Investigations on the occurrence of fatty muscular dystrophy in Austrian slaughter pigs

Lukas Schwarz (  Lukas.Schwarz@vetmeduni.ac.at ) University Clinic for Swine, University of Veterinary Medicine Vienna https://orcid.org/0000-0002-47164913 Carolin Schöner University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien Rene Brunthaler University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien Herbert Weißenböck University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien Tanja Bernreiter-Hofer University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien Barbara Wallner University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien Andrea Ladinig University of Veterinary Medicine Vienna: Veterinarmedizinische Universitat Wien


Results
The two muscle samples were investigated histopathologically and diagnosed with fatty muscular dystrophy. The results of routine histopathology were con rmed by dystrophin-speci c immunohistochemistry. Sex of the two affected animals was determined retrospectively using a PCRbased protocol and resulted in one male and one female pig. A survey to determine the prevalence of fatty muscular dystrophy of pork revealed that this phenomenon gets frequently observed in Styria, but also occurs in Upper Austria and Lower Austria. Mostly gluteal and lumbal muscles were affected and approximately 20% -40% of the affected muscles were replaced by fat.

Conclusions
Fatty muscular dystrophy or muscular steatosis, as it was sometimes called in early literature, seems not to be an uncommon and rare event. As it was detected in both sexes, our observations are different to the described case in Japan, where only one male individual was affected. To avoid further increase of such cases (fatty muscular dystrophy), it would be useful to clarify the cause. First, whether the cause is environmental or genetic, and in case it is genetic it would be key to disentangle the underlying genomic architecture. Having causal variants described -one could think about integrating this information (depending on the mode of inheritance and the number of loci involved) in the breeding program of pigs. Furthermore, the proportion of non-Austrian pig genetics used for commercial pig production in Austria should be reviewed in order to be able to make reliable statements about the spread of the disease not only in Austrian pig breeds, but also in pig breeds worldwide. Background Page 3/12 Meat and carcass inspection at slaughter houses is sometimes challenging for responsible veterinarians, as abnormalities and pathoanatomical changes of organs and tissues cannot always be assessed via visual diagnosis. Once a rare and undetermined abnormality occurs, meat inspecting veterinarians should put effort in nding the cause of this abnormality. Especially muscular abnormalities may stress veterinarians in making decisions whether to discard the carcass or affected muscle parts or not. Using simple methods, such as histology followed by literature search may give valuable hints for uncommon and rare muscular abnormalities.
In humans, dystrophic muscular diseases are well described and investigated [1][2][3][4]. The two forms, Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), are distinguished by the severity of the clinical course of affected patients [1]. While clinical symptoms of DMD, such as starting signs of locomotive disorders and immobility, start very early in life, symptoms of BMD usually emerge later and have a milder course, which leads to a longer survival of the affected individuals [1]. For diagnosing both forms of the disease, a combination of disease-related pedigree records and laboratory tests are available. Additionally, a muscle biopsy may be investigated for the presence and abundance of dystrophin, either immunohistochemically or via Western-blot [3,4]. For studying DMD and BMD of humans, animal models were established in mice and Golden Retriever dogs [4].
In veterinary medicine, dystrophic muscular diseases affect different species. Although the mouse model is the most common animal model used for human research, pigs are more comparable to the human body [5]. Therefore, a BMD pig model was developed and can be used for studying disease progression and future therapy development [6]. Another research group established an alternative pig DMD model by genetically modifying the exon 52 of the dystrophin gene [7].
Despite dystrophic muscular diseases exist in domestic pigs, it is not common to spontaneously observe this pathological phenotype in pigs intended for meat production. This is because commercially used pigs do not reach the age at which pathological and clinical abnormalities are usually observed.
Historically, there are several pathological descriptions of abnormalities of striated muscles in pigs, which probably all relate to the same or a similar clinicopathological entity: muscular steatosis, progressive primary myopathy, pseudohypertrophic atrophy, lipomatous pseudohypertrophy, interstitial lipomatosis, lipomatous muscular dystrophy, myosclerosis, hyperplasia or atrophia lipomatosa [8].
In 2013, Japanese researchers published a case of BMD-like myopathy in a slaughter pig. Further investigations using immunohistochemical staining of dystrophin, PCR-based sex determination and histopathological examination con rmed this case to be homologous to human BMD as the affected pig was a male [9]. Sex determination was performed, as the authors suggested a hereditary disease in pigs similar to human BMD, which is linked to the human X-chromosome [9,10].
In 2019, a veterinarian responsible for meat inspection at a Styrian slaughterhouse observed severe abnormalities of gluteal muscles in two slaughter pigs from a single farm. These muscles were sent to the University Clinic for Swine, University of Veterinary Medicine Vienna, Vienna, for further investigations. The aim of this study was to gain information on muscular abnormalities in slaughter pigs in Austria based on histopathological and genetic investigations of muscles tissue from the two affected pigs from Styria as well as a survey across several meat inspecting veterinarians

