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Glanders is a zoonotic disease of equids caused by the bacterium Burkholderia mallei, responsible for considerable economic loss. This study aimed to describe the clinical manifestations, pathological findings, and also bacteriological and molecular methods for agent detection in naturally infected animals (16 adult horses and one fetus) detected by serological survey from three glanders outbreaks. Of the 16 horses, 6 (37.5%) did not show clinical signs. After necropsy,samples of organs, lymph nodes, lesions and secretions were collectedfor histopathology, bacterial isolation, and PCR. The clinical and gross alterations mainly comprised nasal and respiratory forms in the three outbreaks, and less of the cutaneous form. All tested animals were positive by PCR (100%, n=17) on at least one sample. Of 121 samples analyzed 8.2% (10/121) isolated B. mallei and 41.3% (50/121) were polymerase chain reaction (PCR) positive. This work highlights the importance of combining diagnostic techniques, such as histopathology microbiological culture and PCR,to confirm cases and characterize the morbidity of glanders as well as considering seropositive animals without glanders clinical signs as potential carrier animals affecting disease control programmes., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024. Published by Elsevier Inc.)

Fungal and bacterial culture is currently the primary method for pathogen detection and identification. Next-generation sequencing is a powerful method for detecting and identifying the presence of microbial DNA in samples. We evaluated the correlation between fungal and bacterial culture with next-generation sequencing in equine uterine samples. Fungal cultures (n=63) were evaluated based on their culture results. In culture positive (n=16) samples, next-generation sequencing identified the same organism in 10 samples (62.5%), 5 samples did not identify fungal agents, and 1 sample identified other species of fungal agents. In no growth samples (n=42), next-generation sequencing did not identify fungal agents in 37 samples (88.1%), 4 samples had a potential fungal pathogen identified, and one sample identified only non-pathogenic fungal organisms. Fungal culture and next-generation sequencing had an 80% agreement and moderate correlation by Kappa coefficient (0.508). Bacterial culture (n=57) was also evaluated based on bacterial culture results. In bacterial culture Positive samples (n=32), next-generation sequencing identified the same organism in 25 (78.1%) samples, and identified different organisms from the remaining 7 samples. In bacterial no growth samples (n=14), next-generation sequencing detected bacterial presence in 5 samples, and 9 samples had no bacterial DNA identified. The agreement between bacterial culture and next-generation sequencing was 74% with a moderate correlation by Kappa coefficient (0.46). In conclusion, culture and next-generation sequencing have moderate correlation, and next-generation sequencing has the potential as a diagnostic option for enhancing pathogen detection for equine endometritis., Competing Interests: Declaration of competing interest SHC received honoraria as a speaker at a veterinary conference from MicrogenVet., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alpha-fetoprotein (AFP) concentrations have been reported in healthy foals and proposed as a biomarker of sepsis in foals born from mares with experimentally induced placentitis. This study aimed to describe the diagnostic and prognostic value of plasma AFP in foals spontaneously affected by different diseases. The study included all foals less than 72 h old that were diagnosed with either: (1) prematurity (PRE), when born prior to 320 days of gestation with immature physical characteristics; (2) sepsis (SEP), in the presence of both positive blood culture and SIRS or (3) neonatal encephalopathy (NE), with evidence of hypoxic-ischemic injury. Data from healthy foals (H; n=20) were obtained from a previous study. Foals received a complete physical and hematochemical evaluation and blood culture sample collection at hospital admission. Forty-six foals with an average age of 16 h were enrolled and divided into: PRE group (n=7); SEP group (n=14); NE group (n=25). AFP was measured in plasma collected at admission using a commercially available immunoassay validated for horses. AFP was increased in foals in PRE, SEP and NE groups compared with healthy ones (P<0.001) but was not able to discriminate between different diseases and outcomes. Overall, AFP was negatively correlated with foal age (r=-0.6; P<0.001), foal weight (r=-0.3; P=0.048), monocytes count (r=-0.4; P=0.011) and SAA concentration (r=-0.4; P=0.011). AFP appears to be a useful but non-specific indicator of neonatal health, since it upregulates not only in the presence of SIRS and bacteremia, but also during prematurity and hypoxic-ischemic injury., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydropsical conditions in domestic animals are characterized by abnormal fluid accumulation. While their complete pathophysiology remains unclear, factors such as changes in chorioallantoic membranes, placentitis, or fetal abnormalities may contribute. Hydrops present as a medical emergency that can cause complications for the mare. Early diagnosis and treatment are crucial for the mare's prognosis, as fetal survival is often poor. This report describes a case of hydroallantois in a 9-year-old pregnant Quarter mare at 271 days of gestation, leading to dystocia of a non-viable foal with multiple congenital abnormalities. Given the poor prognosis for fetal survival, transcervical gradual fluid drainage and pregnancy termination were performed. The malformed fetus was subsequently delivered and humanely euthanized. Necropsy findings revealed a schistosomus fetus with palatoschisis, a wry nose, prognathism, and unilateral ovarian agenesia. The case underscores the importance of timely intervention in managing hydroallantois. The report contributes to understanding horses' clinical presentation, diagnosis, and management of hydroallantois., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Coxofemoral dysplasia is a condition characterized by the abnormal development and flattening of the femoral head and deformities in the acetabulum, leading to joint instability. Although described in various animal species, it is uncommon and rarely reported in horses. This article presents a case of incapacitating coxofemoral dysplasia diagnosed in a four-month-old foal Mangalarga Marchador, which exhibited lameness in the hind limbs, thoracolumbar kyphosis, gluteal muscle atrophy, and pronounced sensitivity during pelvic limb flexion. The clinical diagnosis indicative of bilateral coxofemoral dysplasia was confirmed through post-mortem examination. This case report highlights the importance of awareness and monitoring for early detection of coxofemoral dysplasia in foals., Competing Interests: Declaration of competing interest None of the authors have any financial or personal relationships that could improperly influence or bias the content of the paper., (Copyright © 2024. Published by Elsevier Inc.)

Serum protein electrophoresis (SPE) is widely used to evaluate protein changes associated with several pathologies, aiming to assist clinical diagnosis. This study determines, for the first time, SPE profile changes in West Nile virus-naturally infected horses. Sixty horses with different clinical and infection status (encephalitis, asymptomatic infection, and immunologically naïve) were included. Total protein concentrations were determined via an automated biuret method and SPE was performed using a cellulose acetate membrane-based automated system. Statistical analysis revealed significantly higher total protein between both infected horse groups (encephalitis, asymptomatic) and naïve controls. Horses with encephalitis had higher globulin and α2-globulin levels than the other two groups (asymptomatic, naïve), along with lower albumin percentage and albumin-to-globulin ratio. Furthermore, γ-globulin levels were significantly higher in asymptomatically infected compared to control horses. These findings highlight the diagnostic value of SPE profile determination, assisting the clinicians, and providing the grounds for further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier Inc. All rights reserved.)

