New distribution patterns of Dirofilaria immitis in Italy.

In recent decades, the number of autochthonous cases and foci of Dirofilaria immitis in dogs from southern regions has increased considerably, suggesting that the distribution of the species is not limited to northern Italian regions. This epidemiological picture emerges from case reports or studies in specific locations where outbreaks of heartworm disease have occasionally been reported together with the presence of mosquito vectors. To obtain a more comprehensive picture of the current distribution of D. immitis in southern Italy, a multicenter cross-sectional survey of canine filariasis was conducted. Owned and sheltered dogs (n = 1,987) were included in the survey regardless their breed, attitude and/or sex. All included dogs were older than 1 year and had no history of chemoprophylactic treatment against filarioses. A blood sample was collected from enrolled dogs and screened by modified Knott's test and, when positive, tested using D. immitis specific ELISA rapid test (SNAP 4DX, IDEXX). The overall microfilaremia prevalence was 17% (n = 338) being single-species infection (92.6%) more common that mixed (7.4%). Remarkably, D. immitis was the most frequent species detected with an overall prevalence of 11.4% (n = 227), followed by Dirofilaria repens (n = 74; 3.7%), and Acanthocheilonema reconditum (n = 12; 0.6%). Sheltered dogs were significantly more infected by D. immitis, as well as mongrel dogs and animals housed in rural areas. Data here reported indicate that D. immitis is largely present in southern Italy, raising awareness about the necessity of proper screening and chemoprophylactic treatments in exposed animals.

Integration of Microscopic, Serologic and Molecular Techniques for Detection of Filarial Parasites in Dogs in Malaysia.

PURPOSE: Canine filariosis in domestic dogs caused by several species of filarids is an emerging vector-borne disease and the spread of this disease remains a global veterinary and public health concern. However, information regarding these filarids and their epidemiological patterns remains scarce in Malaysia. The present study aimed to determine the infection rate and associated risk factors of filarial parasites in dogs in Malaysia. METHODS: A total of 399 dog blood samples were collected from veterinary hospitals and animal shelters in Malaysia to determine the infection rate and associated risk factors via a combination of microscopic, serologic and molecular diagnostic techniques. RESULTS: Two species of canine filariae identified in this study were Dirofilaria immitis (6.5%) and Brugia pahangi (1.3%), and their infections were associated with cross breed, medium size and short hair (p < 0.05). CONCLUSIONS: A new pair of primers was developed to complement the recovery of the 12S rRNA gene fragment of filarial parasites. This study represents the first molecular evidence of B. pahangi in dogs in Malaysia.

Probe-based qPCR as an alternative to modified Knott's test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests.

BACKGROUND: Current recommendations for diagnosis of Dirofilaria immitis infection in dogs rely on the detection of antigen produced largely by adult females coupled with the visualization of microfilariae (mf) in the circulation via a microfilaria detection test (MFDT). It is hypothesized that qPCR assays used in parallel with antigen detection tests will perform better in detecting mf than modified Knott's test (MK), when combined with antigen detection. This study compares probe-based qPCR and MK techniques for mf detection used in parallel with the DiroCHEK® antigen test to screen for heartworm infection in shelter dogs. METHODS: Matching blood and serum samples were collected from 300 shelter dogs in Brazos and Harris counties, Texas, USA. Blood was assessed for the presence of mf via MK and the presence of D. immitis DNA by a species-specific probe-based qPCR assay. Serum samples were tested for the presence of heartworm antigen using DiroCHEK® before and after immune complex dissociation (ICD) via heat treatment. In addition, the performance of each diagnostic test was evaluated via Chi-square test, Cochran's Q test, and post hoc analysis. RESULTS: Qualitatively, MK detected mf in 22.0% (66/300) of samples, 55 of which were morphologically identified as D. immitis and 11 as Acanthocheilonema reconditum. The range of heartworm mf was 28 to 88,803 mf/ml (median: 6627.5). Real-time PCR detected D. immitis DNA in 20.7% (62/300) of samples. Heartworm antigen was detected in 24.7% (74/300) of samples pre-ICD, and in 29.3% (88/300) post-ICD. When comparing tests, the Chi-square and McNemar's tests showed that the difference between positive and negative proportions was statistically significant. The Cochran test showed the difference in the distributions of cases and non-cases was significant when individual tests were combined (χ2 = 62.3, df = 3, P < 0.0001) and when parallel methods were combined (χ2 = 43.1, df = 4, P < 0.0001). CONCLUSION: Considering individual and combined test performances, practicality, and efficient use of bench time, this heartworm-specific probe-based qPCR method is a viable option as a mf detection test to be used in parallel with antigen tests for canine heartworm infection in diagnostic and research settings.

Development of a multiplex qPCR-based approach for the diagnosis of Dirofilaria immitis, D. repens and Acanthocheilonema reconditum.

BACKGROUND: Dirofilaria immitis, D. repens and Acanthocheilonema reconditum are the main causative agents of zoonotic canine filariosis. METHODS: We developed a combined multiplex approach for filaria and Wolbachia detection using the 28S-based pan-filarial and 16S-based pan-Wolbachia qPCRs, respectively, involving a fast typing method of positive samples using triplex qPCR targeting A. reconditum, D. immitis and D. repens, and a duplex qPCR targeting Wolbachia of D. immitis and D. repens. The approach was complemented by a duplex qPCR for the differential diagnosis of heartworms (D. immitis and Angiostrongylus vasorum) and pan-filarial cox1 and pan-Wolbachia ftsZ PCRs to identify other filarial parasites and their Wolbachia, respectively. A total of 168 canine blood and sera samples were used to validate the approach. Spearman's correlation was used to assess the association between filarial species and the strain of Wolbachia. Positive samples for both the heartworm antigen-test after heating sera and at least one DNA-positive for D. immitis and its Wolbachia were considered true positive for heartworm infection. Indeed, the presence of D. repens DNA or that of its Wolbachia as well as A. reconditum DNA indicates true positive infections. RESULTS: The detection limit for Wolbachia and filariae qPCRs ranged from 5 × 10-1 to 1.5 × 10-4 mf/ml of blood. When tested on clinical samples, 29.2% (49/168) tested positive for filariae or Wolbachia DNA. Filarial species and Wolbachia genotypes were identified by the combined multiplex approach from all positive samples. Each species of Dirofilaria was significantly associated with a specific genotype of Wolbachia. Compared to the true positives, the approach showed excellent agreement (k = 0.98-1). Unlike D. immitis DNA, no A. vasorum DNA was detected by the duplex qPCR. The immunochromatographic test for heartworm antigen showed a substantial (k = 0.6) and a weak (k = 0.15) agreements before and after thermal pre-treatment of sera, respectively. CONCLUSIONS: The proposed approach is a reliable tool for the exploration and diagnosis of occult and non-occult canine filariosis. The current diagnosis of heartworm disease based on antigen detection should always be confirmed by qPCR essays. Sera heat pre-treatment is not effective and strongly discouraged.