The breeding of sheep that are less susceptible to flystrike is an attractive proposition for reducing its impact on sheep production. If genetic variation associated with decreased susceptibility to flystrike could be identified, then this might provide greater accuracy for making breeding selections to reduce the prevalence of the disease.

In 2010, a single-nucleotide polymorphism (SNP) association study was undertaken by Smith et al. (2010) on sheep from the fleecerot and flystrike resistant and susceptible flocks in Australia. Among the genes identified as potentially being associated with fleecerot susceptibility was the fatty-acid binding protein 4 (FABP4) gene (FABP4). Given that fleecerot has been identified as an important predisposing factor to flystrike (Norris et al., 2008) and that a study by Raadsma (1991) reported a strong genetic correlation (r > 0.9) between the two conditions, there is justification for testing whether FABP4 is also associated with the occurrence of flystrike.

The fatty-acid binding proteins are hydrophobic ligand-binding cytoplasmic proteins, and are thought to be involved in lipid metabolism through the binding and intracellular transport of long-chain fatty-acids (Furuhashi and Hotamisligil, 2008; Tsuda et al., 2009). Expression of FABP4 has been reported in adipocytes and macrophages (Furuhashi and Hotamisligil, 2008). In skin, FABP4 has been shown to be localized in the sebaceous glands (Watanabe et al., 1997), and it has been suggested to regulate their activity by modulating lipid-signaling and/or lipid metabolism in sebocytes (Lin and Khnykin, 2014).

Tsuda et al. (2009) revealed that FABP4 was strongly expressed in phosphatase-and-tensin (Pten)-null keratinocytes. In this context, FABP4 has been suggested to selectively enhance the activities of peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear hormone receptor family, and one that regulates genes involved in sebaceous tissue differentiation (Michalik and Wahli, 2007). Keratinocyte-specific Pten-null mice display distinct phenotypes, which includes having wrinkled skin and ruffled and shaggy hair. Histological examination revealed that these mice had acanthosis, sebaceous gland hyperplasia, and accelerated hair follicle morphogenesis (Suzuki et al., 2003). The gene, FABP4, is therefore implicated in the development of these phenotypes. Given the loss of waxes and hydrophobicity is thought to be a major contributing factor in the development of fleecerot (Norris et al., 2008), then FABP4 variation may also affect flystrike susceptibility in sheep. Furthermore, the FABP4 protein is also released from adipocytes and macrophages, and it is a proinflammatory signaling protein, with the level of FABP4 protein increasing in the blood of people with the skin inflammatory condition psoriasis (Baran et al., 2017). Inflammation is observed in skin tissues affected by fleece rot.

Yan et al. (2012) studied sequence variation in two regions of FABP4. In the first, which spanned parts of exon 2 and intron 2, five unique sequences were observed. These variants were a consequence of three nucleotide substitutions and one nucleotide deletion in intron 2. In the second region of the gene, which spanned parts of exon 3 and intron 3, four different sequences were detected, which come about as a consequence of four nucleotide substitutions.

To ascertain whether variation in FABP4 is associated with variation in susceptibility to flystrike in New Zealand (NZ) sheep, animals with and without flystrike were identified at shearing time. Genetic variation within FABP4 was analyzed using PCR-SSCP analyses, and associations with the occurrence of fleecerot and flystrike explored. Any association identified might provide a better understanding of biological role of FABP4 in variation in susceptibility to flystrike.

Smith et al. (2010) identified five SNPs in ovine FABP4 from Merino sheep that are associated with fleecerot. Three of these SNPs were also identified by Yan et al. (2012, 2018) and in this study too. In Region-1, Yan et al. (2012) detected five variant sequences of FABP4 (named A₁ -E₁) and four variant sequences (A₂-D₂) for Region-2 in 483 NZ sheep of various breeds. Variants A₁, B₁ and C₁ were the most common and observed in all breeds. Variant D₁ was observed in all the breeds except Poll Dorset sheep and E₁ was only observed in two breeds (Poll Dorset and Corriedale). In this study four of the FABP4 Region-1 variants were detected (A₁-D₁), with E₁ not being found. More recently, Yan et al. (2018) reported the five previously identified Region-1 variants (A₁, B₁, C₁, D₁ and E₁) and three previously identified Region-2 variants (A₂, B₂ and C₂) were detected in NZ Romney sheep, describing fourteen different haplotypes spanning the two regions: A₁-A₂, A₁-B₂, A₁-C₂, B₁-A₂, C₁-A₂, C₁-C₂, D₁-A₂, B₁-B₂, C₁-B₂, D₁-C₂, E₁-B₂, E₁-A₂, D₁-B₂ and B₁-C₂.