A 4.5-month-old Standardbred colt presented for neck swelling and fever. Endoscopy and contrast radiography identified a full thickness esophageal perforation. Surgical intervention with fasciotomies and placement of an esophageal feeding tube was elected. Intensive postoperative treatment was carried out with broad-spectrum antibiotics, gastroprotectants and probiotics, calculated feeding plan, esophagostomy and fasciotomy site lavages, care, and cleaning. Complications included recurrent bilateral pneumothorax and development of an incomplete fistula at the esophagostomy site following removal of the feeding tube, necessitating primary closure. One year following presentation, the colt appears to have made a full recovery with an excellent body and muscle condition score and undergoing athletic training. Anticipated price of sale as a yearling was achieved. This study highlights a viable treatment route for youngstock with esophageal perforation that necessitate an esophagostomy. Despite intensive dietary needs of growing foals, appropriate nutritional requirements and normal growth rates can be achieved in such cases., Competing Interests: Declaration of competing interest There are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A 16-year-old horse, 400 kg, male, Quarter horse gelding was examined for a cardiac murmur that had not been previously heard. Physical examination revealed a regularly irregular pulse and a grade III/VI, decrescendo, diastolic murmur with a point of maximum intensity over the left heart base. Base-apex standard electrocardiographic examination at rest showed sinus rhythm with second-degree atrio-ventricular blocks. Echocardiography identified the presence of four aortic valve cusps, two equal larger and two unequal smaller cusps. Color flow Doppler examination showed a diastolic regurgitant jet emerging from the central region of closed aortic valve. Based on clinical and echocardiographic findings a diagnosis of mild to moderate aortic valve insufficiency secondary to quadricuspid aortic valve was made. This report describes a rare congenital heart defect that can be detected by transthoracic echocardiography in the horse., Competing Interests: Declaration of competing interest All authors declare that there is no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Placental infection is an important cause of late-term pregnancy loss and neonatal diseases in horses. Detection of changes in blood parameters especially during early placentitis could improve the diagnostic accuracy, treatment decision, and potential outcomes. The objectives of this 2-part study were to identify differences in circulating immunological, inflammatory, and hormonal parameters between mares with natural ascending placentitis and control mares; evaluate each and combination of parameters as predictors of placentitis; and determine how these parameters indicate severity of placentitis. Reproductive examination and blood sampling were prospectively performed on pregnant mares in a natural setting. Study 1 enrolled mares diagnosed with early stage of ascending placentitis based on ultrasonographic findings (n = 12), and gestationally age-matched mares with healthy pregnancies as controls (n = 12). Blood samples were classified as pre-onset (before) and early onset (at the time of ultrasonographic changes) of placentitis. There were no detected statistically significant differences between groups and timepoints in immunological and inflammatory parameters, including peripheral lymphocyte subpopulations, cytokine, and serum amyloid A concentrations. The placentitis group showed a reduced (P = 0.01) DHP/20α-DHP ratio when compared to the control group at the early onset timepoint. Plasma estradiol-17β concentration <359 pg/mL predicted ascending placentitis with acceptable accuracy (area under the curve, AUC = 0.71). Combined albumin <3.7 g/dL, estradiol-17β < 499 ng/mL, and DHP <33 ng/mL predicted 100 % of cases of ascending placentitis. In study 2, samples were classified according to the presence and severity of the abnormal ultrasonographic findings as mild (n = 11) and moderate-severe (n = 23), and gestationally age-matched with samples from control mares (n = 34). Mares with moderate-severe ascending placentitis had increased (P = 0.03) plasma 20α-DHP concentration and reduced (P = 0.03) DHP/20α-DHP ratio when compared to control mares. Our results suggest that the early events of ascending placentitis detected by ultrasonographic findings include hormonal alterations of feto-placental metabolism measurable in the mare's circulation, yet without obvious systemic immunological and inflammatory changes. Additional studies are warranted to further assess how hormonal markers and cutoff values could guide decisions for timely therapeutic intervention., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Toxoplasmosis is one of the most common parasitic zoonoses and represents a significant health risk for humans, especially for immunodeficient patients. The main transmission route is by oral uptake of oocysts and consumption of undercooked meat of infected animals. Different species have been evaluated as possible reservoirs of the parasite, but few studies have been carried out to examine the role of horses in transmission of the disease. Given the proximity of these animals to humans and the widespread consumption of their meat in many countries, including the Mediterranean basin, it is important to determine the prevalence of T. gondii infection in this species. In this study, blood samples from 105 horses were collected and the presence of T. gondii was evaluated by serological and molecular methods. Antibodies against T. gondii of 12 horses (11.43%) were detected by enzyme-linked immunosorbent assay (ELISA), whereas 29 horses (27.62%) showed positive for PCR. Seroprevalence was related to use of the animals, being higher in horses used for dressage than in others. Purebreds had higher seroprevalence than crossbred animals. No differences between breed, sex or age were found. The results of this study confirm the presence of T. gondii infection in horses, highlighting the need to analyse the meat of this species before human consumption and to control of this infection in horses, as they could be an important reservoir of this zoonotic parasite., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF phosphorylated neurofilament heavy protein (pNfH) concentrations in support of an EPM diagnosis was also evaluated. 172 horses were divided into three groups: EPM-positive horses (EPM+, n=42), neurological non-EPM horses (n=74) confirmed with non-EPM neurological diseases (cervical vertebral compressive myelopathy, equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy), and control horses (control, n=56) without neurological signs and neurological abnormalities on histology. Logistic regression was used to compare EPM diagnostic regimens. Specifically, EPM+ horses were compared with neurological non-EPM horses showing neurological signs. To consider diagnostic utility, post-test probabilities were calculated by titer. When differentiating between EPM and other neurological diseases, the combination of serum and CSF SarcoFluor testing added more information to the model accuracy than either test alone. Using serum and CSF for pNfH in support of an EPM diagnosis did not identify cutoffs with statistically significant odds ratios but increased the overall model accuracy when used with the IFAT. Utilization of IFAT titers against S. neurona in serum and CSF result in a high post-test probability of detecting EPM+ horses in a clinical setting., Competing Interests: Declaration of Competing Interest P.S.P, W.A.S., C.J.F., M.A., P.A.C., A.P., M.P., K.W., and N.P. work for the School of Veterinary Medicine, University of California-Davis, which markets the SarcoFluor test for diagnostic purposes., (Copyright © 2024. Published by Elsevier B.V.)

