Wolbachia Endosymbiont of the Horn Fly (Haematobia irritans irritans): a Supergroup A Strain with Multiple Horizontally Acquired Cytoplasmic Incompatibility Genes.

The horn fly, Haematobia irritansirritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between $1 billion and $2.5 billion (U.S. dollars) annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, TX, and Alberta, Canada, but it has also been identified in wild-caught samples from Canada, the United States, Mexico, and Hungary. Reassembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularized 1.3-Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1,249 coding genes, 34 tRNAs, 3 rRNAs, and 5 prophage regions. Comparative genomics and whole-genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggested that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicated that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggested two phylogenetically distinct CI loci and acquisition of another cifB homolog from phylogenetically distant supergroup A Wolbachia strains, suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies.IMPORTANCE Horn flies, Haematobia irritans irritans, are obligate hematophagous parasites of cattle having significant effects on production and animal welfare. Control of horn flies mainly relies on the use of insecticides, but issues with resistance have increased interest in development of alternative means of control. Wolbachia pipientis is an endosymbiont bacterium known to have a range of effects on host reproduction, such as induction of cytoplasmic incompatibility, feminization, male killing, and also impacts vector transmission. These characteristics of Wolbachia have been exploited in biological control approaches for a range of insect pests. Here we report the assembly and annotation of the circular genome of the Wolbachia strain of the Kerrville, TX, horn fly (wIrr). Annotation of wIrr suggests its unique features, including the horizontal acquisition of additional transcriptionally active cytoplasmic incompatibility loci. This study provides the foundation for future studies of Wolbachia-induced biological effects for control of horn flies.

Quantitative proteomics reveals significant variation in host responses of cattle with differing buffalo fly susceptibility.

Background: Control of buffalo flies (Haematobia irritans exigua, BFs) relies mainly on chemical methods; however, resistance to insecticides is widespread in BF populations. Breeding for resistance to BFs represents a possible alternative, but direct phenotyping of animals is laborious and often inaccurate. The availability of reliable diagnostic biomarker(s) to identify low BF carrier cattle would facilitate rapid and accurate selection for genetic improvement. However, limited information is available regarding differences amongst cattle in host responses to BF infestation. Methods: This study investigated the variation in Brangus cattle serum proteomic profiles before (naïve) and after peak BF exposure, in low (LF) and high BF burden (HF) cattle. Cattle were phenotyped for susceptibility based on BF counts on multiple dates using visual and photographic techniques. The relative abundance of serum proteins in cattle before and after exposure to BFs was analysed using sequential window acquisition of all theoretical fragment ion mass spectrometry (SWATH-MS). Results: Exposure to BFs elicited similar responses in HF and LF cattle, with 79 and 70 proteins, respectively, showing significantly different abundances post exposure as compared to their relevant naïve groups. The comparison of serum samples from naïve HF and LF cattle identified 44 significantly differentially abundant (DA) proteins, while 37 significantly DA proteins were identified from the comparison between HF and LF cattle post-exposure to BFs. Proteins with higher abundance in naïve LF cattle were enriched in blood coagulation mechanisms that were sustained after exposure to BFs. Strong immune response mechanisms were also identified in naïve LF cattle, whereas these responses developed in HF cattle only after exposure to BF. High BF cattle also showed active anticoagulation mechanisms in response to BF exposure, including downregulation of coagulation factor IX and upregulation of antithrombin-III, which might facilitate BF feeding. Conclusion: Underlying differences in the abundance of proteins related to blood coagulation and immune response pathways could potentially provide indirect indicators of susceptibility to BF infestation and biomarkers for selecting more BF-resistant cattle.

The Development of Cutaneous Lesions in Tropically Adapted Beef Cattle Is Associated with Hypersensitive Immune Response to Buffalo Fly Antigens.

