Diagnostic value of serum amyloid A in differentiating the inflammatory disorders in wild birds.

Rescued wild birds have very high rates of inflammatory diseases; however, there have been limitations in assessing them sensitively. Few studies have examined acute-phase proteins in wild birds. In this study, serum amyloid A (SAA) was evaluated as an inflammatory indicator along with traditional indices such as white blood cell count, albumin, and albumin/globulin ratio. In total, 291 samples from 139 birds of six avian species were analyzed. All samples were divided into four groups (severe, moderate, mild injuries, and clinically healthy) based on clinical examinations and evaluated by group. SAA levels were measured using an anti-chicken SAA ELISA kit and compared with leukocyte counts, albumin concentrations, and albumin/globulin ratios. Differences among groups were evaluated using the Kruskal-Wallis test, followed by a post hoc test using Dunn's multiple comparisons with SPSS V27. Statistical significance was set at a value of p of <0.05. The median concentration and interquartile range (ng/mL) of SAA in each group were 78.10 ng/mL (50.71-109.30), 31.15 ng/mL (19.85-49.24), 9.68 ng/mL (4.71-22.06), and 3.10 ng/mL (1.50-6.00). We observed a significant difference in the mean levels between the groups (p < 0.001), with the same results observed across species. All four indices showed significant differences in mean values between the groups (p < 0.001). In addition, SAA showed rapid changes in periodically collected samples, reflecting either a positive treatment response or the onset of subclinical diseases. SAA can be used to detect inflammatory conditions and asymptomatic disease in wild birds and is helpful in accurately identifying current health status, which is essential for successful treatment and release.

Surveillance of wild animals carrying infectious agents based on high-throughput screening platform in the Republic of Korea.

BACKGROUND: Infectious diseases transmitted by wild animals are major threats to public health. This study aimed to investigate the potential of rescued wild animals that died of unknown causes as reservoirs of infectious agents. From 2018 to 2019, 121 dead wild animals (55 birds and 66 mammals) were included in this study. All wild animals died during treatment after anthropogenic events. After deaths of animals, necropsies were performed and trachea, lungs, large intestine (including stool), and spleen were collected to determine causes of deaths. A high-throughput screening (HTS) quantitative polymerase chain reaction (qPCR) designed to detect 19 pathogens simultaneously against 48 samples in duplicate was performed using nucleic acids extracted from pooled tissues and peripheral blood samples. If positive, singleplex real-time PCR was performed for individual organs or blood samples. RESULTS: The HTS qPCR showed positive results for Campylobacter jejuni (10/121, 8.3%), Campylobacter coli (1/121, 0.8%), Mycoplasma spp. (78/121, 64.5%), and Plasmodium spp. (7/121, 5.7%). Singleplex real-time PCR confirmed that C. jejuni was detected in the large intestine but not in the blood. C. coli was only detected in the large intestine. Mycoplasma spp. were detected in all organs, having the highest proportion in the large intestine and lowest in the blood. Plasmodium spp. was also detected in all organs, with proportions being were similar among organs. CONCLUSIONS: This study shows that wild animals can become carriers of infectious agents without showing any clinical symptoms.

Antioxidant Effects of Myo-Inositol Improve the Function and Fertility of Cryopreserved Boar Semen.

During cryopreservation, sperm undergoes structural and molecular changes such as ice crystal formation, DNA fragmentation, and reactive oxygen species (ROS) production, leading to decreased sperm quality after thawing. Antioxidants play a crucial role in preventing these damages, both in vivo and in vitro. One potent antioxidant is myo-inositol, known for its protective effects on sperm against ROS. This study aimed to investigate the protective effect of myo-inositol on cryopreserved boar semen. The semen was diluted, cooled, and cryopreserved using a BF5 extender. It was then divided into five groups: control and different concentrations of myo-inositol (0.5, 1, 1.5, and 2 mg/mL). The post-thaw evaluation included assessments of motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), caspase activity, gene expression, ROS levels, apoptosis, and IVF with treated semen. Results showed that myo-inositol at 0.5 mg/mL improved motility, acrosome integrity, and fertilization ability. It also reduced the expression of pro-apoptotic genes and increased SMCP expression. Lower concentrations also demonstrated improved viability and reduced apoptosis and ROS levels. In conclusion, myo-inositol treatment during cryopreservation improved sperm quality, reduced apoptosis and ROS levels, and enhanced fertility rates in boar semen.

