Validation of a new immunofluorescence antibody test for the detection of Leishmania infantum infection in cats.

The prevalence data of Leishmania infantum infection in cats are characterized by a large variability mainly attributed to the differences in diagnostic techniques. In the absence of consensus about the method of choice for diagnosing feline leishmaniosis, the performance of a new immunofluorescence antibody test (IFAT) was herein analytically described by the comparison with IFAT commonly used for the diagnosis of canine leishmaniosis (i.e., IFAT-OIE) and a laboratory enzyme-linked immunosorbent assay (ELISA). Sera of cats living in visceral leishmaniosis-endemic (n = 105) and visceral leishmaniosis-non-endemic (n = 50) areas were tested by the above methodologies and real-time PCR (qPCR). The most frequent result was represented by triple negativity to the three tests (IFAT-OIE, ELISA, and qPCR) in 42.9% and 80% cats from endemic and non-endemic areas, respectively. Bayes latent class analysis gave an output probability of 34.1% (posterior standard deviation, psd = 5.4%) of true L. infantum cases (TCL) which represent the true estimated prevalence of infection. The sensitivity of each variable contributing to define the TCL was 24% (psd = 6.3%) for qPCR, 78.8% (psd = 8.7%) for ELISA and 91.8% (psd = 5.2%) for IFAT-OIE. The probability to be a TCL was 94.5% for the sample from an endemic area. The cross-validation of the new IFAT by a logistic model correctly identified as positive 80.7% of subjects defined as TCL and negative 89.9% as not TCL, respectively, by the Bayesian model. The study results estimate a good accuracy of the IFAT in predicting cats exposed to L. infantum. Therefore, this procedure may be beneficial for screening cat populations for a better understanding of the epidemiology of feline leishmaniosis.

Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds.

BACKGROUND: Bovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs. RESULTS: The sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively). CONCLUSIONS: The ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance.

Identification of the most effective serovars to be included in the MAT antigen panel to optimize the serodiagnosis of Leptospira infection in Northern Italy.

The microscopic agglutination test (MAT) assay is adopted as a world-wide reference test for the serodiagnosis of leptospirosis in humans and animals. One of the main limitations of MAT is the lack of sensitivity and serodiagnostic antigens should be periodically updated with locally circulating serovars in order to optimise its performance. The aim of this study was to determine the need to implement the antigen panel currently adopted in Northern Italy for the diagnosis of Leptospira infection in dogs. For this purpose, a group of 288 dogs with and without clinical signs potentially consistent with Leptospira infection or found to have an increased C-reactive protein (CRP) serum concentration, sampled in 2013-2016 in Northern Italy, were tested by MAT comparing the results obtained with a nine antigens panel (Australis-Bratislava, Ballum-Ballum, Canicola-Canicola, Grippotyphosa-Grippotyphosa, Icterohaemorrhagiae-Copenhageni, Icterohaemorrhagiae-Icterohaemorrhagiae, Sejroe-Hardjo, Pomona-Pomona and Tarassovi-Tarassovi serovars) routinely adopted and a panel expanded to 27 antigens. In general, the antigen panel currently adopted in Northern Italy for the routine MAT assay resulted adequate for the diagnosis of Leptospira infection in dogs. The main exception concerns the Sejroe serogroup, with the Saxkoebing and Sejroe serovars that were more effective than Hardjo for diagnosis in dogs and whose inclusion in the antigen panel is recommended. Among other antigens evaluated in this study, Cynopteri serovar was detected with high frequency but its pathogenic role in dogs and as public health threat deserve further investigation.

Synanthropic and Wild Animals as Sentinels of Zoonotic Agents: A Study of Leptospira Genotypes Circulating in Northeastern Italy.

