Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Biomolecular Analysis of Canine Distemper Virus Strains in Two Domestic Ferrets (Mustela putorius furo).

Canine distemper is a contagious and severe systemic viral disease that affects domestic and wild carnivores worldwide. In this study, two adult female ferrets (Mustela putorius furo) were evaluated for cutaneous lesions. Scab, fur, and swab samples from the external auditory canal, cutaneous lesions, and scrapings were analyzed. Canine distemper virus (CDV)-positive samples underwent RT-PCR/RFLP with the restriction enzyme PsiI, and the hemagglutinin gene sequence was obtained. According to the restriction enzyme and sequence analyses, the viral strains were typed as CDV field strains that are included within the Europe lineage and distinct from those including vaccinal CDV strains. The sequence analysis showed the highest nucleotide identity rates in older Europe lineage CDV strains collected from dogs and a fox in Europe. This study is the first to report on CDV infection in ferrets in southern Italy and contributes to the current knowledge about natural CDV infection in this species. In conclusion, vaccination remains crucial for preventing the disease and counteracting cross-species infection. Molecular biology techniques can enable the monitoring of susceptible wild animals by ensuring the active surveillance of CDV spread.

A Review on the Immunological Response against Trypanosoma cruzi.

Chagas disease is a chronic systemic infection transmitted by Trypanosoma cruzi. Its life cycle consists of different stages in vector insects and host mammals. Trypanosoma cruzi strains cause different clinical manifestations of Chagas disease alongside geographic differences in morbidity and mortality. Natural killer cells provide the cytokine interferon-gamma in the initial phases of T. cruzi infection. Phagocytes secrete cytokines that promote inflammation and activation of other cells involved in defence. Dendritic cells, monocytes and macrophages modulate the adaptive immune response, and B lymphocytes activate an effective humoral immune response to T. cruzi. This review focuses on the main immune mechanisms acting during T. cruzi infection, on the strategies activated by the pathogen against the host cells, on the processes involved in inflammasome and virulence factors and on the new strategies for preventing, controlling and treating this disease.

Persistence of DNA from canine parvovirus modified-live virus in canine tissues.

Canine parvovirus (CPV-2) modified-live virus vaccine strain can replicate in lymphoid tissues and intestinal mucosa after administration, being shed through canine faeces. Detection of vaccine strains has been reported in the bloodstream and faeces, potentially interfering with molecular diagnostic tests. The persistence of these strains in canine tissues has not yet been described. With this aim, canine tissues were tested during a molecular survey to screen for the presence of canine enteric viruses. Tissue samples from 165 dead dogs were tested by a conventional PCR assay. Positive samples and five commercial vaccines were subjected to sequence analysis. Vaccinal strains were detected and virus load was measured by using a set of real-time PCR assays using minor-groove binder (MGB) probes. Seventy-five dogs (45.4%) tested positive for CPV-2. Strains from 70 dogs were characterised as field variants. The presence of CPV sequences of vaccine origin was observed in the spleen, intestine, and mesenteric lymph nodes of five young dogs. Vaccinal strains were detected from 12 to 24 days after the last vaccine administration. Viral loads comprised between 6.3 × 102 and 9.95 × 104 DNA copies/10 µl of template. This study confirms that CPV vaccinal strains can be detected in canine tissues after vaccination, so post-mortem diagnosis of CPV infection needs further molecular analyses to assess the viral type (vaccine or field strains). The present study updates the current information on the persistence of CPV vaccine strains in canine tissues and their possible interference with molecular assays.

Study on the Canine Adenovirus Type 1 (CAdV-1) Infection in Domestic Dogs in Southern Italy.

Canine adenovirus type 1 (CAdV-1) is the causative agent of a systemic and potentially fatal viral disease of domestic and wild canids. In Italy, CAdV-1 infection has also been occasionally described in dogs, but information on the epidemiology and its genomic features is still limited. A study was conducted on 291 dogs suspected of infectious gastrointestinal disease. Samples collected from dogs in southern Italy between 2017 and 2020 were analyzed. Virological and histopathological assays were carried out. The presence of CAdVs and other canine viral enteropathogens was investigated, and sequence and phylogenetic analyses were performed. CAdV-1 was detected in six (2.1%) dead stray dogs alone or in mixed infections with other viruses. Gross lesions and histopathological findings referred to CAdV infection were observed, also involving the central nervous system tissues. All inoculated samples were successfully isolated. Sequence analysis evidenced divergences with the circulating strains previously described in Italy and a closer relation with older CAdV-1 strains collected from other countries, suggesting a genetic heterogeneity of CAdV-1 in Italy. The evidence of the circulation of CAdV-1 and its genomic features allows us to have more in-depth knowledge of the epidemiology and evolution of the CAdV-1 genomic variants.