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Raksha Vasantrai Bhoora reports financial support was provided by National Research Foundation. Alicia Venter reports financial support was provided by AgriSETA. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A clinical case of an adult horse with invasive, ulcerative, proliferative, pyogranulomatous disease of the skin (tumor) in the shoulder region is presented. The mass had a granulomatous and crater-shaped appearance, with serosanguinous discharge and the presence of fistulas with caseous material. The tumor was removed by surgery and sent to the laboratory for diagnosis. Histopathology was performed using Grocott-Gomori methenamine silver stain. The presence of necrotic material, fibrosis, infiltrated cells, and brown-colored hyphae, characteristic of members of the genus Pythium, were observed. To identify the infecting species, conventional PCRs for the amplification of the ITS-1 was carried out. Histopathological and PCR tests confirmed infection by a Pythium insidiosum strain closely associated with previous records from the US and Central America. Our report represents the first molecularly confirmed case of equine pythiosis in Mexico., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Placentitis is an important cause of reproductive losses in the equine industry. Many cases of clinical placentitis are not diagnosed until late in the course of the disease, and for this reason there is variability in the timing of intervention, diagnostic measurements, and treatment protocols. An 8-year-old multiparous Dutch Warmblood mare that was recently exposed to EHV-1 in the herd of origin presented for routine foaling management. Placentitis was diagnosed upon intake, and medical treatment was initiated. The mare delivered a term foal, and diagnostics for infectious etiology were unrewarding. While there were obvious clinical signs supporting a diagnosis of placentitis, histopathologic examination did not reveal active inflammatory lesions in the chorioallantois; however, severe funisitis was present. This report reviews early diagnosis and management of placentitis, demonstrates an array of classic and subtle pathologic lesions seen on microscopic examination, and discusses pathophysiology of placentitis., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tapeworm infection in horses can cause serious health concerns, and recent data have documented treatment failures in the most common species, Anoplocephala perfoliata. The threat of anthelmintic resistance in A. perfoliata is of particular concern because of poor diagnostic performance of standard egg counting techniques for detecting this parasite. This study compared the performance of three diagnostic techniques 1) Mini-FLOTAC, 2) Cornell-Wisconsin, and 3) Proudman and Edwards used to detect and quantify A. perfoliata eggs in naturally infected horses. Eighteen adult female horses from the University of Kentucky's historic parasitology herd were included in this study. Fecal samples were collected from all horses at five collection time points two weeks apart and analyzed with the three techniques. A total of 90 samples were collected and 270 counts determined in the study. The proportions of positive samples determined by the three techniques were significantly different from each other (p<0.05): Mini-FLOTAC (16%), Cornell-Wisconsin (47%), and Proudman and Edwards (70%). The Proudman and Edwards technique counted consistently higher numbers of tapeworm eggs compared to the other two techniques throughout the study [p < 0.05]. Total raw counts of tapeworm eggs across the study for each technique were 16, 88, and 410 for the Mini-FLOTAC, Cornell-Wisconsin, and Proudman and Edwards, respectively. This study demonstrated that the Proudman and Edwards technique was superior in diagnosing A. perfoliata infection. Future work needs to assess this technique's potential for Fecal Egg Count Reduction Testing (FECRT)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Implantable loop recorders (ILRs) are increasingly used in equine cardiology to detect arrhythmias in the context of collapse, poor performance or monitoring for recurrence of atrial fibrillation (AF). However to date, the ILR has never been reported to be used with a remote monitoring functionality in horses, therefore the arrhythmia is only discovered when a clinician interrogates the ILR using dedicated equipment, which might delay diagnosis and intervention. This case report describes the use of an ILR with remote monitoring functionality in a horse with recurrent AF. The remote monitoring consisted of a transmission device located in the stable allowing daily transmission of arrhythmia recordings and functioning messages to an online server, available for the clinician to evaluate without specialised equipment. The ILR detected an episode of paroxysmal AF approximately three months after implantation. Seven months after implantation, initiation of persistent AF was seen on an episode misclassified by the ILR as bradycardia, and the horse was retired. This report shows the feasibility and benefits of remote monitoring for ILRs in horses, but also the shortcomings of current algorithms to interpret the equine electrocardiogram., Competing Interests: Conflict of Interest Statement The authors do not have any conflict of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A 23-year-old domestic donkey (Equus asinus) referred for severe respiratory distress due to suspected equine asthma. Ultrasound of the chest revealed bilateral irregular pulmonary consolidation and pleural effusion. Airway endoscopy and tracheal wash cytology showed severe neutrophilic inflammation and bacterial culture was positive for Streptococcus equi subsp. zooepidemicus. Despite aggressive treatment, the donkey died in 48 hours. On post-mortem examination, the lung was whitish, collapsed, and firm, with fibrotic multifocal nodular areas. Pleural effusion and pleuritis were detected. Histologically, the lung architecture was markedly replaced by interstitial fibrosis. The histological features observed were suggestive of a severe chronic fibrosing interstitial pleuropneumonia with type 2 pneumocyte hyperplasia and intralesional syncytial cells. Pulmonary fibrosis was associated with the presence of asinine gammaherpesvirus 2 and 5 infection, confirmed by PCR and sequence analysis. The macroscopic and histological pattern of fibrosis was diffuse and interstitial, and the nodular lesions were consistent with spared lung parenchyma, instead of the canonical nodular distribution of the fibrosis observed in equine multinodular pulmonary fibrosis. Asinine herpesviral pulmonary fibrosis is uncommon, but should be considered by clinicians in the list of differentials in donkeys with chronic respiratory signs., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Horses are intermediate hosts of Sarcocystis spp. capable of forming cysts in their musculature. This study aimed to detect sarcocysts and investigate the presence of nucleic acids from Sarcocystis spp. in samples of striated muscles from horses in the State of Rio Grande do Sul, Brazil, necropsied at the Veterinary Pathology Laboratory of the Federal University of Santa Maria. A total of 108 samples were collected from 24 horses and examined through direct examination. Microscopic tissue cysts were observed in three samples: tongue (2) and esophagus (1) from two animals. Extractions were performed on the found cysts and tissues, even though sarcocystosis detection was not present. DNA samples were subjected to Nested-PCR using Tg18s primers, and the amplified products were subjected to Restriction Fragment Length Polymorphism (RFLP) using DdeI and HpaII enzymes. DNA belonging to Sarcocystis spp. was amplified in tissues from 91.7% (22/24) of the equines, and 67.6% (73/108) of the samples tested positive in the Nested-PCR reaction. The tissues with the highest detection frequency were: diaphragm 92.3% (12/13), gluteal muscle 77.2% (17/22), and esophagus 66.7% (4/6). In RFLP, Sarcocystis spp. was detected in 21 tissues from 11/22 equines, and cysts, identified through nucleotide sequencing, were determined to be S. bertrami. S. neurona was identified in 11 samples from 7/22 animals, with co-infection detected in 5/22 cases. The high detection rate indicates a concerning circulation of the protozoan, particularly the zoonotic S. bertrami found in all tissues, which are commonly exported for human consumption., Competing Interests: Declaration of Competing Interest The authors have no financial or proprietary interests in any material discussed in this article., (Copyright © 2023. Published by Elsevier B.V.)