Leptospirosis is an infectious disease widely reported in veterinary practice and a worldwide zoonosis. In Northeastern Italy, different serogroups and genotypes of Leptospira have been described in ill dogs, the most commonly detected being Icterohaemorragiae (ICT) ST 17, Australis (AUS) ST 24 and ST 198, Pomona (POM) ST 117 and ST 289, and Sejroe (SEJ) ST 155. However, there is little information available on the environmental exposure to Leptospira of wild and synanthropic animals. The aim of this study was to identify the circulating genotypes in potential reservoirs to fill this gap of knowledge. Between 2015 and 2022, 681 animal carcasses collected by the Public Veterinary Service were analyzed for Leptospira with a real-time PCR-based screening test, while positive samples were genotyped by multi-locus sequence typing analysis. To carry out our study, we tested 330 hedgehogs, 105 red foxes, 108 Norway rats, 79 mice, 22 coypus, 10 bank voles, 13 grey wolves, 5 common shrews and 9 greater mouse-eared bats. Five sequence types (STs) common in dogs were also found in wild animals: ST 24, ST 198, ST 17 and ST 155 in hedgehogs, ST 17 and ST 24 in foxes, ST 17 in rats, ST 17 and ST 155 in mice, and ST 117 in a wolf. In addition, to the best of the authors' knowledge, this is the first Italian report of SEJ ST 197 in a bank vole. Furthermore, this study described a previous survey conducted in 2009 on coypus (30 animals from the province of Trento and 41 from the province of Padua), referring to a serological positivity (L. Bratislava) without any molecular detection of Leptospira. This study on Leptospira in synanthropic and wild animals highlighted the importance of increasing our epidemiological knowledge of leptospirosis and its zoonotic risks.

Feline Susceptibility to Leptospirosis and Presence of Immunosuppressive Co-Morbidities: First European Report of L. interrogans Serogroup Australis Sequence Type 24 in a Cat and Survey of Leptospira Exposure in Outdoor Cats.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. The role of the cat as a susceptible host and potential environmental reservoir of Leptospira is still not well understood, due to the lack of obvious clinical signs associated with Leptospira spp. infection in this species. This study aims to describe the first European detection of Leptospira interrogans serogroup Australis ST 24 in a young outdoor cat with a severe comorbidity (feline panleukopenia virus). In addition, the results of a preliminary study conducted in 2014-2016 are presented (RC IZSVE 16/12), which reports an investigation of Leptospira exposure of outdoor cats in Northeast Italy by means of serological investigation and molecular evaluation of urine. The animals included in the survey are part of samples collected during active and passive surveillance (diagnostic samples). The study reported a seroprevalence of 10.5% among outdoor cats and the serogroups identified were Grippotyphosa, Icterohaemorrhagiae, Bratislava, Canicola and Ballum. Symptomatic cats reported high MAT titres (ranging from 1:800 to 1:1600) towards antigens belonging to the serovars Grippotyphosa (1:800), Bratislava (1:1600), Icterohaemorrhagiae (1:200) and Copenhageni (1:200-1:800). In one subject, urine tested positive for Leptospira PCR. Cats with high antibody titres for Leptospira and/or positivity on molecular test suffered from immunosuppressive comorbidities (feline immunodeficiency virus and feline leukaemia virus; feline herpesvirus and lymphoma; hyperthyroidism). The overall prevalence of serum antibodies against Leptospira found in free-ranging cats (10.53%, 95% CI: 4.35-16.70%) and the identification of L. interrogans ST 24 in a young cat with immunosuppressive disease (feline panleukopenia virus) suggest the possibility of natural resistance to clinical leptospirosis in healthy cats. In a One Health perspective, further studies are needed to better define the pathogenesis of leptospirosis in cats and their epidemiological role as environmental sentinels or possible carriers of pathogenic Leptospira.

Are Small Animal Practitioners Occupationally Exposed to Leptospirosis? Results of a Serological Survey.