Identification and Molecular Characterization of a Divergent Asian-like Canine Parvovirus Type 2b (CPV-2b) Strain in Southern Italy.

Canine parvovirus type 2 (CPV-2) is an infectious agent relevant to domestic and wild carnivorans. Recent studies documented the introduction and spread of CPV-2c strains of Asian origin in the Italian canine population. We investigated tissue samples from a puppy collected during necropsy for the presence of viral enteropathogens and all samples tested positive only for CPV-2. The full coding sequence of a CPV-2b (VP2 426Asp) strain was obtained. This virus was related to CPV-2c strains of Asian origin and unrelated to European CPV-2b strains. The sequence had genetic signatures typical of Asian strains (NS1: 60Val, 545Val, 630Pro; VP2: 5Gly, 267Tyr, 324Ile) and mutations rarely reported in Asian CPV-2b strains (NS1: 588N; VP2: 370Arg). Phylogenetic analyses placed this strain in well-supported clades, including Asian CPV-2c-like strains, but always as a basal, single-sequence long branch. No recombination was observed for this strain, and we speculate that it could have originated from an Asian CPV-2c-like strain that acquired the 426Asp mutation. This study reports the first evidence of an Asian-like CPV-2b strain in Italy, confirming the occurrence of continuous changes in the global CPV-2 spread. Since positive convergent mutations complicate data interpretation, a combination of phylogenetic and mutation pattern analyses is crucial in studying the origin and evolution of CPV-2 strains.

Agreement between In-Clinics and Virus Neutralization Tests in Detecting Antibodies against Canine Distemper Virus (CDV).

Core vaccinations and specific antibody titer evaluations are strongly recommended worldwide by all the vaccination guidelines. Virus neutralization (VN) is considered the gold standard for measuring antibody titer against canine distemper virus, but it is complex and time consuming, and the use of in-clinics tests would allow to obtain quicker results. The aim of this study was to evaluate the agreement of the commercial in-clinics VacciCheck test compared to VN. A total of 106 canine sera were analyzed using both methods. The best agreement was obtained using a protective threshold of ≥1:32. VacciCheck showed 95.5% sensitivity, 87.2% specificity, and 92.5% accuracy. The Cohen's kappa coefficient between methods was 0.84 (CI 95% 0.73 to 0.95), revealing an optimal agreement between the two methods (p = 0.0073). The evaluation of discordant results reveal that most samples had less than 1.5 dilution difference, and that usually did not affect the classification as protected or non-protected. Results also suggest that, in dubious cases, especially when a protective result is expected, retesting is advisable. In conclusion, VacciCheck may be considered as a reliable instrument that may help the clinician in identifying the best vaccine protocol, avoiding unnecessary vaccination, and thus reducing the incidence of adverse effects.

Detection and Molecular Characterization of Two Gammaherpesviruses from Pantesco Breed Donkeys during an Outbreak of Mild Respiratory Disease.

Equid and asinine gammaherpesviruses (GHVs; genus Percavirus) are members of the Herpesviridae family. Though GHVs have been reported in horse populations, less studies are available on gammaherpesviral infections in donkeys. This study reports the co-infection with two GHVs in Pantesco breed donkeys, an endangered Italian donkey breed. Samples (n = 124) were collected on a breeding farm in Southern Italy from 40 donkeys, some of which were healthy or presented erosive tongue lesions and/or mild respiratory signs. Samples were analysed by using a set of nested PCRs targeting the DNA polymerase, glycoprotein B, and DNA-packaging protein genes, and sequence and phylogenetic analyses were performed. Twenty-nine donkeys (72.5%) tested positive, and the presence of Equid gammaherpesvirus 7 and asinine herpesvirus 5 was evidenced. In 11 animals, we found evidence for co-infection with viruses from the two species. Virions with herpesvirus-like morphology were observed by electron microscopic examination, and viruses were successfully isolated in RK-13-KY cell monolayers. The histological evaluation of tongue lesions revealed moderate lympho-granulocytic infiltrates and rare eosinophilic inclusions. The detection of GHVs in this endangered asinine breed suggests the need long-life monitoring within conservation programs and reinforces the need for further investigations of GHV's pathogenetic role in asinine species.

