Caprine Arthritis-encephalitis Virus Infection Research Articles

Epizootiological serosurvey on small ruminant lentiviruses: Prevalence, age and breed effects

AbstractIn this study, the prevalence of small ruminant lentivirus, i.e. Maedi-Visna and caprine arthritis encephalitis virus infections in sheep and goat populations were investigated through epizootiological serosurveillance in the Aegean region located in western Türkiye in 2018. A total of 1,840 serum samples (595 goats, 1,245 sheep) were collected from 92 villages in the region and examined by indirect ELISA. Thirty-nine goat and 304 sheep sera were found positive for small ruminant lentivirus antibodies. The prevalence was 24.42% in sheep and 6.55% in goats. An average of 18.64% was found in small ruminants in the Aegean region. The prevalence of small ruminant lentiviruses was found to be higher in Pirlak and Pirlak hybrid sheep and in Saanen and Saanen hybrid goats (P < 0.05). The prevalence was found to increase with age, as it was higher in animals older than 48 months than in the age groups of 0–24 or 25–48 months (P < 0.05). Moreover, the small ruminant lentivirus prevalence was higher in sheep than in goats (P < 0.0001). With the epizootiological approach, this study is expected to contribute to the control and prevention of small ruminant lentivirus infections.

A Combined Approach for Detection of Ovine Small Ruminant Retrovirus Co-Infections.

Jaagsiekte retrovirus (JSRV)-induced ovine pulmonary adenocarcinoma (OPA) is an important ovine respiratory disease in Switzerland. Furthermore, ovine lungs with OPA frequently exhibited lesions suggestive of maedi-visna virus (MVV) or caprine arthritis encephalitis virus (CAEV) infection, indicating that co-morbidities might occur. Lungs and pulmonary lymph nodes were sampled from suspected OPA cases, inflammatory lung lesions and control lungs (total of 110 cases). Tissues were (a) processed for histology and immunohistochemistry (IHC), and (b) underwent DNA extraction and real-time PCR for JSRV, MVV and CAEV. Peptide sequences were used to generate virus-specific customized polyclonal antibodies. PCR-positive OPA cases and formalin-fixed and paraffin-embedded MVV- and CAEV-infected synovial cell pellets served as positive controls. Fifty-two lungs were histologically diagnosed with OPA. Histological evidence of MVV/CAEV infection was detected in 25 lungs. JSRV was detected by PCR in 84% of the suspected OPA cases; six were co-infected with MVV and one with CAEV. MVV was detected by PCR in 14 cases, and four lungs were positive for CAEV. Three lungs had MVV/CAEV co-infection. In IHC, JSRV was detected in 91% of the PCR-positive cases, whereas MVV and CAEV immunoreactivity was seen in all PCR-positive lungs. Although PCR showed a higher sensitivity compared to IHC, the combined approach allows for investigations on viral cell tropism and pathogenic processes in co-morbidities, including their potential interdependency. Furthermore, an immunohistochemical tool for specific differentiation of MVV and/or CAEV infection was implemented.

Serological Evidence of Caprine Arthritis Encephalitis in North Shewa Zone, Ethiopia: Clinical Case Analysis.

IntroductionCaprine arthritis encephalitis (CAE) is a chronic debilitating and economically important viral disease of goats. It is mainly manifested as encephalitis in kids and polyarthritis in adult goats. The present study was conducted to determine the rate of morbidity and mortality due to clinical diseases attributed to infection by Caprine arthritis encephalitis virus (CAEV) and to determine the serological status of CAEV in goat in North Shewa, Ethiopia.MethodsA cross-sectional serological study and a longitudinal clinical case study were conducted. A total of 257 serum samples have been collected from apparently health and clinical cases attributed to CAE infection and tested with the usage of indirect enzyme-linked immunosorbent assays to screen antibodies against CAE. Records have been statistically analyzed by using the chi-square test.ResultsDuring five consecutive years of longitudinal clinical study, a total of 195 clinical diseases of chronic pneumonia, nerve problems, clinical mastitis, and arthritis occurred with prevalence of 99 (50.8%), 57 (29.2%), 27 (13.9%), and 12 (6.2%), respectively. Chronic pneumonia was the highest cause of goat morbidity (50.8%) and mortality (100.0%). Of the total samples tested from clinical cases, 7 (58.3%) were sero-positive for the presence of antibodies against CAEV. The overall seroprevalence of CAE was 4.7%. There has been a significant difference (p < 0.05) in sero-positivity among management system, breeds, and age groups of goats. However, there was no significant variation in sero-positivity between the sexes (p > 0.05) of goats.ConclusionThis finding indicates that CAEV infection exists in the goat flocks in examined localities in Ethiopia. This disease poses serious animal health problems that constrain production with the presence of apparent clinical signs. Further investigations need to be done to explore the seroconversion of CAEV in small ruminants and the associated factors to plan an appropriate eradication program and prevent transmission.