This study investigated the role of cattle immune responses in the pathogenesis of buffalo fly (Haematobia irritans exigua) (BF) lesions. Brangus steers phenotyped for lesion development were divided into three groups: high lesion susceptibility (HL), low lesion susceptibility (LL) and no lesions (NL), based on lesion severity scores. Each steer was injected intradermally with different concentrations of BF, Onchocerca gibsoni (Og), and Musca domestica (Md) antigens. At 1 h post-injection, wheal areas at BF injection sites were found to be significantly larger in HL than NL cattle, but there were no significant differences (p < 0.05) found between either the HL or NL cattle and LL cattle. At 24, 48, and 72 h post-injection, the skinfold thickness response to both BF and Md antigens was significantly greater in the HL group than the NL group. However, skin thickness was significantly greater for the BF antigens than the Md antigens (p < 0.05). There were no significant differences found between the LL and NL animals in response to the BF antigens at any time, and no significant differences were determined between any of the lesion groups in response to the Og antigens. Histological examination of skin sections taken from the BF antigen injection sites in HL cattle at 72 h post-injection revealed necrosis of the epidermis and superficial dermis, along with severe eosinophilic inflammation. This study suggests that differences in the hypersensitivity to BF antigens underlie differences amongst the cattle in their susceptibility to the development of BF lesions, and breeding for immune-related biomarkers may assist in selecting more BF lesion-resistant cattle.

Pathology and pathogenesis of cutaneous lesions in beef cattle associated with buffalo fly infestation.

Haematobia irritans exigua, commonly known as buffalo fly, is the major hematophagous ectoparasite of north Australian cattle herds. Lesions associated with buffalo fly infestation are generally alopecic, hyperkeratotic, or scab encrusted wounds with variable hemorrhagic ulceration. Buffalo flies can transmit a filarial nematode, Stephanofilaria sp., which has been implicated in the pathogenesis of buffalo fly lesions, but Stephanofilaria infection has not been detected in all lesions suggesting that other causal factors may be involved. This study characterized the pathology of buffalo fly lesions to identify the role of Stephanofilaria in lesion development, as well as to identify other potential agents. Lesion biopsies were collected from north and south Queensland and tested for the presence of Stephanofilaria by qPCR. Each lesion was scored grossly (0-4) for hemorrhage, ulceration, exudation, and alopecia. Lesions were also scored microscopically (0-4) for epidermal and dermal damage and inflammatory characters. Stephanofilaria infection was detected in 31% of lesion biopsies. Grossly, Stephanofilaria-infected lesions had significantly larger lesion area and higher scores for alopecia and hyperkeratosis than lesions where no nematodes were found (P < 0.05). Histologically, epidermal, dermal, and adnexal damage was significantly higher in Stephanofilaria infected lesions than lesions without nematodes. Eosinophils, macrophages, and lymphocytes were significantly more abundant in Stephanofilaria positive lesions as compared to negative lesions. This study also noted bacterial infection with colonies of coccoid bacteria, observed in skin sections from 19 lesions. Grossly, lesions with bacterial infection had significantly higher ulceration scores compared to Stephanofilaria positive lesions, and histologically epidermal disruption was significantly greater in bacteria-infected lesions. We found no evidence of bacteria or Stephanofilaria infection in 49% of the lesions assessed and tissue damage patterns and eosinophilic inflammation suggested hypersensitivity to buffalo fly feeding as a possible cause of these lesions. These findings suggest that although the presence of Stephanofilaria infection may increase the severity of lesion pathology, it is not essential for lesion development. These outcomes also suggest a potential role of bacteria and hypersensitivity in pathogenesis of some lesion. A better understanding of buffalo fly lesion etiology will contribute to the optimal treatment and control programmes.

Detection and distribution of Stephanofilaria sp. in buffalo flies and buffalo fly lesions in north Australian beef cattle.

Buffalo flies (Haematobia irritans exigua) are ectoparasites of major animal health and production concern in north Australian beef herds. Skin lesions associated with buffalo fly infestation, cause hide damage and welfare issues and are manifested as dermatitis or ulcerated areas found most commonly near the medial canthus of the eye, along the lateral and ventral neck and on the abdomen. Buffalo flies can transmit a nematode, Stephanofilaria sp., which has been considered the main aetiological agent for buffalo fly lesions, but the role of nematodes in the development of the lesions has not been defined. To investigate the geographical distribution of Stephanofilaria, swabs were taken from the surface of lesions and buffalo flies were netted from the backs of beef cattle from 20 properties located in northern, central and southern Queensland. Swabs and buffalo flies were then tested for the presence of Stephanofilaria by qPCR. In northern and central Queensland, all properties except one, tested positive for the presence of Stephanofilaria in either buffalo flies or swabs, or in both. The infection rate varied amongst sites ranging from 0% to 100% in lesions and 0-34% in female buffalo flies. No nematodes were found in male buffalo flies. In contrast, none of the 66 lesion swabs or 1220 buffalo flies collected from southern Queensland tested positive for Stephanofilaria infection despite the frequent occurrence of lesions in the herds from which samples were collected. These findings suggest that infection with Stephanofilaria, although frequently detected, is not essential for the development of buffalo fly lesions and other factors may contribute to the initiation of lesions. This study also confirmed the potential for using surface swabs as a quicker and less invasive means of sampling lesions than dermal biopsies when testing for the presence of Stephanofilaria by qPCR, but further studies will be required to estimate the sensitivity of this technique. Understanding the pathogenesis of buffalo fly lesions will aid the development of optimal treatment and control strategies.