Equine endometrial degeneration is diagnosed by microscopy of an endometrial biopsy but it is uncertain if findings in a single biopsy represent the state of the entire endometrium. Previous studies have compared samples from multiple sites but conclusions are inconsistent. Further clarification is therefore needed. In this study, presence and characteristics of endometrial degeneration were compared in two full thickness specimens from the ventral base of both uterine horns, obtained post mortem from cyclic mares (n = 82). Hematoxylin and eosin-stained sections were blinded evaluated for periglandular fibrosis, glandular nests, dilated glands, glandular dilation, excessive lymphatic vessels, and lymphatic lacunae. Each section was also assigned to a modified Kenney and Doig categorization. Statistical analysis included multiple logistic regression models for fraction of mares with disagreement in the modified Kenney and Doig category and fraction of mares with disagreement for each parameter, multiple generalized linear regression models for fraction of parameters with disagreement, and equivalence tests for agreement of a cumulative score of degenerative parameters. Possible effect of age, mare type and estrous cycle stage were included in the analyses. For the Kenney and Doig categorization, 49% of the mares had good agreement, 46% had moderate agreement and 5% had poor agreement between the two biopsies. Disagreement in the Kenney and Doig categorization between the two biopsies increased with age (OR = 1.1, P = 0.009). Disagreement within each parameter ranged from 10% of mares (glandular nests), to 31% (periglandular fibroblasts) and 37% (lymphatic lacunae). Disagreement for changes in endometrial glands increased with age (OR = 1.15-1.16, P < 0.040). Several mares (33%) had disagreement in at least two parameters, and the number of parameters with disagreement increased with age (HR = 1.04, P = 0.010). When tested for equivalence, the cumulative scores for paired endometrial samples did not differ for mares younger than 18 years (P = 0.011). In conclusion, the degenerative changes were not uniformly distributed, revealed by a disagreement between paired endometrial samples for individual parameters and for the Kenney and Doig categorization, which increased with age. This suggests that conclusions based on a single biopsy, particularly in mares over 17 years of age, should be interpreted with caution, and evaluation of two biopsies should be considered., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Effects of general anesthesia with controlled ventilation on the respiratory system have had limited evaluation in horses. A prospective observational study was performed with eleven client-owned horses undergoing elective surgery. Physical examination, auscultation with a rebreathing bag, complete blood cell count, lung ultrasound imaging, tracheal endoscopy imaging and transendoscopic tracheal wash were conducted before and 24 hours after anesthesia. Lung ultrasound imaging was also repeated just after recovery. A significant increase in blood neutrophil count between pre- and post-anesthesia (P=0.004) was observed. There was an increase in ultrasonographic score of the lungs at recovery (left P=0.007, right P=0.017). The score of the dependent lung was higher than the independent lung at recovery time (P=0.026) but no difference was observed 24 hours after anesthesia. The tracheal mucus score was higher after anesthesia (P=0.001); severe local inflammation was present in several horses at the site of endotracheal tube cuff. Neutrophil count was significantly higher on tracheal wash fluid cytology after anesthesia (P=0.016), without any significant changes on bacterial load. Increased tracheal mucus score and neutrophil count in tracheal wash samples were observed after general anesthesia in healthy horses without clinical evidence of pneumonia (fever, cough). Tracheal inflammation secondary to endotracheal intubation and cuff inflation was, therefore, suspected. Elective surgery without complications can induce inflammation of the trachea and changes in ultrasound images of the lungs in healthy horses and should be considered when evaluating respiratory system after a general anesthesia., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Equine colic is an important condition associated with acute abdominal pain and one of the leading causes of death in horses. As such, objectively evaluating pain is of interest for attending veterinarians. Pain scales for assessment are present, but no single pain-specific biomarker has been reported. The aim of this study was to determine if substance P (SP) could be a reliable biomarker to reflect pain and serve as a parameter to predict outcome in equine colic. The hypothesis was that horses displaying severe colic signs present with higher values of SP in contrast to those with mild colic signs. Thirty warmblood horses, aged between 3 and 20 years were recruited; evenly distributed (10 horses each) in three colic groups (mild, moderate, severe). To classify the colic signs, the horses were graded by the Equine Acute Abdominal Pain Scale (EAAPS). Clinical examination and EAAPS were performed at arrival in the hospital. Blood samples were collected four times in hourly intervals commencing from arrival. For comparison, already established parameters for prognosticating equine colic (heart rate, serum cortisol, and blood lactate concentration) were also measured. The assumption of increasing SP concentrations along with pain could not be confirmed. SP did not show any association with heart rate, cortisol, lactate, or EAAPS. Whereas the established parameters increased according to the EAAPS, SP remained stable in individual horses regardless of clinical signs, treatment, and disease progression. Consequently, SP was not a reliable parameter to reflect painful conditions or to predict outcome in equine colic., Competing Interests: Declaration of Competing Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2023. Published by Elsevier Inc.)

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field., Competing Interests: Declaration of Competing Interest The LumiraDx instrument was supplied by Shionogi & Co., Ltd. and LumiraDx Ltd. to the Equine Research Institute, Japan Racing Association. None of the authors had any other conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, EP S+PCR- horses and control horses (EP seronegative and PCR negative). It also investigated potential haemato-biochemical markers to aid in distinguishing true EP carriers alongside molecular and serological tests. A comprehensive haematology and biochemistry profile was conducted on 410 sera and EDTA blood samples, comprising 130 EP positives by real-time PCR and competitive ELISA (cELISA) (carriers), 130 EP negatives by real-time PCR but positive to cELISA (S+PCR-) and 150 EP negative horses to real-time PCR and c-ELISA (controls). Our study confirmed that a haematological and biochemistry profile could help to differentiate between EP carriers/S+PCR- from healthy horses. Carriers and S+PCR- horses showed significant increases in the white blood cell count (WBC), high total proteins (TP) and total globulins (GLOB) concentration, and liver function markers compared to controls. Additionally, the evaluation of uric acid (UA) suggested oxidative stress in carrier horses. However, no useful haemato-biochemical diagnostic markers were identified to aid the challenging differentiation of EP carriers and S+PCR- horses, highlighting the need for improvement in molecular/serological diagnosis for these horses., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Eliazar Camino reports financial support was provided by Complutense University of Madrid. Eliazar Camino reports a relationship with Complutense University of Madrid that includes: funding grants., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Congential amastia, a medical condition in which mammary tissue fails to develop, was detected in a 3-year-old Quarter Horse mare. The dam of the mare was also afflicted with amastia, suggesting that the condition was due to an inherited genetic mutation as noted in other species. In addition, on presentation the mare had a purulent vaginal discharge secondary to a pyometra., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aim of the present study was to carry out a serological survey to identify the seroprevalence of Lawsonia intracellularis in six Thoroughbred farms in the Southern region of the state of Rio Grande do Sul, Brazil. During 2019 and 2020, blood samples from 686 Thoroughbred horses were obtained from six different breeding farms. Horses were divided into groups according to age: (1) broodmares (>5 years), (2) two-year-old foals, (3) yearlings, and (4) 0-6 months-old foals. Blood samples were collected by venipuncture of the external jugular vein. The detection of antibodies (IgG) against L. intracellularis was performed by Immunoperoxidase Monolayer Assay. The detection of specific antibodies (IgG) against L. intracellularis in the evaluated population was 51%. The highest detection (86.8%) of IgG was in the broodmares category, while the lowest (5.2%) was in foals of 0-6 months of age. Regarding the farms, the Farm 1 had the highest (67.4%) prevalence of seropositivity against L. intracellularis, while Farm 4 had the lowest (30.6%). There was no record of clinical manifestation of Equine Proliferative Enteropathy in the sampled animals. The results of this study show the high seroprevalence