Identification of trypanosomatids and blood feeding preferences of phlebotomine sand fly species common in Sicily, Southern Italy

In this study, the presence of Leishmania DNA and blood feeding sources in phlebotomine sand fly species commonly present in Sicily were investigated. A total of 1,866 female sand flies including 176 blood fed specimens were sampled over two seasons in five selected sites in Sicily (southern Italy). Sergentomyia minuta (n = 1,264) and Phlebotomus perniciousus (n = 594) were the most abundant species at all the sites, while three other species from the genus Phlebotomus (i.e., P. sergenti n = 4, P. perfiliewi n = 3 and P. neglectus n = 1) were only sporadically captured. Twenty-eight out of the 1,866 (1.5%) sand flies tested positive for Leishmania spp. Leishmania tarentolae DNA was identified in 26 specimens of S. minuta, while the DNA of Leishmania donovani complex was detected in a single specimen each of S. minuta and P. perniciosus. Interestingly, seven S. minuta specimens (0.4%) tested positive for reptilian Trypanosoma sp. Blood sources were successfully identified in 108 out of 176 blood fed females. Twenty-seven out of 82 blood sources identified in fed females of P. perniciosus were represented by blood of wild rabbit, S. minuta mainly fed on humans (16/25), while the sole P. sergenti fed specimen took a blood meal on rat. Other vertebrate hosts including horse, goat, pig, dog, chicken, cow, cat and donkey were recognized as blood sources for P. perniciosus and S. minuta, and, surprisingly, no reptilian blood was identified in blood-fed S. minuta specimens. Results of this study agree with the well-known role of P. perniciosus as vector of L. infantum in the western Mediterranean; also, vector feeding preferences herein described support the hypothesis on the involvement of lagomorphs as sylvatic reservoirs of Leishmania. The detection of L. donovani complex in S. minuta, together with the anthropophilic feeding-behaviour herein observed, warrants further research to clarify the capacity of this species in the transmission of pathogens to humans and other animals.publishersversionpublishe

First report of Stephanostomum cesticillus (Molin, 1858) Looss, 1899 in Merluccius merluccius (Linnaeus, 1758) from the Tyrrhenian Sea (Southern Italy)

The genus Stephanostomum consists of digenean trematodes found in many marine teleosts. In this study, Stephanostomum cesticillus (Molin, 1858) Looss, 1899 metacercariae were identified in European hake (Merluccius merluccius, Linnaeus, 1758) caught in the Tyrrhenian Sea. The metacercariae were found encapsulated in the muscle, close to the spine and gills. Out of 131 specimens, 111 (P = 84.7%, mI = 25.1 mA = 21.3) were infected by digenean trematode metacercariae. Morphological and histological evaluations were carried out. The members of this genus are characterised by a double crown of spines close to the oral sucker. For identification of the parasite, molecular analysis was performed via 28S and 18S ribosomal DNA (rDNA) genes. Partial rDNA sequences of Stephanostomum highly matched to S. cesticillus for the percentage of similar identity from the nucleotide database of BLAST. The present survey reports the presence of S. cesticillus metacercariae in M. merluccius for the first time. Our results improve current knowledge on hake parasites to better understand the distribution of S. cesticillus in M. merluccius caught in the Mediterranean Sea and shed light on the life cycle of the parasite adding other possible hosts

Aspiration Pneumonia with Prominent Alveolar Mineralization in a Dairy Cow

A 2-years-old Jersey breed cow showed severe respiratory distress and prolonged lateral recumbency about 1 week after parturition. The cow was regularly vaccinated against the main respiratory pathogens and was given a calcium salt solution per os shortly after calving in order to prevent milk fever. Despite therapy with broad-spectrum antibiotics and anti-inflammatory drugs, the cow spontaneously died one week later and was necropsied. On gross examination, a severe, acute, diffuse fibrinonecrotic tracheitis was observed. In addition, the cranioventral portions of both lungs appeared firm and severely congested, while the pleural surface was covered by a discrete amount of fibrinous exudate. Microscopically, the following lesions were observed: tracheal hemorrhages, acute, fibrinonecrotic and suppurative tracheitis, pulmonary hemorrhages, fibrinous bronchopneumonia and fibrinous pleuritis. Noteworthy, multiple foci of mineralization were observed, scattered throughout the lung parenchyma and occasionally within the tracheal mucosa. The presence of calcium deposits was confirmed by means of Von Kossa staining method. Based on clinical history, clinical signs and pathological findings, aspiration pneumonia caused by the accidental inhalation of liquid calcium salt supplement was diagnosed. The present case report highlights the relevance of the staff training to optimize animal production and welfare

Do Different LED Colours Influence Sand Fly Collection by Light Trap in the Mediterranean?

