Varroa destructor resistance to tau-fluvalinate: relationship between in vitro phenotypic test and VGSC L925V mutation.

BACKGROUND: Varroa destructor is a parasitic mite of the honey bee, Apis mellifera. Its presence in colonies can lead to a collapse within a few years. The use of acaricides has become essential to manage the hive infestation. However, the repeated and possibly incorrect use of acaricide treatments, as tau-fluvalinate, has led to the development of resistance. The in vitro phenotypic test allows the proportion of susceptible or resistant individuals to be known following an exposure to an active substance. In Varroa mites, resistance to tau-fluvalinate is associated with the presence of mutations at the position 925 of the voltage-gated sodium channel (VGSC). RESULTS: Here, we compared the results obtained with an in vitro phenotypic test against tau-fluvalinate and those obtained with an allelic discrimination assay on 13 treated and untreated Varroa populations in France. The correlation between the phenotype and the genetic profile rate is found to be 0.89 Varroa mites having resistant phenotypic profile have a probability of 63% to present the L925V mutation (resistance detection reliability). However, 97% of the Varroa mites having the susceptible phenotype do not present the L925V mutation (susceptible detection reliability). CONCLUSION: The L925V mutation explains most of the resistance to tau-fluvalinate in V. destructor in the populations tested. However, other mutations or types of resistance may also be involved to explain the survival of Varroa mites in the phenotypic test. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens.

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.

Rapid Identification of Tropilaelaps Mite (Mesostigmata: Laelapidae) Species Using a COI Barcode-HRM.

Tropilaelaps mite (Mesostigmata: Laelapidae) is an ectoparasite of bees present, to date, only on the Asian continent. In the context of the threat of Tropilaelaps's introduction into new regions, accurate, rapid, and sensitive detection of the Tropilaelaps spp. is essential. In the present study, we developed a novel molecular method for bee mite's identification, which consists of a new real-time PCR method. A high-resolution melting analysis (HRM) was then performed on the amplified products to differentiate the species. PCR amplification was applied on the cytochrome c oxidase subunit I gene (580 bp). Short fragments from the most variable regions of this gene were identified in silico to amplify and discriminate among the four Tropilaelaps species. Four reference plasmids were synthesized to characterize species by well-distinguished melting curves. The method was then validated in terms of its specificity and sensitivity using a panel of 12 specimens. The results showed that an HRM method can be applied for the intended objective: for rapid and simultaneous identification of Tropilaelaps species. To our knowledge, this study reports the first direct HRM assay developed for the genome of a bee mite, specific for Tropilaelaps species. This COI barcode-HRM technique could be a promising tool for mite species identification.

Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison.

The Small Hive Beetle (Aethina tumida Murray, 1867) is an invasive scavenger of honeybees. Originally endemic in sub-Saharan Africa, it is regulated internationally in order to preserve the areas still free from this species. To ensure the reliability of official diagnoses in case of introduction, an inter-laboratory comparison was organised on the identification of A. tumida by morphology and real-time PCR. Twenty-two National Reference Laboratories in Europe participated in the study and analysed 12 samples with adult coleopterans and insect larvae. The performance of the laboratories was evaluated in terms of sensitivity and specificity. Sensitivity was satisfactory for all the participants and both types of methods, thus fully meeting the diagnostic challenge of confirming all truly positive cases as positive. Two participants encountered specificity problems. For one, the anomaly was minor whereas, for the other, the issues concerned a larger number of results, especially real-time PCR, which probably were related to inexperience with this technique. The comparison demonstrated the reliability of official diagnosis, including the entire analytical process of A. tumida identification: from the first step of the analysis to the expression of opinions. The performed diagnostic tools, in parallel with field surveillance, are essential to managing A. tumida introduction.

Complete Mitochondrial Genome Sequence of Aethina tumida (Coleoptera: Nitidulidae), a Beekeeping Pest.

We report here the full mitochondrial genome sequence of Aethina tumida, a Nitidulidae species beetle, that is a pest of bee hives. The obtained sequence is 16,576 bp in length and contains 13 protein-coding genes, 2 rRNA genes, and 22 tRNAs.

First Description of Infection of Caprine Herpesvirus 1 (CpHV-1) in Goats in Mainland France.

