Serological diagnosis of canine leishmaniosis: comparison of three commercial ELISA tests (Leiscan®, ID Screen® and Leishmania 96®), a rapid test (Speed Leish K®) and an in-house IFAT

BACKGROUND: Speed Leish K(®) is used as a serological screening test for Leishmania infection prior to vaccination. Limited comparative serological studies with Speed Leish K(®) have been performed. The aim of this study was to evaluate the diagnostic performance of four commercially available serologic tests including ELISAs (Leiscan(®), ID Screen(®) and Leishmania 96(®)), a rapid test (Speed Leish K(®)) and an in-house IFAT for the detection of specific antibodies against Leishmania infantum antigen in dogs in different states of infection. METHODS: Sick infected dogs (n = 36), healthy infected dogs (n = 18), L. infantum seropositive dogs with low to high levels of antibodies (n = 53), dogs seropositive to other pathogens (to evaluate cross reaction) (n = 14) and uninfected dogs from a non-endemic area (n = 50) and from an endemic area (n = 32) were analysed by the serological methods mentioned above. RESULTS: The sensitivity was as follows: ID Screen(®) (0.953), Leiscan(®) and Leishmania 96(®) (0.925), IFAT (0.869) and Speed Leish K(®) (0.636). The maximum specificity (1.000) was attained for all diagnostic tests except the Leishmania 96(®) (0.896) and IFAT (0.917). The accuracy was as follows: ID Screen(®) (0.975), Leiscan(®) (0.961), Leishmania 96(®) (0.911), IFAT (0.892) and Speed Leish K(®) (0.808). In relation to the area under the ROC curve (AUC-ROC), the maximum value was attained with the ID Screen(®) (0.993) closely followed by Leiscan(®) (0.990), then, Leishmania 96(®) (0.962), IFAT (0.926) and Speed Leish K(®) (0.818). For the Kappa index, the best result was obtained by the ID Screen(®) (0.951) followed by Leiscan(®) (0.921), Leishmania 96(®) (0.822), IFAT (0.783) and Speed Leish K(®) (0.622). Statistically significant differences were found between the AUC-ROC of quantitative serological tests and the only qualitative rapid test evaluated. There were also statistically significant differences between AUC-ROC of the ELISAs (ID Screen(®) and Leiscan(®)) and IFAT. CONCLUSIONS: Leiscan(®) and ID Screen(®) had superior diagnostic performance measures than IFAT and all quantitative serological tests were superior when compared to Speed Leish K(®). Thus, Speed Leish K(®) may be considered a less valuable screening test prior to vaccination as it may result in vaccination of seropositive dogs and in some cases seropositive sick dogs

Detection of a Novel Chlamydia Species in Invasive Turtles

Trachemys scripta is a turtle species native to Central America. Since the 1950s, pond sliders have been imported worldwide as companion animals, but have often ended up in foreign ecosystems with great ecological consequences. Moreover, both autochthonous and invasive species of turtles can be carriers of pathogens, including Chlamydiaceae. In the present study, pulmonary tissues collected from four Trachemys scripta were tested with a 23S-targeting real-time PCR (rPCR) specific for the Chlamydiaceae family. The turtles were hosted in a rescue center for wild exotic animals located in northeastern Italy, and were found dead after the hibernation period. Two out of four individuals resulted positive in rPCR for the presence of Chlamydiaceae. Further characterization of this positivity was performed by phylogenetic analysis of the 16S rRNA and outer membrane protein A genes. The phylogenetic tree showed that these chlamydial strains are identical to a novel Chlamydia reported in 2017 in Polish freshwater turtles, and closely related to Chlamydia pneumoniae and to other chlamydial strains found in reptiles. This first finding evidences the presence of this Chlamydia strain in Italian turtles, but further studies will be necessary to confirm the presence and the strain pathogenicity and to evaluate its prevalence in the local turtles’ population

Persistence of Leptospira borgpetersenii Serovar Hardjo in Refrigerated Raw Milk: A Transmission Risk of Leptospirosis to Humans

Leptospira borgpetersenii serovar Hardjo (LH) is an important infectious agent of reproduction pathologies and lactation decline in cattle, with a possible zoonotic role. To figure out the potential zoonotic risk for human raw-milk consumption, the present study aims at assessing the persistence and viability of LH in refrigerated raw milk over a 10-day period, which is set as the maximum time range for raw-milk domestic consumption. A negative sample of fresh raw milk was contaminated with an LH strain (2 × 108 Leptospires/mL) and analyzed by a rrs (16S) gene targeting real-time PCR (rPCR) protocol for LH DNA at days 1, 2, 3, 6, 7, 9, and 10. Seven aliquots of the same sampling time were inoculated into a semisolid EMJH media for bacterial culture. All aliquots tested positive in both rPCR and culture, which demonstrates that raw milk does not alter the detectability and viability of LH, respectively. The analytical sensitivity (LoD, limit of detection) determined for the rPCR (103 Leptospires/mL) was repeatable during the study, whereas it gradually decreased when it came to the bacterial culture. This study demonstrates that bovine raw milk might be a potential vehicle of infection by LH, even when storage conditions are strictly respected

Serological diagnosis of canine leishmaniosis : comparison of three commercial ELISA tests (Leiscan ®, ID Screen ® and Leishmania 96 ®), a rapid test (Speed Leish K ®) and an in-house IFAT

Speed Leish K ® is used as a serological screening test for Leishmania infection prior to vaccination. Limited comparative serological studies with Speed Leish K ® have been performed. The aim of this study was to evaluate the diagnostic performance of four commercially available serologic tests including ELISAs (Leiscan ®, ID Screen ® and Leishmania 96 ®), a rapid test (Speed Leish K ®) and an in-house IFAT for the detection of specific antibodies against Leishmania infantum antigen in dogs in different states of infection. Sick infected dogs (n = 36), healthy infected dogs (n = 18), L. infantum seropositive dogs with low to high levels of antibodies (n = 53), dogs seropositive to other pathogens (to evaluate cross reaction) (n = 14) and uninfected dogs from a non-endemic area (n = 50) and from an endemic area (n = 32) were analysed by the serological methods mentioned above. The sensitivity was as follows: ID Screen ® (0.953), Leiscan ® and Leishmania 96 ® (0.925), IFAT (0.869) and Speed Leish K ® (0.636). The maximum specificity (1.000) was attained for all diagnostic tests except the Leishmania 96 ® (0.896) and IFAT (0.917). The accuracy was as follows: ID Screen ® (0.975), Leiscan ® (0.961), Leishmania 96 ® (0.911), IFAT (0.892) and Speed Leish K ® (0.808). In relation to the area under the ROC curve (AUC-ROC), the maximum value was attained with the ID Screen ® (0.993) closely followed by Leiscan ® (0.990), then, Leishmania 96 ® (0.962), IFAT (0.926) and Speed Leish K ® (0.818). For the Kappa index, the best result was obtained by the ID Screen ® (0.951) followed by Leiscan ® (0.921), Leishmania 96 ® (0.822), IFAT (0.783) and Speed Leish K ® (0.622). Statistically significant differences were found between the AUC-ROC of quantitative serological tests and the only qualitative rapid test evaluated. There were also statistically significant differences between AUC-ROC of the ELISAs (ID Screen ® and Leiscan ®) and IFAT. Leiscan ® and ID Screen ® had superior diagnostic performance measures than IFAT and all quantitative serological tests were superior when compared to Speed Leish K ®. Thus, Speed Leish K ® may be considered a less valuable screening test prior to vaccination as it may result in vaccination of seropositive dogs and in some cases seropositive sick dogs

Molecular Investigation of Recent Canine Parvovirus-2 (CPV-2) in Italy Revealed Distinct Clustering

Canine parvovirus Type 2 (CPV-2) is a worldwide distributed virus considered the major cause of viral gastroenteritis in dogs. Studies on Italian CPV-2 are restricted to viruses circulating until 2017. Only one study provided more updated information on CPV-2 but was limited to the Sicily region. No information regarding the circulation and genetic characteristics of CPV-2 in Northeast Italy has been made available since 2015. The present study investigated the genetic characteristics of CPV-2 circulating in the dog population of Northeast Italy between 2013 and 2019. The VP2 gene of 67 CPV-2 was sequenced, and phylogenetic analysis was performed to identify patterns of distribution. Phylogenetic and molecular analysis highlighted unique characteristics of Northeast Italian CPV-2 and interestingly depicted typical genetic clustering of the Italian CPV-2 strains, showing the existence of distinct CPV-2 genetic groups. Such analysis provided insights into the origin of some Italian CPV-2 genetic clusters, revealing potential introductions from East European countries and the spread of CPV-2 from South/Central to North Italy. This is the first report that describes the genetic characteristics of recent Italian CPV-2. Tracking the genetic characteristics of CPV-2 nationally and globally may have impact on understanding the evolution and distribution of CPV-2, in particular in light of the current humanitarian emergency involving Ukraine, with the massive and uncontrolled movement of people and pet animals

IFAT and ELISA phase I/phase II as tools for the identification of Q fever chronic milk shedders in cattle

Q fever is a widespread zoonotic disease caused by Coxiella burnetii. In cattle the bacterial shedding can persist without symptoms for several months and the shedders identification is a critical issue in the control of the infection at herd level. Following the example of the human protocols for the assessment of Q fever infection status, the aim of this study was the evaluation of the antibody response dynamics to phase I and phase II antigens in C. burnetii shedder dairy cows by means of a phase-specific serology, to verify the suitability of the investigated tools in recognising milk shedders. A total of 99 cows were monitored during time and classified on the basis of serological and PCR results in five groups identifying different shedding patterns. The 297 sera collected in three sampling times were tested by means of ELISA IgG for differential phase I and phase II antibodies detection, while a selection of 107 sera were tested by means of phase specific IgM and IgG IFAT. Both ELISA IgG and IFAT IgG highlighted a low reactivity in non-shedder seropositive animals compared to chronic milk shedder animals. ELISA IgG seemed to perform better than IFAT IgG-IgM, showing significant serological differences among groups that allowed recognising specific serological group patterns, in particular for chronic and occasional milk shedders. These results supported the hypothesis that an animal classification based on phase patterns is reasonable, although it needs to be further investigated