of L. intracellularis in Thoroughbred farms in the Southern of Rio Grande do Sul, suggesting a large and continuous exposure to the agent., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A 6-year-old Marwari mare presented with recurrent vulvar growth. The growth was surgically excised, fixed and processed routinely. Microscopically, neoplasm showed proliferation of epithelial and myoepithelial cells with tubulopapillary pattern. On immunohistochemistry, myoepithelial cells showed strong immunoreactivity with smooth muscle actin alpha and p63. On basis of histopathology and immunohistochemistry, tumour was diagnosed as complex apocrine carcinoma. This case report describes first confirm vulvar complex apocrine carcinoma in equines., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Traditional visual lameness assessment is subjective. Ethograms have been developed for evaluating pain and objective sensors to detect lameness. Heart rate (HR) and heart rate variability (HRV) have been used to evaluate stress and pain. The aim of our study was to compare subjective and behavioral lameness scores, a sensor system measuring movement asymmetry, HR, and HRV. We hypothesized that these measures would show related trends. In 30 horses, an inertial sensor system was used to measure movement asymmetries during trot in-hand. A horse was categorized as sound if each asymmetry was less than 10 mm. We recorded riding to observe lameness and evaluate behavior. Heart rate and RR intervals were measured. Root mean squares of successive RR intervals (RMSSD) were calculated. Five horses were categorized as sound and 25 horses as lame by the inertial sensor system. No significant differences were detected between sound and lame horses in the ethogram, subjective lameness score, HR, and RMSSD. Overall asymmetry, ethogram, and lameness score had no significant correlation with each other, whereas overall asymmetry and ethogram correlated significantly with HR and RMSSD during certain phases of the ridden exercise. The main limitation of our study was the small number of sound horses detected by the inertial sensor system. The association between gait asymmetry and HRV suggests that the more gait asymmetry a horse shows during trot in-hand, the more pain or discomfort it probably experiences when ridden with a higher intensity. The threshold for lameness used by the inertial sensor system may require further evaluation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Heterotopic mineralization in equine distal limbs has been considered an incidental finding and little is known about its imaging features. The study aimed to identify heterotopic mineralization and adjacent pathology in the fetlock region with cone-beam (CB) computed tomography (CT), fan-beam (FB) CT, and low-field magnetic resonance imaging (MRI). Images from 12 equine cadaver limbs were examined for heterotopic mineralization and adjacent pathology and verified by macro-examination. Retrospective review of the CBCT/MR images from 2 standing horses was also included. CBCT and FBCT identified twelve mineralization's with homogeneous hyperattenuation: oblique-sesamoidean-ligament (5) without macroscopic abnormality; deep-digital-flexor-tendon (1) and suspensory-branch (6) with macroscopic abnormalities. MRI failed to identify all mineralization's, but detected suspensory branch splitting, and T2 and STIR hyperintensity in 4 suspensory-branches and 3 oblique-sesamoidean-ligaments. Macro-examination found corresponding disruption/splitting and discoloration. All modalities identified 7 ossified fragments showing cortical/trabecular pattern: capsular (1), palmar sagittal ridge (1), proximal phalanx (2) without macroscopic abnormality, and proximal sesamoid bones (3). On MRI, fragments were most identifiable on T1 images. All abaxial avulsions had suspensory-branch splitting on T1 images with T2 and STIR hyperintensity. Macro-examination showed ligament disruption/splitting and discoloration. Suspensory-branch/intersesamoidean ligament mineralization's were identified by CBCT in standing cases; 1 had associated T2 hyperintensity. Both CT systems were generally superior in identifying heterotopic mineralization's than MRI, while MRI provided information on soft tissue pathology related to the lesions, which may be important for management., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A 21-year-old Quarter Horse mare presented with a chronic, progressively worsening left pelvic limb lameness of 3 weeks duration. The initial examination identified a consistent lameness at a walk. Neurological examination showed sensory and gait abnormalities consistent with left femoral nerve dysfunction. The horse minimally advanced the leg cranially and had a shortened stride length at the walk. During the stance phase, the heels of the left hind foot did not contact the ground and the horse quickly took weight off of the limb. Diagnostic imaging (ultrasound and nuclear scintigraphy) examinations did not reveal a cause. Severe lymphocytosis was identified on complete blood cell count (69,600 cells /uL; reference range: 1,500-4,000 cells/uL), suggestive of lymphoma. Postmortem examination revealed focal swelling of the left femoral nerve. Multiple masses were found in the stomach, large colon, adrenal gland, mesentery, heart, and meninges. The entire left pelvic limb was dissected and did not reveal other causes of the gait deficit. Histologic evaluation of the left femoral nerve revealed disseminated intermediate cell size B cell lymphoma, with an immunophenotype suggestive of plasmacytoid differentiation. These lymphocytes infiltrated the femoral nerve at the location of the focal nerve swelling, in addition to other peripheral nerves. This case highlights a horse with an atypical diagnosis of femoral nerve paresis caused by direct neoplastic lymphocyte infiltration, deriving from disseminated B cell lymphoma with plasmacytoid differentiation (neurolymphomatosis). Though rare, disseminated lymphoma with direct nerve infiltration should be considered in horses with peripheral neuropathies., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Contagious equine metritis (CEM) detection by PCR is recognized by the European Union according to Commission Implementing Regulation (EU) No 846/2014, and real-time PCR is now recommended by the World Organisation for Animal Health Terrestrial Manual at the same level as the culture method. The present study highlights the creation of an efficient network of approved laboratories in France in 2017 for CEM detection by real-time PCR. The network currently consists of 20 laboratories. A first proficiency test (PT) was organized by the national reference laboratory for CEM in 2017 to evaluate the performance of the early network, followed by annual proficiency tests organized for ongoing periodic assessment of network performance. Results of the 5 PTs organized from 2017 to 2021 are presented, during which 5 real-time PCRs and 3 DNA extraction methods were used. Overall, 99.20% of the qualitative data corresponded to expected results and the R-squared of global DNA amplification calculated for each PT varied from 0.728 to 0.899. DNA extraction is also an important step in the analytical process, and results were more favorable with direct lysis compared to column extraction. Focusing on the most commonly used PCR (PCR 1: 86.4% of results) showed lowest cycle threshold values with direct lysis compared to column and magnetic bead extractions, and with magnetic bead extraction compared to column extraction, but neither of these differences were statistically significant., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Theileria equi is a tick-borne intracellular apicomplexan protozoan parasite that causes equine theileriosis (ET). ET is an economically important disease with a worldwide distribution that significantly impacts international horse movement. Horses are an essential part of the economy in Xinjiang which is home to ∼10% of all the horses in China. However, there is very limited information on the prevalence and genetic complexity of T. equi in this region. Blood samples from 302 horses were collected from May to September 2021 in Ili, Xinjiang, and subjected to PCR examination for the presence of T. equi. In addition, a Bayesian latent class model was employed to estimate the true prevalence of T. equi, and a phylogenetic analysis was carried out based on the 18S rRNA gene of T. equi isolates. Seventy-two horses (23.8%) were PCR positive. After accounting for the imperfect PCR test using a Bayesian latent class model, the estimated true prevalence differed considerably between age groups, being 10.8% (95%CrI: 5.8% - 17.9%) in ≤ 3-year-old horses and 35.7% (95%CrI: 28.1% - 44.5%) in horses that were > 3 year-old. All T. equi isolates had their 18S rRNA gene (430bp) sequenced and analyzed in order to identify whether there were multiple genotypes of T. equi in the Xinjiang horse population. All of the 18S rRNA genes clustered into one phylogenetic group, clade E, which is thus probably the dominant genotype of T. equi in Xinjiang, China. To summarize, we monitored the prevalence of T. equi in horses of Xinjiang, China, with a focus on the association between age and the occurrence of T. equi by Bayesian modelling, accompanied by the genotyping of T. equi isolates. Obtaining the information on genotypes and age structure is significant in monitoring the spread of T. equi and studying the factors responsible for the distribution., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In