Occurrence of Human Enteric Viruses in Shellfish along the Production and Distribution Chain in Sicily, Italy.

Contamination of bivalve mollusks with human pathogenic viruses represents a recognized food safety risk. Thus, monitoring programs for shellfish quality along the entire food chain could help to finally preserve the health of consumers. The aim of the present study was to provide up-to-date data on the prevalence of enteric virus contamination along the shellfish production and distribution chain in Sicily. To this end, 162 batches of mollusks were collected between 2017 and 2019 from harvesting areas, depuration and dispatch centers (n = 63), restaurants (n = 6) and retail stores (n = 93) distributed all over the island. Samples were processed according to ISO 15216 standard method, and the presence of genogroup GI and GII norovirus (NoV), hepatitis A and E viruses (HAV, HEV), rotavirus and adenovirus was investigated by real-time reverse transcription polymerase chain reaction (real-time-RT PCR), nested (RT)-PCR and molecular genotyping. Our findings show that 5.56% of samples were contaminated with at least one NoV, HAV and/or HEV. Contaminated shellfish were sampled at production sites and retail stores and their origin was traced back to Spain and several municipalities in Italy. In conclusion, our study highlights the need to implement routine monitoring programs along the whole food chain as an effective measure to prevent foodborne transmission of enteric viruses.

The Effect of a 7 Year-Long Cryopreservation on Stemness Features of Canine Adipose-Derived Mesenchymal Stem Cells (cAD-MSC).

Mesenchymal stem cells (MSCs) are used in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. Their clinical application requires a ready off-the-shelf amount of viable therapeutics doses. For this purpose, it is useful to cryopreserve MSCs to gain a ready and controlled source of abundant autologous stem cells. We evaluated the effect of 7 years cryopreservation using 10% dimethyl sulfoxide (DMSO) with different fetal bovine serum (FBS) concentrations (from 10 to 90%) on different passages of MSCs isolated from canine adipose tissue (cAD-MSCs). The study aimed to evaluate the most adequate cell passage and FBS percentage for the long-term cryopreservation of cells by maintaining the stemness features. Phenotype morphology, cell viability, osteogenic and adipogenic differentiation potentials, proliferative potential and expression of pluripotency markers were analyzed in thawed cells and compared with fresh ones. We demonstrated that cells cryopreserved with at least 80% FBS maintain unaltered the stemness characteristics of the freshly isolated cells. In particular, cells of P0-P1 passages have to be expanded in vitro and subsequently cryopreserved and cells of P2-P4 passages should be considered in the studies on therapeutic application and in vitro study of cAD-MSCs.

Serological and Molecular Evidence of Bartonella henselae in Stray Cats from Southern Italy.

Bartonella henselae is a slow growing and facultative intracellular pathogen mainly transmitted by arthropod vectors adapted to domestic and wild mammalian reservoir hosts. Since cats are the major source of the B. henselae infection, this study aimed to evaluate the seroprevalence and the DNA presence in randomly sampled stray cats. Blood samples of 429 cats were collected from shelter of Palermo (Southern Italy) and sera and whole blood were analyzed for the presence of antibodies against B. henselae by indirect immunofluorescence assay (IFA) and by real-time polymerase chain reaction (PCR), respectively. Two hundred and three sera (47.3%) were positive to IFA and 148 blood samples (34.5%) to real-time PCR. Based on serological results, the evaluation of the potential risk factors (sex, age, coat color) was carried out. The multivariate analysis indicated that cats more than 12 months old were more likely to be seropositive to B. henselae than cats <12 months. These data will add useful information to the understanding of the spread of B. henselae in stray cats in Southern Italy.

Hepatitis E Virus Occurrence in Pigs Slaughtered in Italy.

