Chronic progressive lymphoedema in a Friesian horse: clinical aspects and diagnostic approach – case report

Chronic Progressive Lymphoedema (CPL) is a disabling disease of draft horses that affects lymphatic system function typically in the distal limbs. Deformities of distal limbs, especially of the pastern, with fibrosis and skin nodular lesions are consequences of disease progression. A 15-year-old Friesian stallion presented for evaluation with history of forelimb enlargement and nodule formations distal to the carpus for four years. Simple radiographs showed soft tissue nodular lesions and venous contrasted radiography showed intense enlargement of II common digital palmar vein of both forelimbs. Tissue culture tests revealed Streptococcus equi and Proteus mirabilis isolation and skin scraping test identified Chorioptes bovis mites. Histological examination revealed perivasculitis and lymph vessels distention. History, clinical and histological findings, and complementary exams suggested CPL diagnosis. We were unable to find previously published cases describing this disease in Brazil, where the increasing number of draft horses requires attention to this problem. The correct and early diagnosis substantially delays disease progression. Therefore, we highlight the need for nationwide propagation of these data to ensure better diagnosis and early treatment of future CPL cases.


INTRODUCTION
Lymphatic system functions include maintaining tissue fluid homeostasis and removing cellular debris.Lymph is a protein-rich fluid that occupies lymphatic capillaries and is passively transported through the lymphatic system to blood capillaries by muscle contraction.The capillary filtration dysfunction between blood vessels, lymphatic vessels and interstitium results in lymph stasis or lymphoedema.Lymphoedema reduces tissue perfusion and oxygenation ending in metabolic waste augmentation that compromises immunity and skin barrier integrity (Skobe and Detmar, 2000;Keyser, 2014).
Due to the scarcity of draft breeds in Brazil, equine veterinarians are frequently unfamiliar with CPL, which can result in misdiagnosis and retard correct support treatment.Therefore, this study aims to report for the first time detailed clinical and complementary findings of a CPL case in a draft horse in Brazil.

CASUISTRY
A 15-year-old Friesian stallion weighing 450 Kg presented with a history of lameness and chronic lesions in both forelimbs first observed four years previously.The horse had already been admitted at other facilities and after radiographic and histological exams the final diagnosis was not stated.The horse had been treated for habronemosis with ivermectin-and trichlorfonbased products; furthermore, ozone therapy, antibiotic and non-steroidal anti-inflammatory drugs were unsuccessfully used.Due to the lack of diagnosis and progression of disease with intense lameness and severe weight loss, the horse was referred to our facility.
At admission the clinical evaluation revealed a heart rate of 44 bpm, respiratory rate of 19 mpm, pale pink mucous membrane, 37.7°C rectal temperature and normal gut motility.The musculoskeletal evaluation revealed severe fibrous tissue deposition at distal limbs ranging from pastern to carpus.Multiple ulcerated or non-nodular lesions, skin folds and subcutaneous tissue were visible under the hair (Fig. 1).Lesions were firm in consistency and unpainful when palpated.The same nodular aspect and skin folds were observed when hind limbs were examined except for the ulcerative lesions that were absent in hind limbs.Additionally, all four limbs showed skin crusts along the lesions and hair loss dorsal and caudal to carpus joints (Fig. 2) Occasionally, discrete itching was observed when the horse scratched the lesions against objects.
The horse presented left forelimb lameness (score 4 to 5/5 according to AAEP), alternating from severe lameness to complete limb dysfunction.Additionally, the left forelimb hoof showed chronic deformity with overgrown heels.Based on CPL suspicion, lesions were biopsied for histopathological exam, bacterial and fungal culture of deep tissue.Skin scraping, contrasted (venography) and simple radiograph were obtained, in addition to complete blood count, hepatic and renal blood tests.
The blood tests revealed hyperfibrinogenemia, normal erythrogram and leukogram, low levels of creatinine (1.08 mg/dL), hypoalbuminemia (2 mg/dL) and hyperglobulinemia (5.9 g/dL) (Kaneko et al., 1997).Tissue culture was negative for fungus, whereas the aerobic culture found growth of Streptococcus equi and Proteus mirabilis, both sensitive to cephalosporins.From the skin scraping test, Chorioptes bovis was identified.The histology of tissue biopsies showed large epidermal and superficial dermic erosion areas with ulceration and necrosis associated with polymorphonucleated (neutrophil and eosinophil) infiltrate.Bacterial myriads and serocellular scabs were also observed.When epidermic tissue was integrated, acanthosis was observed, as well as multifocal dermal fibroplasia associated with neovascularization, mild edema, and lymphocytic infiltrate with perivascular pattern on blood and lymphatic vessels and with stasis of deep lymphatic vessels (Fig. 3).
Radiograph exams revealed multiple soft tissue nodulations and skin folds in all limbs.On the left forelimb we observed exostosis on the dorsal aspect of the distal phalanx, distal interphalangeal joint osteoarthritis and enthesophytes on common digital extensor tendon insertion with osseous proliferation and remodeling of the distal phalanx extensor process.Venography showed severe dilatation of digital palmar vein (Fig. 4).
To identify and treat skin lesions, both forelimbs were shaved from the carpus to the hoof and a compressive bandage was applied on the area.Additionally, Firocoxibe (0.1mg/Kg IV SID), intravenous regional perfusion with ceftriaxone was started in left forelimb (2g IV, every other day) and doramectin 0.3mg/kg SC (Dectomax®, doramectin, Zoetis Brazil, Brazil) was administered.For topical use, selenium sulfate 1.5% shampoo and fipronil 0.25% (Frontline®, fipronil, Merial, France) for limbs were indicated.Two days after the beginning of treatment, lameness score for the left forelimb improved but worsened again a few days later, alternating between mild (3/5) to severe lameness (4/5).
Five days after admission the horse developed a surgical colic syndrome with large colon impaction with a fecalith in small colon.After surgery the clinical parameters deteriorated, and the horse died because of postoperative complications.

