Anopheles sacharovi in Italy: first record of the historical malaria vector after over 50 years.

BACKGROUND: Anopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. METHODS: Investigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker. RESULTS: Out of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing. CONCLUSIONS: The discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered.

Rickettsial Infection in the COVID-19 Era: The Correlation between the Detection of Rickettsia aeschlimannii in Ticks and Storytelling Photography of a Presumable Human Rickettsiosis Case.

Rickettsia aeschlimannii infection is an emerging human tick-borne disease with only a few recorded cases. We reported a presumable autochthonous case of rickettsiosis in an Italian cattle breeder associated with a Hyalomma marginatum bite. Rickettsia aeschlimannii DNA was detected in both the tick specimen from the patient and the grazing cattle close to his farm.

Linguatula serrata (Fröhlich, 1789) in Gray Wolf (Canis lupus) from Italy: A Neglected Zoonotic Parasite.

Linguatula serrata, Frohlich, 1789, is a cosmopolitan zoonotic worm-like parasite of carnivores and other vertebrates including herbivores and omnivores. The adult form of the parasite typically inhabits the upper respiratory system, nares, and frontal sinuses of dogs, wolves, and cats. Infective eggs may be spread by sneezing, nasal secretions, and stool. The immature stages of the parasite are localized in the visceral organs of intermediated hosts, usually ruminants or rodents, and they are orally transmitted to predators during the ingestion of infested viscera. This paper reports the morphological identification and the molecular characterization of L. serrata specimen collected from a gray wolf in the Apulia region (southern Italy) and it also provides epidemiological information on this rarely reported zoonosis.

Molecular Characterization of Anopheles algeriensis Theobald, 1903 (Diptera: Culicidae) Populations from Europe.

Anopheles algeriensis Theobald, 1903, considered a competent vector of Plasmodium parasites, is a mosquito species widely distributed in the Mediterranean area but rare in Northern and Central Europe. The disappearance of its suitable breeding sites in Italy is having a detrimental effect on the occurrence of this species once common along the Southern coasts and on the islands. Recently, molecular investigations have renewed interest in this species, highlighting a genetic heterogeneity among European populations. In this study, An. algeriensis populations from Italy, Germany, Romania, and Sweden were analyzed by molecular typing of the intergenic transcribed spacer 2 (ITS2). The mitochondrial cytochrome c oxidase subunit I (COI) was also analyzed from specimens collected in Southern Italy. With the aim of investigating the population structure of this species, the obtained data were compared to all publicly available ITS2 and COI sequences of An. algeriensis, adding specimens from Spain and Portugal. The analyses of both markers indicate a split between Iberian populations (Spain for ITS2 and Spain/Portugal for COI) and those from the rest of Europe, revealing two cryptic species. The analysis of the COI barcode revealed a third clade representing a cryptic species present in Danube Delta (Romania). The high levels of genetic divergence among the clades of An. algeriensis indicate that this taxon represents a species complex, potentially harboring several distinct cryptic species.

Release of DNA from Dermanyssus gallinae during the Biting Process.

Dermanyssus gallinae is a hematophagous ectoparasitic mite that usually infests poultry, but is also known for occasionally attacking other animals and humans. It represents a major problem for poultry systems all over the world, with detrimental effects for both production and animal welfare. Despite the significance of D. gallinae, very little is known about the biting process to date. Therefore, this study has aimed to verify if mite DNA is injected into the host skin during the blood meal. Mite DNA has been detected by seminested PCR from infested chicken skin and quantified by real-time PCR. Furthermore, its localization within the host tissue has been checked by fluorescent in situ hybridization. Results showed that a very little amount of D. gallinae DNA can be released by mites, suggesting that the latter do not introduce whole or partially destroyed cells into the host, but rather it injects traces of nucleic acids, possibly together with merocrine secretions.

Detection of Coxiella burnetii DNA in sheep and goat milk and dairy products by droplet digital PCR in south Italy.