In Europe, foodborne transmission has been clearly associated to sporadic cases and small clusters of hepatitis E in humans linked to the consumption of contaminated pig liver sausages, raw venison, or undercooked wild boar meat. In Europe, zoonotic HEV-genotype 3 strains are widespread in pig farms but little information is available on the prevalence of HEV positive pigs at slaughterhouse. In the present study, the prevalence of HEV-RNA positive pigs was assessed on 585 animals from 4 abattoirs located across Italy. Twenty-one pigs (3.6%) tested positive for HEV in either feces or liver by real-time RT-PCR. In these 21 pigs, eight diaphragm muscles resulted positive for HEV-RNA. Among animals collected in one abattoir, 4 out of 91 plasma tested positive for HEV-RNA. ELISA tests for the detection of total antibodies against HEV showed a high seroprevalence (76.8%), confirming the frequent exposure of pigs to the virus. The phylogenetic analyses conducted on sequences of both ORF1 and ORF2 fragments, shows the circulation of HEV-3c and of a novel unclassified subtype. This study provides information on HEV occurrence in pigs at the slaughterhouse, confirming that muscles are rarely contaminated by HEV-RNA compared to liver, which is the most frequently positive for HEV.

Current status and risk factors of canine parvovirus type 2 in North Central Nigeria.

Since its emergence in Nigeria, canine parvovirus type 2 (CPV-2) infection has posed problems to dog breeding and requires constant awareness and monitoring. In this study, the status, the assessment of extrinsic risk factors of parvoviral infection in dog kennels in North Central Nigeria, and isolation of the CPV-2 were carried out. Potential risk factors were considered during sampling: age, breed, sex, location, vaccination and health status, using well-structured questionnaires on dog owners with experience of CPV-2 infection. There was high prevalence which depended on age, breed, location, clinical status of the dog while vaccination status of the dogs did not influence the prevalence. CPV-2 vaccination compliance by the breeders and management system of the kennels were also observed as risk factors. Isolation of CPV-2a and -2c strains from Nigeria for further study has been reported. The spread of CPV-2 in Nigeria is increasing, hence needs for continual epidemiological monitoring and review.

Seroprevalence of Borrelia burgdorferi in Stray Dogs from Southern Italy.

Borrelia burgdorferi is a bacterial pathogen transmitted by Ixodes ticks and is responsible for Lyme disease in both humans and dogs. The aim of this work was to evaluate B. burgdorferi diffusion among stray dogs in Palermo (Sicily, Italy) by serological methods in order to study the risk factors associated with the infection. Serum and blood samples of 316 dogs were collected from a shelter in Palermo, and were analyzed for the presence of antibodies against B. burgdorferi by indirect immunofluorescence assay (IFA), and of the ospA gene by real-time PCR, respectively. Seventeen sera (5.4%) were positive for the antibodies via IFA and one blood (0.3%) for ospA via real time PCR. On the basis of serological results, the evaluation of the potential risk factors (sex, age, breed and coat color) was carried out. The multivariate analysis indicated that male sex is a factor significantly associated with B. burgdorferi seropositivity. This study confirms that male dogs have a higher risk of developing the disease than females, and represents the first investigation on the spread of B. burgdorferi among stray dogs in Sicily.

In Vitro Biocompatibility Evaluation of Nine Dermal Fillers on L929 Cell Line.

OBJECTIVE: Biomaterial research for soft tissue augmentation is an increasing topic in aesthetic medicine. Hyaluronic acid (HA) fillers are widely used for their low invasiveness and easy application to correct aesthetic defects or traumatic injuries. Some complications as acute or chronic inflammation can occur in patients following the injection. Biocompatibility assays are required for medical devices intended for human use, in order to prevent damages or injuries in the host. In this study, nine HA fillers were tested in order to evaluate their cytotoxicity and their effects on L929 cell line, according to the UNI EN ISO 10993 regulation. METHODS: Extracts were prepared from nine HA fillers, and MTS viability assay was performed after 24 h, 48 h, and 72 h of exposure of cells to extracts. Cells cultured with HA filler extracts were monitored for up to 72 h, counted, and stained with haematoxylin/eosin in order to evaluate the cell proliferation rate and morphology. RESULTS: None of the filler tested showed a cytotoxic effect. Two samples showed a higher vitality percentage and higher cell number while two samples showed a lower vitality percentage and lower cell number at 72 h. CONCLUSION: Data obtained suggest that although examined fillers are not cytotoxic, they show different effects on the in vitro cell proliferation rate. In vitro studies of medical devices could lead to important implications since these could aid to predict effects about their in vivo application. These easy and rapid assays could be useful to test new materials intended for human use avoiding animal tests.