DISCUSSION
CPL has high occurrence in draft horse breeds, especially, Clydesdales, Shires, and Belgians (Cock et al., 2006a) and, recently, Friesian was included as a susceptible breed, thus suggesting genetic content for disease development (Cock et al., 2003;Affolter, 2013;Keyser et al., 2014;Affolter et al., 2020).A previous study observed minor elastin content in dermis biopsies of susceptible draft horse breeds when compared to other draft horse breeds with no previous reports of CPL, such as Percheron (Cock et al., 2006b).In this study, the stallion was a Friesian horse, previous reported as a susceptible breed.The gender is not related to disease manifestation, although male horses frequently develop disease when they are older, with accelerated progression (Keyser et al., 2014).
CPL symptoms frequently begin at the age of two years; however, long hair in limb extremities of draft horses hamper early detection of disease signs and the lesions are identified when itching starts during secondary infection (bacterial or parasitic) (Affolter, 2013;Affolter et al., 2020).In the present case report the late manifestation or identification may be a consequence of intense hair coverage on distal limbs or late manifestation in males, both of which are associated with lack of knowledge about this disease in Brazil (Keyser et al., 2014).Chronic and progressive, as the name suggests, CPL affects distal limbs causing progressive enlargement, soft tissue deformation and aberrant skin aspect.When the disease progresses and affects dermis, subcutaneous and adjacent tissues, the entire limb can lose its original shape (Keyser et al., 2014).In the late stages the disease culminates in fibrosis, skin nodulations and folds, first on the pastern, then can ascend to the carpus or tarsus (Affolter, 2013).We found, in this case, macroscopic findings compatible with late-stage CPL.
Forelimbs presented more evolved lesions reaching the carpus region whereas in the hind limbs, nodulation and fibrosis were palpated, but restricted to half to distal metatarsus.The grade of previously proposed lesion ratings varies according to the number and thickness of skin folds, limb enlargement and number and region of fibrous nodulations (Cock et al., 2006a).All four limbs of the presented horse fitted into the most severe grade (grade 4), according to the authors above.
CPL is a multifactorial disease that covers genetic and environment interaction (Keyser et al., 2014).Before CPL was accepted as a syndrome, uncured distal-limb lesions in horses were diagnosed as chronic pastern dermatitis, which is characterized by crusty, hyperkeratotichyperplastic, nodular, or warty dermatitis.Currently, the term CPL is correctly used given the lymphatic compromise in this disease (Keyser et al., 2014).In our case, pastern dermatitis was first suspected.However, the lack of response to conventional treatment highlighted the possibility of some unknown components.
Ulceration observed on skin folds was associated with skin trauma resulting in erosion, ulceration, and hemorrhaging.The integrity loss of skin barrier perpetuates the inflammation, impedes lymph drainage, and contributes to secondary infection and lymphoedema progression (Affolter et al., 2020).
The left forelimb lameness may have more than one cause, since there was deep soft tissue infection and osteoarthritis signs in distal interphalangeal joint, with bone remodeling of the second phalanx and a large enthesophyte on a common extensor tendon insertion of the distal phalanx (Ramos et al., 2020).Mechanical movement restriction was also described as a consequence of intense fibrosis of the limb, nodulation and skin folds, which when surrounding the entire region, can restrict movement and result in lameness (Keyser et al., 2014).Although, functional lameness is described as caused by fibrosis, the same lesion grade was observed in other limbs without apparent lameness, suggesting that fibrosis was not the main cause in our case.
Fibrinogen is an unspecific acute phase protein that guides the diagnosis and prognosis of inflammatory process in horses, and its elevation is associated with tissue lesion of either infectious or inflammatory cause (Campbell et al., 1981;Alonso et al., 2020).The related horse had marked hyperfibrinogenemia (800mg/dL) caused by the chronic inflammatory condition associated with CPL.