Application of topical rocuronium bromide dosing by ocular size in four species of wild birds.

BACKGROUND: Rocuronium bromide has been evaluated as a mydriatic agent in birds, but the species applied were limited and the dose and effect were variable. OBJECTIVE: This study aims to evaluate the efficacy of topical rocuronium bromide as mydriatics in 4 species according to horizontal palpebral fissure length: Feral pigeon (Columba livia), Common kestrel (Falco tinnunculus), Northern boobook (Ninox japonica), and Eurasian eagle owl (Bubo bubo). METHODS: A total of 32 birds (8 for each species) were included as pre-releasing examination. Rocuronium bromide was instilled in one randomly selected eye of each bird based on palpebral fissure length criteria (0.5 mg/50 µL for pigeons, 1 mg/100 µL for kestrels and boobook owls, and 2 mg/200 µL for eagle owls). The contralateral eye was used as control and treated with normal saline. After instillation of the drug, pupil diameter, pupillary light reflex, intraocular pressure, heart rate, and respiratory rate were evaluated at 10 min intervals up to 180 min and at 30 min intervals up to 360 min. RESULTS: Statistically significant mydriasis was obtained in all birds (p < 0.001). However, in boobook and eagle owls, marked mydriasis persisted until 360 min. Side effects including corneal erosion and lower eyelid paralysis were common, which was observed in 26/32 birds. Blepharospasm was also noted during this study. No systemic adverse signs were observed. CONCLUSIONS: Rocuronium bromide could be a good mydriatics option for 4 species of birds, however, further studies are needed to find lowest effective dose to reduce drug-related side effects.

Case report: Generalized lymphatic anomaly of multiple abdominal organs in a young dog.

A 9-month-old, female Pomeranian dog presented with vomiting and lethargy. Ultrasonography revealed multilobulated anechoic round shape structures at the ovarian and uterine locations. Through computed tomography scan, an extensive non-contrast multilobulated fluid-filled mass suspected of originating from the walls of the ovary, uterus, urinary bladder and rectum was observed. Ovariohysterectomy and urinary bladder biopsy were performed. Histopathological examination revealed numerous cystic lesions lined by plump cuboidal cells believed to be of epithelial origin. Immunohistochemical staining showed that the cyst-like lesions lining cells were strongly positive for lymphatic vessel endothelial hyaluronan receptor 1. Based on these results, lesions were identified as generalized lymphatic anomaly (GLA), in which lymphangiomas develop in multiple organs. After 6 months follow-up, the size of the cysts remaining in the region of the bladder did not undergo much change. GLA should be included in the differential diagnosis when multiple cystic lesions are interspersed in multiple organs.

The presence of anthracosis is associated with the environmental air quality of zoo, wildlife, and companion animals in Jeollabuk-do Province, South Korea.

OBJECTIVE: To determine pulmonary anthracosis in zoo, wildlife, and companion animals of Jeollabuk-do Province, South Korea. ANIMALS: A total of 350 animals of 61 different species, belonging to 3 classes (mammals: n = 38; avian: 21; and reptiles: 2) from different habitats in Jeollabuk-do Province, were examined. PROCEDURES: Gross lung examination and tissue sampling were done at postmortem, and histopathological analysis was microscopically done on hematoxylin and eosin-stained slides. RESULTS: Macroscopic analysis of anthracotic lung tissue revealed minute (pinpoint size) spots and black pigmentation in a scattered and/or coalescing fashion. The presence of carbon particles was noted in 154 (44%, 154/350) cases. Based on habitation, zoo animals had the highest frequency of anthracosis in the lung (55.2%, 69/125), followed by companion animals (45.2%, 56/124) and wildlife animals (28.7%, 29/101). There was an association between habitation and the presence of anthracosis (P < .05). CLINICAL RELEVANCE: This study revealed evidence that the presence of anthracosis is associated with the environmental air quality of zoo, wildlife, and companion animals in Jeollabuk-do Province, South Korea. Air pollution may affect the respiratory health of the endangered species at the Jeonju Zoo as well as the human population. Continuous monitoring of particulate matter and establishing policies that control industrialization around the province would enable quick action to curb any potential respiratory health risks to animals kept in the urban cities of the province.