Histological, immunohistochemical investigations and PCRbased sex determination
Histologically, tissue samples from both affected pigs showed severe diffuse in ltration of the interstitium with fat tissue. The diameters of muscle bres were highly variable and groups of severely atrophic bres were observed, especially at the borders to fat tissue. Several bres lost their striation and became homogenized and segmentally fragmented. In some muscle cells the nuclei became centralized. Sporadically mild non-purulent in ltration around small interstitial arteries was found. No obvious signs of muscle bre disruption were seen (Fig. 1a). Compared to control sections ( Fig. 1b), immunohistochemical staining of dystrophin revealed marked alterations of its expression in the sarcolemma (Fig. 1c), ranging from reduction to complete absence of immunostaining. In one of the muscles, dystrophin expression was focally dislocated to the inside of muscle bres (Fig. 1d). Summarized, the results of histopathological and immunohistochemical examination strongly supported the diagnosis fatty muscular dystrophy.  According to results of the PCR-based sex determination one affected pig was male, the other one female.

Survey of Austrian meat inspecting veterinarians
Fourteen veterinarians gave feedback on their observations about fatty muscular dystrophy using our survey. The survey resulted in a response rate of 14.9% (14/94). Results of the survey are shown in Table  1. but also veterinarians of Lower Austria and Upper Austria reported at least once an indication of fatty muscular dystrophy. Most often, fatty muscular dystrophy was observed in slaughterhouse 1, with frequencies of 4-times a year to twice a month. In the remaining slaughterhouses, observations of fatty muscular dystrophy lesions were observed sporadically or were even single observations. In most cases the relative extent of fat in ltration in affected muscles parts was 20-40%, in some cases up to 80%.
Clinical abnormalities were not reported in any individual during inspection of live animals before slaughtering. Out of all responses, four veterinarians never had observed fatty muscular dystrophy. Unfortunately, we cannot determine whether all of the non-responding persons (80/94) never observed fatty muscular dystrophy or just were uninterested in participating in the survey. Over the past few years (2018-2020), approximately ve million pigs were slaughtered per year in Austria [11].