Arq. Bras. Med. Vet. Zootec., v.74, n.4, p.693-700, 2022
In the absence of hepatic lesion observed by the normal range for liver enzymes in blood tests, we attribute the hypoalbunemia (Muñoz et al., 2010), lower creatinine values (Diago and Señaris, 2020) and hyperglobulinemia (Crisman et al., 2008;Muñoz et al., 2010) to malnutrition and chronic inflammation.Low creatinine levels are associated with an excessively catabolic muscle state in situations such as intense inflammatory response, malnutrition, and lack of physical activity (Diago and Señaris, 2020).The weight loss marker is associated with lameness severity, since chronic pain results in movement restriction and consequently limits food intake (Tamzali, 2006).
Although Staphyloccus sp. and Dermatophilus congolensis are the most common agents isolated from CPL skin lesions (Affolter, 2013), in the present study Streptococcus equi subsp.zooepidemicus and Proteus mirabilis were isolated.These findings did not indicate climatic or environmental interference, given that all the agents cited above are opportunistic pathogens and are found in healthy skin microbiota (Timoney, 2004;Barracco and Bisno, 2006;Westgate et al., 2011;Weese and Yu, 2013;Drzewiecka, 2016;Haag et al., 2019).Considering the characteristics of secondary infection and the commensal presence of these agents in microbiota, our findings are justified despite the less usual isolation.
Histopathologic findings characterize the presence of bacterial dermatitis and fibrous dermatitis and support the suspicion of CPL with the presence of perivasculitis and enlarged lymphatic vessels (Cock et al., 2003;Affolter, 2013).Complementary exams assist in finalizing the diagnosis, but predominantly based on history, breed, and clinical findings.The initial symptoms can be confused with other pastern dermatitis caused by mites, fungi and/or bacterial infections; nonetheless, these infections cannot cause severe symptoms as observed in CPL (Keyser et al., 2014).
Radiographic exams permit early detection of skin thickness, tissue folds and nodulations (Keyser et al., 2014).In this case, due to advanced disease, nodulations were easily identified.Severe fibrosis resulted in lymph stasis and lymphatic vessel dilatation, usually observed by contrasted radiography of limb lymphatic vessels.Unfortunately, since we could not accomplish this exam, sedation was prioritized for regional perfusion of antibiotics.Before regional antibiotic perfusion, angiography was performed and revealed intense blood vessel enlargement.Similar to what happens to the lymphatic vessels, limb enlargement and fibrosis reduce blood return resulting in vessel dilatation (Cock et al., 2003).Angiographic findings support CPL diagnosis.
Previous studies raised the possibility that affected horses could have higher levels of antielastin antibodies, which in humans are quantified using an enzyme-linked immunosorbent assay (ELISA) and are important for monitoring severe connective tissue disorder.The ELISA for anti-elastin antibodies was already described for horses (Keyser et al., 2012); however, more recently, the real importance of the anti-elastin quantification for CPL diagnosis was not sustained (Keyser et al., 2015).
The genetic content of the disease is highly suspected; however, this hypothesis is yet to be proven.Recently, genomic associations were evaluated to investigate CPL in Friesians.The results of the genomic study suggest high complexity and other than the studied nucleotide polymorphisms (Affolter et al., 2020).The absence of specific tests for CPL diagnoses highlights the importance of clinical and radiographic findings to identify the disease.