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that is responsible for Q fever, a common zoonosis which is present virtually worldwide. This microorganism infects a wide range of wild and domestic mammals, but the main reservoirs are cattle, goats and sheep, which also represent sources of human infection. A potential route of transmission of this pathogen to humans is the consumption of C. burnetii-contaminated raw milk or dairy products derived from contaminated raw milk, although the role of these foods as possible infection sources is controversial. The aims of this study were (i) to apply two ddPCR based assays targeting the C. burnetii IS1111 and icd genes for the detection and quantification of C. burnetii DNA, and (ii) to evaluate the occurrence of C. burnetii DNA in raw milk and raw milk products from sheep and goats in Apulia and Basilicata regions of Southern Italy. Of 413 milk and cheese samples tested, 78 were positive for the presence of C. burnetii DNA (18.9%), specifically, 68 of 285 milk samples (23.9%) and 10 of 128 cheese samples (7.8%) The presence of both IS1111 and icd genes was detected in only 2 (2.6%) of the 78 positive samples, while the remaining 76 (97.4%) were positive only for IS1111. C. burnetii DNA was specifically detected by the ddPCR method, whereas no cross-amplification was observed with the DNA of other foodborne bacterial pathogens. The sensitivity of the ddPCR method was determined as 0.35 and 0.56 copies/µL for IS1111 and icd genes, respectively. The findings of this study demonstrate the presence of C. burnetii DNA in a significant proportion of raw milk and dairy products. Although there is no conclusive epidemiological evidence that C. burnetii infection occurs via food, the presence of this organism in raw milk and dairy products made of raw milk should be considered a potential hazard. ddPCR is a useful tool to investigate the quality and safety of food products due to its sensitivity and precision, and could be applied to routine testing.

Genetic Evidence of the Black Death in the Abbey of San Leonardo (Apulia Region, Italy): Tracing the Cause of Death in Two Individuals Buried with Coins.

The Abbey of San Leonardo in Siponto (Apulia, Southern Italy) was an important religious and medical center during the Middle Ages. It was a crossroads for pilgrims heading along the Via Francigena to the Sanctuary of Monte Sant'Angelo and for merchants passing through the harbor of Manfredonia. A recent excavation of Soprintendenza Archeologica della Puglia investigated a portion of the related cemetery, confirming its chronology to be between the end of the 13th and beginning of the 14th century. Two single graves preserved individuals accompanied by numerous coins dating back to the 14th century, hidden in clothes and in a bag tied to the waist. The human remains of the individuals were analyzed in the Laboratorio di Antropologia Fisica of Soprintendenza ABAP della città metropolitana di Bari. Three teeth from each individual were collected and sent to the Istituto Zooprofilattico Sperimentale di Puglia e Basilicata to study infectious diseases such as malaria, plague, tuberculosis, epidemic typhus and Maltese fever (Brucellosis), potentially related to the lack of inspection of the bodies during burial procedures. DNA extracted from six collected teeth and two additional unrelated human teeth (negative controls) were analyzed using PCR to verify the presence of human DNA (ß-globulin) and of pathogens such as Plasmodium spp., Yersinia pestis, Mycobacterium spp., Rickettsia spp. and Brucella spp. The nucleotide sequence of the amplicon was determined to confirm the results. Human DNA was successfully amplified from all eight dental extracts and two different genes of Y. pestis were amplified and sequenced in 4 out of the 6 teeth. Molecular analyses ascertained that the individuals buried in San Leonardo were victims of the Black Death (1347-1353) and the data confirmed the lack of inspection of the corpses despite the presence of numerous coins. This study represents molecular evidence, for the first time, of Southern Italy's involvement in the second wave of the plague pandemic.

Entomological Surveillance in Former Malaria-endemic Areas of Southern Italy.