Discussion
The results of this study show for the rst time the incidence of muscular dystrophic diseases in swine in Austria, based on observations by meat inspecting veterinarians in slaughterhouses. Comparing our ndings with the results in a Japanese study [9], histopathological alterations of the two investigated muscles from Styria were equal to the ones described there [9]. In both of our cases, severe fatty in ltration of affected muscles, degenerative hyalination and centrally placed cell nuclei could be observed. Furthermore, the reduced expression of dystrophin in the sarcolemma could be con rmed by immunohistochemistry. DMD could be excluded in the Japanese as well as in our study, due to the fact that dystrophin was not totally lacking and no malfunctions of the muscular apparatus of live animals could be observed [1,9]. Our ndings in the two pigs from Styria differ at least in parts from the Japanese ndings as we could not detect myophagy, loss of muscle bre structure, necrosis and brosis. This may be due to a different underlying mechanism causing muscular dystrophy in the Austrian compared to the Japanese case -either genetically determined or based on another yet unknown aetiology. It has been shown, that even different mutations in the dystrophin gene can result in variable properties of the protein and accordingly diverse histologic characteristics. For example, in a minipig the mutation of the central rod domain of the dystrophin gene led to mild symptoms [6], whereas a modi cation of the gene at exon 52 in another pig resulted in severe DMD [7]. Moreover, a case in the United States reported a mutation of exon 41 of the dystrophin gene in a pig which, additionally to classical ndings of muscle dystrophy of striated muscles, showed porcine stress syndrome [12]. As we do not yet know the cause of fatty muscular dystrophy in Austrian pigs, we can just speculate why our cases differ from the Japanese ndings. Summarizing all recently described cases, obviously more than one mechanism exists which may end up in different muscular dystrophy or fatty muscular dystrophy, respectively.
Fatty muscular dystrophy is often caused by mutations in the dystrophin gene, which in humans as in pigs is located on the X-chromosome. X-chromosomal location of a gene causes a gonosomal mode of inheritance [9]. Assuming also a disease causing alteration in the dystrophin gene, we speculate that sows which carried an impaired X-chromosome could have reproduced affected piglets even when they were inseminated with semen of unaffected (homozygous for the wildtype allele) boars due to the gonosomal recessive inheritance which consequently developed fatty muscular dystrophy. Such matings would result in a majority of healthy siblings, but theoretically 25% of the male piglets may also end up in fatty muscular dystrophy, despite the boar was no carrier of a dystrophin gene mutation. Horiuchi et al.
(2014) suggested, analogous to human BMD and based on just one investigated case, that only male pigs were affected [9]. As in our case one male and one female pig were affected by fatty muscular dystrophy, we hypothesize, that the mother sow and boar used for insemination may have been carriers of an impaired X-chromosome. As we did not investigate the genetic background of the two Austrian pigs, we can just speculate whether the cause of fatty muscular dystrophy was based on a hereditary disease or if it has an absolutely different aetiology. However, the occurrence of fatty muscular dystrophy in a female pig could be more important than expected. Supposing fatty muscular dystrophy in Austrian pigs is a hereditary disease based on a recessive mutation on the dystrophin gene in both, male and female pigs, this would be alarming as it would imply a high allele frequency in the Austrian swine population. Next it would be indicated to nd out the origin of affected animals for the determination of the genetic background and the in uencing environment. This may be followed by a deep analysis of inheritance and estimation of heritability of the trait. Based on the afore mentioned steps one may put effort in determining responsible loci in the genome for establishing a molecular genetic test which probably could be used in swine breeding for eradication of fatty muscular dystrophy.
Based on the results of the survey it can be concluded, that fatty muscular dystrophy is not only a regional problem in Styria, as the pathophenotype was reported by meat inspecting veterinarians also in Lower Austria and Upper Austria. Nevertheless, most observations were reported from Styrian veterinarians. We assessed the reports from Styria as relevant, as all responding meat inspecting veterinarians regularly and more than once a week inspect meat. Interestingly, in slaughterhouse one, observations of animals affected by fatty muscular dystrophy were reported once to twice a month.
Hypothesizing that Austrian observations of fatty muscular dystrophy results from a hereditary disease background, one may speculate that the higher incidence in Styria is due to the establishment of carriers in local gilt and boar breeding stock. For this reason, further research is necessary to investigate whether fatty muscular dystrophy in Austria is genetically determined and if so, the underlying genetic basis should be resolved to detect carriers in Austrian swine breeds which serve as multiplying factors. Moreover, rst a cost-bene t-analysis has to be performed to nd out whether fatty muscular dystrophy in Austria is an economical or health related problem of pigs that would justify the time and effort of further research.
Another explanation for the accumulated occurrence in Styria could be, that the relatively low response rate of our survey may have substantially biased the situation. Despite it seems that reported cases primarily originated from Styria, we can just hypothesize about the reason for this observation.

Conclusion
To conclude, fatty muscular dystrophy was diagnosed for the rst time in Austria and was more often observed in Styria compared to other federal states. As we just can speculate about the causative mechanism of fatty muscular dystrophy, an in-depth analysis is indicated to elucidate the prevalence of fatty muscular dystrophy not only in the Austrian swine population, but also in the pig population worldwide. Further studies are needed to investigate the cause for the observed pathophenotype in order to develop strategies to eradicate fatty muscular dystrophy.