Role of Staphylococcus agnetis and Staphylococcus hyicus in the Pathogenesis of Buffalo Fly Skin Lesions in Cattle.

Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. IMPORTANCE Skin lesions in cattle associated with feeding by Haematobia fly species are a significant welfare issue in Australia, North and South America, and Europe. The development of these lesions has been attributed to a number of causal factors, but the exact etiology and pathogenesis were unclear. This study characterized Staphylococcus agnetis and Staphylococcus hyicus strains from cattle skin lesions and in vector flies and demonstrated their role in the pathogenesis of these lesions. These findings will aid the development of targeted and more effective treatment and control strategies for lesions associated with fly infestation in cattle.

Development and Validation of Novel PCR Assays for the Diagnosis of Bovine Stephanofilariasis and Detection of Stephanofilaria sp. Nematodes in Vector Flies.

BACKGROUND: Stephanofilaria spp. nematodes are associated with cutaneous lesions in cattle and other livestock and mammalian wildlife species. In Australia, Haematobia irritans exigua, commonly known as buffalo fly (BF) transmits a well-described but presently unnamed species of Stephanofilaria, which has been speculatively implicated in the aetiology of BF lesions. The sensitivity of current techniques for detecting Stephanofilaria spp. in skin lesions and vector species is low, and there is no genomic sequence for any member of the genus Stephanofilaria currently available in sequence databases. METHODS: To develop molecular assays for the detection of the Australian Stephanofilaria sp., skin biopsies were collected from freshly slaughtered cattle with typical lesions near the medial canthus. Adult nematodes and microfilariae were isolated from the biopsies using a saline recovery technique. The nematodes were morphologically identified as Stephanofilaria sp. by scanning electron microscopy. DNA was extracted and the internal transcribed spacer 2 (ITS2) region of rDNA, and the cytochrome c oxidase subunit 1 (cox1) region of mtDNA was amplified and sequenced. Stephanofilaria sp. specific polymerase chain reaction (PCR) and qPCR assays (SYBR Green® and TaqMan™) were developed and optimised from the novel ITS2 sequence obtained. The specificity of each assay was confirmed by testing against nematode species Onchocerca gibsoni and Dirofilaria immitis, as well as host (bovine) and BF DNA. RESULTS: Scanning electron microscopy of the anterior and posterior ends of isolated nematodes confirmed Stephanofilaria sp. A phylogenetic analysis of the cox1 sequence demonstrated that this species is most closely related to Thelazia callipaeda, a parasitic nematode that is a common cause of thelaziasis (or eyeworm infestation) in humans, dogs, and cats. Both conventional and qPCR assays specifically amplified DNA from Stephanofilaria sp. Conventional PCR, TaqMan™, and SYBR Green® assays were shown to detect 1 ng, 1 pg, and 100 fg of Stephanofilaria DNA, respectively. Both qPCR assays detected DNA from single Stephanofilaria microfilaria. CONCLUSION: Molecular diagnostic assays developed in this study showed high specificity and sensitivity for Stephanofilaria sp. DNA. The availability of an accurate and sensitive PCR assay for Stephanofilaria will assist in determining its role in the pathogenesis of cattle skin lesions, as well as in understanding its epidemiological dynamics. This assay may also have application for use in epidemiological studies with other species of Stephanofilaria, most particularly closely related S. stilesi, but this will require confirmation.

Use of COI, CytB and ND5 genes for intra- and inter-specific differentiation of Haematobia irritans and Haematobia exigua.

A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.