In the context of the observed associations with Region-1 variant A₁ in this study, it must, therefore, be noted that A₁ could potentially be present in the sheep studied in one of three haplotypes across these two regions (A₁-A₂, A₁-B₂, and A₁-C₂), although the haplotypes were not established here as that require nucleotide sequencing of all 844 sheep. Variant C₁ was also found in three haplotypes (C₁-A₂, C₁-C₂, and C₁-B₂) by Yan et al. (2018), albeit in different breeds. Variants A₂ and B₂ occurred in haplotypes with all five Region-1 variants, and C₂ with all except E₁ (Yan et al., 2018). It is therefore not surprising that associations were not detected with Region-2 of the gene and future research focused on association studies using haplotypes is merited.

The SNPs described in FABP4 in this study were all found in introns. Although introns do not code for amino acids, some SNPs in introns, silent substitutions in coding sequences and variation in regions flanking coding sequences can have a functional role, and/or affect gene expression. For example, SNPs in introns have been reported to affect primary transcript splicing efficiency, the stability of mRNA, and the translation of mRNA (Le Hir et al., 2003); and silent substitutions in coding regions (typically in position three of the codon) have also been shown to affect mRNA stability and pre-mRNA splicing (Duan et al., 2003; Supek et al., 2014). The FABP4 SNPs identified do not fall into any known RNA splicing sites, however, they may be linked to sequence variation in other regions of FABP4 that is of structural or functional importance. This has also been suggested by Smith et al. (2010) and Yan et al. (2012, 2018).

Given that the aim of this study was to determine if there was an association between the presence of flystrike and variation in ovine FABP4, then it is notable that sheep with the A₁ variant were less likely to have flystrike than those without A₁. It is, however, impossible to say if any given sheep carrying the A₁ variant will, or will not get flystrike, as some sheep in this study carrying A₁ also had flystrike and some sheep carrying two copies of A₁ were also struck, with them accounting for 2.5% of the struck genotypes. It must be remembered in this context that the analyses are only finding associations rather than causative mutations.

The first associations between SNPs in or near FABP4, and fleecerot, were described by Smith et al. (2010). However, given there is a high genetic correlation (r > 0.9) between fleecerot and flystrike, it is perhaps not that surprising that an association has been found with flystrike susceptibility. The susceptibility of sheep to fleecerot and flystrike can also be assessed through the measurement of indicator traits, including assessment of physical characteristics and chemical characteristics. In the context of the latter, wax and suint levels affect the wettability of the wool (Belschner, 1937; Norris et al., 2008), and thus susceptibility to disease. In fine wool sheep, bright (high luster), high yielding (but greasy) wool, which is white in color, is the most consistent wool type associated with fleecerot resistance, and wool of this type is highly heritable (Hayman, 1953; James et al., 1984; Raadsma, 1987). Fiber diameter is another highly heritable trait that has been associated with resistance to fleecerot (Raadsma, 1993), with a lower fiber diameter resulting in a greater resistance in Merino sheep (James and Ponzoni, 1992). Given these observations, the wax levels in the wool are possibly influenced by FABP4, and this could explain why variation in FABP4 appears to be associated with fleecerot and flystrike resilience. In this context, exploring the relationship between FABP4 expression and wool wax levels, along with other wool traits, could shed light on FABP4’s role in flystrike resilience, although it has been reported that the phenotypic correlations between fleecerot, flystrike and other fleece characteristics, are typically low (Raadsma, 1987).