Methods
This study was performed in two different parts. Part one contained in-depth diagnostics of skeletal muscle samples of two slaughter pigs from Styria and part two was a survey among meat inspecting veterinarians at Austrian slaughterhouses.
Muscle samples from slaughter pigs Two gluteal muscle samples were sent for further diagnostics to the University Clinic for Swine, University of Veterinary Medicine Vienna, Austria, by a veterinarian responsible for meat inspection. Cubic pieces with an edge length of approximately 1.5 cm of each muscle were placed in 10% buffered formalin for histological and immunohistochemical investigations. Additionally, tissue of each muscle was stored at -20°C for potential further investigations.

Histological and immunohistochemical investigations
After 24 h of xation, the samples were embedded in para n wax, sectioned at ~ 3 µm and stained with hematoxylin and eosin. Dystrophin immunohistochemistry with a monoclonal antibody (dilution 1:50; Lab Vision, Fremont, CA, USA) was performed with an automated immunostainer (Thermo Autostainer 360-2D; Thermo-Fisher Scienti c, Fremont, CA, USA) using the Ultravision LP detection system (Thermo-Fisher) and DAB as chromogen (Thermo-Fisher). As positive control skeletal muscle from an unaffected pig was used. For negative control the primary antibody was replaced by the respective immunoglobulin isotype.

DNA isolation and PCR-based sex determination
Genomic DNA was isolated from 25 mg muscle tissue using Nexttec DNA Isolationskit (nexttec, Germany). PCR sex determination was performed as described by Blanes et al. (2016) including positive controls from pigs with con rmed sex and a no-template control [13]. PCR fragment sizes were visualized on a 1.5 % Agarose gel containing Atlas clearsight DNA dye (Atlas, Estonia).

Survey among Austrian meat inspecting veterinarians
For the determination of the frequency of fatty muscular dystrophy or muscular steatosis a questionnaire was prepared. This questionnaire was sent to meat inspecting veterinarians in Lower and Upper Austria, Carinthia and Styria. Additionally, the survey was extended to operators of slaughter houses and meat processing companies with the goal to forward the questionnaire to the responsible veterinarians. The survey was conducted from May 2019 until November 2019 and the questionnaire was sent in total to 94 different recipients via e-mail. To increase the return rate of lled questionnaires we decided to keep it short in regards to the numbers of questions and points asked. Due to data protection reasons no personrelated data were collected. In case of submission of additional personalized data, this was anonymized. The questionnaire rst asked if fatty muscular dystrophy has been observed at all. In case of a rmation, consecutive questions were on the frequency of the observations, which muscle parts have been observed to be affected, the relative extent of affected muscle parts and if any clinical signs were observed during clinical examination of live animals at the slaughterhouse (Additional File 1). For explanatory reasons a description of fatty muscular dystrophy or muscular steatosis respectively was sent with the questionnaire (Additional File 2). This was necessary to exclude the misinterpretation of other muscular diseases as fatty muscular dystrophy.
Data were only descriptively analysed using Microsoft Excel (Microsoft O ce 365, USA) due to the low response rate.
LS and CS wrote the rst draft of the manuscript and proof-read it, LS initiated the cooperation with BW and supervised diploma student CS and this study, searched for literature and nalized the manuscript. BW performed the PCR-based sex determination of the muscle samples and gave valuable input in writing the manuscript. RB and HW were responsible for histopathological examination and dystrophin immunohistochemistry. TBH was responsible for organization of the transfer of the samples to the university clinic, literature study and proof read the manuscript. AL and BW gave important support on writing the manuscript and proof read the manuscript.

Figure 1
Histopathological and immunohistochemical investigations. legend: (a) Skeletal muscle of a pig with fatty muscular dystrophy with diffuse in ltration of the interstitium (asterisk) with fat tissue and one fragmented muscle ber (cross) (Hematoxylin and eosin (H&E), bar=160 µm). (b) Immunhistochemical staining of unchanged skeletal muscle of a control pig with continuous dystrophin expression in the sarcolemma (Immunohistochemistry (IHC), bar=80 µm). (c) Immunhistochemical evidence of reduction to complete loss (arrowhead) of dystrophin expression in an affected pig (IHC, bar=80 µm). (d) Dislocation of the dystrophin expression to the inside of muscle bres (arrowhead) (IHC, bar=80 µm).

Supplementary Files
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