevalence is low in both hosts (5.9-6.0%). No significant differences in individual prevalence were detected between age groups. C. xiaoi and the zoonotic C. parvum and C. ubiquitum were identified. In sheep, C. parvum was the predominant species and its prevalence increased with age, in contrast to C. xiaoi; C. ubiquitum was an occasional finding in adults. In goats, C. xiaoi and C. ubiquitum were the most frequent species and slightly more prevalent in adults than in post-weaned kids, in contrast to C. parvum. Subtyping analysis of C. parvum isolates revealed the presence of IIaA15G2R1 and IIaA14G2R1 in sheep, whereas IIaA13G1R1 and IIdA17G1 were restricted to goats; only the C. ubiquitum XIIa subtype 3 was found. Although the prevalences detected are low, these values are probably underestimated due to, amongst others, the cross-sectional design of the study and the intermittent oocyst-excretion of post-weaned and adult small ruminants. Thus, these animals may play an important role in the appearance of cryptosporidiosis outbreaks in humans and domestic ruminant neonates and therefore should be considered as a potential threat to animal production and human health.

Prospective Clinical Study of Zirconia Full-coverage Restorations on Teeth Prepared With Biologically Oriented Preparation Technique on Gingival Health: Results After Two-year Follow-up.

OBJECTIVES: To evaluate the clinical behavior of one-piece complete-coverage crowns and fixed partial dentures (FPDs) on teeth with vertical preparation without finish line biologically oriented preparation technique (BOPT). METHODS AND MATERIALS: This prospective study included 52 patients requiring treatment with restorations in the esthetic region: 74 crowns and 27 FPDs. The sample included a total of 149 teeth that were prepared vertically without finish line. The sample was divided into two groups: one-piece crowns and FPDs, all with zirconia cores, feldspathic ceramic veneer, and a 0.5-mm prosthetic finish line of zirconia. All procedures were carried out at the University of Valencia from 2013 to 2014. The following parameters were evaluated over a two-year follow-up: oral hygiene, periodontal state, gingival thickening, gingival margin stability, the presence of complications, and restoration survival rate. Patient satisfaction with treatment was assessed by means of a visual analogue scale (VAS). RESULTS: Two years after treatment, 80.5% of treated teeth remained free of gingival inflammation and bleeding. Mean gingival thickening was 0.41 ± 0.28 mm for one-piece crowns and 0.38 ± 0.36 mm for FPDs. Gingival margin stability was 100%, but 2% of the sample presented biological complications. The VAS patient satisfaction scores were eight out of a maximum score of 10. CONCLUSIONS: Two years after treatment, vertical preparation without finish line produces gingival thickening, margin stability, and optimal esthetics. Neither crowns nor FPDs presented any mechanical complications.

Hypoderma actaeon: an emerging myiasis in roe deer (Capreolus capreolus).

Subcutaneous larvae of Hypoderma spp. (Diptera: Oestridae) were detected in the dorsal region in 10 roe deer, Capreolus capreolus (Artiodactyla: Cervidae), hunted in central Spain between January and March 2016. All larvae were found in the inner side of the hide during the skinning of the animals. The study of the morphological features of eight larvae of different stages collected from two animals allowed the identification of Hypoderma actaeon Brauer. The small size (4-5 mm) of some of the first instars suggests that the internal lifecycle of H. actaeon may be exclusively subcutaneous. This is the first confirmation of H. actaeon in roe deer; however, further studies to assess the spread of the parasite and to follow the evolution of this myiasis in roe deer are needed.

Modulatory effects of interferon-γ and interleukin-4 on cellular immune responses against Hypoderma lineatum antigens.

This study investigates the in vitro modulatory effects of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on both proliferative bovine T cell responses and IL-10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL-10 production, which, in general, did not revert after the addition of IFN-γ and IL-4. In the absence of antigens, IL-4 induced significant inhibition of mitogen-induced T cell responses. Exogenous IFN-γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN-γ potentiates their anti-proliferative activity; by contrast, IL-4 had no consistent effects on proliferative responses to Hypoderma. IL-4 provoked an increment of IL-10 levels in supernatants of HyB-stimulated cells. In conclusion, exogenous IFN-γ and IL-4 were unable to counteract the suppressor effects of H. lineatum antigens.

Histological and immunohistochemical characterization of Hypoderma lineatum (Diptera: oestridae) warbles.

Hypoderma larvae are tissue invading parasites which spend several months migrating within the host tissues before completing their development in the sub-dermal tissues of the back. Subcutaneous stages of the parasite produce an inflammatory reaction in the skin called "warbles", as well as holes through which larvae breathe. In order to elucidate the microscopical structure of the warbles, three hides from warbled cows were collected in a slaughterhouse in Lugo (NW, Spain) between March and May 2012. A total of 60 skin samples, including warbles at different phases of development, were chosen for histopathological and immunohistochemical examination. Microscopic lesions were classified into three groups, according to the predominance and distribution of different cell populations. In warbles containing living or recently dead larvae with apparently well preserved cuticle (type 1), plasma cells were observed in high number. However, macrophages and lymphocytes were the predominant cells in granulomas (type 2) formed in relation to remnants of the dead parasite, containing or not remains of the altered cuticle. Scars (type 3) were characterized by granulation tissue. Immunohistochemistry showed that B lymphocytes and IgG(+) cells were predominant in the lesions, as long as the cuticle of the larvae is intact. On the other side, CD3(+) lymphocytes increased once cuticle is destroyed and a granuloma is formed. Macrophages, revealed by CD68(+), MAC387(+) and lysozyme immunolabelling, were detected in all types of lesions, but they were more abundant in type 2 and scarce in scars. These cells appeared isolated around the intact larvae or forming aggregates around its remains in the granuloma. Moreover, a strong immunolabelling against MAC387 antibody was registered in the squamous epithelium covering the breathing pore. This finding may be associated with the expression of calprotectin, a molecule involved on the healing process of the skin after larvae outcome. Our results suggest the predominance of a humoral response inside the warble as long as larvae are intact. Once they are destroyed, cellular response occurred, isolating and destroying the remains of the larvae until healing process completes and scars with low numbers of inflammatory cells appear.