Antimicrobial resistance of commensal Escherichia coli and Enterococcus faecalis isolated from clinically healthy captive wild animals in Seoul zoo.

Despite the importance of antimicrobial resistance, only a few studies on the antimicrobial susceptibility on wild animals have been conducted owing to their population, accessibility, and characteristics. The objective of this study was to investigate the prevalence and characteristics of antimicrobial resistance pattern in Escherichia coli and Enterococcus faecalis isolated from the feces of captive wild animals in a zoo. A total of 61 captive wild animals were included in this study. E. coli was isolated from 58 of the 61 animals and E. faecalis was isolated from 29 animals. Among the isolated E. coli strains, ampicillin exhibited the highest resistance rate (27/29, 93.1%). Of these, 18 strains (18/29, 62%) showed multidrug resistance. The multilocus sequence typing (MLST) test showed that only ST155 was detected twice, while the other 16 strains showed different ST types. Among the E. faecalis strains, two were susceptible to all tested antimicrobials, whereas the remaining 27 strains showed resistance to one or more antimicrobials. Nine strains (9/27, 31%) showed multidrug resistance. Among the E. faecalis strains, resistance to quinupristin/dalfopristin was the highest at 96.3% (26/27), while the MLST of the nine MDR strains showed no predominant ST. Genetic association with human isolates or livestock products was observed in the isolated ST types. This indicates that antibiotic resistance in the zoo is responsible for the use of antibiotics and the partial horizontal transmission between humans and animals through feeding or contact.

Application of a modified nictitating membrane flap in a fledgling feral pigeon (Columba livia domestica) with bullous keratopathy.

A fledgling feral pigeon with systemic protozoal infection was referred with corneal protrusion in the right eye after being treated for a corneal ulcer for 12 days. Ophthalmic examination revealed a corneal bulla covering almost the entire central cornea and preventing the eyelids from closing. Gelatinous corneal stroma with numerous heterophils surrounding the corneal bulla was also detected on cytologic examination. Bullous keratopathy and melting keratitis in the right eye were diagnosed. Temporary tarsorrhaphy with topical eye drops was prescribed for a week; however, the bulla persisted. A modified nictitating membrane flap was created under general anesthesia and maintained for 2 weeks. The corneal bulla resolved, and corneal thickness was restored. The pigeon was presented again with recurrence of the corneal bulla in the right eye after 45 days. Alternative surgical options were recommended; however, the pigeon was euthanized because the protozoa-induced intra-oral and intra-aural masses caused malformation of the beaks, which made voluntary feeding impossible.

Distribution and epidemiological relatedness of methicillin-resistant Staphylococcus aureus isolated from companion dogs, owners, and environments.

This study aimed to investigate the distribution and epidemiological relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates from companion dogs, owners, and residential environments of 72 households. Sampling was performed twice from January to June 2018 and a total of 2,592 specimens were collected. The specimens collected from each household were streaked on CHROM agar S. aureus and the colonies grown on the medium were further identified using a mass spectrometry microbial identification system. Antimicrobial susceptibility testing, Panton-Valentine-Leukocidin (PVL) gene PCR, staphylococcal cassette chromosome mec (SCCmec) typing, Staphylococcus aureus Protein A (spa) typing, pulsed-field gel electrophoresis (PFGE), and multi-locus sequence typing (MLST) were conducted to evaluate the phenotypic and genotypic characteristics of the MRSA isolates. A total of 65 S. aureus strains (2.5%) were isolated and 49 (1.9%) of 65 strains were MRSA displaying cefoxitin-resistance with mecA carriage. MRSA strains were isolated from dogs (n=6, 9.2%), owners (n=27, 41.5%), and residential environments (n=16, 24.6%), respectively. Overall prevalence of non-duplicated MRSA was 16.7% (12/72 households) at household level. ST72-SCCmec IVc MRSA clones predominantly appeared in MRSA-positive families. Furthermore, PFGE analyses showed that ST72-SCCmec IVc-t324 is shared between dog owners and dogs. To our knowledge, this is the first study to report the sharing of ST72 MRSA between dogs and their owners.

Identification of Zoonotic Tick-Borne Pathogens from Korean Water Deer (Hydropotes inermis argyropus).

Korean water deer (Hydropotes inermis argyropus) are widespread in the Republic of Korea (ROK). Mostly, Korean water deer are essential hosts for maintaining ticks and tick-borne diseases (TBDs). Here, we investigated the prevalence of tick-borne pathogens (TBPs) among rescued Korean water deer. Anaplasma phagocytophilum (21.4%, 6/28), Anaplasma capra (14.3%, 4/28), Babesia capreoli (3.6%, 1/28), and Coxiella burnetii (3.6%, 1/28) were detected, but Borrelia burgdorferi, Ehrlichia, Rickettsia, and Theileria infections were not found. A. phagocytophilum was the most commonly detected pathogen, and co-infection with A. capra and B. capreoli was also noted in one Korean water deer. To our knowledge, this is the first article of B. capreoli infection in Korean water deer in the ROK. The infecting isolate of A. phagocytophilum was genetically characterized by 16S ribosomal RNA (rRNA) gene and ankyrin-related protein (ankA) gene. Although the 16S rRNA gene alone may not be informative enough to delineate distinct host species, ankA-based phylogeny revealed a high identity of Korean water deer sequences with those of the causative agent of human granulocytic anaplasmosis. A. capra was detected by using citrate synthase gene (gltA), heat-shock protein (groEL), and major surface protein 4 (msp4) genes. Phylogenetic tree based on these gene markers revealed that there were at least two distinct variants within A. capra circulating in the ROK. One variant originated from different hosts including humans, ticks, goats, and sheep, whereas the other variant was reported recently in Korean water deer in the ROK. Consequently, these sequences were identified to belong to a zoonotic species. Sequencing analysis of the 18S rRNA gene revealed that our isolate belonged to B. capreoli and was distinct from Babesia divergens and Babesia venatorum. Moreover, our isolate showed 92.2% homology with B. capreoli sequences, indicating that these differences may be attributed to the different tick species that transmit B. capreoli or to different host species. Genotyping and phylogenetic analysis of C. burnetii based on 16S rRNA and IS1111 genes revealed that our isolate was grouped with several strains of C. burnetii and was genetically distant from Coxiella-like bacteria isolates. The present results highlight that Korean water deer act as potential reservoir hosts for zoonotic TBPs, and thus play an important role in the transmission of TBDs in humans, animals, and livestock.

Biofilm production of coagulase-negative staphylococci isolated from rescued wild animals in the Republic of Korea.

Biofilm production is a well-known causative factor of catheter- and medical device-related sepsis. Its high prevalence in coagulase-negative staphylococci (CoNS) has recently been reported. Information on biofilm production in CoNS isolated from wild animals is lacking. Herein, we studied the biofilm formation capabilities of CoNS isolated from rescued wild animals in the Republic of Korea. Swab samples were collected from the conjunctiva, nasal cavity, perianal area, and rectum for mammals while the sampling was done from the conjunctiva, oral mucosa, pericloacal area, and cloaca for birds. Isolation of CoNS was based on morphological and biochemical analyses along with molecular typing. Biofilm production was analyzed using 96-well plate based quantitative adherence assays. The studies demonstrated that CoNS of mammalian origin have higher biofilm-producing ability (70.4%) than the isolates from birds (62.5%). In particular, all methicillin-resistant (MR) CoNS isolated from mammals were capable of biofilm formation while only 63.3% of MR CoNS isolated from birds could produce biofilms. The MR CoNS isolated from mammals also had a significantly higher ability to form biofilms (100%) than methicillin susceptible CoNS (60.0%) than those isolates from birds. The findings show that wild animals may act as reservoirs as well as possible transmitters of biofilm-mediated antibiotic resistant genes.

Molecular genomic characterization of severe fever with thrombocytopenia syndrome virus isolates from South Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne emerging infectious disease caused by the SFTS virus (SFTSV) and is a threat to public health due to its high fatality rate. However, details on tick-to-human transmission of SFTSV are limited. In this study, we determined the whole-genome sequence of a South Korean SFTSV strain (CUK-JJ01), compared it to those of other recent human SFTSV isolates, and identified the genetic variations and relationships among the SFTSV strains. The genome of CUK-JJ01 was consistent with the genome of other members of the genus Phlebovirus, including the large (L), medium (M), and small (S) segments of 6368, 3378, and 1744 nucleotides, respectively. Based on amino acid sequences of the M and S segments, which are used to distinguish the six SFTSV genotypes, CUK-JJ01 was classified as genotype B. Segment analysis revealed that the L, M, and S segments were 97.49%, 97.18%, and 97.94% similar to those of KAJNH2/2013/Korea, ZJZHSH-FDE/2012/China, and KADGH/2013/Korea, respectively. Currently, only few studies on SFTSV have been conducted in Korean population and most were limited to serological analysis. Although the present study has limitations in terms of number of sample analyzed, the findings may serve as basis to understand the transmission and spread of SFTSV, as well as for the development of diagnostic and detection methods for viral recombinants by comparing the whole genome sequence of SFTSV isolates from South Korea and that of foreign isolates.

Epicoccum nigrum-induced respiratory infection in a wild Eurasian scops owl (Otus scops).

A wild adult Eurasian scops owl (Otus scops), which was unable to fly, was rescued. Physical examination revealed a sticky exudate around the glottis. Heterophilic leukocytosis was identified through complete blood count, and radiography revealed a marked elevated density of posterior air sacs and inner cavities in both sides of the humerus and femur. Fungal cultures of samples taken from the owl suggested a respiratory fungal infection. Through molecular typing, the fungus was identified as Epicoccum nigrum. The owl was treated with oral itraconazole and broad-spectrum antibiotics. After one month, the inner cavities of pneumatic bones were slightly distinguishable by radiography and the owl started to fly well. Two months later, the air sac and all pneumatic bones displayed normal appearance.

Prevalence and genotypes of Enterocytozoon bieneusi in wildlife in Korea: a public health concern.

BACKGROUND: Enterocytozoon bieneusi is a unicellular microsporidian fungal pathogen that infects a broad range of animal hosts, including wild and domestic animals and humans. The infection burden of this parasite in wild animals in Korea is largely unknown. In this study, the occurrence and genotypes of E. bieneusi were investigated in wild animal populations in Korea. METHODS: A total of 157 fecal samples (97 from Korean water deer, 48 from raccoon dogs and 12 from other taxa) were collected from wild animals at five wildlife centers in Korea. Genomic DNA was extracted from the samples and screened by nested-PCR targeting the internal transcribed spacer (ITS) region of rRNA, followed by sequence analysis to determine the genotype(s) of E. bieneusi. RESULTS: The overall prevalence of E. bieneusi was 45.2% (71/157), with rates of 53.6% (52/97) in Korean water deer, 35.4% (17/48) in raccoon dogs and 16.7% (2/12) in other taxa. We detected seven ITS genotypes, including one known (genotype D) and six new genotypes (Korea-WL1-Korea-WL6). Phylogenetically, all detected genotypes clustered with counterparts belonging to group 1, which includes isolates from different animal hosts and humans, suggesting their zoonotic potential. CONCLUSIONS: Our survey results indicate that E. bieneusi circulates widely in wild animals in Korea. These findings address the role of wildlife as a potential source of microsporidiosis in domestic animals and humans.

First Detection of New Delhi Metallo-ß-Lactamase-5-Producing Escherichia coli from Companion Animals in Korea.

The gene for New Delhi metallo-ß-lactamase-5 (NDM-5) in Escherichia coli has been identified in many countries mainly from human clinical specimens. The isolates carrying this gene are even more rarely isolated from companion animals. In this study, four carbapenem-resistant isolates were recovered from four dogs in Korea. All isolates carried blaNDM-5 and exhibited resistance to meropenem and imipenem, and were susceptible to colistin. Epidemiological analysis showed that all four isolates were sequence type 410 (ST410) and shared 99% similarity as determined by pulsed-field gel electrophoresis analysis. Among the four isolates, the Z0117EC0033 strain was randomly selected for whole-genome sequencing, composed of a 4.7Mb circularized chromosome carrying the CMY-2 gene and two plasmids. The first plasmid of the IncFIB type had 83 coding sequences (CDS) in ca. 74 kb. The second smaller plasmid of the IncX3 type had 57 CDS and carried only the blaNDM-5 gene in ca. 46 kb. The plasmid structures were highly similar (> 99%) to those of the NDM-5 human-like IncX3 plasmid. This is the first report of carbapenemase-producing Enterobacteriaceae from companion animals in Korea. The human-like blaNDM-5 IncX3 plasmid identified in this study suggests a potential transmission route of the NDM-5 plasmid between humans and companion animals.

Analysis of Five Arboviruses and Culicoides Distribution on Cattle Farms in Jeollabuk-do, Korea.

Arthropod-borne viruses (Arboviruses) are transmitted by arthropods such as Culicoides biting midges and cause abortion, stillbirth, and congenital malformation in ruminants, apparently leading to economic losses to farmers. To monitor the distribution of Culicoides and to determine their relationship with different environmental conditions (temperature, humidity, wind speed, and altitude of the farms) on 5 cattle farms, Culicoides were collected during summer season (May-September) in 2016 and 2017, and analyzed for identification of species and detection of arboviruses. About 35% of the Culicoides were collected in July and the collection rate increased with increase in temperature and humidity. The higher altitude where the farms were located, the more Culicoides were collected on inside than outside. In antigen test of Culicoides against 5 arboviruses, only Chuzan virus (CHUV) (2.63%) was detected in 2016. The Akabane virus (AKAV), CHUV, Ibaraki virus and Bovine ephemeral fever virus (BEFV) had a positive rate of less than 1.8% in 2017. In antigen test of bovine whole blood, AKAV (12.96%) and BEFV (0.96%) were positive in only one of the farms. As a result of serum neutralization test, antibodies against AKAV were generally measured in all the farms. These results suggest that vaccination before the season in which the Culicoides are active is probably best to prevent arbovirus infections.

PREVALENCE AND PHYLOGENETIC ANALYSIS OF AVIAN HAEMOSPORIDIA IN WILD BIRDS IN THE REPUBLIC OF KOREA.

Avian blood parasites, including Plasmodium spp. and Haemoproteus spp., are found worldwide but only limited information of their occurrence is available in the Republic of Korea (ROK). We determined the prevalence of Plasmodium and Haemoproteus and their phylogenetic characteristics in wild birds in ROK. Blood samples were collected from 118 wild birds of 27 species in the Chonbuk Province, ROK. While 43 (36%) were positive for avian haemosporidia on microscopic examination of blood smears, 53 (45%) were positive by PCR targeting the cytochrome b gene. By direct sequencing of PCR amplicons, 47 (89%) were identified as Haemoproteus spp. and 6 (11%) as Plasmodium spp. Phylogenetic analysis using the cytochrome b gene revealed that resident and migrant birds have very similar genetic lineages of both parasites in ROK, suggesting the possibility that migrant birds may act as a mediator for the parasite among Asian countries.

Sepsis Caused by Streptococcus suis Serotype 2 in a Eurasian River Otter ( Lutra lutra) in the Republic of Korea.

An adult male Eurasian river otter ( Lutra lutra) was diagnosed with systemic infection. Microbiologic findings identified Streptococcus suis serotype 2, clonal complex 28, and sequence type 629. Genetic analysis strongly suggested the transmission of S. suis isolate from pigs to wild animals through environmental contamination.

Polymorphism in the serotonin transporter protein gene in Maltese dogs with degenerative mitral valve disease.

Degenerative mitral valve disease (DMVD) is the most commonly acquired cardiac disease in dogs. This study evaluated the relationship between genetic variations in the serotonin transporter (SERT) gene of Maltese dogs and DMVD. Genomic DNA was extracted from blood samples collected from 20 client-owned DMVD Maltese dogs and 10 healthy control dogs, and each exon of the SERT gene was amplified via polymerase chain reaction. The resulting genetic sequences were aligned and analyzed for variations by comparing with reference sequences; the predicted secondary structures of these variations were modeled and cross-verified by applying computational methods. Genetic variations, including five nonsynonymous genetic variations, were detected in five exons. Protein structure and function of the five nonsynonymous genetic variations were predicted. Three of the five polymorphisms were predicted to be probable causes of damage to protein function and confirmed by protein structure model verification. This study identified six polymorphisms of the SERT gene in Maltese dogs with DMVD, suggesting an association between the SERT gene and canine DMVD. This is the first study of SERT mutation in Maltese dogs with DMVD and is considered a pilot study into clinical genetic examination for early DMVD diagnosis.