Microbiological, serological and molecular diagnosis of abscess of withers in equine in the State of Pará

There are several infectious diseases that affect equines and can cause large economic losses in this sector and damage to animal welfare. In this species, abscessing lesions of a purulent character preferentially in the withers are strongly suggestive of brucellosis. However, few studies have been carried out on the disease in this species, making its identification and control difficult due to the lack of standardization in the interpretation of results. Therefore, samples of whole blood, serum and purulent secretion were collected from three horses. The animals came from the state of Pará. The diagnostic methods selected were the Rose-Bengal plate test (RBPT) and Polymerase Chain Reaction (PCR), for the negative molecular results for brucellosis, microbiological analysis were chosen. Although part of the samples were positive for brucellosis in serological tests, all of them showed negative results for PCR. In bacterial cultivation, species were isolated Streptococcus equi subsp. zooepidemicus, Enterococcus faecalis e Staphylococcus sciuri subsp. lentus., which showed resistance to antibacterials, especially the E. faecalis isolate classified as MDR (multi drug resistant). In conclusion, the best form of diagnosis is the joint evaluation of several tests, both screening and confirmatory, in order to obtain a definitive diagnosis with assertiveness.

There are several infectious diseases that affect horses and can cause great economic losses in this sector and damage to the well-being of animals, including diseases of zoonotic potential such as glanders, leptospirosis and brucellosis (Barcelos et al., 2017;Rocha et al., 2019). In equines, purulent abscess lesions in bursae, ligaments, tendons, synoviums and joints, preferably in the withers are strongly suggestive of brucellosis, and reproductive disorders in this species are rare. Thus, economic damages resulting from brucellosis are considered lower when compared to other species of zootechnical interest, such as cattle and pigs in which brucellosis is considered a reproductive disease (Silva et al., 2018).
The isolation and identification of bacteria from cervical lesions is the gold standard for diagnosis, however the difficulty of this procedure and limitation for large-scale use has made the use of techniques such as the Rose-Bengal plate test (RBPT) and Standard Tube agglutination Test (SAT) more frequent due to the speed of the result and the low cost (Fernandes & Gradela, 2014;Gharekhani et al., 2020). The most widely used serological method is the RBPT (recommended by PNCEBT) and in which the presence of agglutination, will classify the animal as reagent to the test, being able to be submitted to confirmatory tests. Non-reactive animals will be considered negative and positive animals will be destined for sanitary slaughter (Fernandes & Gradela, 2014).
Conventional serological methods have low sensitivity during early stages of the disease and reduced specificity in areas where the disease is endemic (Asaad & Alqahtani, 2012). In addition, the occurrence of nonspecific reactions, the lack of standardization in the interpretation of results and false positive results in the serological study of equines has been alerted (Brasil, 2006;Pinho et al., 2014;Paim et al., 2015). Thus, the importance of differential microbiological diagnosis in cases of abscesses in the withers and synovitis in equines is evidenced in order to avoid economic losses and risks to public health ( Silva et al., 2018). The present study aimed to identify the etiological agents of withers abscesses in equines from the state of Pará and to assess their susceptibility to antimicrobial drugs.

Material and Methods
The study was carried out from withers abscess samples from three horses of different origins, two from a property in Marajó and one from the metropolitan region of Belém. The animals were known to be positive in the RBPT performed in the field, according to the MAPA recommendations (Brasil, 2006), however, for confirmatory effect, the samples reacting to RBPT were submitted to retesting the conventional RBPT, according to the procedure adopted by Ribeiro et al., (2003) and to the Polymerase-mediated Chain Reaction (PCR) (Paim, et al., 2015).
The blood was collected by venipuncture of the jugular vein in a sterile tube with EDTA for PCR and in a sterile tube without anticoagulant for serology. Samples from Marajó were transported in a thermal box with recyclable ice at -10 ° C and the from Belém was sent directly to the laboratory (from veterinary microbiology at the Universidade Federal Rural da Amazônia), subsequently they were transferred to 1.5 µL eppendorfs and kept in the ultra-freezer at -80°C. The secretions were collected from puncture with a sterile syringe and kept in sterile tubes between 6°C and 10°C.
After the negative PCR assay result were the samples of purulent secretion was inoculated in base agar plus 5% sheep blood and MacConkey agar, being in a bacteriological incubator at 37°C for 24 hours to isolate the etiological agent according by Ikram, (2005). The classification and identification of the genus of the microorganisms was carried out by through of the morphotintorial characteristics to the Gram staining, biochemicals and the macroscopy of the colonies, regarding the species, were identified by the automated method VITEK® 2.