Bakhtiarizadeh et al. (2013) used Lori-Bakhtiari and Zel sheep to investigate the differences in FABP4 expression in fat-tail tissue and visceral adipose tissue. Their results suggested that the expression of FABP4 was higher in the fat-tail of Lori-Bakhtiari sheep, than expression in either the fat-tail or visceral tissue of the Zel sheep. The higher level of expression of FABP4 in the fat-tail tissue of Lori-Bakhtiari sheep was related to there being more fatty-acid transportation into the fat-tail compared with the Zel sheep. If fat is a factor that affects flystrike resilience in sheep, then increased expression of FABP4, leading to more fatty-acid transport into different areas of the body could be an underlying mechanism for the variation in resilience. It would be interesting to look at the frequency of FABP4 variants A₁-E₁ in Lori-Bakhtiari and Zel sheep to see if they have a higher frequency of the A₁ variant, especially as this variant has also been found to occur at a high frequency in a line of sheep that were deliberately bred for increased fatness (Yan et al., 2012). The Lori-Bakhtiari and Zel sheep could also be investigated to ascertain their resilience to flystrike in the context of FABP4 variation.

Yan et al. (2012) found a vast difference between fat and lean lines of sheep and variation in FABP4. Sheep carrying the FABP4 A₁ variant were predominantly in the fat line, while sheep carrying the C₁ variant was associated with the lean line. In the present study, sheep with the A₁ variant were the least likely to get flystrike. While the level of expression (if any) of this variant was not ascertained in either study, it might nevertheless speculated that sheep with a higher carcass fat content, may also have an increased or different lipid content in their wool. Wool wax contains fatty acids, and these coat the wool fibers and skin, where they inhibit bacterial growth by lowering the pH of the skin surface (Lambers et al., 2006). It might, therefore, be worthwhile, to investigate whether FABP4 affects wool wax production (qualitatively and quantitatively) and whether this affects the likelihood of fleecerot and/or flystrike occurring. However, if the FABP4 A₁ variant decreases flystrike susceptibility and increases the fat content in meat, then farmers would have to decide if they want to selectively breed sheep for both these traits. This could cause conflict with consumer demand for healthier sheep that produce leaner meat. In some markets, the current consumer demand is for leaner meat due to the common perception that fat is linked to obesity and cardiovascular disease (Volk, 2007; Pethick et al., 2011), and this demand is met by producing leaner slaughter animals on-farm (Pethick et al., 2011).

Fatty acid-binding protein 4 has also been shown to modulate inflammatory responses in macrophages (Furuhashi and Hotamisligil, 2008). In macrophages lacking FABP4 (FABP4 -/-) there are several signaling pathways that are suppressed, including the production of cytokines such as tumor-necrosis factor –α (TNF-α), interleukin 1 β (IL β) and IL6 (Makowski et al., 2005; Furuhashi and Hotamisligil, 2008). It is of interest that inflammatory cytokines and T cell-dependent cytokine IL-2 and IFN–γ are produced in the skin during flystrike (Bowles et al., 1994). FABP4 also acts to coordinate functional interactions between macrophages and adipocytes in the adipose tissue (Furuhashi and Hotamisligil, 2008). The adipocytes sit near the skin surface, thus these cells could play a role in the inflammatory response to flystrike in the skin. The “fatter” type sheep, as shown by Yan et al. (2012) to have a high frequency of A₁ variant, may therefore have greater resilience to flystrike by having an improved inflammatory response.

While the results of this study suggest that variation in FABP4 might be used as a gene-marker for flystrike resilience in sheep and that breeding for sheep that carry the A₁ variant may decrease disease prevalence, the pleiotropic nature of the gene, including its potential effect on other important production traits, suggests considerably more research needs to be undertaken.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethical review and approval was not required for the animal study because the research we undertook was retrospective/observational (not experimental), with the animals analyzed being located on private farms where they are part of commercial sheep farming operations. They were not part of experimental flocks, and the flocks were not structured, or created for experimental purposes. The blood collection approach we employed falls within the allowable practices specified in Section 7.5 Animal Identification of the Animal Welfare (Sheep and Beef Cattle) Code of Welfare 2010, which is a code of welfare issued under the Animal Welfare Act 1999 (New Zealand Government).

HZ and JH contributed to conception and design of the study. LB collected the data, organized the database, performed the experiments, and wrote the first draft of the manuscript. LB, CF, and RF performed the statistical analysis. HZ, RF, and JH contributed to the manuscript revision. All authors read and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.