Clinical, Pathological and Molecular Findings in Caprine Arthritis – Encephalitis Virus Infection in Damascus Goats

Abstract Caprine Arthritis-Encephalitis Virus Infection was presented with clinical, pathological, and molecular findings in dairy goats from a breeding unit in Turkey. According to history, 50 of 700 goats and kids showed the clinical signs including anorexia, mastitis, swelling of the joints, lameness, dyspnea and head pressing in the past 2 months. Fifteen and 6 animals were examined clinically and pathologically, respectively. Clinical examination revealed depression, weakness, pneumonia, ataxia and paresis in 15 goats. Histological lesions were composed by a varying degree of interstitial pneumonia, nonsuppurative leukoencephalomyelitis, interstitial nephritis and interstitial myocarditis. Immunohistochemical examinations showed the presence of viral antigen in tubular epithelium, in glial cells and macrophages. In conclusion, this is the first report documenting pathological and molecular changes of Caprine Arthritis-Encephalitis Virus Infection in Turkey in goats.

First study on the molecular prevalence of caprine arthritis encephalitis virus in goats in Babylon, Iraq.

Background and Aim:Caprine arthritis encephalitis virus (CAEV) is a virus that affects goats all over the world and causes enormous economic losses; as a result, screening for the disease is a priority, especially in Iraq. The present study aimed to estimate the prevalence of CAEV in infected goats using the précised PCR method in Babylon, Iraq.Materials and Methods:A total of 85 blood samples from goats aged 1 month to ≥6 years were analyzed for CAEV infections using molecular methods. The polymerase chain reaction primer was designed to amplify a 573 bp region of the proviral pol gene.Results:The CAEV tests revealed that five out of 85 goats were positive for CAEV. There were no significant differences in CAEV infection according to goat sex and significant differences according to age.Conclusion:Based on these results, the present study is the first molecular survey to confirm the current CAEV genome in an Iraqi goat flock.

Low colostrum immunoglobulin G concentration in Saanen dams with seropositive to caprine arthritis encephalitis virus infection without affecting kid growth rate

Low colostrum immunoglobulin G concentration in Saanen dams with seropositive to caprine arthritis encephalitis virus infection without affecting kid growth rate

Caprine Arthritis Encephalitis Virus Is Associated with Renal Lesions.

Caprine arthritis encephalitis virus (CAEV) is a monocyte/macrophage-tropic lentivirus that primarily infects goats resulting in a well-recognized set of chronic inflammatory syndromes focused on the joint synovium, tissues of the central nervous system, pulmonary interstitium and mammary gland. Clinically affected animals generally manifest with one or more of these classic CAEV-associated tissue lesions; however, CAEV-associated renal inflammation in goats has not been reported in the peer-reviewed literature. Here we describe six goats with chronic, multisystemic CAEV infections in conjunction with CAEV-associated renal lesions. One of the animals had CAEV antigen-associated thrombotic arteritis resulting in infarction of both the kidney and heart. These goats had microscopic evidence of inflammatory renal injury (interstitial nephritis) with detectable renal immunolabeling for CAEV antigen in three of six animals and amplifiable proviral sequences consistent with CAEV in all six animals. Cardiac lesions (vascular, myocardial or endocardial) were also identified in four of six animals. Within the viral promoter (U3) region, known transcription factor binding sites (TFBSs) were generally conserved, although one viral isolate had a duplication of the U3 A region encoding a second gamma-activated site (GAS). Despite the TFBS conservation, the isolates demonstrated a degree of phylogenetic diversity. At present, the clinical consequence of CAEV-associated renal injury is not clear.

Agreement between ECG values obtained in the sternal recumbent position and right lateral recumbency in goats

The aim of the study was to analyze if body positioning during electrocardiogram recording has an influence on the results in goats. Forty-seven Polish White Improved and Polish Fawn Improved goats from a large dairy herd selected for sanitary culling due to caprine arthritis-encephalitis virus (CAEV) infection, ranging from 2-12 years (median 5.5 years) were included in the study. ECG records were obtained from each goat in the sternal recumbent position and in right lateral recumbency using ASPEL AsCARD Mr. Silver machine with a frequency range of 0.05-150 Hz. The signals from leads I, II, III, aVR, aVL and aVF were recorded for 30 seconds. The mean values of P, QRS, T, PQ, QT, P-R duration (in sec) and P, Q, R, S, T waves amplitude (mV) were measured and analyzed. Values obtained in both positions did not differ significantly.

Study of metalloproteinases in the blood of goats experimentally infected with caprine encephalitis arthritis virus

Caprine arthritis encephalitis is a lentiviral disease that leads to considerable losses in goat farming. In the acute phase of viral infection, though antiviral antibodies are produced by the host’s immune system, they are not sufficient to be detected by serological tests. Acute infections begin with an incubation period, during which the viral genome replicates and host innate responses are initiated. Matrix metalloproteinases (MMPs) are enzymes that play an important role in the physiological and pathological processes of tissue remodeling. The present study aimed to evaluate the expression of MMPs and their activity in the blood serum of goats experimentally infected with caprine arthritis encephalitis virus (CAEV). Five dairy goats, aged 3-4 years, were intravenously inoculated with CAEV Cork strain (titer: 105-6 TCID50/mL) after being tested negative for CAEV thrice at consecutive intervals of 30 days using western blot analysis and nested-PCR. The study included three stages: S1 or pre-infection stage; S2 or seroconversion stage, corresponding to the occurrence of first seroconversion; and S3 or post-seroconversion stage, corresponding to 23 weeks after seroconversion. Zymography was performed for the samples using gelatin zymography gels (12.5%), which were subjected to electrophoresis at 170V, 1A, and 300W for 50-70 min. The density of MMP-2 was found to be lower at S1 (1456.20 pixels) than that at S2 and S3 (1943.80 and 2104.40 pixels, respectively) (P &lt; 0.05); and the density of MMP-9 was found to be lower at S3 (133.60 pixels) than that at S1 and S2 (359.60 and 370.60 pixels, respectively). The density of proMMP-2 was low at S1 and S3 (130.45 and 145.20 pixels, respectively). On the other hand, the density of proMMP-9 was statistically different between S1 and S3 (89.22 vs. 415.60 pixels). Both proMMP-2 and proMMP-9 were absent at S2. Thus, MMP-2 and MMP-9 exhibited opposite behaviors depending on the stage of infection. As the greatest activity of MMP-2 was detected at stage S3, we suggest that MMP-2 can be used as a biomarker for complementary diagnosis of acute CAEV infection. In addition, the presence of proMMP-13 can be used to indicate active viral infection.

The Vif protein of caprine arthritis encephalitis virus inhibits interferon production.

Caprine arthritis-encephalitis (CAE) is a chronic progressive infectious disease caused by caprine arthritis-encephalitis virus (CAEV) that seriously threatens the goat industry. Chronic infection and life-long multi-tissue inflammation are the typical features of the disease. Innate antiviral immunity is essential for the host defense system that rapidly recognizes and eliminates invading viruses. Interferon β (IFN-β) is important for innate immunity and regulates immunity against a broad spectrum of viruses. To investigate the details of the IFN-β response to CAEV infection, the effects of six viral proteins and the molecular mechanisms by which they affect IFN-β production were analyzed. Overexpression of DU and Vif promote virus proliferation and inhibit the production of IFN-β. qRT-PCR and luciferase reporter assays showed that overexpression of Vif inhibits the expression of luciferase under the control of the ISRE, NF-κB or IFN-β promoter but does not affect the expression of IFN-β activated by IRF3, indicating that Vif negatively regulates IFN-β production by affecting upstream signal transduction of IRF3. Amino acids 149-164 of Vif were found to be necessary for the inhibitory effect of IFN-β production. Our results indicate that CAEV evades surveillance and clearance by intracellular innate immunity by downregulating IFN-β production.

Seroprevalence of small ruminant caprine arthritis encephalitis lentivirus among goats from selected small ruminant farms in Selangor, Malaysia.

Background and Aim:Caprine arthritis encephalitis (CAE) is an important viral disease of small ruminants particularly in dairy goats with severe social and economic implication. Hence, this study was designed to determine the seroprevalence of CAE virus (CAEV) among goat population in selected small ruminant farms in Selangor and the risk factors associated with the occurrence of the disease.Materials and Methods:Blood samples were collected from a total of 91 goats selected at random. Blood serum was harvested and used for competitive enzyme-linked immunosorbent assay test to detect antibodies against CAE virus.Results:The result obtained showed that 8/91 (8.8%) of the goats were seropositive for CAEV. In addition, biosecurity management, source of origin and sex of the animal were observed to be important risk factors associated with the occurrence of CAE in goats.Conclusion:The findings of this study affirmed that the seroprevalence of CAEV infection among goat population in small ruminant farms in Selangor, Malaysia, is low. However, there is need to institute strict control measures such as testing and culling positive animals or separation of infected animals from those that tested negative to the disease for effective eradication of the disease.

Prevalence of CAEV infections in goat herds

The aim of the study was to analyze the occurrence of caprine arthritis encephalitis virus (CAEV) in white improved goats, colored improved goats, Saanen goats, Alpine goats and Boer goats, raised in three regions of Poland. The analysis of CAEV infection levels was carried out in goat herds from the provinces in mid-Eastern Poland. The analysis included four goat herds. In total, they encompassed 251 herds of the following breeds: white improved goat, colored improved goat, Saanen goat, Alpine goat and Boer goat. The identification of the infected animals and the assessment of the epidemiological condition of the herds was carried out using serological analysis of blood serum samples. The assay of CAEV-specific antibodies in blood serum was performed using ELISA test. As a result of the observations it was established that with regards to the provinces, the prevalence level ranged from 35.40% in Mazowieckie province to 81.10% in Lubelskie province, and the average infection rate of animals in the studied region was 51.60%. As far as the breeds are concerned, it was found that the observed infection rate of the Saanen goat population was significantly higher (p ≤ 0.05) than the rates for improved white goat (48.90%) and Alpine goat (50.00%). In relation to the colored improved goat, the calculated statistical difference was greater (p ≤ 0.01), and the percentage of infected animals was 32.10%. The analysis of the infection rate in the Boer goat herd revealed that 66.70% of animals are seropositive, and the statistical analysis did not show any significant differences in prevalence compared to other studied breeds. To sum up, it should be noted that the improvement of the situation may be attributed to consistent examination of goats and development of CAEV-elimination programs for herds. When creating programs aimed at the elimination of the virus it is also important to remember to raise the awareness of the farmers with regards to the detrimental effect of CAEV infections..

Blood and milk polymorphonuclear leukocyte and monocyte/macrophage functions in naturally caprine arthritis encephalitis virus infection in dairy goats

The exact influence of caprine arthritis encephalitis virus (CAEV) infection on blood and milk polymorphonuclear leukocytes (PMNLs) and monocyte/macrophages of goats remains unclear. Thus, the present study sought to explore the blood and milk PMNL and monocyte/macrophage functions in naturally CAEV-infected goats. The present study used 18 healthy Saanen goats that were segregated according to sera test outcomes into serologically CAEV negative (n=8; 14 halves) and positive (n=10; 14 halves) groups. All milk samples from mammary halves with milk bacteriologically positive outcomes, somatic cell count ≥2×106cellsmL−1, and abnormal secretions in the strip cup test were excluded. We evaluated the percentage of blood and milk PMNLs and monocyte/macrophages, the viability of PMNLs and monocyte/macrophages, the levels of intracellular reactive oxygen species (ROS) and the nonopsonized phagocytosis of Staphylococcus aureus and Escherichia coli by flow cytometry. In the present study, a higher percentage of milk macrophages (CD14+) and milk polymorphonuclear leukocytes undergoing late apoptosis or necrosis (Annexin-V+/Propidium iodide+) was observed in CAEV-infected goats; we did not find any further alterations in blood and milk PMNL and monocyte/macrophage functions. Thus, regarding our results, the goats naturally infected with CAEV did not reveal pronounced dysfunctions in blood and milk polymorphonuclear leukocytes and monocytes/macrophages.

Diagnosis of Caprine Arthritis Encephalitis Virus infection in dairy goats by ELISA, PCR and Viral Culture.

For preventive and control strategies of Caprine Arthritis Encephalitis Virus (CAEV) infection in dairy goats, performance of the available diagnostic tests was described as one of the most important and necessary aspects. The study aimed at evaluating the diagnostic test performance, including PCR, ELISA and viral culture, for CAEV infection in dairy goats in Thailand. Blood samples of 29 dairy goats from five low- to medium-prevalence herds and one very low-prevalence herd were collected for PCR and ELISA methods. The performance of these two diagnostic methods was evaluated by comparing with cytopathic effects (CPE) in the co-cultivation of CAEV and primary synovial cells. Results indicated that sensitivity, specificity were, respectively, 69.6%, 100%, for PCR; and 95.7%, 83.3% for ELISA. The PCR assay tended to have lower sensitivity and higher specificity than ELISA. When multiple tests were applied, parallel testing provided sensitivity and specificity of 98.7% and 83.3%, while series testing showed sensitivity and specificity of 66.6% and 100% respectively. These results indicated that combination of ELISA and PCR provided some advantages and possibly offered optimal methods to detect CAEV-infected goats. Kappa value of the agreement between PCR and ELISA test was 0.34, indicating fair agreement. Regarding the possibility of antigenic variation between CAEV strains used in both PCR and ELISA assays, the actual circulating CAEV strain should be reviewed in order to develop and enhance the diagnostic tests using the CAE viral antigens derived from specific local strains of Thailand.

Influence of Caprine Arthritis Encephalitis Virus infection on milk production of French Alpine goats in Croatia

We have investigated the influence of infection with Caprine Arthritis Encephalitis Virus (CAEV), developed clinical arthritis and recorded subclinical mastitis on the length of lactation, milk yield and its composition per lactation. The study was carried out on the sample of 808 goats of French Alpine breed from production farms in Croatia. Samples of blood were taken for serological tests and milk samples for subclinical mastitis detection. Presence of clinical arthritis was noted by inspection and palpation of the joints. Lactation and production data were collected by Croatian Agricultural Agency. The influence of infection status for CAEV and clinical arthritis, separately, were tested on milk yield, length of lactation and milk composition (fat, protein and lactose) using GLM procedure for models including the year of measurement, lactation number of the animal and subclinical mastitis record. CAEV was serologically confirmed in 53.72 % of the total number of goats. From the total sample 23.08 % were infected with CAEV with confirmed subclinical mastitis of bacteriological etiology. In addition, 22.47 % of the total sample had clinical arthritis. Of the total sample 16.95 % were CAEV infected and with diagnosed clinical arthritis. Influence of virus infection was confirmed on all of the analyzed traits, with lower production results. Clinical arthritis had significant influence on decrease in most of analyzed traits, exceptions being total milk, fat amount (%) and lactose content (%) and amount (kg). Diagnosed subclinical mastitis was not found to be significant influence in models of duration of lactation, or fat, protein and lactose content (%).

Development of a recombinase polymerase amplification lateral flow dipstick (RPA-LFD) for the field diagnosis of caprine arthritis-encephalitis virus (CAEV) infection

Caprine arthritis-encephalitis (CAE) in goats is a complex disease syndrome caused by a lentivirus. This persistent viral infection results in arthritis in adult goats and encephalitis in lambs. The prognosis for the encephalitic form is normally poor, and this form of the disease has caused substantial economic losses for goat farmers. Hence, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed in the present study for detecting the proviral DNA of caprine arthritis-encephalitis virus (CAEV). Under the optimal incubation conditions, specifically, 30min at 37°C for RPA followed by 5min at room temperature for LFD, the assay was found to be sensitive to a lower limit of 80pg of total DNA and 10 copies of plasmid DNA. Furthermore, there was no cross-reaction with other tested viruses, including goat pox virus and bovine leukemia virus. Given its simplicity and portability, this RPA-LFD protocol can serve as an alternative tool to ELISA for the primary screening of CAEV, one that is suitable for both laboratory and field application. When the RPA-LFD was applied in parallel with serological ELISA for the detection of CAEV in field samples, the RPA-LFD assay exhibited a higher sensitivity than the traditional method, and 82% of the 200 samples collected in Taiwan were found to be positive. To our knowledge, this is the first report providing evidence to support the use of an RPA-LFD assay as a specific and sensitive platform for detecting CAEV proviral DNA in goats in a faster manner, one that is also applicable for on-site utilization at farms and that should be useful in both eradication programs and epidemiological studies.

Seroprevalence and risk factors of exposure to caprine arthritis-encephalitis virus in southern Spain

The aim of the present transversal descriptive study was to determine the exposure and risk factors associated with caprine arthritis-encephalitis virus (CAEV) in southern Spain. A total of 3,312 serum...

First isolation and nucleotide comparison of the gag gene of the caprine arthritis encephalitis virus circulating in naturally infected goats from Argentina.

Caprine arthritis encephalitis virus (CAEV) has been reported in different countries worldwide, based on serological and molecular detection. In Argentina, the prevalence of CAEV infections is increasing, with goats showing symptoms associated mostly with cachexia and arthritis. Although in Argentina the virus has been detected by serology, it has never been isolated or characterized. Thus, the objectives of this work were to isolate and analyze the nucleotide sequences of the gag gene of Argentine CAEV strains and compare them with those of other SRLVs previously reported. Nucleotide sequence comparison showed homology with CAEV-Co, the CAEV prototype. Phylogenetic analyses showed that the Argentine strains clustered with genotype B, subtype B1. Because the molecular characterization of the gag region is suitable for phylogenetic studies and may be applied to monitor the control of SRLV, molecularly characterizing the Argentine CAEV strains may help develop a proper plan of eradication of CAEV infections.

A census to determine the prevalence and risk factors for caprine arthritis-encephalitis virus and visna/maedi virus in the Swiss goat population

In Switzerland, viruses belonging to two different phylogenetic groups of small ruminant lentiviruses (SRLV) are currently circulating: the caprine arthritis-encephalitis virus (CAEV) and visna/maedi virus (VMV). In the past two decades, a mandatory national control program has led to a very low prevalence of seropositivity, while completely eliminating CAE as a clinical manifestation. However, in order to reduce the high costs and effort associated with this program, adjustments based on the most recent epidemiological knowledge are needed. The purpose of this study was to estimate the seroprevalence of CAEV and VMV using the newest diagnostic tools available, and to identify potential risk factors for infection with these viruses in Switzerland. For the prevalence estimation, a census was carried out including 10,696 farms with a total of 85,454 goats. Blood samples were analysed using a 3-step serological testing algorithm consisting of Chekit ELISA, Western Blot and SU5 ELISA. A risk factor analysis was conducted using logistic regression models built with data obtained from a mail questionnaire, and serological results from the census. The apparent herd-level prevalences were 0.38%, 2.77%, and 3.04% for CAEV, VMV and SRLV, respectively. Animal-level prevalences were 0.06% for CAEV, 0.55% for VMV, and 0.61% for SRLV. No statistically significant risk factors associated with CAEV or VMV infection were identified. However, the proportional high number of CAEV seropositive dwarf goats, in relation to their population size, could indicate that these hobby breeds may slip through some of the official controls. For an infection with SRLV, a medium herd size (7–40 goats) was found to be protective, compared with smaller (OR=1.90, p=0.034) and larger herds (OR=1.95, p=0.038). In conclusion, considering that all CAEV positive animals were culled, these results imply that CAEV is no longer actively spreading and has successfully been controlled in Switzerland. However, given the uncertain pathogenic potential of VMV in goats, future surveillance should also be taking into account the not insignificant number of VMV circulating in the Swiss goat population.

High prevalence of caprine arthritis encephalitis virus (CAEV) in Taiwan revealed by large-scale serological survey.

In this study, a large-scale serological survey of caprine arthritis encephalitis virus (CAEV) infection was conducted between March 2011 and October 2012. 3,437 goat blood or milk samples were collected from 65 goat farms throughout Taiwan. A commercial ELISA kit was used to detect antibodies against CAEV. The overall seropositive rate was 61.7% (2,120/3,437) in goats and in 98.5% (64/65) of goat farms. These results provide the first large-scale serological evidence for the presence of CAEV infection, indicating that the disease is widespread in Taiwan.