Malaria still represents a potential public health issue in Italy, and the presence of former Anopheles vectors and cases imported annually merit continuous surveillance. In areas no longer endemic, the concurrent presence of gametocyte carriers and competent vectors makes re-emergence of local transmission possible, as recently reported in Greece. In October 2017, due to the occurrence of four suspected introduced malaria cases in the province of Taranto (Apulia region), entomological investigations were performed to verify the involvement of local anopheline species. In 2019-2020 entomological surveys were extended to other areas historically prone to malaria between the provinces of Taranto and Matera and the province of Foggia (Gargano Promontory). Resting mosquitoes were collected in animal shelters and human dwellings, larvae were sampled in natural and artificial breeding sites, and specimens were both morphologically and molecularly identified. A total of 2228 mosquitoes were collected, 54.3% of which were anophelines. In all the investigated areas, Anopheles labranchiae was the most widespread species, while Anopheles algeriensis was predominant at the Gargano sites, and Anopheles superpictus and Anopheles plumbeus were recorded in the province of Matera. Our findings showed a potentially high receptivity in the surveyed areas, where the abundance of the two former malaria vectors, An. labranchiae and An. superpictus, is related to environmental and climatic parameters and to anthropic activities.

Study on the Role of the Common House Fly, Musca domestica, in the Spread of ORF Virus (Poxviridae) DNA under Laboratory Conditions.

ORF virus (Poxviridae) is the causative agent of contagious ecthyma (soremouth), a disease primarily affecting sheep and goats worldwide, but also humans exposed to disease-ridden animals. Pathogens are shed with scabs, and infection mainly occurs by direct contact. Although the disease is relatively benign and self-limiting, the morbidity rate is high in livestock with subsequent significant financial and economic impact. The aim of the study was to experimentally investigate the potential for the housefly, Musca domestica, to act as a mechanical vector of the virus. Homogenate of crusted scabs from ORFV-positive sheep (Italy, Apulia) were used to infect laboratory-reared flies. Flies walking on viral mixture and flies inoculated on their wings were individually placed in Falcon tubes and the ORFV DNA was searched by PCR on tube walls; flies were fed on the same homogenized crusts and their crop and spots (vomit and feces) molecularly examined for ORF DNA at 2, 4, and 6 h. All of the flies (100%) used in the experiments were able to pick up and transmit the viral genome to contact surfaces; 60% were found ORF virus (DNA)-positive in both spots and crop. These results suggest that M. domestica could play a role as potential mechanical vector and/or reservoir in the epidemiology of the ORF virus infection. Thus, house fly management should be considered in the measures to control the disease in ovine-caprine farms.

Discrimination of Bacillus cereus Group Members by MALDI-TOF Mass Spectrometry.

Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) technology is currently increasingly used in diagnostic laboratories as a cost effective, rapid and reliable routine technique for the identification and typing of microorganisms. In this study, we used MALDI-TOF MS to analyze a collection of 160 strains belonging to the Bacillus cereus group (57 B. anthracis, 49 B. cereus, 1 B. mycoides, 18 B. wiedmannii, 27 B. thuringiensis, 7 B. toyonensis and 1 B. weihenstephanensis) and to detect specific biomarkers which would allow an unequivocal identification. The Main Spectra Profiles (MSPs) were added to an in-house reference library, expanding the current commercial library which does not include B. toyonensis and B. wiedmannii mass spectra. The obtained mass spectra were statistically compared by Principal Component Analysis (PCA) that revealed seven different clusters. Moreover, for the identification purpose, were generated dedicate algorithms for a rapid and automatic detection of characteristic ion peaks after the mass spectra acquisition. The presence of specific biomarkers can be used to differentiate strains within the B. cereus group and to make a reliable identification of Bacillus anthracis, etiologic agent of anthrax, which is the most pathogenic and feared bacterium of the group. This could offer a critical time advantage for the diagnosis and for the clinical management of human anthrax even in case of bioterror attacks.

Case Report: Molecular Detection of Dirofilaria repens in an Italian Patient after a Stay in Tanzania.

