Development of a New Selective Plating Medium for Rhodococcus equi

A new selective plating medium for Rhodococcus equi containing ceftazidime (20 mg/l) and novobiocin (25 mg/l) on a Mueller-Hinton agar basis is described. It proved to be less inhibitory for R. equi than selective plating media devised earlier and grew only very few other nocardioform bacteria.     

Potential tick vectors for Theileria equi in Israel

Theileria equi Mehlhorn and Schein, 1998 (Piroplasmida: Babesiidae) is an important tick-borne pathogen of horses that is highly endemic in many parts of the world, including Israel. The present study evaluated the potential roles of five hard tick species [Hyalomma excavatum Koch, 1844; Hyalomma marginatum Koch, 1844; Rhipicephalus turanicus Pomerantsev 1936; Rhipicephalus annulatus Say, 1821; Haemaphysalis parva (Neumann, 1897) (all: Ixodida: Ixodidae)], previously found to infest horses in Israel, in acting as vectors for piroplasmosis. For this, DNA was extracted from whole ticks and, when possible, from the salivary glands in each species (n = 10–59). Polymerase chain reaction amplification and sequencing of the 18S rRNA gene were used to detect T. equi in 48 of the 127 ticks (37.8%) and in 21 of the 90 extracted salivary glands (23.3%) in all five species. All but two sequences were classified as T. equi genotype A; the remaining two were classified as genotype D. The findings of this study point to Ha. parva and R. annulatus as potential novel vectors of T. equi, and suggest that parasite genotype selection occurs within the tick vector.     

Expression of Virulence-Associated Antigens of Rhodococcus equi Is Regulated by Temperature and pH

We recently reported that there are two different virulence-associated antigens correlated with virulence levels in Rhodococcus equi isolates from AIDS patients: virulent R. equi that kills mice with 10⁶ cells expresses 15- to 17-kDa antigens and intermediately virulent R. equi that kills mice with 10⁷ cells expresses a 20-kDa antigen. Environmental parameters were evaluated for their effects on the expression of these virulence-associated antigens in virulent R. equi strains by immunoblotting using monoclonal antibodies in this study. Expression of these two virulence-associated antigens of R. equi was regulated by pH and temperature; the antigens were produced maximally when the isolates were grown at 38 C and pH 6.5, but were not produced when grown at 38 C and pH 8, nor at temperatures below 30 C. The 20-kDa antigen was found to be located on the cell surface, as were the 15- to 17-kDa antigens, and showed susceptibility to proteolysis by trypsin. These results indicate that expression of the virulence-associated antigens of R. equi is dependent on the environmental conditions.     

Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.     

Dividing the pie: differential dung pat size utilization by sympatric Haematobia irritans and Musca autumnalis

Horn flies [Haematobia irritans (Diptera: Muscidae) (L.)] and face flies [Musca autumnalis (Diptera: Muscidae) De Geer] use the same larval resource, but their interactions are poorly studied. Dung pats (n = 350) were core sampled in the summers of 2012 and 2013 from irrigated pastures in Pomona, California, U.S.A. (34°03′N, 117°48′W) and held for face fly and horn fly emergence. Surface areas and estimated weights were recorded for each whole pat. Almost half (42.0%) of the pat cores yielded neither fly, 29.7% yielded horn flies only, 12.9% yielded face flies only and 15.4% yielded both flies. Of the fly‐positive pats, surface area and mass were larger for face fly‐occupied pats, whereas horn fly‐occupied pats were smaller. Pats shared by the two species were intermediate. Horn flies per positive core were unaffected by the absence/presence of face flies, but half as many face flies emerged when pats were co‐inhabited by horn flies. Face flies inhabited larger pats, which might better resist heating and drying, to which they are susceptible; horn flies inhabited a broad pat size range. Horn fly tolerance of lower dung moisture probably allows horn flies to colonize and survive in a wide range of pats in dry areas like southern California.     

Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population

In Switzerland, the prevalence and incidence of equine piroplasma parasite (EPP) infections are unknown. In order to obtain a first insight into the prevalence, a representative sample of 689 sera of horses from Switzerland was serologically tested for the presence of antibodies directed against T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). A total of 50 (7.3%) horses were seropositive for EPP: overall, the seroprevalence of T. equi was significantly higher than that of B. caballi (p = 0.002). The seropositivities in indigenous horses (animals bred and raised in Switzerland) and in imported horses were 4.8% (11/230) and 8.5% (39/459), respectively. Unlike in indigenous horses, where no significant difference in seroprevalences could be observed between the two parasite species, the seroprevalence of T. equi was significantly higher (p < 0.001) than that of B. caballi in imported horses. Horses imported from France, Spain and Portugal exhibited a significantly higher seroprevalence, and horses imported from Germany a significantly lower seroprevalence of EPP compared to indigenous horses. There were no associations between sex, age, weight loss, surgery or blood transfusions with T. equi and B. caballi seroprevalences.The overall seroprevalence of 7.3% clearly shows that infection with EPP is a threat to the health of the horses in Switzerland. With the presumed expansion of permissive tick vectors, EPP infections will potentially increase in importance in the future. Therefore, continuous monitoring is mandatory.     