A Cross Sectional Study on Serological Prevalence of Ehrlichia canis and Rickettsia conorii in Different Canine Population of Sicily (South-Italy) during 2017-2019.

Vector-borne pathogens such as Erlichia canis and Rickettsia conorii are widespread in the Mediterranean basin. Rhipicephalus sanguineus, is considered the main vector in Mediterranean climatic areas. Seroprevalence in dogs is variable in relation to environmental factors, presence of vectors, and dogs' management. We investigated the seroprevalence in Sicilian dogs during 2017-2019, considering temporal as well as spatial variations, and different canine population. A total of 11,009 sera were analyzed: 7568 and 3441 sera were tested to detect antibodies to E. canis and to R. conorii, respectively, by immunofluorescence assay. The rainfall average in the sampling sites during the three-year period was also considered. Statistical analyses were performed using chi-square tests for association between two or more variables. We reported a prevalence of 29.6% and 53.6% for E. canis and R. conorii, respectively. Significant temporal variation was found in R. conorii, while significant difference was found considering canine population and spatial variation regarding both pathogens. Our study updates the previous results of E. canis and R. conorii seroprevalence in dogs in Sicily, and confirms the wide distribution of these pathogens. In addition, we considered, for the first time, three different variables to identify the areas and the canine populations most exposed to risk.

Nearly full-length genome characterization of canine parvovirus strains circulating in Nigeria.

Canine parvovirus type 2 (CPV-2) emerged suddenly in the late 1970s as pathogen of dogs, causing a severe and often fatal gastroenteric disease. The original CPV-2 was replaced by three antigenic variants, CPV-2a, CPV-2b and CPV-2c, which to date have gained a worldwide distribution with different relative proportions. All previous studies conducted in Africa were based on partial VP2 gene sequences. The aim of this study was to provide a genome analysis to characterize the CPV strains collected in Nigeria, Africa. Rectal swab samples (n = 320) were collected in 2018 and tested by means of an immunochromatographic assay. Among the 144 positive samples, 59 were selected for further analyses using different molecular assays. The results revealed a high prevalence of CPV-2c (91.5%) compared to the CPV-2a variant (8.5%). The VP2 gene sequences showed a divergence from the strains analysed in 2010 in Nigeria and a closer connection with CPV strains of Asian origin. The non-structural gene analysis evidenced amino acid changes never previously reported. The molecular analysis based on genomic sequences evidenced a geographical pattern of distribution of the analysed strains, suggesting a potential common evolutionary origin with CPV of Asian origin. This study represents the first CPV molecular characterization including all the encoding gene sequences conducted in the African continent and contributes to define the current geographical spread of the CPV variants worldwide.

Cytotoxicity Evaluation of Endodontic Pins on L929 Cell Line.

OBJECTIVE: The aim of this study was to evaluate the cytotoxic potential of a type of endodontic pin on L929 cell line according to the UNI EN ISO 10993/2009 rule. METHODS: L929 cells were used for the assays; extracts were prepared from three different-diameter endodontic pins, made of epoxy resin and fiberglass matrix and from Reference Materials (ZDEC, ZDBC, and HDP films). MTS assay was performed after 24 h, 48 h, and 72 h of exposure of L929 cells to pin and Reference Material extracts, 5% phenol solution, and control reagent. Cells cultured with different media containing extracts were monitored for up to 72 h and stained with haematoxylin/eosin. RESULTS: Pins of different diameters had no cytotoxic effects on L929 cells at 24 h, 48 h, and 72 h (all values >70%). Cells cultured in medium containing pin extracts grew without any differences compared to the control cells. CONCLUSION: The endodontic pins tested showed no cytotoxic effects and did not induce changes in morphology for up to 72 h.