the present review, the authors, based on the multiple functions performed by the liver, analyze the multiple biochemical and hematological changes as an expression of altered liver function in the horse. The liver performs important metabolic functions related to the synthesis, degradation, and excretion of various substances. Modification of these functions can be evaluated and diagnosed by determining serum concentrations of several serum analytes, including enzymes and other endogenous substances. Hepatocellular enzymes, such as sorbitol dehydrogenase-SDH and glutamate dehydrogenase-GLDH, are released following hepatocellular necrosis. Hepatobiliary enzymes, such as γ-glutamyl transferase-GGT, increase in response to necrosis, cholestasis, and other alterations in bile conducts. Serum concentrations of mainly endogenous and exogenous substances that the liver should synthesize or eliminate, such as proteins (albumin and globulins), bile acids, urea, glucose, total and direct bilirubin, and coagulation factors, and fibrinogen should be included in the liver function test profile. The interpretation of laboratory tests of liver function will allow the diagnosis of functional loss of the organ. Some of the analytes considered provide information on the prognosis of liver disease. This review will provide an accurate and objective interpretation of the common biochemical and hematological tests in use in the diagnosis of equine hepatic disease patients, aiding still further the veterinary activity on the applied equine clinical cases., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The interpretation of the blood count is essential to help the equine clinician in the diagnosis, prognosis, patient management, and control of equine diseases. Hematologic alterations often reflect the condition of the individual or an overall response to a pathological situation. A thorough clinical examination of the patient is essential to correctly interpret the hematological results. The most common abnormalities in the erythrogram are mainly anemia and polycythemia. The frequent causes of anemia in horses are acute and chronic blood loss, hemolytic anemia, and anemia caused by chronic disease. Evaluation of leukogram, including a total white cell count, a differential cell count, absolute numbers of specific leukocytes can help identify abnormalities that may suggest specific diseases such as a viral or bacterial infection, inflammatory disorders or even a neoplastic process. The platelet count is most often used to monitor or diagnose conditions that cause too much bleeding related with thrombocytopenia; it can be due to multiple mechanisms such as reduction of thrombopoiesis (myeloptisis, myelofibrosis, myeloproliferative disease, and idiopathic medullary aplasias or due to the effect of mielosuppresive drugs), increased peripheral destruction of platelets (immune mediated thrombocytopenia), consumption (dissemined intravascular coagulation) sequestration of the spleen and loss of platelets by idiopathic origin., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Among the recognized neurologic diseases in horses, equine protozoal myeloencephalitis (EPM) has been reported around the world and still presents challenges in diagnosis and treatment. Horses can present with clinical neurologic signs consistent with EPM while testing negative for the two main causative agents, Sarcocystis neurona or Neospora hughesi, and may still be clinically responsive to anti-parasitic drug therapy. This context led to our hypothesis that another protozoal parasite, Toxoplasma gondii, which is known to cause toxoplasmosis in other mammalian species, is a potential pathogen to cause neurologic disease in horses. To evaluate this hypothesis, serum and cerebrospinal fluid (CSF) were collected from 210 horses presenting with clinical signs compatible with EPM, and the indirect immunofluorescent antibody test (IFAT) was used to detect antibody titers for T. gondii, S. neurona, and N. hughesi. Additionally, the serum to CSF titer ratio was calculated for T. gondii, S. neurona, and N. hughesi infections, suggesting intrathecally-derived antibodies for each of the three agents if the serum:CSF ratio was ≤ 64. There were 133 (63.3%) horses positive for serum T. gondii antibodies using a cutoff titer of 160, and 31 (14.8%) positive for CSF T. gondii antibodies using a cutoff titer of 5. Overall, 21 (10.0%) of EPM-suspect horses had a serum:CSF ratio ≤ 64 for antibodies for T. gondii, while 43 (20.5%) and 8 (3.8%) horses had a serum to CSF ratio ≤ 64 for antibodies for S. neurona and N. hughesi, respectively. A total of 6 (2.9%) animals presented evidence of concurrent intrathecally-derived antibodies for T. gondii and at least one other apicomplexan parasite in this study. Signalment and clinical signs were not different across the groups aforementioned. These data provide evidence of intrathecal production of anti-T. gondii antibodies, indicative of T. gondii infection in the brain and/or spinal cord of horses with EPM-like disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Leishmania infantum infections have long been described in humans and dogs worldwide, but characterization of equine cases remains scarce. We describe the clinical evolution of a natural L. infantum infection to contribute to the diagnostic knowledge and epidemiology of equine leishmaniasis (EL). An auction-acquired four-year-old Mangalarga Marchador mare from Pernambuco state, presented a few subcutaneous nodules on the head and neck upon arrival at the purchaser's stud at Bahia state, in November of 2019. They progressed to multiple ulcerated and non-ulcerated nodules and spread to both right limbs in seven weeks. Hematology revealed anemia, lymphocytosis, monocytosis, and elevated plasma fibrinogen. Histopathology of the biopsied nodules identified a granulomatous dermatitis with macrophages containing Leishmania amastigotes. PCR detected Leishmania in skin lesions, but not in blood or spleen aspirate samples; ITS1 PCR-RFLP and DNA sequencing confirmed L. infantum species. A topical antiseptic and insect-repellent therapy and a monthly follow-up were established. All lesions improved progressively, without specific anti-Leishmania treatment, and 14 months later there was a consistent resolution. This first description of EL by L. infantum in an endemic area is relevant to emphasize the need for epidemiological studies, and to enhance clinicians' awareness for differential diagnosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Equine endocrine disease is an important area for equine research, requiring an appropriate case definition for inclusion and criteria for exclusion from disease. Defining a case for research may be different from criteria for clinical diagnosis. Further, clinical diagnosis recommendations have been changing regularly, making this area challenging for equine scientists. This review discusses the diagnosis of major equine endocrine diseases, pituitary pars intermedia dysfunction, equine metabolic syndrome and insulin dysregulation, focusing on the most appropriate diagnostic methods for research case definitions. Different diagnostic methods, including use of reference intervals and clinical decision limits, will be discussed with their relative merits for use in case definition for research., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Trypanosoma evansi, a hemoflagellate protozoan, leads to wasting disease, surra in livestock animals causing huge economic losses. Currently, the preferred assay for surra diagnosis is whole cell lysate (WCL) based ELISA, which requires the use of rodents for WCL preparation. To avoid use of laboratory animals, we used recombinant DNA technology to express T. evansi invariable surface glycoprotein (ISG) in E. coli. The potential of recombinant ISG65 (rISG65) as a diagnostic antigen was investigated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The results indicated that rISG65 reacted with horse T. evansi positive serum giving two bands of approximately 48 kDa and 96 kDa. T. evansi-specific antibodies were detected as early as 10 and 14 days post infection using immunoblot and indirect ELISA, respectively using rISG65 antigen. No cross-reactivity was observed in ELISA and immunoblot with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions were observed between 30 and 100 kDa in T. evansi isolate of horse origin indicating the existence of multiple copies of ISG protein in a single trypanosome. The recombinant ISG has proven to be good candidate antigen to be used in ELISA for serodiagnosis of T. evansi infection in different animals., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mares' subfertility represents a complex diagnostic and therapeutic challenge and both clinical and subclinical endometritis are considered major causes of impaired fertility. Thanks to its properties, ozone has a big potential as a treatment for equine endometritis. Therefore, the aim of this study is to describe the safety and the effects on endometrium and reproductive parameters of mares of a commercial ozone foam preparation (Riger Spray®). Twenty-four mares were treated during estrus: ozone group with an intrauterine instillation of ozone foam preparation (OG, n=16) and control group with 20 ml of lactated Ringer's solution (CG, n=8). Samples for endometrial cytology were collected before the ozone treatment (T0), after 24 h (T1), after one week (T2), two weeks (T3), and when the subsequent estrous phase was detected (T4). Furthermore, samples for histological examination and uterine swab for bacteriological examination were collected at T0 and T4. At T1, a statistically significant increase of endometrial inflammation in the OG mares compared to T0 (P<.05) and to CG at same time point (P<.05) was observed, but it was already resolved at T2. No differences in endometrial inflammation in CG, biopsy grade before and after the treatment in the two groups, number of mares pregnant at the end of the season and number of mares pregnant at the first cycle were observed. However, the number of inseminations required for pregnancy tended to be lower (P=.0711) in the OG (1.69±0.06) than in CG mares (2.60±0.89)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This review is aimed to (i) appraise the literature on the use of molecular techniques for the detection, quantification and differentiation of gastrointestinal helminths (GIH) of equids, (ii) identify the knowledge gaps and, (iii) discuss diagnostic prospects in equine parasitology. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we retrieved 54 studies (horses: 50/54; donkeys and zebras: 4/54) from four databases. Polymerase chain reaction (PCR) was employed in all of the studies whereas PCR amplicons were sequenced in only 18 of them. Other techniques used (including modifications of PCR) were reverse line blot, quantitative (q)PCR, restriction fragment length polymorphism, nested-PCR, PCR-directed next-generation sequencing, Southern blotting, single strand conformation polymorphism, PCR-enzyme linked immunosorbent assay, matrix-assisted laser desorption/ionisation-time of flight and random amplification of polymorphic DNA. Most of the studies (53/54) used nuclear ribosomal RNA (including the internal transcribed spacers, intergenic spacer, 5.8 S, 18 S, 28 S and 12 S) as target loci while cytochrome c oxidase subunit 1 and random genomic regions were targeted in only three and one studies, respectively. Overall, to date, the majority of molecular studies have focused on the diagnosis and identification of GIHs of equids (i.e. species of Anoplocephala, Craterostomum, cyathostomins, Oesophagodontus, Parascaris, Strongylus, Strongyloides and Triodontophorus), with a recent shift towards investigations on anthelmintic resistance and the use of high-throughput nemabiome metabarcoding. With the increasing reports of anthelmintic resistance in equid GIHs, it is crucial to develop and apply techniques such as advanced metabarcoding for surveillance of parasite populations in order to gain detailed insights into their diversity and sustainable control. To the best of our knowledge, this is the first systematic review that evaluates molecular investigations published on the diagnosis and quantification of equid GIHs and provides useful insights into important knowledge gaps and future research directions in equid molecular parasitology., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Boehringer Ingelheim did not have any role in the design or content of this manuscript., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Two equine patients presented separately with severe abdominal distention, colic, lethargy, and decreased appetite. An ante-mortem diagnosis of lymphoma was reached in each case based on peritoneal fluid cytology. Due to a poor prognosis, the horses were humanely euthanized. Post-mortem examination with histology and immunohistochemistry confirmed both cases as lymphoma: alimentary B-cell lymphoma of the distal jejunum and cecum in one case, and T-cell lymphoma of the cecum in the second case. Both cases exhibited extensive metastasis with peritoneal and pleural serosae covered in small nodules and plaque like masses consistent with lymphomatosis. These cases document a unique presentation of lymphoma in equine patients presenting as peritoneal lymphomatosis with ascites., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study aimed to determine the occurrence of hemoplasmas and tick-borne pathogens (TBP) (Theileria equi, Babesia caballi, and Ehrlichia sp.) in horses and ticks' salivary glands, and determine the factors associated with exposure/infection in a rural settlement in southern Brazil. Blood samples from 22 horses were screened for anti-T. equi and anti-Ehrlichia sp. antibodies by an indirect fluorescent antibody test (IFAT) assays. Samples were also tested by PCR assays for T. equi and B. caballi (18S rRNA and rap-1 genes, respectively), hemoplasmas (16S rRNA gene), and Ehrlichia sp. (dsb gene). Ticks were removed from the animals (inspection) and the environment (flannel trawling and dry ice traps), and morphologically identified. Additionally, salivary glands DNA was extracted from 28 adult ticks infesting the animals and four nymphs from the environment, and further screened for Ehrlichia sp. and hemotropic Mycoplasma sp. Anti-T. equi and anti-Ehrlichia sp. antibodies were detected in 40.91% (nine/22; 95% CI: 23.26-61.27) and 31.81% (seven/22; 95% CI: 16.36-52.68) horses, respectively. Theileria equi, B. caballi, and hemotropic Mycoplasma sp. DNA was detected in 59.09% (13/22), 4.55% (one/22), and 50% (11/22) horses, respectively. All horses tested negative in the PCR for Ehrlichia sp. All sequences showed ≥99% identity with multiple T. equi, B. caballi, and Mycoplasma ovis sequences deposited in GenBank database. Adult ticks were identified as Dermacentor nitens (44/47; 93.62%) and Rhipicephalus microplus (three/47; 6.38%). Ticks' salivary glands were negative for Ehrlichia sp., while 39.29% from adults (11/28) and 50% from nymphs (two/four) from the environment were positive for hemotropic Mycoplasma sp. This is the first report of M. ovis infection in horses from Brazil and the first detection of hemoplasma DNA in salivary glands of D. nitens and R. microplus ticks. Further studies are needed to elucidate the vector competence of ticks to transmit hemoplasmas., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

An 8-year-old mare mule was presented with a facial mass and history of anorexia, unilateral epistaxis and purulent nasal discharge. The facial mass was encountered from the ventral to the lateral right canthus of the eye up to the facial crest. Thoracic ultrasonography showed five rounded masses. A standing CT under sedation was performed revealing a huge extension and infiltration on surrounding tissues. Due to the extension, type of lesions, outcome and suspicion of metastasis, the owner chose to euthanize the patient. Necropsy confirmed the presence of tumoral lesions spread into the lungs, spleen, adrenal glands, ovary, oral cavity, and right frontal sinus. The microscopic examination was consistent with hemangiosarcoma. Hence, hemangiosarcoma is a differential diagnosis to rule out  in future similar cases. This clinical description did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The study examined and compared the sensitivity of culture and a quantitative PCR assay for screening equine semen for the presence of Taylorella equigenitalis (CEMO). Chilled semen samples, both raw and treated with extender, from two stallions were spiked with the organism at seven or 23 days postejaculation and prepared in serial dilutions. Culture of the 7-day raw semen readily detected CEMO at all dilutions, but extended semen yielded counts that were two log cycles lower at equivalent dilutions, with the organism being nearly undetectable at the maximal dilutions. By contrast, PCR sensitivity was not affected by extender, but for 7-day-old raw semen, PCR detection declined abruptly three log dilutions earlier than detection by culture. The more aged 23-day-old semen proved less satisfactory for spiking, with detection of CEMO by culture failing in three of the four samples due to overgrowth with commensal organisms. However, PCR performance was similar in both the 23- and 7-day spiking series. The detection limit by PCR is estimated at between 10 4 and 10 5 cfu/mL. Typical CEMO concentrations in the semen of colonized stallions are not widely reported but where natural semen contamination has been investigated, the organism was present at this order of magnitude. The reliability of detecting CEMO infection using semen samples by either method is discussed., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