A 35-year-old man was admitted to a hospital in the south of Italy because of a periocular nodule and subpalpebral edema. The patient reported having been stayed in Tanzania five months before. Hematologic parameters were within the normality range, the Acanthocheilonema viteae ELISA did not detect significant levels of antifilarial IgG, and no further symptoms were described. The surgical inspection of the nodule led to the isolation of two filarioid parasites, identified as Dirofilaria repens by scanning electron microscope (SEM), and then by molecular assays. Knott's test did not reveal microfilaremia, whereas loop-mediated isothermal amplification and PCR detected D. repens DNA. The patient was treated with doxycycline, and he was found no more positive at the follow-up.

Identification and characterization of Orf viruses isolated from sheep and goats in Southern Italy.

Orf virus (ORFV; Family: Poxviridae) is the causative agent of contagious ecthyma, or Orf disease in sheep, goats and other domestic or wild ruminants with a worldwide distribution. The disease is endemic in Italy, but few data are available about its distribution and epidemiology. In the present study we analysed 32 clinical samples, obtained from crusted scab lesions of 5 goats and 27 sheep, from 19 suspected outbreaks of contagious ecthyma in Apulia and Basilicata regions between 2012 and 2014. Negative staining electron microscopy (EM) and polymerase chain reaction (PCR) targeting the late transcription factor gene (VLTF-1) were used to identify the virus. Isolation was also attempted on BHK-21 cell line. PCR was proved to be more sensitive than EM, as it detected the virus in 28 out of 32 samples, whereas the EM detected it only in 26 out of the 32 samples. The majority of isolated strains forms a monophyletic group; these isolates, according to the VLTF-1 sequencing,  are high related to ORFV strains previously shown to circulate in Southern Italy.

Spotted fever group rickettsiae associated with ixodid ticks in wild environment in Southern Italy.

Ixodidae ticks are vectors and reservoirs of several species of rickettsiae, and tick-borne rickettsioses are reported worldwide. This study was aimed to verify the distribution of spotted fever group rickettsiae associated with ticks in a wild environment, the National Park of Gargano, where there is proximity between wild and domestic animals, and which is within an endemic area for rickettsiosis. Ticks were collected from animals or vegetation, morphologically identified and tested by a PCR targeting the 17kDa gene, and by a loop-mediated isothermal amplification (LAMP) targeting ompB gene. Out of 34 tested tick pools, 2 from Dermacentor marginatus, 1 from Ixodes ricinus, and 1 from Rhipicephalus turanicus resulted positive. Nucleotide sequences of amplicons showed high similarity with sequences from Rickettsia slovaca, Rickettsia raoultii, Rickettsia helvetica, and Rickettsia felis. The overall calculated infection rate was 26.19 per 1,000, while it rose up to 107.77 when only D. marginatus was considered. The results highlight the association among Ri. slovaca, Ri. raoultii, D. marginatus and wild boars from which infected ticks were collected. Finally, the study shows the low efficacy of the previously described LAMP method for the detection of Rickettsia spp., when compared to PCR, making urgent the development of most effective LAMP protocols.

Microfilaricidal efficacy of a single administration of Advocate(®) (Bayer Animal Health) in dogs naturally infected with Dirofilaria immitis or Dirofilaria repens.

The present study evaluated the microfilaricidal efficacy of a single application of the spot-on containing imidacloprid 10%/moxidectin 2.5% (Advocate(®), Bayer Animal Health) in dogs naturally infected either by Dirofilaria immitis or Dirofilaria repens. Dogs living in north-eastern and central-southern Italy, endemic for D. immitis and D. repens respectively, were randomly screened. Sixteen animals, eight infected with D. immitis and eight with D. repens, and fulfilling inclusion criteria were enrolled. Dogs infected with D. immitis received an adulticide treatment prior to the study and Advocate(®) 3 weeks after. The animals were divided in blocks of two (1:1, T1:T2) animals each, where Day 0 (D0) had an interval of 15days to compare T2 vs. T1 dogs during the first fortnight of examination (i.e. T2 dogs acted as control animals at each examination). At baseline (Days -15 and 0 for T2 and T1 dogs, respectively) the animals had a range of microfilaraemia of 180-99.700mff/ml (D. immitis) and 60-750 mff/ml (D. repens). All animals received a topical administration of Advocate(®) at D0 and were examined for microfilariae with microscopic and molecular tests at D15, D30, D60 and D90. All animals scored negative for mff at the first control post-treatment and throughout the study, with the exception of two D. immitis- infected animals that had a 2 mff/ml count at D15, and then become negative from Day 30 onwards. No adverse events were observed. The present study demonstrates the safety and the high microfilaricidal efficacy (99.97% and 100% for D. immitis and D. repens, respectively) of a single dose of moxidectin contained in Advocate(®) in naturally infected dogs.

Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples.

Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/µl (corresponding to about 50 copy of COI gene per µl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic.

Coxiella-like endosymbiont associated to the "Anatolian brown tick" Rhipicephalus bursa in Southern Italy.

Several different ticks have been reported to harbor microbes related to Coxiella burnetii, the agent of the Q fever. Rhipicephalus bursa is an important vector of tick-borne diseases in livestock in Mediterranean area; it is also abundant in ovi-caprine farms with C. burnetii infection, in Southern Italy. 60 females of Rh. bursa (15 pools) and 40 their eggs (2 pools) were screened for C. burnetii by a conventional PCR targeting the insertion sequence IS1111 and by Loop mediated isothermal amplification assay (LAMP) targeting com1 gene. One of 15 tick pools (1/15) and both egg pools (2/2) were found positive by LAMP assay and negative by PCR targeting IS1111 gene. 16S rRNA gene was amplified by PCR from the LAMP-positive pools, amplicons were sequenced and found 95% similar to the corresponding sequences from C. burnetii. This let us to hypothesize the presence of a new Coxiella-like endosymbiont associated with Rh. bursa which could be vertically transmitted, described here for the first time. The lack of detection of IS1111 in Coxiella endosymbiont of Rh. bursa could be related to the possible absence of the Pathogenicity island of C. burnetii, to which IS1111s are associated.

Oral hirudiniasis in a stray dog, first report in Italy.

In June 2014, a male stray dog was recovered at Ente Nazionale di Protezione Animali (ENPA) kennel of Manfredonia, Apulia region, showing oral bleeding and physical prostration. The dog fell in a water canal and was trapped. During the clinical examination, a specimen of leech was revealed into its oral cavity. The parasite, probably entered by drinking unfiltered and contaminated water, has been identified as an adult of aquatic leech Limnatis nilotica. Leeches could overrun wide variety of animals, and few reports about blood sucking leech infestations in mammals are available in literature. This paper describes here the first oral hirudiniasis in a dog in Italy and highlights the possibility of human nasopharyngeal leech-related infection in Apulia region.

Molecular detection of Coxiella burnetii using an alternative loop-mediated isothermal amplification assay (LAMP).

Q fever, caused by Coxiella burnetii, is a worldwide zoonosis with important consequences for human and animal health. In livestock, the diagnosis, using direct and indirect techniques, is challenging even if to tackle coxiellosis in domesticated animals a rapid diagnosis is crucial. In the recent years, new molecular methods have been developed to overcome these issues. Several polymerase chain reaction (PCR) assays have been studied, but loop mediated isothermal amplification (LAMP) has not been fully developed. This new methodology is emerging due to simplicity and speed in diagnosis of microbial diseases. In this study, we design a new LAMP assay against C. burnetii targeting the com1 gene as an actual alternative to conventional PCR. The assay was specific to C. burnetii reactive with sensitivity comparable to standard PCR. The application of the com1 LAMP on 10 clinical samples from water buffalo, sheep, and goats, previously tested positive, confirmed the presence of C. burnetii. To our knowledge, this study is the first report of LAMP targeting C. burnetii in Europe and the results also suggest that it may be an useful and cost-effective tool for the clinical and epidemiological surveillance of Q Fever.