Biological control of <Emphasis Type="Italic">Musca domestica</Emphasis> and <Emphasis Type="Italic">Stomoxys calcitrans</Emphasis> by mass releases of the parasitoid <Emphasis Type="Italic">Spalangia cameroni</Emphasis> on two Norwegian pig farms

House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans (L.)) (Diptera: Muscidae) are nuisance pests on livestock farms. In the present study we tested the effect of biweekly mass release of Spalangia cameroni Perkins (Hymenoptera: Pteromalidae), the most common parasitoid of house flies and stable flies in southern Norway, on pig premises with scattered fly breeding sites. The study spanned two successive summer periods, with two control and two release units each year. Spalangia cameroni suppressed both house flies and stable flies in one release unit during the first year, and stable flies in one release unit the second year. The apparent lack of success in fly control in the other release units was likely due to immigration of flies in two of the trials and high temperatures in one trial. Recommendations concerning release of S. cameroni in similar pig production premises are given. Handling editor: Torsten Meiners.     

Infection with Haemoproteus iwa affects vector movement in a hippoboscid fly—frigatebird system

Haemosporidian parasites, which require both a vertebrate and invertebrate host, are most commonly studied in the life stages occurring in the vertebrate. However, aspects of the vector's behaviour and biology can have profound effects on parasite dynamics. We explored the effects of a haemosporidian parasite, Haemoproteus iwa, on a hippoboscid fly vector, Olfersia spinifera. Olfersia spinifera is an obligate ectoparasite of the great frigatebird, Fregata minor, living among bird feathers for all of its adult life. This study examined the movements of O. spinifera between great frigatebird hosts. Movement, or host switching, was inferred by identifying host (frigatebird) microsatellite genotypes from fly bloodmeals that did not match the host from which the fly was collected. Such host switches were analysed using a logistic regression model, and the best‐fit model included the H. iwa infection status of the fly and the bird host sex. Uninfected flies were more likely to have a bird genotype in their bloodmeal that was different from their current host's genotype (i.e. to have switched hosts) than infected flies. Flies collected from female birds were more likely to have switched hosts than those collected on males. Reduced movement of infected flies suggests that there may be a cost of parasitism for the fly. The effect of host sex is probably driven by differences in the sex ratio of bird hosts available to moving flies.     

Viability of Rhodococcus equi and Parascaris equorum Eggs Exposed to High Temperatures

There is great concern about the potential pathogen contamination of horse manure compost spread in the same fields horses graze in. To ensure that pathogen destruction occurs, temperatures need to be sufficiently high during composting. Here, we investigated the survival rate of two marker organisms, Rhodococcus equi and Parascaris equorum eggs, exposed to temperatures potentially encountered during horse manure composting. Our results show that the time required to achieve a 1 log10 reduction in R. equi population (D-value) are 17.1 h (±1.47) at 45°C, 8.6 h (±0.28) at 50°C, 2.9 h (±0.04) at 55°C and 0.7 h (±0.04) at 60°C. For P. equorum eggs we show that at 45 and 50°C, 2 log10 reduction of viability is reached between 8 and 24 h of incubation and that it takes less than 2 h at 55 and 60°C to achieve a viability reduction of 2 log10. These results are useful for identifying composting conditions that will reduce the risk of environmental contamination by R. equi and P. equorum eggs.     