Background: Equine granulocytic anaplasmosis (EGA) is a tick-borne disease caused by Anaplasma (A.) phagocytophilum. In Germany, this pathogen is transmitted primarily by Ixodes ricinus. There is limited knowledge about its prevalence in horses in Germany. The aim of this retrospective study was to analyze the results of serological and molecular testing for A. phagocytophilum in horses which were done in a commercial laboratory in Germany over fourteen years. Additionally, risk factors were evaluated, and hematological abnormalities were addressed in horses with positive PCR results., Methods: This retrospective study examined results of direct (Polymerase chain reaction [PCR]) and indirect (immunofluorescence antibody test [IFAT]) detection methods for A. phagocytophilum in horses on samples provided by German veterinarians and processed by the commercial laboratory LABOKLIN from 2008 to 2021. In horses with positive test results, a Complete Blood Count (CBC) and Serum Amyloid A (SAA) were also analyzed where possible., Results: In total, 1217/4834 horses tested positive (PCR: 190/1246 horses, 15.2%; IFAT: 1036/3849 horses, 26.9%). Seasonality and location, as classified by federal state, had a statistically significant impact on PCR results (P < 0.001 for both). In horses with positive PCR results, hematological abnormalities were detected in 112/118 horses (95%), with thrombocytopenia (86%) and anemia (52%) representing the most common findings. The remaining 6/118 horses (5%) showed no hematological abnormalities on CBC. SAA was measured in 35 horses with positive PCR results, which exclusively showed marked elevation., Conclusions: The seasonality of A. phagocytophilum infections confirmed by PCR testing was consistent with known peaks in vector activity in Germany. The high rate of horses with positive PCR results when compared to dogs and cats may be due to a lack of ectoparasite prophylaxis. Infections with A. phagocytophilum should be considered as a differential diagnosis in horses with cytopenia on CBC and SAA elevation, especially in the summer and after any possible tick exposure., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ingo Schaefer reports a relationship with Laboklin Laboratory for Clinical Diagnostics GmbH & Co that includes: employment. Elisabeth Mueller reports a relationship with Laboklin Laboratory for Clinical Diagnostics GmbH & Co that includes: employment. Declaration of interest Elisabeth Müller is the CEO of the commercial laboratory LABOKLIN (Bad Kissingen, Germany) and Ingo Schäfer an employee of the laboratory. This has not influenced the results of our study in any way., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ultrasound remains a mainstay in proximal suspensory ligament (PSL) evaluation. Despite recent improvements facilitating earlier diagnosis/treatment, needs exist for (1) characterization of normal ultrasonographic PSL cross-sectional area (CSA) in specific breeds/disciplines, (2) improved detection of early/subtle changes in the PSL, and (3) an understanding of suspensory ligament (SL) morphology change from work-related stress. The objectives of this study were to establish normal PSL CSA in Quarter Horses (QH) used for cutting via angle contrast ultrasonography, ultrasonographically monitor SL morphology as horses progress through training, and assess the impact of PSL disease on performance (Lifetime Earnings - $LTE). One hundred ten 2-year-old cutting horses had PSL ultrasound examination on all limbs, and examinations were repeated 2 years later on 51 remaining horses. PSL CSA at select locations was compared between time points and entire examinations subjectively graded by an equine radiologist. $LTE was obtained from the National Cutting Horse Association for each horse and analyzed by abnormality number, location, and development over time. A total of 267 PSL ultrasound examinations across both times points were measured and graded. Normal CSA was established by zone for forelimbs and hindlimbs. The prevalence of abnormal limbs increased between the 2-year-old and 4-year-old years, forelimb abnormalities were statistically more prevalent than hindlimb abnormalities at both time points, and osseous attachment abnormalities were statistically more prevalent than ligament abnormalities at both times points. No significant differences in PSL CSA change over time were seen in any group and there was no significant difference in $LTE among or between groups., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cystic hygroma (hygroma cysticum) is a malformation that has not yet been described as a cause of early pregnancy loss in equines. The condition is a congenital anomaly occurring during embryogenesis due to a failure in which the primitive lymphatic sac does not reach the venous system at the jugular vein, resulting in a lymphatic stasis that starts in the neck region and continues to the rest of the body. From 2015 to 2020, a total of 5,730 ultrasound examinations were performed in mares from 43 different horse farms and embryo transfer farms when sexing pregnancies. In 12 pregnant mares, a suspected fetal cystic hygroma was diagnosed via transrectal ultrasound performed from day 52 to 75 of pregnancy. Six fetuses were collected and fixed to conduct histopathological and karyotyping. Macroscopic and microscopic analysis supported the suggested diagnosis being the first description of cystic hygroma in equine fetuses and concluded as a cause of pregnancy loss around 65 days of gestation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Endometritis is a relevant cause of subfertility in mares. However, the accurate diagnosis, essential for effective treatment, can be difficult due to the variability of results and interpretations resulting from different examination methods and sample collection techniques. The present work compared gynecological evaluation methods and sample collection techniques to diagnose endometritis in subfertile mares. Forty animals with a history of subfertility were selected for gynecological evaluation using clinical methodologies, such as perineal conformation, transrectal palpation and ultrasonography, vaginoscopy, and digital examination of the cervix. In addition, we performed laboratory analyses, including uterine microbiological culture and endometrial cytology and histology, of which the latter is the gold standard for the diagnosis of endometritis. Samples were collected for microbiological culture and endometrial cytological evaluations using three different techniques: a commercial cytobrush/swab collector, low-volume uterine flush, and a new tested technique, by flush the fragment resulting from the endometrial biopsy. Transrectal palpation and ultrasound showed the best results among clinical examinations. However, they were less efficient in laboratory tests of endometrial cytology and uterine microbiological culture, in which the latter showed the highest sensitivity and specificity for endometritis compared with endometrial histology. The use of multiple results from different methods has also proved to be an effective alternative for diagnosis. Among the techniques used to collect endometrial material for cytology and microbiological culture, the most effective and practical in this study was the commercial cytobrush/swab collector., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Equine idiopathic haemorrhagic cystitis (EIHC) is a recently described form of aseptic cystitis in horses in which there is no discernible underlying cause. This case report describes a 9-year-old Thoroughbred gelding that presented with stranguria, pollakiuria, and haematuria. Cystoscopy revealed ulceration and haemorrhage of the bladder mucosa, diffuse mural hyperaemia and marked urine sedimentation. Histopathological evaluation of the bladder revealed chronic active ulcerative neutrophilic, lymphoplasmacytic, and eosinophilic cystitis. There was no bacterial or fungal growth upon culture but polymerase chain reaction (PCR) testing and sequencing for equine herpesvirus-1 (EHV-1) on bladder mucosa was positive. Conservative therapy with broad spectrum antimicrobials and non-steroidal anti-inflammatory therapy yielded complete resolution of clinical signs with significant improvement of macroscopic lesions in 14 days. Although a positive EHV-1 PCR suggests a viral cause, the horse's clinical signs, histology and recovery rate are more consistent with equine idiopathic haemorrhagic cystitis (EIHC). Neutrophilic and lymphoplasmacytic inflammation is a known feature of EIHC but eosinophilic infiltrates have not been previously described. The significance of the eosinophilic involvement is not certain; however, their presence has been associated with fungal, viral, parasitic, and immune-mediated aetiologies in other body systems. This is the first report of a horse with possible EIHC in Australia, as well as the first case with eosinophilic infiltrates and testing positive for EHV-1., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Lower airway cytology has been proposed as a complementary diagnostic method to confirm the presence and quantifying the severity of exercise-induced pulmonary hemorrhage (EIPH). Bronchoalveolar lavage is usually preferred over tracheal wash (TW), yet the need for sedation imposes as a limitation for active sport horses in addition to be a highly invasive technique. This study aims to evaluate the diagnostic value of TW with Total Hemosiderin Score (THS) for monitoring EIPH in active Thoroughbred racehorses. A sampling of 47 randomly selected Thoroughbreds were endoscopically examined for EIPH grading 30 to 60 minutes after competing in an official race and then classified as Group 1 (EIPH-negative), Group 2 (EIPH-positive) or Group 3 (furosemide users). Tracheal wash fluids (TWFs) were collected from 24 to 30 hours later and smears were stained for differential cell count and hemosiderophage grading and counting to calculate THS. Differential cell counts were compared by the median test. Comparisons between mean THS values by EIPH grade and by group were compared by the Mann-Whitney test and ANOVA, respectively. Test performance criteria were determined with a contingency table. It was not possible to establish a THS cut-off point but statistical results showed that, at THS stand view, all groups had similar results despite their EIPH grades. Most animals showed no signs of neutrophilic inflammation, but haemosiderophages were found even on a first-time runner from Group 1. Thus, TW can detect evidence of lung bleeding even on horses with no history of EIPH. The implementation of TW analysis to diagnose EIPH in racehorses is promising, as TW is both low-cost and a less invasive tool., (Copyright © 2022 Elsevier Inc. All rights reserved.)