The purpose of this study was to investigate the epidemiological situation of the caprine herpesvirus 1 (CpHV-1) infection in nine districts in mainland France, mostly in the south, near Italy or Spain, where high seroprevalence has been observed. Two more central areas were also included in the study. The serosurvey was carried out in 9564 goats (275 herds) using bovine herpesvirus 1 (BoHV-1) glycoprotein B and E ELISAs. To confirm the presence of specific CpHV-1 antibodies, some of the samples were tested in neutralization assay. Results demonstrate, for the first time, CpHV-1 infection in goat herds on the French mainland. The analysis found cases of alphaherpesviruses infection in each district studied, with different levels of seroprevalence observed within each district (ranging from 0.2% to 31.56% at an individual level and from 9% to 46.2% for herd seroprevalence). Moreover, in the Alpes-Maritimes district, the seroprevalence seemed to be higher in older goats (79.45% of animals 6 years old or more) than in younger animals (40.99% of one-year-olds). This result suggests frequent virus re-excretion and circulation in herds. Results analysis also shows that the seroprevalence was higher when the herd size increased. In addition, the first French CpHV-1 strain was isolated from nasal swabs taken on an infected goat. The data reported herein demonstrate that CpHV-1 circulates in mainland France, which should henceforth be taken into consideration in cases of unexplained abortion in goats.

Impact of IS1111 insertion on the MLVA genotyping of Coxiella burnetii.

Q fever epidemiological investigations of the likely sources of contamination may involve Coxiella burnetii MLVA for direct and rapid typing from clinical samples. However, little information is available with regards to PCR amplification failures in C. burnetii MLVA typing. This paper focuses on difficulties encountered with MLVA loci that may impact the interpretation of MLVA data and shows that some loci may constitute hotspots for mutational events. MLVA genotyping, using 17 different loci, was used on vaginal swabs (VS) from clinically infected animals as described elsewhere (Chmielewski et al., 2009). Amplicons of interest were sequenced and identified using the BLAST software by comparison with sequences available in GenBank. All VS samples produced MLVA patterns. However, amplification failures or unexpected sizes amplicons (>to 1.5 kbp), making the interpretation of MLVA complicated, were also observed. Sequencing of these amplicons revealed the presence of IS1111 element insertion. In this C. burnetii MLVA study some difficulties encountered with genotyping are highlighted and the role of IS1111 element in genome plasticity is confirmed. Finally, the need for the selection of a set of VNTRs for an efficient MLVA scheme and the question of standardization and harmonization for comparable MLVA typing data are raised again.

Pestiviruses infections at the wild and domestic ruminants interface in the French Southern Alps.

In alpine pasture, interspecies transmission has recently been incriminated in the epidemiology of pestivirus infection. The aim of this study was to investigate pestivirus infections in wild and domestic ruminants sharing pastures in the French Southern Alps. Animal sera were screened for pestivirus antibodies against the pestivirus NS3 protein by a commercial blocking enzyme linked immunosorbent assay (ELISA). All 38 domestic herds tested were positive for pestivirus-specific antibodies. Individual sero-prevalence reached 76.5% (95% confidence interval [95% CI]: [74.2-78.8%]) of the 1383 sheep tested. For wild ruminants, 38.7% (95% CI: [33.8-43.9%]) of the 369 chamois tested, 28.7% (95% CI: [17.4-38.1%]) of the 72 roe deer, and 22.2% (95% CI: [6.5-37.9%]) of the 27 mouflons were seropositive. Virus screening was carried out on spleen samples from hunted wild animals (n=160) and from 15 domestic ruminants (clinically suspected to be persistently infected animals), by a conventional reverse transcription-polymerase chain reaction (RT-PCR). Three pestivirus strains were isolated from the sheep samples positive by RT-PCR. The viruses were classified in the BDV-3, BDV-Tunisian and BDV-6 genotypes. For the first time, one strain (RUPI-05 strain) was isolated from an alpine chamois and clustered in the BDV-6 genotype, showing in the 5'-UTR region 92% of identity with the ovine isolate from the same area. Thus, an active circulation of pestiviruses was demonstrated in both wild and domestic ungulates from the French Southern Alps. The results suggest that interspecies transmission between sheep and chamois probably occur.

Experimental infection of pregnant Pyrenean chamois (Rupicapra pyrenaica) with border disease virus subtype 4.

Border disease virus (BDV) causes high mortality in Pyrenean chamois (Rupicapra pyrenaica) on the French and Spanish sides of the Pyrenees Mountains. We investigated the pathology induced by BDV in pregnant chamois via experimental infection. Three females were inoculated during the second third of pregnancy with a BDV-4 subgroup strain isolated from a wild Pyrenean chamois during an acute epizootic. A fourth pregnant chamois and one nonpregnant ewe were kept as negative controls. Animals were monitored to assess clinical signs, hematology, viremia, and serology. Postmortem examinations included necropsy, histopathology, and quantification of viral RNA in organs. Pregnancy was unsuccessful in all inoculated animals. One died 24 days postinoculation (dpi) without showing any precursory clinical signs. The second animal had profuse diarrhea from 13 dpi to its death at 51 dpi. The third aborted at 46 dpi and was euthanized at 51 dpi. All animals were viremic from 4 dpi until death. Neutralizing antibodies against BDV-4 were detected from 12 dpi. Necropsies showed generalized lymphadenomegaly, associated in one case with disseminated petechial hemorrhages in the digestive tract. Seventy-eight of 79 organs from inoculated adults and their fetuses had detectable viral RNA. The main histologic lesions in adults were mild lymphohistiocytic encephalitis associated with moderate or moderately severe lymphoid depletion. Control animals remained negative for virus (in blood and organs), antibody, and lesions upon postmortem examination. BDV infection during pregnancy in Pyrenean chamois causes severe disease leading to abortion, then death. Despite 100% fetal death following inoculation, viral RNA was recovered from all organs of infected fetuses, suggesting that persistently infected offspring could be born. Our results may help explain the reported decrease in chamois populations in several areas and suggest that great care must be taken when interpreting infection status for wildlife.