CPL is an incurable disease whose treatment involves intense management changes to detain disease progression.There are three major principles: to treat the secondary infection, to take care of the skin, and to promote lymph transport.Even achieving the three steps, treatment can be disappointing with frequent infection recurrence (Keyser et al., 2014).Despite the bad prognosis, in our case, the owner chose to maintain animal's life and institute the support treatment.To contain bacterial infection and parasitic infestation, regional perfusion was started in the cephalic vein with ceftriaxone, and to minimize lymphedema the limbs were bandaged.The severe lameness precluded controlled exercise, which is essential to improve lymphatic circulation.Muscle activity acts as a pump to propel lymph through lymphatic vessels, so the combination of compressive bandaging, massage and exercise is imperative to improve lymph drainage in horses (Affolter, 2013), resulting in reducing limb enlargement and improving movement (Keyseret al., 2014).
Clipping the limb enables visualization of lesions covered by hair and improvement of topical treatment contact.There are frequent descriptions of an owner's reluctance to clip a draft horse's distal limb hair given its importance as a breed characteristic.We highlight the necessity of clipping the affected area to improve diagnoses and lesion treatment (Affolter, 2013).For parasite control two doramectin doses were planned at a 14-day interval as described by Rendle et al. (2007) and topical administration of fipronil (Littlewood, 2000;Rendle et al., 2007;Affolter, 2013) with routine bathing with selenium-sulfate-based shampoos acting as a keratolytic (Affolter, 2013;Keyser et al., 2014).Unfortunately, due to the colic episode with unfavorable progression we could not evaluate the treatment's effectiveness in controlling CPL.Although the animal's death prevented the treatment follow-up, we emphasize the importance of this case description, given the lack of CPL records in Brazil and the difficulty in diagnosing the disease for lack of CPL knowledge by local equine veterinarians.Describing clinical findings along with diagnostic strategies can facilitate early disease detection.

Figure 1 .
Figure 1.Lesion aspect of chronic progressive lymphoedema in a Friesian stallion: Lateral aspect of left forelimb during admission (A) and same limb after clipping (B); In a closer approximation, respectively, after admission (C) and after clipping (D).Observe deformities, nodulations, and granulomatous wounds where ulceration has occurred.Cranial and medial aspects of forelimbs after hair removal indicate multiple nodulations (E).

Figure 2 .
Figure 2. Lesion aspect of chronic progressive lymphoedema in a Friesian stallion: Caudal aspect of forelimbs (A) Detailed caudal view of the right hind limb evidencing caudal tarsus with intense fibrous tissue and alopecia before clipping (B) and after clipping (C), respectively; and cranial view of left tarsus, showing alopecic area (D).

Figure 4 .
Figure 4. Radiographic images of Friesian horse showing CPL: Simple latero-medial radiography of right and left forelimbs (A and B), and right and left hind limbs (C and D), respectively.And contrasted radiograph in latero-medial left forelimb (E) demonstrating II common digital palmar vein enlargement (arrows).