Haematobia irritans parasitism of F1 yak × beef cattle (Bos grunniens × Bos taurus) hybrids

The horn fly Haematobia irritans (Diptera: Muscidae) is a blood obligate ectoparasite of bovids that causes annual losses to the U.S. beef cattle industry of over US$1.75 billion. Climate warming, the anthropogenic dispersion of bovids and the cross‐breeding of beef cattle with other bovid species may facilitate novel horn fly–host interactions. In particular, hybridizing yaks [Bos grunniens (Artiodactyla: Bovidae)] with beef cows (Bos taurus) for heterosis and carcass improvements may increase the exposure of yak × beef hybrids to horn flies. The present paper reports on the collection of digital images of commingled beef heifers (n = 12) and F1 yak × beef hybrid bovids (heifers, n = 7; steers, n = 5) near Laramie, Wyoming (～ 2200 m a.s.l.) in 2018. The total numbers of horn flies on beef heifers and F1 yak × beef heifers [mean ± standard error (SE): 88 ± 13 and 70 ± 17, respectively] did not differ significantly; however, F1 yak × beef steers had greater total horn fly abundance (mean ± SE: 159 ± 39) than female bovids. The present report of this experiment is the first such report in the literature and suggests that F1 yak × beef bovids are as susceptible as cattle to horn fly parasitism. Therefore, similar monitoring and treatment practices should be adopted by veterinarians, entomologists and producers.     

Horizontal Transfer of the Tetracycline Resistance Gene <Emphasis Type="Italic">tetM</Emphasis> Mediated by pCF10 Among <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the House Fly (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 × 10⁻⁵ to 4.5 × 10¹. Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.     

Distribution and activity of Drosophila suzukii in cultivated raspberry and surrounding vegetation

Spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), may utilize wild ‘Himalaya’ blackberry (HB) Rubus armeniacus Focke or other non‐crop plants as refugia and possibly exploit adjacent field margins before colonizing cultivated fruiting crops. Studies were conducted to determine the role of field margins containing HB and their effect on D. suzukii activity, density and distribution in an adjacent commercial red raspberry crop. One‐ha plots adjacent to field margins containing HB or known non‐host (NH) grass crops were established in 2011 and 2012 and replicated three times. Each plot contained two transects with monitoring traps for D. suzukii in the field margin (0 m) and spaced approximately 10 (crop boundary), 40, 70 and 100 m into the adjacent crop (n = 10 traps/plot). Field margin vegetation was treated with a 10% chicken egg white mark solution weekly from pre‐harvest until the end of harvest using a cannon sprayer. Adult D. suzukii were collected from traps weekly and analysed for the presence of the egg white mark using an egg white‐specific enzyme‐linked immunosorbent assay (ELISA). During both years, marked flies and total flies were captured in higher numbers in HB field margins, whereas virtually no flies were captured in field margins containing no known alternative host. Similarly, more flies were captured in the crop near HB than near NH. Spatial Analysis by Distance IndicEs (SADIE) and mean D. suzukii trap captures additionally displayed significantly higher fly densities in the raspberry field near HB than near NH. These results suggest that HB may contribute to elevated D. suzukii populations and pest pressure in comparison with field margins containing no known alternate host vegetation for D. suzukii. Having closely adjacent non‐crop alternate host landscapes may result in increased D. suzukii pest pressure.     

Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.     

Genomic and phenomic differentiation of Rhodococcus equi and related strains

16S rDNA sequence and pyrolysis mass spectrometric analyses were carried out on representatives of Rhodococcus equi and marker strains of genera that encompass mycolic acid containing actinomycetes. The R. equi strains formed a monophyletic clade within the evolutionary radiation occupied by members of the genera Nocardia and Rhodococcus. The 16S rDNA sequence data also showed R. equi to be an heterogeneous taxon. This heterogeneity was underscored by the pyrolysis mass spectrometric data. These observations are in line with those of previous studies where similar profiles of relatedness were found between pyrolysis mass spectral data and the results of DNA:DNA pairing and numerical phenetic studies.     

Suppression of female melon fly,Zeugodacus cucurbitae,with cue‐lure and fipronil bait stations through horizontal insecticide transfer

Melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), is an important quarantine tephritid fruit fly with resident populations established in Hawai'i, USA. In the male‐annihilation approach, male flies are targeted using dispensers with cue‐lure (C‐L) and insecticides, typically organophosphates. The efficacy of the male annihilation approach is thought to be limited to individual male flies, contacting the lure and the pesticide, after which they die. Alternative classes of insecticides, such as fipronil, have been investigated for use in male‐annihilation. We hypothesized that ingestion of fipronil by male flies could lead to horizontal transfer and mortality in female flies. Horizontal insecticide transfer extends pesticide control beyond the individual contacting the toxicant through indirect contact via food sharing or other mechanisms. We tested the possibility for horizontal transfer of fipronil from male to female Z. cucurbitae through field and laboratory studies. Two repeated field trials were conducted to compare the numbers of female flies collected in fields treated with Amulet C‐L (0.34% fipronil active ingredient) bait stations, sanitation, and spot treatments of GF‐120 Fruit Fly Bait to numbers collected in fields where sanitation and spot‐treatments were used without Amulet C‐L. In fields with Amulet C‐L bait stations in conjunction with sanitation and weekly protein bait spot treatments of GF‐120 Fruit Fly Bait, female captures were significantly lower than those in field plots treated with weekly protein bait spot treatments and sanitation. In subsequent laboratory studies, all females died within 6 h after direct exposure to male flies that had access to Amulet C‐L for 1–4 min. The possibility that male regurgitant could be a mechanism for horizontal transfer and subsequent female mortality was determined by collecting regurgitated droplets from fipronil‐fed male flies and feeding them to males and females. Both male and female flies exposed to regurgitant from fipronil‐fed male flies or droplets containing fipronil had higher mortality than the male and female flies that were exposed to regurgitant or droplets with only the C‐L compound or sugar solution. Thus, female flies do experience mortality from exposure to regurgitant from males that have fed on fipronil laced solutions. This provides evidence of at least one mechanism of horizontal transfer of insecticide in tephritid fruit flies. These findings are discussed in the context of Z. cucurbitae integrated pest management programs in Hawai'i.     