Leptospirosis is a worldwide zoonosis frequently responsible for clinical disease in dogs and rarely reported in human people. The risk of human exposure to Leptospira has been investigated in a sample population working in the northeast of Italy, a geographical area with high endemicity of canine leptospirosis. Two-hundred twenty-one human serum samples were analyzed for Leptospira microagglutination test (MAT): 112 clinical freelance small animal practitioners (exposed subjects) and 109 people not occupationally exposed to Leptospira-infected animals (unexposed subjects) were voluntarily enrolled. Despite the previously reported serological detection of antibodies vs. Leptospira in people in different Italian regions, this study did not detect any reactivity in the investigated population. This study shows that veterinarians do not appear to be at a greater risk of leptospirosis than the reference population. This may be due to both veterinarian awareness of the Leptospira zoonotic risk and the efficiency of the preventive measures and management of patients. Moreover, it could be the result of the relatively low excretion of Leptospira in symptomatic dogs, which can be considered as an environmental sentinel for Leptospira presence rather than a vehicle of transmission.

Outbreak of Leptospira borgpetersenii Serogroup Sejroe Infection in Kennel: The Role of Dogs as Sentinel in Specific Environments.

Kennels may represent high-risk environments for the diffusion of Leptospira infection in dogs and consequently a threat to public health. This study describes an outbreak of Leptospira infection in a kennel in Italy in 2020, both with clinically ill and asymptomatic dogs. Fifty-nine dogs, including three ill dogs, were tested for Leptospira spp. infection by the microscopic agglutination test (MAT) and real-time qPCR. Multi-locus sequence typing (MLST) analysis was used to genotype the identified leptospires. Thirty of the fifty-nine (50.9%) dogs had MAT titer and/or molecular positivity indicative of Leptospira infection. Twenty-two of the fifty-nine (37.3%) dogs exhibited seropositivity against at least one serovar belonging to the Sejroe serogroup, and MLST analysis identified L. borgpetersenii serogroup Sejroe (Leptospira ST155) as responsible for the outbreak. Up to now, Sejroe serogroup infection was sporadically reported in dogs. The extension of the MAT antigen panel to several serovars belonging to the serogroup Sejroe could be useful in the diagnosis of canine leptospirosis. Dogs may serve as sentinel of leptospires in specific environments, and surveillance of Leptospira infection in kennels is strongly recommended even when the correct vaccine prophylaxis is administered, because the vaccines currently available are not able to protect from all of the serogroups.

One-Year Surveillance of SARS-CoV-2 Exposure in Stray Cats and Kennel Dogs from Northeastern Italy.

Dogs and cats are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). During the pandemic, several studies have been performed on owned cats and dogs, whereas limited data are available on the exposure to stray animals. The objective of this study was to investigate the exposure to SARS-CoV-2 of feral cats and kennel dogs in northeastern Italy, through serological and molecular methods. From May 2021 to September 2022, public health veterinary services collected serum, oropharyngeal, and rectal swab samples from 257 free-roaming dogs newly introduced to shelters, and from 389 feral cats examined during the routinely trap-neutered-return programs. The swabs were analyzed for viral RNA through a real-time reverse transcriptase PCR (rRT-PCR), and sera were tested for the presence of the specific antibody against SARS-CoV-2 (enzyme-linked immunosorbent assay). Serology was positive in nine dogs (9/257) and three cats (3/389), while two asymptomatic cats tested positive to rRT-PCR. One cat turned out to be positive both for serology and molecular analysis. In addition, this study described the case of a possible human-to-animal SARS-CoV-2 transmission in a cat that travelled in close contact to a COVID-19-positive refugee from Ukraine. This study shows that SARS-CoV-2 can infect, in natural conditions, stray cats and kennel dogs in northeastern Italy, although with a low prevalence.

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.

Environmental Exposure of Wild Carnivores to Zoonotic Pathogens: Leptospira Infection in the First Free Living Wolf (Canis lupus Linnaeus, 1758) Found Dead in the Friuli Venezia Giulia Region.