Results and Discussion
All blood samples were reactive to the RBPT carried out in the field and when submitted to the re-test in the laboratory they presented the same results. Purulent secretions were also evaluated by the RBPT and only the animal sample from the metropolitan region was laboratory reagent, but none of the blood and secretion samples tested positive for Brucella spp. when applied to PCR assay (Figure 1). Result similar has been found by Meirelles-Bartoli & Mathias (2020), which was a sensitivity of 99.6% and a relative specificity of 83.9% of the RBPT (screening test) were found in relation to confirmatory tests, however Ribeiro et al., (2003) warns of the occurrence of nonspecific reactions, so that the serological diagnosis of brucellosis is more reliable when obtained through varied tests. Research, Society and Development, v. 10, n. 9, e34910917754, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17754 Another relevant point is the lack of standardization in the interpretation of serological tests of the disease in horses, since the PNCEBT does not have detailed rules for serodiagnostic methods for equine brucellosis, causing difficulties in terms of sanitary conduct, control actions and prophylaxis of the disease in this species, as reported Ribeiro et al., (2008) andPinho et al., (2014).
Due to the negative results in PCR for brucellosis, another etiology for abscesses was sought, opting for microbiological examination. The three samples showed positive growth on 5% sheep blood agar plates incubated at 37°C for 24 hours and were classified according to colony size, shape, color, hemolysis and presence of catalase.
Evidencing the importance of microbiological diagnosis in cases of withers abscesses and synovitis in the equine species, already documented by several authors such as Ribeiro et al., (2003) and Karthik et al., (2016), who recommend making the differential diagnosis for bacteria: Arcanobacterium pyogenes, Actinomyces bovis, Escherichia coli, Pseudomonas aeruginosa, Proteus mirablis and for the genus Pasteurella spp., Corynebacterium spp., Streptococus spp. e Staphylococus spp. which can also be isolated from fistulous abscesses in equines.
With light microscopy it was observed the presence of Gram positive cocci organized in chains (pearl necklace aspect) or pairs obteined in pure culture, which were identified by automated equipment due to their biochemical characteristics in the species Streptococcus equi, subsp. zooepidemicus, Enterococcus faecalis e Staphylococcus sciuri subsp.
lentus. (Figure 2). Enterococcus are present as commensals in the gastrointestinal tract of most mammals, and according to Cortés-Delgadillo, (2007) they can behave as opportunistic pathogens causing infections, such as cases of diarrhea in foals and suppurative lesion conditions in adult equine in accordance with results of the present work. Silva et al., (2013) characterizes Staphylococcus sciuri subsp. lentus as part of the commensal microbiota of mammalian skin, being isolated from healthy and sickness equine. However, Kengkoom & Ampawong, (2017) described the first case of subcutaneous abscess and chronic dermatitis in rats associated with S. sciuri infection in Thailand. As in the report by Pessoa et al.,(2014), in which samples were collected from 28 abscesses, which no animal presented reactive serology for Development, v. 10, n. 9, e34910917754, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17754 6 brucellosis. The nodule samples were then sown and the growth of Staphylococcus spp. It was obtained. However, little is known about the pathology and epidemiology of S. sciuri infections in animals.
The antibiotics of selection for microorganisms of the genus Streptococcus spp. they belong to the penicillin group (penicillin G, oxacillin, ampicillin, amoxicillin + clavulanate) and third generation cephalosporins (cefotaxime). Sulfonamides, although recommended as the first choice, were not used because infections with large amounts of debris do not respond well to treatment with this chemotherapy as documented by Spinosa et al., (2017).
Isolate of Streptococcus equi subsp. zooepidemicus was susceptible to cephalosporin and all penicillins used, except ampicillin, corroborating previous research of Pansani et al., 2016 that detected in equine samples 100% of the isolates of the subspecies sensitive to penicillin, 40% resistant to ampicillin, however 35% presented resistance to cefotaxime, differently our study (Table 1).  For the species Enterococcus faecalis the antibiotics of first choice are combinations of beta-lactams and aminoglycosides or cephalosporins aiming at the synergistic effect of these drugs, since these bacteria have intrinsic resistance to various antimicrobial therapies and the ability to acquire more resistance by genes introduced by plasmids and other resistance strategies as reported by Herdan et al., (2012).
The antibiotics of choice for microorganisms of the genus Staphylococcus spp. they are the groups of penicillins (penicillin G, ampicillin and oxacillin), macrolides (azithromycin) and sulfonamides, however sulfas were not used for the test due to their limited action on sample agents such as those of the present study. The S. sciuri strain was also resistant to multiple drugs in the classes of penicillins, macrolides, aminoglycosides and quinolones, so that all antimicrobials of first choice would be ineffective in combating this strain. Result in agreement by Salaberry et al., 2016, in which isolates of Staphylococcus spp.
However, the microorganism has demonstrated sensitivity in vitro to drugs indicated as alternatives to antibiotic therapy in MDR cases by the FDA Clinical Indications, (2019).

Conclusions
Although withers abscesses are still one of the main indicative symptoms of brucellosis in the equine species, it is of utmost importance to carry out different types of tests to compare the results, both screening and confirmatory ones; molecular, microbiological or serological, aiming to arrive at the definitive diagnosis with assertiveness and obtain success in the treatment of the disease when it is possible, avoiding the dissemination of multiresistant genes in bacteria. Thus, avoiding economic losses, risks to public health and the unnecessary sacrifice of animals.