Carbon nanotubes catalyzed UV-trigger production of hyaluronic acid from Streptococcus equi

Hyaluronic acid (HA) has great importance in biomedical applications. In this work, a novel nanoparticle-based method that stimulates the hyaluronic acid (HA) production by the bacteria Streptococcus equi subsp. Zooepidemicus has been reported. CNTs with diameters of 40–50 nm and lengths of about 20 mm were used at four different concentrations (0, 10, 25, 50, and 100 μg) to the bacteria and determined the mass of the produced HA in dependence on the exposure time under UV-irradiation. The results clearly showed that the exposure for one minute with low power UV light (254 nm) and 100 µg (CNTs) treatments steadily increased HA production from the control (0.062 g/L) to the highest value (0.992) g/L of HA. The incubation of the streptococci with CNTs led to an increase of the HA production by a factor of 4.23 after 300S exposure time under UV light, whereas the HA production was no significant enhancement under visible light. It is explained that the CNTs nanoparticle-stimulated increase of the HA production with the internalization of the nanoparticles by the bacteria since they “serve as co-enzymes” under induced mutation by UV-irradiation. Transformation process was carried out and showed that the major protein band of Streptococcus equi was observed in the Streptococcus DH5α. RAPD analysis indicates that the amplified DNA fragments and the percentage of polymorphism was similar between Streptococcus equi and Streptococcus DH50α. The chemical structure and molecular weight of the photoproduced HA from Streptococcus equi was similar to the chemical structure of the standard sample.     

Stomoxys calcitrans,mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re‐emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re‐examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non‐infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.     

Microsatellite loci in the house fly,Musca domestica L. (Diptera: Muscidae)

Genomic libraries from house flies enriched for (CA)₁₅ and (CAG)₁₀ repeats were constructed by using biotinylated probes. Twenty‐five loci were isolated and evaluated for polymorphisms in wild flies representing two geographically diverse populations. Fourteen of 19 dinucleotide loci, and one of six trinucleotide loci were polymorphic. One hundred and twenty‐seven alleles were detected, 39 of which were private. Average number of alleles per polymorphic locus was 8.4 ± 2.5 and average heterozygosity was 72 ± 4%. F_ST by the private allele method was 0.73. Three of 15 loci showed significant heterozygote deficiencies, attributed to null alleles. Five of 15 loci were amplified in the face fly, Musca autumnalis.     

Testing a candidate parasitoid in the glasshouse: control efficacy of <Emphasis Type="Italic">Aphaereta debilitata</Emphasis> (Hymenoptera: Braconidae) against shore fly populations

Biological control efforts against the shore fly Scatella tenuicosta Collin, a pest of commercial glasshouses, have had limited success. The ability of one of its parasitoids, Aphaereta debilitata Morley, to control shore fly populations was investigated on lettuce crops within six experimental glasshouse units, over 26 weeks. The six shore fly populations, either with or without a single release of 150 mixed-sexed parasitoids, were estimated by rearing from harvested pots each week. The presence of the parasitoid significantly reduced shore fly numbers from a mean of 30.3–13.2 adult flies emerging per pot. The level of damage to each lettuce was assessed using a scoring system (0–5: no damage to heavy damage, respectively). The introduction of parasitoids reduced the median damage score of the lettuces from 2 to 1 over the study, representing a considerable change in the marketability of the crop. This study shows that single inoculative releases of A. debilitata can quickly establish within a glasshouse and significantly reduce crop damage. This suggests that the parasitoid may represent a valuable addition to an IPM strategy for shore flies.