Leptospirosis is a worldwide-spread zoonosis causing disease and death in dogs and in humans. A Leptospiral infection has been recorded in several wild carnivore species in Europe, but tissue pathological changes were not commonly described. The Grey wolf (Canis lupus) has been expanding its distribution range in north-eastern Italy during the last decade. A young wolf, representing the first individual handled in the region, was found road-killed and then submitted to necropsy. Pathological changes included erosive lesions of gingival mucosa, mild liver enlargement, and multifocal degenerative-necrotic areas along with hyperemic reactive lesions; multifocal interstitial nephritis and multifocal lung hemorrhages were observed. A Polymerase Chain Reaction (PCR) able to detect pathogenic species of Leptospira performed on a kidney sample was positive. Serological reactions for serogroup Gryppotyphosa (1:6400), Pomona (1:800), and Icterohaemorrhagiae (1:200) were evidenced by MAT. Genotyping by Multilocus Sequence Typing (MLST) performed on detected Leptospira characterized it as belonging to Sequence Type (ST) 117, which refers to L. kirschneri, serogroup Pomona, serovar Mozdok. Regardless of the role of Leptospira infection as an eventual predisposing factor to the road killing of this wolf, to the best of the authors' knowledge, this is the first report of Leptospira-induced pathology in a wolf in Europe. Surveys on Leptospira infection in free-ranging wildlife species should be pursued in order to achieve further epidemiological knowledge on the circulation of the Leptospira strain.

Do modified live virus vaccines against bovine viral diarrhea induce fetal cross-protection against HoBi-like Pestivirus?

Bovine Pestivirus heterogeneity is a major challenge for vaccines against bovine viral diarrhea (BVD). In breeding herds, fetal protection is a high priority issue. To some degree, fetal infections in vaccinated heifers have been attributed to the antigenic diversity of bovine Pestiviruses. The purpose of this study was to assess fetal protection against a divergent bovine Pestivirus (Hobi-like Pestivirus, HoBiPeV) with a commercially available modified live vaccine (MLV) claiming fetal protection against BVDV 1 and BVDV 2 up to one year after the first inoculation. Five vaccinated and four unvaccinated heifers were challenged by intranasal inoculation with the HoBiPeV Italy-1/10-1 strain between 82 and 89 days after insemination, i.e. between 4 and 6 months after vaccination. At challenge, neutralizing antibody titers to HoBiPeV in vaccinated heifers were low or even undetectable. Of the four unvaccinated heifers, one control animal aborted (fetus not available) and the remaining three gave birth to HoBiPeV positive calves. Among the heifers of the vaccinated group, one aborted the fetus in the sixth month of pregnancy, which tested Pestivirus negative, while three others gave birth to healthy, HoBiPeV negative calves; the remaining heifer delivered one HoBiPeV positive calf. The results suggest that the BVDV vaccine might be able to elicit a partial fetal protection against HobiPeV, even in absence of a strong specific antibody response.

Detection of New Leptospira Genotypes Infecting Symptomatic Dogs: Is a New Vaccine Formulation Needed?

Leptospirosis in dogs has been largely described worldwide, and epidemiological studies have been mainly based on serological data. This study aims to detect and genotype leptospires affecting symptomatic dogs in Northeast Italy between 2013 and 2019. Overall, 1631 dogs were tested using real-time PCR, and leptospires from 193 dogs were subjected to Multilocus Sequence Typing and a Multiple Loci Variable-number Tandem Repeat Analysis. Leptospires were successfully isolated from 15 symptomatic dogs. Six distinct Sequence Types (STs) were found for 135 leptospires, with 3 STs characterizing Leptospira interrogans (ST17, ST198 and ST24), 2 STs characterizing Leptospira kirschneri (ST117 and ST289) and 1 ST characterizing Leptospira borgpetersenii (ST155), revealing the circulation of the serogroups Icterohaemorrhagiae, Australis, Sejroe and Pomona. The Multiple Loci Variable-number Tandem Repeat Analysis of 17 samples did not result in any additional discrimination. Genotypes were compared with those of strains present in the historical internal database, and possible transmission chains were identified from rat, mouse, hedgehog and pig. This work highlights the importance of molecular methods in revealing and identifying circulating Leptospira strains, and it also encourages the evaluation of the ability of commercially available vaccines to reduce the disease burden among dogs.

Canine leishmaniosis in the Italian northeastern Alps: A survey to assess serological prevalence in dogs and distribution of phlebotomine sand flies in the Autonomous Province of Bolzano - South Tyrol, Italy.

The Autonomous Province of Bolzano-South Tyrol (APB), located in the northernmost territory of the Italian eastern Alps, is still considered non-endemic for canine leishmaniosis (CanL) despite clinical cases being observed and a competent Leishmania infantum vector (Phlebotomus perniciosus) having been recorded since 2008. A serological survey of leishmaniosis among a randomly-selected subpopulation of registered owned dogs was carried in 2018, followed by entomological investigations performed in 2019 and driven by canine survey results. A total of 457 resident dogs from all over the APB territory were examined through IFAT for antibodies against L.infantum, of which 63 (13.8%) tested positive. Thirty-five seropositive cases (7.7%) were considered autochthonous to APB, i.e. dogs born and lived in the province, or imported dogs with no travel history in the past 5 years. Most of these animals showed an antibody titre at the threshold level of 1:40, suggesting a low degree of parasite transmission/contacts. In 2 autochthonous cases with moderately high IFAT titre, the infection was confirmed by nested-PCR in peripheral blood. Thirty-one georeferenced sites were monitored for sand flies by means of interception (sticky papers) and attraction (CDC miniature light traps) collection devices. Traps were set during summer approximately on monthly basis, and extended up to October for positive sites. Only 2 sites were found positive for a total of 317 phlebotomine specimens collected by sticky traps, which included a previously known P. perniciosus-endemic site near Bolzano town. Sergentomyia minuta was by far the most prevalent (98.1%) and the only recorded sand fly species in the most northerly Italian site ever investigated (Coldrano municipality in Venosta valley). For the first time, Leishmania serology and n-PCR positive dogs autochthonous to APB were identified, however the spread of sand flies competent for L. infantum transmission could not be demonstrated in several places where endemic seropositive cases were recorded. APB can be considered a territory of low CanL endemicity, however awareness and continuous monitoring are needed to detect changes in the epidemiological status of the zoonosis.

Survival of Rickettsia conorii in artificially contaminated whole and leukoreduced canine blood units during the storage period.

BACKGROUND: The ability of tick-borne agents to survive in stored blood bags is a key factor for their transmissibility by blood transfusion. The aim of this study was to evaluate the survival and potential infectivity of Rickettsia conorii (RC) in artificially contaminated canine whole blood (WB) and in leukoreduced whole blood (LR-WB) during the storage period. METHODS: RC was cultured on L929 cells. We used a one-week 25-cm2 flask with 70-80% of L929 infected cells to prepare the bacterial inoculum by pelleting cells and suspending the pellet in the donors' serum. We infected five 100 ml WB units with RC within 2 h from the collection and maintained it at room temperature for 4 h prior to refrigeration. We filtered 50 ml of each WB bag to obtain leukoreduced WB (LR-WB) at day 1 post-infection (dpi). We checked WB and LR-WB bags at 1, 4, 7, 14, 21, 28, 35 dpi for RC presence and viability through real-time PCR (rPCR) for DNA and mRNA, respectively, and by isolation. Identification of isolates was confirmed by indirect immunofluorescence and rPCRs. RESULTS: RC survived for the entire storage period in both whole and leukoreduced blood. All bags contained viable bacteria until 7 dpi; RC viability generally decreased over time, particularly in LR-WB bags where the isolation time was longer than in WB. Viable bacteria were still isolated at 35 dpi in 3 WB and 3 LR-WB. CONCLUSIONS: Leukoreduction reduced but did not eliminate RC in infected units. The survival and infectivity of RC in canine blood during the storage period may represent a threat for recipients.

Surveillance of Infectious Bovine Rhinotracheitis in marker-vaccinated dairy herds: Application of a recombinant gE ELISA on bulk milk samples.

Infectious Bovine Rhinotracheitis (IBR) occurs worldwide, requiring significant resources for eradication programs or surveillance purposes. The status of infection is usually detected by serological methods using the virus neutralization test (VNT) or enzyme-linked immunosorbent assay (ELISA) on individual sera. The gE DIVA (Differentiating Infected from Vaccinated Animals) vaccines approach, adopted in order to reduce the virus circulation and prevent clinical signs, have tightened the range of available methods for the serological diagnosis. Different gE blocking ELISA could be performed to detect specific antibodies in sera of infected or whole virus-vaccinated animals but with less sensitivity if applied to bulk milk samples, especially in marker-vaccinated herds. A new rec-gE ELISA was recently developed in Italy and applied with good performances on blood serum samples. The present paper focuses on the application of a rapid protocol for purification/concentration of immunoglobulin G (IgG) from bulk milk and on the use of the new rec-gE indirect ELISA. The study involved three different partners and 225 herds (12,800 lactating cows) with different official IBR diagnostic statuses. The diagnostic specificity of the method was demonstrated closed to 100% while the diagnostic sensitivity was strictly related to the herd-seroprevalence. Considering 2.5% as the limit of detection of within-herd seropositivity prevalence, the diagnostic sensitivity showed by the proposed method was equal to 100%. A single reactivation of a whole strain vaccine in an old cow was detected inside a group of 67 lactating cows, showing the field applicability of the method.

Seroprevalence study of the main causes of abortion in dairy cattle in Morocco.

Sera from 221 cattle were collected in 25 farms in Morocco to investigate the evidence and circulation of some of the main bovine abortive agents in the dairy cattle farming, where abortions are often reported. All sera were examined for brucellosis, 176 for neosporosis, 88 for leptospirosis, and 42 for Bovine viral diarrhoea (BVD/MD), Bovine Herpesvirus 1 (BHV-1) (Infectious bovine rhinotracheitis, IBR/IPV), and Bovine Herpesvirus 4 (BHV-4) infections (at least 1 sample per herd). Abortions were reported in 23 (10.4%) of the 221 tested cattle. Antibodies against the investigated pathogens were detected in all samples tested, with an overall seroprevalence of 33.48% for Brucella, 9.09% for Leptospira, 8.52% for Neospora, 37.71% for BVDV, 50% for BHV-1, 9.52% for BHV-4. As for Leptospira antibodies against serovars Hardjo, Pomona, and Tarassovi were identified. Mixed infections were common. The lack of evidence of non-infectious factors epidemiologically related to abortions suggested that the investigated agents are to be considered important risk factors in the dynamic of the abortion syndrome, even if further investigations are necessary to identify the abortion cause. Particular attention should be paid on brucellosis, considering the high seroprevalence and its zoonotic relevance.

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

Exposure to vector-borne pathogens in candidate blood donor and free-roaming dogs of northeast Italy.

BACKGROUND: Many vector-borne pathogens including viruses, bacteria, protozoa and nematodes occur in northeast Italy, representing a potential threat to animal and human populations. Little information is available on the circulation of the above vector-borne pathogens in dogs. This work aims to (i) assess exposure to and circulation of pathogens transmitted to dogs in northeast Italy by ticks, sandflies, and mosquitoes, and (ii) drive blood donor screening at the newly established canine blood bank of the Istituto Zooprofilattico Sperimentale delle Venezie. METHODS: Blood samples from 150 privately-owned canine candidate blood donors and 338 free-roaming dogs were screened by serology (IFA for Leishmania infantum, Ehrlichia canis, Anaplasma phagocythophilum, Babesia canis, Rickettsia conorii, R. rickettsii), microscopic blood smear examination, and blood filtration for Dirofilaria spp. All candidate donors and seropositive free-roaming dogs were tested by PCR for L. infantum, E. canis, A. phagocythophilum, Babesia/Theileria and Rickettsia spp. The dogs had no clinical signs at the time of sampling. RESULTS: Overall, 40 candidate donors (26.7 %) and 108 free-roaming dogs (32 %) were seroreactive to at least one vector-borne pathogen. Seroprevalence in candidate donors vs free-roaming dogs was: Leishmania infantum 6.7 vs 7.1 %; Anaplasma phagocytophilum 4.7 vs 3.3 %; Babesia canis 1.3 vs 2.7 %; Ehrlichia canis none vs 0.9 %; Rickettsia conorii 16 vs 21.3 % and R. rickettsii 11 vs 14.3 %. Seroreactivity to R. rickettsii, which is not reported in Italy, is likely a cross-reaction with other rickettsiae. Filariae, as Dirofilaria immitis (n = 19) and D. repens (n = 2), were identified in free-roaming dogs only. No significant differences were observed between candidate donors and free-roaming dogs either in the overall seroprevalence of vector-borne pathogens or for each individual pathogen. All PCRs and smears performed on blood were negative. CONCLUSIONS: This study demonstrated that dogs are considerably exposed to vector-borne pathogens in northeast Italy. Although the dog owners reported regularly using ectoparasiticides against fleas and ticks, their dogs had similar exposure to vector-borne pathogens as free-roaming dogs. This prompts the need to improve owner education on the use of insecticidal and repellent compounds in order to reduce the risk of arthropod bites and exposure to vector-borne pathogens. Based on the absence of pathogens circulating in the blood of healthy dogs, the risk of transmission of these pathogens by blood transfusion seems to be low, depending also on the sensitivity of the tests used for screening.

A sero-survey of major infectious causes of abortion in small ruminants in Morocco.

A serological survey was conducted to estimate the seroprevalence of 5 major abortive infections in 13 sheep flocks and 10 goat herds in 2 regions of Morocco. A total of 308 from aborted females (202 ewes and 106 does) and 197 sera (97 ewes and 99 does), were tested for brucellosis, chlamydiosis, Q fever, toxoplasmosis, and for 9 major serovars of Leptospira. An average abortion rate of 12.10% was found in ewes and 10.26% in does. The serological analyses revealed the presence of all 5 abortive infections, both in sheep and in goats. Ten (43%) herds/flocks were positive to brucellosis, 21 (91%) to chlamydiosis, 17 (74%) to toxoplasmosis, 13 (57%) to Q fever, and 5 (22%) to leptospirosis. Leptospira spp. serovars Copenhageni and Grypothyphosa were found in a single sheep flock, while Tarassovi and Copenhageni were detected in 4 goat herds. Of the 23 investigated herds/flocks, 22 (96%) showed mixed infections. The findings of this study confirmed the possible involvement of the 5 selected abortive infections in abortion outbreaks occurring in the investigated regions. Further investigations are needed to better understand the aetiology of infectious abortions in herds and flocks within investigated regions.

Molecular characterization by MLVA of Coxiella burnetii strains infecting dairy cows and goats of north-eastern Italy.

Q fever is a worldwide zoonotic disease caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. In ruminants, shedding into the environment mainly occurs during parturition or abortion, but the bacterium is shed also in milk, vaginal mucus, stools and urine. In Italy few surveys have been conducted and reported seroprevalence values ranged between 10% and 60%, even if few human cases have been described. Genotyping of bacteria is crucial for enhancing diagnostic methods and for epidemiological surveillance. The objective of this study was to investigate genotypic differences of C. burnetii genotypes directly in 34 samples, collected during a 3-years survey among 11 dairy cattle and 11 goat farms in the north-eastern part of Italy using a 6-locus multiple loci variable number of tandem repeat analysis (MLVA) method. The samples analysed included 13 bulk tank milk (BTM), 6 individual milk, 11 vaginal swabs and 4 foetal spleens. MLVA-type 2 was determined as the most prevalent in cattle in this study. C. burnetii strains circulating in the studied cattle population are very similar to genotypes previously described, while genotypes from goats showed an important variability. Further investigation are needed to understand the reason of this pattern.