Outbreak of Q fever, Florac, Southern France, Spring 2007.

INTRODUCTION: In May 2007, five patients with Q fever-like symptoms were reported in an agricultural educational center in the rural southern French town of Florac. An investigation was undertaken to identify the outbreak source and risk factors for infection, and to implement control measures. MATERIALS AND METHODS: We undertook active case finding. Patients were defined as individuals with an unexplained fever of ≥38.5°C who lived in, worked in, or visited Florac between April 1 and June 30, 2007. Patients were confirmed by a positive Q fever serology test. A cross-sectional survey with a seroprevalence component was carried out in the educational center and surrounding area. A standardized questionnaire on known risk factors for the infection was used and serological testing was carried out on finger prick blood specimens from participants. The veterinary services investigated local herds within a 5-mile radius using polymerase chain reaction and serological tests. RESULTS: One hundred twenty-two people were included in the cross-sectional survey. Eighteen serologically confirmed acute cases were identified, of whom 12 were from the educational center. The statistical analysis showed an independent association between acute infection and living or working near an area where manure had been spread (p = 0.0.042) and male gender (p = 0.022). Frequenting the educational center's canteen was also associated with infection (p = 0.008) among staff and students. The veterinary investigations identified 11 of the 26 tested flocks of goats and sheep as seropositive for Coxiella burnetii, including 2 ovine flocks located northwest of Florac that had high shedding levels of the bacterium. DISCUSSION: The observed excess of cases of Q fever in Florac, an area endemic for this infection, in spring 2007 could be explained by an aerial transmission from infectious ovine flocks situated close to the town. All local herd owners were re-educated about the risks and prevention practices for Q fever.

Molecular epidemiology of Q fever in Poland.

Coxiella burnetii is the etiologic agent of Q fever, a worldwide distributed zoonosis, accountable for serious health problem both for humans and animals. The exposure to C. burnetii infected animals and their products is the main risk factor for Q fever in humans. Several outbreaks of Q fever have been described in Poland which sources were recognized to be related to imported animals and their products or to wildlife using serological methods. Moreover, some of them have been confirmed by isolation of C. burnetii strains. In this study, multispacer sequence typing (MST) and multiple loci variable number tandem repeats (VNTR) analysis (MLVA) have been used to characterize C. burnetii strains isolated in Poland. A total of two sequence types (MST) and four MLVA types were identified among 6 C. burnetii isolates examined. This study highlighted the usefulness of these methods in the improvement of epidemiological investigations of Q fever loci on the Polish territory.

Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats.

Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection.

Modified vaccinia virus Ankara as a vaccine against feline coronavirus: immunogenicity and efficacy.

Feline infectious peritonitis virus (FIPV) is a coronavirus that induces a fatal systemic disease mediated by an inappropriate immune response. Most previous vaccination attempts against FIPV were unsuccessful because IgG antibodies against the surface protein enhance the infection. However, two studies have shown that poxvirus vectors (vaccinia WR and canarypox) expressing only the FIPV membrane (M) protein can elicit a partially protective immunity which is supposed to be cell-mediated (Virology 181 (1991) 327; International patent WO 97/20054 (1997)). In our study, we report the construction of another poxvirus, the modified vaccinia virus Ankara (MVA), as an expression vector for the FIPV M protein. In this vector, the M gene has been inserted downstream a strong early/late promoter, whereas the two previously described poxviruses expressed the M protein during their early stage only. The immunogenicity of the recombinant MVA-M was evaluated in the murine model which revealed an effect of the vector on the Th1/Th2 balance. The vaccine was then tested in cats to evaluate its efficacy in an FIPV 79-1146 challenge. Vaccinated kittens developed FIPV-specific antibodies after immunization, however, none of them was protected against FIPV. Our results suggest a crucial role for the type of poxviral promoter that must be used to induce an effective immune response against FIPV.

Adverse effects of feline IL-12 during DNA vaccination against feline infectious peritonitis virus.

Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime-boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations - on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant.