Stray cats potentially act as reservoir for zoonotic agents, posing a risk of exposure to humans and domestic cats. The most prevalent Chlamydiaceae species in cats is Chlamydia (C.) felis, which is frequently associated with conjunctivitis and/or upper respiratory disease. The zoonotic potential of C. felis is believed to be relatively low, although exposure is possible through handling infected cats, by contact with their aerosol, and via fomites. Infection is more frequent in conditions of overcrowding, stress, poor hygiene and impairment of the immune system. For this reason, stray cats appear to be particularly susceptible to this pathogen. Aim of the study was to identify the molecular occurrence of Chlamydiaceae in stray and colony cats. Between May 2021 and June 2022, in seven provinces of northeastern Italy, veterinary services officers collected oropharyngeal swabs from 379 stray and colony cats. The samples were screened for Chlamydiaceae by real-time PCR targeting a 23S gene fragment. Positive samples were further analyzed either by a C. felis-specific qPCR or by amplification and sequencing of a 16S rRNA gene fragment. Overall, 7.7% of the cats tested positive for Chlamydia spp., and all were identified as C. felis. Among the positive individuals, only one exhibited respiratory symptoms. The analysis of anamnestic data revealed a significantly higher frequency of C. felis in male intact cats during the spring season, suggesting a potential behavioral aspect of this infection. Although the zoonotic risk of this Chlamydia species is low, it would be prudent to exercise caution when handling stray cats.
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.
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. 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. 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. 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.
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.
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. 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). 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.
SUMMARY Two outbreaks of Leptospira borgpetersenii serovar Hardjo infection in dairy cattle herds were managed through the application of enhanced biosecurity measures, whole-herd antibiotic treatment and vaccination. Micro-agglutination test antibody titres were determined in paired serum samples at 3 weeks (T 1 : n = 125, 97% seropositivity, median 800, range 100–12 800) and 24 weeks (T 2 : n = 110, 88% seropositivity, median 200, range 100–6400) after vaccination and studied in relation to cows' age, herd of origin and sampling time. From T 1 to T 2 , vaccine-elicited antibody titres decreased by 84·7% (95% CI 76·2–90·1). Consistent with increasing immunocompetence in calves (aged <12 months) and immunosenescence in adult cows (aged >36 months) associated with ageing, antibody titres correlated positively with calves' age and negatively with adult cows' age. No cow had cultivable, (histo)pathologically detectable and/or PCR-detectable leptospires in urine or kidney samples after treatment and vaccination. Vaccination together with proper biosecurity measures and chemoprophylaxis are an affordable insurance to control bovine leptospirosis.
Leptospirosis is a widespread disease throughout the world, presenting in severe clinical forms in dogs. The pathogenicity of the different serovars in field infections is not fully documented, and clinical diagnosis is often limited to a combination of serological tests and molecular analyses. The latter, although a fundamental tool, cannot identify the infecting strain without further analysis. This study reports the use of various indirect (microscopic agglutination test, MAT) and direct (microbiological culture, real-time PCR) laboratory techniques, followed by typing protocols (Multi-locus Sequence Typing (MLST), Multiple Loci Variable number tandem repeat Analysis (MLVA), serotyping) that allowed for the identification of the Leptospira serovar Australis in a symptomatic and previously vaccinated dog (vaccine containing heterologous strains). This study reports long-term clinical follow-up (0–640 days) and describes the possible role of the infection in the development of chronic renal failure. This study aims to highlight how a combination of different techniques can be useful to better characterise the environmental circulation of zoonotic agents. Therefore, the identification and isolation of circulating L. strains would facilitate the updating of epidemiological data, enhance the knowledge of pathogenicity and long-term clinical effects, and provide a valuable resource for improving the efficacy of a specific serovar vaccination.