Light traps represent the most used attractive system to collect and monitor phlebotomine sand flies. Recent studies have suggested that light traps can be easily upgraded by the use of light-emitting diode (LED) with positive effects on trap design, weight, and battery life. However, scant data on the effect of different LED colours on the attractiveness to phlebotomine sand fly species are available in literature. In this study, the capture performances of light traps equipped with different LED colours on phlebotomine sand fly species indigenous in the Mediterranean area were evaluated. Phlebotomine sand fly collections were performed using a classical light trap (CLT), equipped with a traditional incandescent lamp, and five Laika 4.0 light traps supplied, each with LED of different colours and wavelengths: (i) white; (ii) red; (iii) green; (iv) blue; (v) UV. Light traps were set for three consecutive nights fortnightly from May to October 2017 and climate data recorded using a meteorological station. A total of 411 phlebotomine sand flies (191 males and 220 females), belonging to three different species, namely, Phlebotomus perniciosus (n= 298, 141 males and 157 females), Sergentomyia minuta (n=110, 48 males and 62 females), and Phlebotomus neglectus (n=3, 2 males and 1 females) were collected. Abundance of capture was influenced by colours of LED and time. The highest number of phlebotomine sand flies was captured on June (P0.05) or LED colour (P>0.05) was recorded for S. minuta and P. neglectus. According to the results of the present study light trap equipped with UV LED can represent an effective tool for the capture of sand fly species in the Mediterranean area

Leishmania infantum in wild animals in endemic areas of southern Italy.

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears. This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area

Rapid Evaporative Ionization Mass Spectrometry-Based Lipidomics for Identification of Canine Mammary Pathology

The present work proposes the use of a fast analytical platform for the mass spectrometric (MS) profiling of canine mammary tissues in their native form for the building of a predictive statistical model. The latter could be used as a novel diagnostic tool for the real-time identification of different cellular alterations in order to improve tissue resection during veterinary surgery, as previously validated in human oncology. Specifically, Rapid Evaporative Ionization Mass Spectrometry (REIMS) coupled with surgical electrocautery (intelligent knife—iKnife) was used to collect MS data from histologically processed mammary samples, classified into healthy, hyperplastic/dysplastic, mastitis and tumors. Differences in the lipid composition enabled tissue discrimination with an accuracy greater than 90%. The recognition capability of REIMS was tested on unknown mammary samples, and all of them were correctly identified with a correctness score of 98–100%. Triglyceride identification was increased in healthy mammary tissues, while the abundance of phospholipids was observed in altered tissues, reflecting morpho-functional changes in cell membranes, and oxidized species were also tentatively identified as discriminant features. The obtained lipidomic profiles represented unique fingerprints of the samples, suggesting that the iKnife technique is capable of differentiating mammary tissues following chemical changes in cellular metabolism

Postmortem Electrical Conductivity Changes of Dicentrarchus labrax Skeletal Muscle: Root Mean Square (RMS) Parameter in Estimating Time since Death

Electric impedance spectroscopy techniques have been widely employed to study basic biological processes, and recently explored to estimate postmortem interval (PMI). However, the most-relevant parameter to approximate PMI has not been recognized so far. This study investigated electrical conductivity changes in muscle of 18 sea bass specimens, maintained at different room temperatures (15.0 °C; 20.0 °C; 25.0 °C), during a 24 h postmortem period using an oscilloscope coupled with a signal generator, as innovative technology. The root mean square (RMS) was selected among all measured parameters, and recorded every 15 min for 24 h after death. The RMS(t) time series for each animal were collected and statistically analyzed using MATLAB®. A similar trend in RMS values was observed in all animals over the 24 h study period. After a short period, during which the RMS signal decreased, an increasing trend of the signal was recorded for all fish until it reached a peak. Subsequently, the RMS value gradually decreased over time. A strong linear correlation was observed among the time series, confirming that the above time-behaviour holds for all animals. The time at which maximum value is reached strongly depended on the room temperature during the experiments, ranging from 6 h in fish kept at 25.0 °C to 14 h in animals kept at 15.0 °C. The use of the oscilloscope has proven to be a promising technology in the study of electrical muscle properties during the early postmortem interval, with the advantage of being a fast, non-destructive, and inexpensive method, although more studies will be needed to validate this technology before moving to real-time field investigations

Identification of phlebotomine sand flies through MALDI-TOF mass spectrometry and in-house reference database

Phlebotomine sand flies are vectors for many pathogens responsible for human and animal diseases worldwide. Their identification at species level is of importance in epidemiological studies and control programmes. MALDI-TOF MS has been increasingly investigated as an alternative approach to the conventional identification of arthropods species. To establish an in-house protein spectra database for a quick and reliable species identification of phlebotomine sand flies, 166 field-caught sand fly specimens, morphologically identified as Phlebotomus perniciosus (no = 56; 26 males and 30 females), Phlebotomus neglectus (no = 4 males), Phlebotomus sergenti (no = 6; 4 males and 2 females) and Sergentomyia minuta (no = 100; 45 males and 55 females), were subjected to MALDI-TOF MS analyses. Out of 166, 149 specimens (89.8%) produced consistent species-specific protein spectra. Good quality database for P. perniciosus and S. minuta were generated; no databases have yet constructed for P. neglectus and P. sergenti due to the low number of specimens examined. The identification of 80 sand flies (no = 20 P. perniciosus; no = 60 S. minuta) were confirmed using the new generated SuperSpectra as validation test. The results reported support the use of MALDI-TOF MS for rapid, simple and reliable phlebotomine sand fly species identification suggesting its usefulness in accurate survey studies, ultimately improving biological and epidemiological knowledge on these important vectors of pathogens